Mammalian Subventricular Zones: Their Roles in Brain Development, Cell Replacement, and Disease

Mammalian Subventricular Zones: Their Roles in Brain Development, Cell Replacement and Disease Mammalian Subventricul...

Author: Steve W. Levison

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Mammalian Subventricular Zones: Their Roles in Brain Development, Cell Replacement and Disease

Mammalian Subventricular Zones: Their Roles in Brain Development, Cell Replacement and Disease

Edited by

Steven W. Levison UMDNJ-New Jersey Medical School Newark, NJ, USA

Library of Congress Control Number: 2005926333 ISBN-10: 0-387-26067-6 (Hardbound) ISBN-13: 978-0387-26067-9

Printed on acid-free paper.

ß2006 Springer ScienceþBusiness Media, Inc. All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer ScienceþBusiness Media, Inc. 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks and similar terms, even if they are not identiWed as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed in the United States of America 10 9

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Contents Synopsis: The mammalian subventricular zone is spatially restricted site in the brain that harbors neural stem cells and other immature neural precursors. This book will provide in depth reviews of the properties of SVZ cells, how they participate in neural development, and how they are affected by and respond to brain diseases or brain injuries. Chapter 1. Cellular Heterogeneity of the Neonatal SVZ and its Contributions to Forebrain Neurogenesis and Gliogenesis Steven W. Levison and James E. Goldman Chapter 2. Extrinsic and Intrinsic Factors Modulating Proliferation and Self-renewal of Multipotential CNS Progenitors and Adult Neural Stem Cells of the Subventricular Zone. Sara Gil-Perotin and Patrizia Casaccia-Bonnefil Chapter 3. Birth, Migration and Function of SVZ-Derived Neurons in the Adult Brain Minoree Kohwi, Rui Pedro Galva˜o and Arturo Alvarez-Buylla Chapter 4. Contributions of the Neocortical SVZ to Human Brain Development Nada Zecevic, Sonja Rakic, Igor Jakovcevski, Radmila Filipovic Chapter 5. Responses of the Adult SVZ to Neuronal Death and Injury Jason G. Emsley and Jeff D. Macklis Chapter 6. Responses of the SVZ to Radiation and Chemotherapy Ami M. Karkar, Radoslaw Rola and John R. Fike Chapter 7. The Subventricular Zone Responds Dynamically to Mechanical Brain Injuries Maria L.V. Dizon and Francis G. Szele

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Chapter 8. Responses of the SVZ to Hypoxia and Hypoxia/Ischemia 242 Ryan J. Felling, H. VanGuilder, Michael J. Romanko and Steven W. Levison Chapter 9. Responses of the SVZ to Demyelinating Diseases B. Nait-Oumesmar, L. Decker, N. Picard-Riera and A. Baron-van Evercooren Chapter 10. Auxiliary Proliferative Zones in the Developing and Adult Central Nervous System: Lessons From Studies on the Effects of Ethanol Michael W. Miller and Marla B. Bruns Index

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Chapter 1 Cellular Heterogeneity of the Neonatal SVZ and its Contributions to Forebrain Neurogenesis and Gliogenesis Steven W. Levison and James E. Goldman

Introduction The vast majority of the cells that comprise the telencephalon are generated from precursors that reside within two germinal regions, the ventricular zone (VZ) and the subventricular zone (SVZ). These regions have been classically defined based upon distinctions in the morphologies of the cells that comprise them. In addition, they produce distinct progeny. Whereas developmental neurobiologists have extensively studied the VZ, it is only within the last 2 decades that the roles of SVZ cells, in both development and cell replacement, have been elucidated. In this chapter, we will contrast the cells of the subventricular zones with those of the ventricular zone, describe the spatiotemporal origins of perinatal dorsolateral SVZ (SVZDL ) cells, and finally review the contributions of perinatal SVZ cells to neurogenesis and gliogenesis, citing studies largely performed on rodents.

The Ventricular vs the Subventricular Zone The cells of the VZ are direct descendants of the primitive neuroectoderm of the neural plate. Like the cells of that neuroepithelium, the cells of the early VZ extend processes across the width of the developing central nervous system. As first described by Sauer (1935), their nuclei undergo interkinetic movements in accordance with cell cycle progression. Thus, at any given time, the nuclei of VZ cells are distributed at different levels within the VZ, and consequently, the ventricular zone is described as a pseudostratified epithelium. The first mature cells produced by the pallial VZ are deeplayer neurons. These cells leave the VZ to migrate apically towards the pial surface using radial glia as their guides (Rakic, 1971; Misson et al., 1991). These cells settle in specific cortical laminae as dictated by their birth date 1

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with earlier-born neurons generally settling into the deeper laminae and later-born neurons migrating past them to colonize more superficial layers (Angevine and Sidman, 1961). In this manner, the layers of the neocortex are established in an inside-out pattern. While this process has been extensively studied, as reviewed in many articles and book chapters (For review see Bayer and Altman, 1991; McConnell, 1991), one important new finding is that radial glia divide in a self-renewing manner and are capable of producing both neurons and astrocytes; hence, they fulfill the criteria of bipotential neural stem cells (Malatesta et al., 2000; Hartfuss et al., 2001; Noctor et al., 2001; Campbell and Gotz, 2002). Not long after the first neurons emerge from the ventricular zone, a second proliferative population becomes discernable at the border between the VZ and the cell-sparse intermediate zone. Whereas the proliferative fraction and prominence of the VZ declines rapidly, the SVZ cell population expands exponentially during the latter third of prenatal development. For instance, in the E16 mouse, over 90% of the SVZ cells are dividing, whereas the majority of the cells in the VZ are leaving the cell cycle (Takahashi et al., 1995). Thus, as the VZ disappears, the SVZ achieves prominence, peaking in size during early postnatal development (Lewis and Lai, 1974; Bayer and Altman, 1991). This secondary proliferative population is referred to by many names in the literature including the subependymal plate, subependymal layer, subependymal zone and the subventricular zone. In this chapter, we refer to both the prenatal and postnatal equivalents of this secondary proliferative population as the subventricular zone. The postnatal equivalent of the SVZ may be referred to as a subependymal zone since these cells are subjacent to the ependymal cell layer. However, as it appears that some VZ cells persist into postnatal development, it seems appropriate to retain the SVZ designation for these cells (Staugaitis et al., 2001). SVZ cells are morphologically distinct from VZ cells, mature neurons and glia. In the following pages we will review in greater detail the types of cells in the SVZ, but by comparison with VZ cells, the SVZ is a region comprising mostly small cells with a layered or stratified organization (His, 1904; Bayer and Altman, 1991). These small cells are highly migratory and usually possess a single, leading process, not necessarily oriented perpendicular to the pial surface. Unlike VZ cells, SVZ cells show no evidence of interkinetic nuclear movements during the cell cycle (Takahashi et al., 1995). As discussed above, the number of cells in the SVZ peaks during the first week after birth in rodents after which the SVZ begins to decrease in size (Thomaidou et al., 1997). However, in both humans and rodents (and probably in all mammals), a residual, mitotically active SVZ persists into adulthood, as will be discussed at greater length later in other chapters in this book.

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Embryonic Origins of SVZ Cells A Subset of SVZ Cells Arise from the Dorsolateral Neuroepithelium of the Telencephalon As a secondary proliferative zone, the SVZ is a relatively late-appearing germinal zone of the developing brain. Surprisingly, there remains no consensus as to where these cells originate. Since the early studies conducted by Privat and Leblond (1972), we have known that the SVZ contains at least two populations of cells. Privat and Leblond characterized one population that stained lightly and contained large oval nuclei, and a second population of cells that had smaller, round nuclei that stained darkly. They surmised that these two cell types had separate origins, but had no means to test their hypothesis. More recently, studies using transgenic mice support separate origins for these two cell populations with one set of SVZ cells being formed from the neuroepithelial cells that surround the ventricles and the other set of SVZ cells being derived from at least 2 other structures. The population of large cells in the SVZ appears to be derived from the dorso-lateral neuroepithelium (Staugaitis et al., 2001). Beginning around E14, the lateral neuroepithelium of the lateral ventricle pinches together along the corticostriatal boundary. As a result, a wedge-shaped region of neuroepithelial cells, identified by their expression of Zebrin II (AldolaseC), is created at the dorsolateral aspect of the lateral ventricle (Staugaitis et al., 2001; Marshall and Goldman, 2002). These Zebrin II-expressing cells possess fine processes and resemble the light-nucleated cells previously described, (Smart, 1961; Privat and Leblond, 1972). They create the dorsal border of the triangular prominence that is the dorsolateral SVZ (SVZDL ) (Figs. 1.1 and 1.2).

A Second Subset of SVZ Cells Arises from the Medial Ganglionic Eminence The medial ganglionic eminence is a subpallial aggregation of SVZ cells that strongly express the distal-less transcription factors (Dlx). Marshall and Goldman (2002) used mice in which the LacZ gene was knocked into the Dlx2 locus in order to test the hypothesis that subpallial-derived Dlx2-expressing cells migrate dorsally to colonize the SVZDL . They found that beginning around E19 in the mouse, cells that are descendants of the MGE and that express Dlx2, migrate dorsally into the wedge- shaped region created by adjacent Zebrin IIþ cells. These immigrants take up residence and proliferate within the mediolateral region of the SVZ. Consequently, the perinatal SVZDL can be described as a shell of dorsolateral VZ derivatives that express Zebrin II, with a core of ventrally derived cells (Marshall and Goldman, 2002).

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Figure 1.1. Formation of the SVZDL . At E10, Zebrin II is expressed by all cells of the telencephalic primordium. (A) At E14, A ZebrinIIþ wedge emerges during corticostriatal sulcus formation. (B) At E16 the Zebrin IIþ wedge is fenestrated by Dlx2þ or PSA-NCAMþ migratory, subpallium-derived cells. PSA-NCAMþ cells are the progeny of Dlx2þ cells. (C) At P0, the total population of Dlx2-expressing cells and their PSA-NCAMþ progeny can be defined by Dlx2/tauLacZ (b-gal) expression. Subpallium derived cells defined by Dlx2/tauLacZ (b-gal) expression form the central region of the perinatal SVZ. (D) At P10 the postnatal SVZ can be divided into two subpopulations of cells based on the expression of Zebrin II or the Dlx2/tauLacZ reporter. Zebrin IIþ residual VZ cells form the outer borders of the SVZ. Cells of subpallial origin, as defined by Dlx2/tauLacZ (b-gal) expression, populate the central SVZ and give rise to astrocytes and oligodendrocytes in the striatum, white matter, and cerebral cortex and olfactory bub neurons. Adapted from Marshall and Goldman, 2002. Copyright (2003) Elsevier Science Ltd.

A Third Subset of SVZ Cells is Derived from the Lateral Ganglionic Eminence The lateral ganglionic eminence (LGE) is another aggregation of SVZ cells that lies along the lateral wall of the ventricle, dorsal to the MGE in the fetal

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Figure 1.2. Zebrin II in the P0 forebrain. Panel A depicts non-radioactive in situ hybridization for Zebrin II of a neonatal SVZ in coronal section, showing intense expression next to the ventricle and in dense patches in the lateral angle. Panels B–F depicts immunofluorescence staining for Zebrin II, reflecting the ISH expression pattern very well. Panel C is immunofluorescence showing cells at the ventricular surface and immediate subventricular area. Panel D is immunofluorescence of cells in the lateral angle of the SVZ. Cells all display a large polygonal to round cell body, and usually a few thin processes. Scale bar, A, 175 mm; B 100 mm; C, D, 10 mm, E, F 200 mm. Adapted from Staugatis et al., 2001.

brain. These cells also express DLX transcription factors. Stenman et al. (2003) examined the expression of several transcription factors within the LGE. They found that the fetal LGE could be subdivided into two regions, a more ventral region that expressed the islet-1 transcription factor and a more dorsal domain that expressed the Er81 transcription factor. These regions appear around by E12.5 and are still recognizable at E16.5, but by birth the population of Islet-1 positive cells within the SVZ has dwindled to a very small percentage. These islet-1 expressing cells are precursors for the projection neurons of the striatum and the reduction in their numbers coincides with the completion of striatal neurogenesis. However, some islet-1 positive cells persist in the perinatal SVZ (Stenman et al., 2003). Whereas the islet-1 population of LGE derived SVZ cells decreases as the brain matures, the Er81þ population persists. Dlx-2þ/Er81þ cells, are prominent within the perinatal SVZDL, comprise a subset of the Dlxþ cell population. Er81þ cells also can be found within interneurons of the olfactory bulb. Further in mice with decreased expression of Er81, there is a corresponding decrease in olfactory bulb interneurons, supporting the conclusion that these Er81þ LGE derivatives are the source of the neuroblasts required for the majority of olfactory bulb interneurons (Stenman et al., 2003).

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A Fourth Subset of SVZ Cells Arise from the Neocortical VZ Another subset of mediolateral SVZDL cells is derived from the neocortical VZ at mid-gestation. As reviewed above, at E14, the neuroepithelium of the VZ creates the triangular prominence of the SVZDL and these lightly staining cells surround the SVZ. Subsequently, these cells are infiltrated by Dlx1/2þ immigrants from the MGE and LGE. But there is evidence for a fourth source of SVZ cells. Studies using replication-deficient retroviruses provide direct evidence that cells in the embryonic neocortical ventricular zone produce SVZDL cells. Injection of retrovirus at E17 initially labels VZ cells and within one day of infection, radially oriented clones of cells spanning both the VZ and the SVZ appear, indicating that cells within these clones originated in the VZ and proliferated within the SVZDL (Acklin and Van der Kooy, 1993). Using time lapse video microscopy of the SVZ, Noctor has provided direct evidence that VZ cells undergo their terminal division with the SVZ (Noctor et al., 2004). These cells clearly stop transiently within the SVZ, and they proliferate within the SVZ, but it remains to be established whether any of these cells colonize the SVZ. The SVZDL is an important source of oligodendrocyte progenitors and there is compelling evidence that there are atleast 2 sources of the oligodendrocyte progenitors within the SVZ. In vitro experiments have shown that murine E10 neocortical VZ cells grown in low concentrations of FGF-2 generate oligodendrocytes (Qian et al., 1997). As the ganglionic eminences have not yet been formed in the E10 mouse, these data suggest that cells originating in the VZ can populate the neocortical SVZDL with oligodendrocyte precursors. Supporting this interpretation, two groups have shown that oligodendrocytes can be generated from neocortical precursors via a sonic hedgehog (SHH) independent pathway. As ganglionic eminence-derived oligodendrocytes require SHH for their development, these studies suggest an alternative source for a subset of oligodendrocyte progenitors in the SVZ (Spassky et al., 2001; Chandran et al., 2003; Kessaris et al., 2004). Additional data to support this view comes from the work of Gorski et al. (2002), who used an Emx1-Cre transgene to activate a conditional lacZ reporter. The Emx1 transcription factor encodes a homeodomain protein whose expression is primarily restricted to cortical subdivisions of the telencephalon, including the SVZ, and it is not detectable in the ventral pallium during early stages of development, They found LacZ expression in cells in the white matter with the morphology of oligodendrocytes. Moreover, these cells stained using the Rip monoclonal antibody, which stains oligodendrocytes and myelin. Assuming that EMX-1 is not acquired by cells that migrate ventro-dorsally, these observations argue for a dorsal derivation of some oligodendrocytes. Thus, these data support at least two origins of the precursors in the SVZDL that can generate oligodendrocyte progenitors; the subpallial derived Dlx-2þ cells and pallial EMX-1þ cells.

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Finally, the SVZ in the human brain is quite large (as documented in Chapter 4 by Nada Zecevic and colleagues) and a significant proportion of SVZ cells in the human forebrain appear to be descendants of pallial VZ cells. While none of these studies provide conclusive evidence, cumulatively these studies provides data to support the view that a proportion of the small migratory progenitors within the SVZ are derived directly from the telencephalic VZ. Whereas there exist elegant studies documenting the production of oligodendrocytes from cells of the entopeduncular region (Spassky et al., 2000), there is no evidence that entopeduncular cells migrate dorsally to seed the SVZ despite attempts to visualize such migration (B. Zalc, personal communication).

The Perinatal SVZ Produces Subsets of Neurons Migration Paths Taken by SVZ Neuroblasts In a classic study by Altman and Das (1966), newborn rats were administered 3 H-thymidine at different time points postnatally in order to identify regions of proliferating cells and also to reveal cell migrations. Between 3 and 6 days after labeling, the percentage of labeled cells in the SVZ did not change. However, beyond 6 days, the number of labeled cells in the SVZ decreased. As the number of labeled cells in the SVZ decreased, the number of labeled cells in the olfactory bulb increased. Altman and Das realized that some of the cell types present in the adult olfactory bulb had not yet formed, specifically the periglomerular and granular cells; therefore, they hypothesized that proliferating cells in the SVZ were migrating to the olfactory bulb where they were becoming periglomerular and granule cells. As will be discussed in greater detail below, more recent studies in which higher spatial resolution can be achieved by infecting SVZ cells with low titers of replication deficient retroviruses confirm that cells stream forward from the SVZ to the olfactory bulb. Furthermore, by combining retroviral labeling with immunofluorescence, the identity of these postnatally generated neurons has been established.

Mechanisms of SVZ Neuroblast Migration SVZ cells possess high levels of PSA-NCAM, a cell surface molecule of the IgG superfamily that facilitates association of adjacent cells expressing this adhesion protein (Edelman and Rutishauser, 1981; Rousselot et al., 1995). The adult rodent brain contains a pathway of PSA-NCAMþ cells that connect the SVZ with the core of the olfactory bulb, a pathway known as the rostral migratory stream (RMS). The cells in the RMS appear in long chains, their processes oriented parallel to the pathway. 3 H-thymidine and BrdU labeling reveals that these PSA-NCAMþ cells are proliferating in the

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RMS. In contrast to migrating cells elsewhere in the forebrain, markers for radial glial cells are not found in the RMS and there are no axons in the RMS. These observations suggest that the chains of cells migrating through the RMS are not guided by radial glia or axons. However, staining for GFAP reveals a meshwork of GFAP positive cells surrounding the chains of PSA-NCAMþ cells. Therefore, neuroblasts migrating from the SVZ to the olfactory bulb do so through the RMS, where flanking GFAP positive cells provide a ‘‘tunnel’’ in which PSA-NCAM positive cells move along one another to reach their target (Fig. 1.3). This glial border also prevents migrating cells from straying outside the RMS. The migration of RMS cells, termed ‘‘chain migration’’, appears to be intrinsic, because isolated SVZ cells will aggregate and migrate in series with each other in a Petri dish (Wichterle et al., 1997).

SVZ Neuroblasts Contribute Interneurons to the Olfactory Bulb Studies conducted on the neonatal rat firmly establish that the SVZ contributes glomerular and periglomerular neurons to the olfactory bulb. When retroviruses are injected into the SVZ of developing rats, the infected cells migrate anteriorly through the RMS. After arriving at the olfactory bulb they disperse radially (Luskin, 1993; Lois et al., 1996) and settle in the periglomerular and granule cell layers (Luskin, 1993). These retrovirally labeled cells develop the morphologies of periglomerular and granule interneurons and express TUJ1 and MAP-2. Additional studies confirm that they

Figure 1.3. Migration of cells from the SVZa in the Rostral Migratory Stream. (A) The path of neuroblasts as they migrate from the SVZa within the RMS to their final destination in the olfactory bulb. (B) The inset from panel A is enlarged to depict the chain migration of the neuroblasts. Migrating neuroblasts form close associations with adjacent cells and they migrate through a tunnel formed by astrocytes. (C) Depicts the RMS in cross section. Copyright Elsevier Ltd (2003).

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express tyrosine hydroxylase or GABA and other markers of olfactory bulb neurons (Betarbet et al., 1996; Baker et al., 2001). Whereas early studies had suggested that the most anterior aspect of the SVZ, the so-called SVZa, was the sole source of OB neurons generated during the perinatal period, later studies show that cells along the full rostro-caudal extent of the adult SVZ provide neuroblasts that stream forward to the olfactory bulb (Doetsch and Alvarez-Buylla, 1996; Suzuki and Goldman, 2003). Previous studies on the perinatal SVZ also had failed to observe cells from caudal regions of the SVZ migrating tangentially through the RMS (Levison et al., 1993a; Young and Levison, 1996), but Suzuki and Goldman used concentrations of retrovirus 10 fold higher than those previously employed. Thus, in the perinatal period, the SVZa is likely to be the predominant source of OB neuroblasts. Time-lapse video microscopy on slices of neonatal rat forebrain after infecting specific regions of the SVZ with a green fluorescent protein (GFP) expressing retrovirus directly showed cells migrating tangentially through the RMS to the olfactory bulb, once they reach the bulb, they migrate radially and then differentiate into cells with the morphologies and laminar positions of olfactory bulb interneurons. While some of the GFP labeled cells occasionally turn within the RMS to migrate to the edge of the stream, they do not cross into adjacent tissue, suggesting important inhibitory interactions at the border (See chapter 3).

Perinatal SVZ Neuroblasts May Contribute Neurons to the Hippocampus Several lines of investigation have shown that the MGE is a source of hippocampal neurons during fetal development and emerging data suggestes that perinatal SVZ cells generate hippocampal interneurons. When DiI crystals are placed in the basal telencephalon of E13.5 mice to label the MGE, labeled cells are detected in the hippocampus 72 hours later (Pleasure et al., 2000). When MGE cells previously labeled with BrdU are transplanted into a host slice, BrdU positive cells are again found in the hippocampus, 50% of which also express GABA. Similarly, when reporter gene expressing NG2þ cells from the neonatal mouse SVZ are transplanted into P2 wild type recipients, the labeled cells migrate within the wall of the lateral ventricle to the hippocampus (as well as to other structures) (Aguirre et al., 2004). Three weeks after transplantation, these cells have colonized the stratum radiatum, stratum lucidum, stratum oriens and stratum pyramidal. They uniformly express GAD-67 as well as other GABAergic markers. Moreover, these cells are functionally integrated into hippocampal circuitry. By performing whole cell patch clamp on the differentiated progeny of the transplanted cells, Aguirre and colleagues showed that these cells fire action potentials when stimulated with depolarizing current pulses. Additionally, all of the cells recorded possess spontaneous inward currents that are blocked by NBQX.

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Figure 1.4. Migratory patterns of perinatal SVZ cells. (A) Coronal plane. Migratory pathways of SVZ cells into the white matter, cortex and the striatum are shown by orange and yellow arrows, respectively. SVZDL neuronal progenitor migration is depicted by purple dots and by short purple arrows. A region where cell accumulate between the corpus callosum and the subcortical white matter is indicated by light orange shades. (B) Sagittal plane. Representative migratory patterns of glial progenitors out of the anterior and posterior SVZ are shown by orange arrows. Migration of neuronal progenitors is shown by light purple arrows. (C) Reconstruction of the SVZ (green), the striatum (blue), and corresponding migration routes of glial progenitors. Migration toward the dorsal cortex (white asterisks), lateral cortex (red asterisks) and frontal cortex (pink asterisks) is shown. Yellow arrows indicate migration into the striatum. The orange ellipsoid shadow shows the cell accumulation layer between the corpus callosum and the subcortical white matter D. (D) Reconstruction of the SVZ (green) and migratory pathway of neuronal progenitors shown in A and B. G, Pathway of glial progenitors; N, pathway of neuronal progenitors. From Suzuki and Goldman, 2003. Copyright Society for Neuroscience, with permission.

Consistent with the requirement for Dlx transcription factors to form neocortical interneurons, GABA positive cells are absent in the hippocampi of Dlx 1/2 null mice (Anderson et al., 1997; Marin et al., 2000). Furthermore, a reduction in total cell number is observed in the hippocampus of knockout mice as compared to wild type. These studies provide compelling data that

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the MGE is a primary source for hippocampal GABAergic interneurons during fetal development, and that there is the potential that the perinatal SVZ also produces new hippocampal interneurons in the mouse brain.

The Perinatal SVZ Produces Subsets of Glial Cells Migration Paths Taken by SVZ Glioblasts Whereas the SVZDL has long been considered a major source of neocortical and striatal glial cells, it is only recently that the routes and means of SVZ cell migration have been revealed. Initial studies on SVZ cell migration and maturation were performed by administering pulses of [3H] thymidine to label dividing cells and then observing the distribution and cell type of the labeled cells during subsequent development. These studies demonstrated that many SVZ cells are mitotically active and that, with time, the number of strongly labeled cells residing in the SVZ decreases. Since cells that morphologically resembled SVZ cells were observed outside of this germinal zone, it was concluded that the SVZ cells migrated out of the germinal zone. Based on both light and electron microscopic observations of labeled cells, the SVZ was regarded as a source of astrocytes and oliogodendrocytes (Smart, 1961; Lewis, 1968; Privat and Leblond, 1972; Paterson et al., 1973b; Paterson, 1983). However, thymidine uptake by glial cells dividing outside of the SVZ and dilution of label with successive mitoses hampers a clear interpretation of these experiments. In addition, the pathways of SVZ cell migration are difficult to assess because the [3H]thymidine labels cells throughout the germinal matrix. For example, the relative contributions of coronal and sagittal migration to the eventual dispersion of glial cells cannot be determined. Later studies used retroviral-mediated gene transfer to enable cells at more precise locations of the SVZ to be labeled, thus permitting SVZ cell migration to be directly visualized. Using this technique, we showed that retrovirally labeled cells originating in the P2 rat SVZDL move laterally to the adjacent striatum, dorsally to the white matter (WM) and dorsally to the neocortical gray matter (Levison et al., 1993a; Levison and Goldman, 1993b; Zerlin et al., 1995; Kakita and Goldman, 1999) (Fig. 1.4). More recently, studies employing time-lapse video microscopy have permitted even greater spatial resolution. Time-lapse video microscopy of GFP expressing SVZ cells have shown that cells emerging from the SVZDL have a simple morphology, with a single process oriented in the direction of travel. SVZ cells leaving the germinal matrix and moving through adjacent tissues migrate in a saltatory fashion, averaging 90 mm=hr (although within the SVZDL they move more slowly) (Kakita and Goldman, 1999). The cells appear to emigrate from the SVZ on radial glia, since they initially move radially and one can image many cells closely apposed to radial glial

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processes (Zerlin et al., 1995). The movement of these cells requires two separate processes—elongation of the leading process and nuclear translocation—which can occur either synchronously or independently. The final localization of SVZ cells changes over time. Thus, SVZDL cells from P2 animals preferentially colonize gray matter whereas P14 SVZDL cells predominantly colonize white matter. Of the progeny from P2 SVZDL cells, approximately 80% of the cells migrate to the neocortex, 10% migrate to the white matter, and the remaining labeled cells migrate to either striatum or to the border of the white matter and neocortex or striatum (border region). In animals injected with retrovirus at P14, 80% of the labeled cells colonize the white matter and the remaining cells migrate to the striatum or the border region (Levison et al., 1993a; Levison and Goldman, 1993b) Since the SVZDL is located adjacent to the white matter, many of the cells leaving the SVZDL encounter nascent white matter tracts. These white matter tracts contain few oligodendrocytes during the first week of postnatal life and they are highly permissive migratory routes for SVZDL cells, enabling the SVZ cell to move millimeters through white matter within a day (Zerlin et al., 1995). Earlier in this chapter we indicated that SVZ neuroblasts use chain migration to colonize the olfactory bulb. However, the gliogenic SVZDL cells do not use chain migration en route to their final destination; rather they use astroglial guides. Retrovirally labeled SVZDL cells are observed aligned along vimentinþ radial cells in sections of the developing brain (Zerlin et al., 1995) and vertical columns of clonally related progeny are also observed later in development. These vertically aligned clones are observed quite laterally in the neocortex, suggesting that the founding SVZ cell first migrates through the white matter and then turns 908 to migrate along a radial glial cell into the neocortex. Studies on P14 rat forebrain (which corresponds to a time when radial glia have transformed into astrocytes) further supports the concept that radial glia are the substrate for the vertical migration of gliogenic SVZDL cells. Retrovirally labeled SVZDL cells in the P14 brain migrate into the subcortical white matter and the striatum, but they are unable to migrate into the neocortex. Thus, without radial glia, the SVZDL cells are restricted from colonizing dorsal forebrain structures. Migration of perinatal SVZDL cells within the medial and lateral axis is developmentally regulated. P2 SVZDL cells migrate extensively along the axonal tracts of the corpus callosum, preferring to move laterally. A few cells migrate medially, along the corpus callosum, into the contralateral hemisphere, where they differentiate into glia (Kakita et al., 2003). In contrast, P14 SVZDL cells preferentially migrate medially into the central or medial corpus callosum, largely avoiding lateral white matter. More cells appear to migrate across the corpus callosum into the contralateral hemisphere (Levison et al., 1993a). Callosal migration may take place on unmyelinated axons, since: (1) there are no radial glial processes that span the callosum; (2) there is a close proximity of migrating cells with axons; and

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(3) the eventual location of the migrating cells seems to coincide closely with the projections of the callosal fibers (Kakita et al., 2003).

Rostro-Caudal Migration of Glial Progenitors is Limited While glial progenitors migrate extensively within the coronal plane, neither neonatal nor juvenile gliogenic SVZ cells migrate far within the rostrocaudal dimension. For instance, in an experiment where we injected two retroviral vectors into the forebrain SVZ at coordinates separated by 1.75 mm along the anteroposterior axis, there was a significant migration of the SVZ cells along the dorso-ventral axis, but only limited mixing of retrovirally labeled cells within the rostro-caudal axis (Fig. 1.5) (Levison et al., 1993a). In contrast, neurogenic SVZ progenitors migrate extensively in the rostro-caudal dimension as described above. Whereas the initial fate mapping studies did not indicate that the SVZa could give rise to glial cells in the neocortex (Luskin, 1993), more recent studies provide evidence that the SVZa does harbor glial progenitors (see

Figure 1.5. Labeling distant SVZs with separate retroviruses reveals limited anterior posterior migration. The BAG retrovirus was injected into the neonatal rat SVZ at the level of the septal nuclei (site a), while the DAP retrovirus was injected in the same animals into the SVZ 1.75 mm caudally and 1 mm laterally (site b). Plotted are the locations of the X-galþ cells (triangles) and the APþ cells (circles) from a representative animal. Every other section was sampled and plotted on the most appropriate coronal section from 1.9 to 2.9 mm lateral to bregma (most medial section in foreground).

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above and Suzuki and Goldman, 2003). The SVZa contains both neuroblasts and glial progenitors, the latter migrating into and colonizing the frontal lobe of the neocortex.

The Perinatal SVZ Produces Oligodendrocytes From the first descriptions of the cells of the SVZ, these cells have been regarded as glial precursors due to their similarities to mature glial cells (Kershman, 1938; Bryans, 1959; Smart, 1961; Blakemore, 1969). Studies using short intervals between 3H-thymidine injection and analysis concluded that most of the cells of the SVZ migrated into neighboring white and gray matter where they differentiated into oligodendrocytes (Altman, 1966; Imamoto et al., 1978). As SVZ cells strongly express GD3 ganglioside, antibodies against this glycolipid were used to trace the differentiation of these cells. GD3þ cells appeared to migrate from the SVZ into the subcortical white matter, and with time, these cells expressed the early oligodendrocyte marker, carbonic anydrase. By EM, these GD3þ cells had the classic morphologies of immature oligodendrocytes (Levine and Goldman, 1988). Studies using stereotactically injected replication-deficient retroviruses confirmed the earlier interpretations. Of those retrovirally labeled P2 SVZDL cells migrating into the corpus callosum, the majority (89.5%), differentiate into cells with the morphology of non-myelinating or myelinating oligodendrocytes (Figs. 1.6–1.8). Approximately half of the oligodendrocytes form clearly visible myelin sheaths within one month. Similarly, at one month of age, approximately half of the retrovirally labeled cells morphologically characterized as oligodendrocytes also express carbonic anhydrase II, which is enriched in mature oligodendrocytes (Levison et al., 1993a). As stated above, SVZ cells also migrate along radial glial cells to the neocortex where they generate myelinating oligodendrocytes as well as non-myelinating oligodendrocytes closely apposed to blood vessels and neurons (‘‘satellite’’ oligodendrocytes) (Figs. 1.7 and 1.8). Interestingly, approximately equal proportions of myelinating and non-myelinating gray matter oligodendrocytes are generated from the perinatal SVZDL (Levison et al., 1999). Furthermore, clusters of oligodendrocytes are frequently observed in the neocortex, indicating that these oligodendrocyte precursors remain mitotically active.

The Perinatal SVZ Produces NG2 Cells In addition to producing mature oligodendrocytes, some SVZDL cells produce cells that retain several properties of immature cells and they continue to divide slowly in the mature brain (Fig. 1.7 and 1.8). These cells, which have been recently termed ‘‘polydendrocytes’’ (Nishiyama et al., 2002), express the NG2 chondroitin sulfate proteoglycan. The observations that SVZ cells produce polydendrocytes is important for two reasons: (1) they demonstrate that

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Figure 1.6. Schematic of Postnatal SVZ Descendants. Replication deficient retroviruses were injected in the subventricular zone of postnatal 2 rats and the types of cells labeled were examined either 2 days post infection (DPI) or 28 DPI. The location of the labeled cells is indicated as well as the types of cells. At 2 DPI (A), the majority of labeled cells had simple unipolar morphologies and most of the labeled cells remained within the SVZ. At 28 DPI, labeled cells could found dispersed throughout the forebrain, and these labeled cells could be classified as protoplasmic astrocytes (asterisks in B), myelinating oligodendrocytes (dark circles in B), or nonmyelinating oligodendrocytes (open circles in B). Camera lucida drawings of a typical protoplasmic astrocyte with an end-foot on a blood vessel (arrow), and of a myelinating oligodendrocyte are provided. Adapted from Levison and Goldman, (1993); and Levison et al. (1993).

cohorts of these cells in the neocortex are derived directly from the dorsolateral SVZ; and (2) they demonstrate that some NG2þ cells proceed to differentiate into oligodendrocytes, whereas others do not. The discovery that SVZ cells can give rise to these polydendrocytes comes from a retroviral lineage tracing experiment in which animals were allowed to survive for extended periods of time to study the local proliferation of those SVZ descendents that migrate into the neocortex. Some gray matter clonal clusters expanded whereas others contracted. A detailed analysis of clones over several months demonstrated that the homogeneous astrocyte clonal clusters in the neocortex shrunk in size, whereas clonal clusters containing oligodendrocytes, and more specifically those neocortical clonal clusters that contain NG2þ cells, expanded. In fact, the average size of a neocortical clonal cluster that contained at least one NG2þ cell doubled between one and three months of age (4.5 cells/cluster – 9.5 cells/cluster, respectively) (Levison et al., 1999). Almost 90% of the cells

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Figure 1.7. Types of cells generated from P2 SVZDL cells. Progenitors that leave the SVZDL and differentiate within the subcortical white matter become either myelinating or non-myelinating oligodendrocytes. Few become astrocytes. Those progenitors that differentiate within the neocortex become myelinating oligodendrocytes as well as satellite oligodendrocytes and cells that label with the NG2 proteoglycan. Additionally, those progenitors that make contact with unensheathed cerebral endothelial cells become protoplasmic astrocytes. Copyright Elsevier Ltd. (2003).

in clonal clusters at one month were NG2þ and at three months, approximately 70% of the cells within these clonal clusters were NG2þ. These data suggested that a subset of SVZDL derived oligodendroglial clonal clusters were composed of NG2þ cells that continued to reproduce, but which did not become myelinating oligodendrocytes. There is some controversy as to whether the NG2þ population represents immature oligodendrocytes or represent a stable population in the mature brain, with physiological features distinct from both myelinating oligodendrocytes and astrocytes (Nishiyama et al., 2002).

The Perinatal SVZ Produces Astrocytes As reviewed above, the SVZDL had long been regarded as a source of oligodendrocytes, and some authors had suggested that it was a source of astrocytes (Altman, 1963; Paterson et al., 1973); however, only more recently has it become widely accepted that the SVZDL is a major source of

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Figure 1.8. Representative examples of descendants of retrovirally labeled P2 SVZDL cells. (A) Two neocortical protoplasmic astrocytes. (B) A perineuronal nonmyelinating oligodendrocyte. (C) A clonal cluster of polydendrocytes. (D) A myelinating oligodendrocyte in the subcortical white matter. (E) A clonal cluster containing 2 protoplasmic astrocytes and 3 non-myelinating oligodendrocytes. (F) A clonal cluster containing 4 protoplasmic astrocytes and a bipolar neuron. Selected images from Levison et al., 1999.

gray matter astrocytes. For example, when replication deficient retroviruses were stereotactically delivered to early postnatal rat SVZ, descendants of these cells with the bushy processes and markers of astrocytes were found in the neocortex and striatum (Luskin et al., 1988; Levison et al., 1993b; Levison and Goldman, 1993a; Zerlin et al., 1995) (Figs. 1.6–1.8). Interestingly, protoplasmic astrocyte differentiation was predominantly observed within the gray matter, with very few cells with astrocytic features observed in the white matter. Within the gray matter 65% of the cells descended from the P2 rat SVZDL could be classified as astrocytes (Levison and Goldman, 1993a). Many of these newly generated astrocytes in the gray matter resided in tightly knit clusters, consistent with the view that astrocyte precursors continue to divide after they reach their final destination. In contrast, only 8.5% of the progeny of the P2 SVZDL cells migrating into the white matter display astrocytic features, indicating that nascent white matter tracts may not be permissive for astrocyte differentiation, or may promote oligodendrocyte differentiation of glial progenitors (Levison and Goldman, 1993a).

Contact Between Migrating SVZ Cells and Blood Vessels is an Early Stage of Astrocyte Development An interaction with blood vessels or the pial surface appears to be one of the first indicators of astrocyte differentiation (Zerlin et al., 1995; Zerlin and

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Goldman, 1997). After leaving the SVZ, many of the migrating SVZ cells still retain their simple, largely unipolar morphology. After contacting a blood vessel with its leading process, the SVZ cell begins to ensheath the blood vessel and it will form the classic end-foot of an astrocyte (Zerlin et al., 1997). During this ensheathment process, the cell slowly extends multiple fine processes into the surrounding parenchyma. The cell also acquires nestin and vimentin immunoreactivity—intermediate filaments characteristic of immature astrocytes that form the cytoskeleton of the cell (Zerlin et al., 1995). This interaction with blood vessels thus constitutes an early stage in astrocyte development. In fact, contact with endothelial cells induces astrocyte differentiation in astrocyte precursors cultured from optic nerve (Mi et al., 2001). In other words, endothelial cells induce the expression of the astrocyte markers, GFAP and S100b, in immature cells. This induction can be neutralized with antibodies to leukemia inhibitory factor (LIF), a growth factor expressed by endothelia (Mi et al., 2001). How astrocyte precursors recognize blood vessels is not known. It may be that SVZ cells acquire the early stages of astrocyte development as they migrate, and that these early changes allow them to bind to blood vessels. Further blood vessel interaction promotes greater astrocyte differentiation. This idea of stages in astrocyte development is supported by finding SVZ cells that have migrated into the cortex that express the astrocyte marker ZII prior to their contact with blood vessels (Staugaitis et al., 2001).

Heterogeneity of Perinatal SVZ Cells Because the SVZ is a geographically delineated region of the brain rather than a group of cells that are descended from a single source, or a group of cells that share specific molecular features, it is difficult to define precisely the types of cells within the SVZ. In this chapter, we have reviewed the types of cells produced by the perinatal SVZ and have suggested that there are different types of SVZ cells. But how strong are the data that there are multiple types of progenitors within the SVZ? The alternative is that there may be only two types of cells whose specification can be directly induced by extrinsic signals. On the basis of the evidence garnered to date, we would argue that there is strong evidence to support the existence of neural stem cells, tripotential, bipotential and unipotential progenitors.

Evidence that Neural Stem Cells Reside in the Perinatal SVZ The evidence that the perinatal SVZ contains neural stem cells is derived largely from extrapolation from studies conducted on the fetal and adult SVZs. Concurrent with the appearance of the SVZ during fetal development, a new population of precursors can be isolated that proliferates as non-adherent

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balls of cells in the presence of epidermal growth factor (EGF), as first described by Weiss and colleagues (Reynolds and Weiss, 1992; Reynolds and Weiss, 1996). These aggregates are termed neurospheres. Neurospheres can be formed from the forebrain beginning around E14 and this capacity persists throughout life (Reynolds and Weiss, 1996; Laywell et al., 2000; Ciccolini, 2001). A sphere can develop from a single cell, in the presence of EGF will generate hundreds of progeny. When such a sphere is dissociated into a single cell suspension, only 5–7% of the dissociated cells are competent to form new neurospheres. Neurospheres have been dissociated in this fashion for many passages, suggesting that the cells that are competent to form these spheres are capable of unlimited self-renewal—a critical property of a neural stem cell. Furthermore, upon withdrawing the growth factors, cells within clonally derived neurospheres differentiate into neurons, oligodendrocytes, and astrocytes, demonstrating the multipotential nature of the originally isolated stem cell. To determine the relative proportion of progenitors vs stem cells in the adult SVZ, the constitutively proliferating population of cells was eliminated using three injections of high doses of 3 H-thymidine every 4 hours to kill the mitotically active population of cells with the SVZ (Morshead et al., 1994). The number of cells left in the subependyma following the 3 H-thymidine kill that remained capable of dividing was then determined using injections of BrdU. At 0.5 days post-kill, less than 1% of the original cells in the subependyma were proliferating; yet this small percentage of proliferating cells is capable of completely replenishing the constitutively proliferating cells over a period of eight days (Morshead et al., 1994). Therefore, these data suggest that the cells that were resilient to mitotic inhibitors are likely the neural stem cells and that they comprise at most 1% of the SVZ cell population. Similar studies have been performed on the adult brain using infusions of cytosine arabinoside (Doetsch et al., 1999). The cell type that persists after anti-mitotic drug treatment resides immediately subjacent to the ependymal layer and shares several features with immature astrocytes, including the expression of GFAP (Doetsch et al., 1999; Laywell et al., 2000) These cells are discussed in much greater detail by Kowhi et al. (Chapter 3, Fig. 1.10).

Evidence that Tri-potential Progenitors Reside in the Perinatal SVZ The neonatal mouse brain contains tri-potential progenitors that express the cell surface markers Lewis X and NG2 and the intracellular protein 2’,3’cyclic nucleotide 3’-phosphodiesterase (CNP) (Belachew et al., 2003; Aguirre et al., 2004). Using several lines of transgenic mice expressing the green fluorescence protein under the control of the CNPase promotor as well as fluorescence-activated cell sorting, Vittorio Gallo’s lab showed that the NG2 þ/CNPaseþ positive cells in the P2 SVZ are rapidly cycling cells that express

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the phenotype of the transit amplifying cells in the adult SVZ characterized as ‘‘type C’’ cells. These NG2þ/CNPaseþ cells in neonatal SVZ express the EGF receptor, the transcription factors Dlx, Ascl1 (formerly Mash1), and Olig2. Moreover, these type C-like cells form neurospheres that can generate GABAergic neurons, as well as astrocytes and oligodendrocytes, and these ‘‘type C-like’’ cells are self-renewing. Additional data supporting the existence of tripotential progenitors in the perinatal SVZ comes from in vitro retroviral lineage tracing experiments. In an analysis of 128 clonal clusters derived from P14 retrovirally infected SVZDL progenitors, approximately 10% of the clonal clusters contained neurons, astrocytes and oligodendrocytes on the basis of reporter gene expression and staining for TuJ1, GFA and O4 (Young and Levison, 1996). Similar results were obtained when the same analysis was performed on SVZ cells from the P2 infected SVZDL (Levison and Goldman, 1997).

Evidence that Bipotential Neuron/Glial Progenitors Reside in the Perinatal SVZ As noted above, the large majority of SVZ cells that migrate into white matter and cortex produce glia. In very rare cases, SVZ cells produce both neurons and astrocytes, or neurons and oligodendrocytes (Fig. 8) (Levison and Goldman, 1993b). Interestingly, these mixed neuron glial clones are observed within two weeks after viral infection, but they do not persist when clonal compositions are assessed after longer survival intervals (i.e. after one month). These observations underscore the relative immaturity of SVZDL cells and their potential to generate neurons and either type of macroglial cell; however, they also indicate that postnatally generated neocortical neurons are relatively short-lived. These neurons may be the ‘‘exception that proves the rule.’’ Further evidence for the existence of bipotential neuron/glial SVZ progenitors is provided by in vitro studies. When a culture medium permissive for neuronal differentiation is employed, approximately 25% of both P2 and P14 SVZDL progenitors generate clones containing at least one neuron (Young and Levison, 1996; Levison and Goldman, 1997). Furthermore, comparing the average size of clonal clusters that contained at least one neuron vs. those that are exclusively non-neuronal, neuron-containing clusters are approximately twice as large as clusters lacking neurons. These results suggest that the progenitors that generate neuron-containing clones are less restricted and that they generate precursors that have a greater proliferative capacity (Young and Levison, 1996). We should note, however, that the culture observations only show that SVZ cells have the potential to generate both neurons and glia, but not that they actually do so in vivo.

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Evidence that Neuron Restricted Progenitors Reside in the Perinatal SVZ As noted above, both the perinatal and adult forebrain SVZ produce periglomerular and granule cell interneurons of the olfactory bulb. These cells begin their journey in the SVZ, emigrate into the RMS, and then migrate into the bulb. Several observations suggest that neuronal fate specification of these precursors begins in the SVZ itself. First, some of the migratory cells in the SVZ express the neuronal beta-tubulin (TuJ1þ) and these cells continue to divide as they migrate (Menezes et al., 1995). Second, retroviral lineage tracing studies using either single viruses, or libraries of viruses with 100–400 unique inserts, demonstrate that all of the cells that enter the RMS differentiate into neurons, not glia (Luskin, 1993; Reid et al., 1999). Neuronal differentiation is a lengthy process; however, the acquisition of neuronal characteristics occurs as they migrate and it continues in the bulb (Carleton et al., 2003). Third, the prenatal LGE-SVZ is populated by immature cells that express the ventral forebrain homeobox protein, dlx. This LGE population also contains a subpopulation of precursors in its dorsal extent, identified by their expression of the transcription factor, Er81 (Stenman et al., 2003). Olfactory bulb granule and periglomerular interneurons also express Er81. Fate mapping of the dlxþ cells in the LGE-SVZ shows that the Er81þ olfactory bulb interneurons originate as dlxþ cells, suggesting that they derive from this specific population within the LGE. Fourth, clonal analyses of SVZ cell differentiation in vitro demonstrate clones that contain only neurons (Young and Levison, 1996; Levison and Goldman, 1997). These various observations do not imply that neuroblasts cannot be induced to change fates, perhaps in pathological conditions such as demyelination, in which neuronal precursors in the RMS are induced to leave the RMS and differentiate into oligodendrocytes (Nait-Oumesmar et al., 1999). However, the cells that enter the RMS appear to be restricted to a neuronal fate in vivo under normal developmental conditions.

Evidence that Glial Restricted Progenitors Reside in the Perinatal SVZ Clonal analyses using retroviral vectors in conjunction with immunofluorescence or electron microscopy have provided evidence that most SVZ progenitors that migrate radially produce either astrocytes or oligodendrocytes, but not necessarily both—in other words the majority of progenitors within the perinatal SVZDL appear to be restricted (Luskin et al., 1993). For instance, of the retrovirally labeled progenitors that exit the P2 SVZDL and migrate into the neocortex, approximately 45% produce homogeneous clonal clusters of protoplasmic astrocytes, and approximately 30% produce homogeneous clonal clusters of oligodendroglia (Levison and Goldman, 1993b). Indeed, due to the relative rarity of mixed oligodendrocyte/astrocyte

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clonal clusters, Luskin and colleagues concluded that these glial lineages had diverged prior to birth. However, three separate studies found that 10–15% of the progenitors within the postnatal day 2 SVZDL produce clonal clusters that contain both oligodendrocytes and protoplasmic astrocytes (Levison and Goldman, 1993b; Levison et al., 1999; Parnavelas, 1999). Whereas the identities of the cells within these clonal clusters were originally classified on the basis of morphological features, staining for cell type specific markers has corroborated the morphological classfications (Levison and Goldman, 1993b; Zerlin et al., 1995; Kakita and Goldman, 1999). Most recently, a retroviral library with 100 unique tags has been used to determine whether there are bipotential glial progenitors in the SVZ. With this more definitive approach, Zerlin and Goldman (2004) found that approximately 15% of retrovirally labeled P2 SVZDL cells do indeed generate both astrocytes and oligodendrocytes in the rat forebrain. Although this study confirmed that all clusters are clonal, it also showed that not all clones are entirely composed of tightly-knit clusters of glia. Glial progenitors continue to divide during their migrations and thus can generate spatially dispersed clones, since the offspring of a given division do not necessarily continue migrating in the same direction. The retrospective nature of retroviral lineage tracing has inherent qualifications. First, cell death during clonal expansion might remove one or more potential lineages. Second, local environmental influences might promote an astrocytic or oligodendrocytic differentiation of a bipotential progenitor. Thus, it is not possible to conclude from these studies as to how many SVZ cells are actually astrocyteoligodendrocyte bipotent progenitors. It is also not possible from these studies to examine the possibility that individual SVZ cells gave rise not only to glia but also to olfactory interneurons. Additional arguments supporting the existence of bipotential glial progenitors derive from in vitro studies. When two retroviruses were injected into the P2 or P14 SVZDL and the infected cells removed and placed in culture, 50% of the labeled progenitors generated clonal clusters that contained both astrocytes and oligodendrocytes (Levison and Goldman, 1993b). Using the combination of A2B5/GFAP/O4, these polygonal astrocytes could be classified as forebrain ‘‘type 1’’ astrocytes (Levison et al., 1993a) (Fig. 1.9). These macroglial progenitors are thus distinct from O-2A progenitors, that generate ‘‘type 2’’ astrocytes and oligodendrocytes in vitro (Raff et al., 1983). Furthermore, mixed glial clones are obtained even under culture conditions that are permissive for neurogenesis, supporting the conclusion that these progenitors are macroglial lineage restricted (Young and Levison, 1996; Levison and Goldman, 1997).

Conclusions We have reviewed in a general way the types of cells that populate the newborn SVZ, but our knowledge is far from complete. Data from multiple

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Figure 1.9. Cellular heterogeneity of the perinatal SVZDL . The neural stem cells reside in a niche subjacent to the ependymal cells that line the ventricle. Evidence suggests that the stem cells extend a single process that interdigitates between two ependymal cells to contact the cerebral spinal fluid. Neural stem cells give rise to transient amplifying precursor cells. These multipotential progenitors generate other, more restricted progenitors, such as bipotential glial progenitors and unipotential glial precursors and neuroblasts. The neuroblasts are typically ensheathed by immature astrocytes.

studies underscore the existence of multiple classes of precursors within the SVZ, but it is likely that the true mosaic nature of the SVZ is not fully appreciated. For instance, carefully performed studies have classified the types of cells within the adult SVZ, and yet, similar studies have not been performed on the perinatal SVZ. It is likely that the composition of the perinatal SVZ is different from that of the adult. From a developmental perspective, there is a clear need to understand which types of progenitors populate the embryonic, fetal and perinatal SVZ, what their characteristics are, and whether different progenitor types are harbored within different domains of the SVZ. As has been reiterated in several review articles, there is truly a need for additional markers for neural precursors. With specific

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Figure 1.10. Clone from a P2 SVZ cell that generated a type 1 astrocyte and a late oligodendrocyte progenitor. X-gal staining (A,B); C. R24 (C); GFAP (D); O4 (E). One day after labeling SVZ cells in vivo with retrovirus, the SVZ region was excised, dissociated and grown in vitro on poly-L-lysine-coated coverslips. At 7 div, the coverslips were processed for X-gal histochemistry and triple-label immunofluorescence for glial lineage antigens. (A) Phase-contrast view of two cells, a polygonal cell (left) and a smaller, process-bearing cell (right) that were members of a 15-cell mixed phenotype clone. (B) The same field viewed with bright-field optics to illustrate X-gal staining. Combining triple-label immunofluorescence with b-gal histochemistry required an abated X-gal reaction product. Though the process-bearing cell is lightly stained for X-gal, it contains distinct granular precipitates of X-gal that are not evident in the surrounding unstained cells. (C) This is viewed with rhodamine epifluorescence. The process-bearing cells is GD3þ, while the polygonal cell is GD3-. (D) The same field viewed with coumarin epifluorescence to show that the polygonal GD3-cell is GFAPþ, and, therefore, a ‘type 1’ astrocyte. (E) The same field viewed with fluorescein epifluorescence to show that the GD3þ process-bearing cell is O4þ and, therefore, an oligodendrocyte progenitor. Bar represents 10 mm. From Levison et al., 1993.

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markers in hand, it will be possible to define how many types of precursors inhabit the SVZs at different developmental time points, how the proportions of different progenitors change during maturation, and across the lifespan.

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Levison, S.W. and Goldman, J.E. (1993). Both oligodendrocytes and astrocytes develop from progenitors in the subventricular zone of postnatal rat forebrain. Neuron 10: 201–12. Levison, S.W. and Goldman, J.E. (1997). Multipotential and lineage restricted precursors coexist in the mammalian perinatal subventricular zone. J. Neurosci. Res. 48: 83–94. Levison, S.W., Young, G.M. and Goldman, J.E. (1999). Cycling cells in the adult rat neocortex preferentially generate oligodendroglia. J. Neurosci. Res. 57: 435–446. Lewis, P.D. (1968). The fate of the subependymal cell in the adult rat brain, with a note on the origin of microglia. Brain 91: 721–736. Lewis, P.D. and Lai, M. (1974). Cell generation in the subependymal layer of the rat brain during the early postnatal period. Brain Res. 77: 520–525. Lois, C., Garcia–Verdugo, J.M. and Alvarez–Buylla, A. (1996). Chain migration of neuronal precursors. Science 271: 978–981. Luskin, M.B. (1993). Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11: 173–189. Luskin, M.B., Parnavelas, J.G. and Barfield, J.A. (1993). Neurons, astrocytes, and oligodendrocytes of the rat cerebral cortex originate from separate progenitor cells: An ultrastructural analysis of clonally related cells. J. Neurosci. 13: 1730–1750. Luskin, M.B., Pearlman, A.L. and Sanes, J.R. (1988). Cell lineage in the cerebral Cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus. Neuron 1: 635–647. Malatesta, P., Hartfuss, E. and Gotz, M. (2000). Isolation of radial glial cells by fluorescent–activated cell sorting reveals a neuronal lineage. Development— Supplement 127: 5253–5263. Marin, O., Anderson, S. and Rubenstein, J.L. (2000). Origin and molecular specification of striatal interneurons. J. Neurosci. 20: 6063–6076. Marshall, C.A. and Goldman, J.E. (2002). Subpallial dlx2–expressing cells give rise to astrocytes and oligodendrocytes in the cerebral cortex and white matter. J. Neurosci. 20: 30–42. McConnell, S.K. (1991). The generation of neuronal diversity in the central nervous system. Annu. Rev. Neurosci. 14: 269–300. Menezes, J.R., Smith, C.M., Nelson, K.C. and Luskin, M.B. (1995). The division of neuronal progenitor cells during migration in the neonatal mammalian forebrain. Mol. Cell Neurosci. 6: 496–508. Mi, H., Haeberle, H. and Barres, B.A. (2001). Induction of astrocyte differentiation by endothelial cells. J. Neurosci. 21: 1538–1547. Misson, J.P., Austin, C.P., Takahashi, T., Cepko, C.L. and Caviness, Jr., V.S. (1991). The alignment of migrating neural cells in relation to the murine neopallial radial glial fiber system. Cereb. Cortex 1: 221–229. Morshead, C.M., Reynolds, B.A., Craig, C.G., McBurney, M.W., Staines, W.A., Morassutti, D., Weiss, S. and Van der Kooy, D. (1994). Neural stem cells in the adult mammalian forebrain: A relatively quiescent subpopulation of subependymal cells. Neuron 13: 1071–1082. Nait–Oumesmar, B., Decker, L., Lachapelle, F., Avellana–Adalid, V., Bachelin, C. and Van Evercooren, A.B. (1999). Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination. Eur. J. Neurosci. 11: 4357–4366.

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Nishiyama, A., Watanabe, M., Yang, Z. and Bu, J. (2002). Identity, distribution, and development of polydendrocytes: NG2–expressing glial cells. J. Neurocytol. 31: 437–455. Noctor, S.C., Flint, A.C., Weissman, T.A., Dammerman, R.S. and Kriegstein, A.R. (2001). Neurons derived from radial glial cells establish radial units in neocortex. Nature 409: 714–720. Noctor, S.C., Martinez–Cerdeno, V., Ivic, L. and Kriegstein, A.R. (2004). Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat. Neurosci. 7: 136–144. Parnavelas, J.G. (1999). Glial cell lineages in the rat cerebral cortex. Exp. Neurol. 156: 418–429. Paterson, J.A. (1983). Dividing and newly produced cells in the corpus callosum of adult mouse cerebrum as detected by light microscopic radioautography. Anat. Anz. 153: 149–168. Paterson, J.A., Privat, A., Ling, E.A. and Leblond, C.P. (1973). Investigation of glial cells in semithin sections III Transformation of subependymal cells into glial cells as shown by radioautography after 3H–thymidine injection into the lateral ventricle of the brain of young rats. J. Comp. Neurol. 149: 83–102. Pleasure, S.J., Anderson, S., Hevner, R., Bagri, A., Marin, O., Lowenstein, D.H. and Rubenstein, J.L. (2000). Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons. Neuron 28: 727– 740. Privat, A. and Leblond, C.P. (1972). The subependymal layer and neighboring region in the brain of the young rat. J. Comp. Neurol. 146: 227–302. Qian, X., Davis, A.A., Goderie, S.K. and Temple, S. (1997). FGF2 concentration regulates the generation of neurons and glia from multipotent cortical stem cells. Neuron 18: 81–93. Raff, M.C., Abney, E.R., Cohen, J., Lindsay, R. and Noble, M. (1983). Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides, and growth characteristics. J. Neurosci. 3: 1289–1300. Rakic, P. (1971). Neuron–glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacus Rhesus. J. Comp. Neurol. 141: 283–312. Reid, C.B., Liang, I. and Walsh, C.A. (1999). Clonal mixing, clonal restriction, and specification of cell types in the developing rat olfactory bulb. J. Comp. Neurol. 403: 106–118. Reynolds, B.A. and Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255: 1707– 1710. Reynolds, B.A. and Weiss, S. (1996). Clonal and population analyses demonstrate that an EGF–responsive mammalian embryonic CNS precursor is a stem cell. Dev. Biol. 175: 1–13. Rousselot, P., Lois, C. and Alvarez–Buylla, A. (1995). Embryonic (PSA) N–CAM reveals chains of migrating neuroblasts between the lateral ventricle and the olfactory bulb of adult mice. J. Comp. Neurol. 351: 51–61. Sauer, F.C. (1935). Mitosis in the neural tube. J. Comp. Neurol. 62: 377–405. Smart, I.H. (1961). The subependymal layer of the mouse brain and its cellular production as shown by radioautography after thymidine–3h injection. J. Comp. Neurol. 116: 325–349.

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Spassky, N., Heydon, K., Mangatal, A., Jankovski, A., Olivier, C., Queraud–Lesaux, F., Goujet–Zalc, C., Thomas, J.L. and Zalc, B. (2001). Sonic hedgehog–dependent emergence of oligodendrocytes in the telencephalon: evidence for a source of oligodendrocytes in the olfactory bulb that is independent of PDGFRalpha signaling. Development 128: 4993–5004. Spassky, N., Olivier, C., Perez–Villegas, E., Goujet–Zalc, C., Martinez, S., Thomas, J. and Zalc, B. (2000). Single or multiple oligodendroglial lineages: a controversy. Glia 29: 143–148. Staugaitis, S.M., Zerlin, M., Hawkes, R., Levine, J.M. and Goldman, J.E. (2001). Aldolase C/zebrin II expression in the neonatal rat forebrain reveals cellular heterogeneity within the subventricular zone and early astrocyte differentiation. J. Neurosci. 21: 6195–6205. Stenman, J., Toresson, H. and Campbell, K. (2003). Identification of two distinct progenitor populations in the lateral ganglionic eminence: Implications for striatal and olfactory bulb neurogenesis. J. Neurosci. 23: 167–174. Takahashi, T., Nowakowski, R.S. and Caviness, Jr., V.S. (1995). Early ontogeny of the secondary proliferative population of the embryonic murine cerebral wall. J. Neurosci. 15: 6058–6068. Thomaidou, D., Mione, M.C., Cavanagh, J.F. and Parnavelas, J.G. (1997). Apoptosis and its relation to the cell cycle in the developing cerebral cortex. J. Neurosci. 17: 1075–1085. Wichterle, H., Garcia–Verdugo, J.M. and Alvarez–Buylla, A. (1997). Direct evidence for homotypic, glia–independent neuronal migration. Neuron 18: 779–791. Young, G.M. and Levison, S.W. (1996). Persistence of multipotential progenitors in the juvenile rat subventricular zone. Dev. Neurosci. 18: 255–265. Zerlin, M. and Goldman, J.E. (1997). Interactions between glial progenitors and blood vessels during early postnatal corticogenesis: blood vessel contact represents an early stage of astrocyte differentiation. J. Comp. Neurol. 387: 537–546. Zerlin, M., Levison, S.W. and Goldman, J.E. (1995). Early patterns of migration, morphogenesis, and intermediate filament expression of subventricular zone cells in the postnatal rat forebrain. J. Neurosci. 15: 7238–7249. Zerlin, M., Milosevic, A. and Goldman, J.E. (2004). Glial progenitors of the neonatal subventricular zone differentiate asynchronously, leading to spatial dispersion of glial clones and to the persistence of immature glia in the adult mammalian CNS. Dev. Biol. 270: 200–213.

Chapter 2 Extrinsic and Intrinsic Factors Modulating Proliferation and Self-renewal of Multipotential CNS Progenitors and Adult Neural Stem Cells of the Subventricular Zone Sara Gil-Perotin1,2 and Patrizia Casaccia-Bonnefil1

Introduction Regulation of cell number in germinal zones of the nervous system is dependent on the interaction of extracellular signals with the ‘‘intrinsic’’ properties of the germinal cells that may vary depending on the developing stage of the organism. During early embryonic development, proliferation of cells occurs along the lumen of the developing neural tube, in an area defined as ‘‘the ventricular zone’’. At this stage, cells proliferate very fast and characteristically give rise to identical daughter cells, via a process identified as ‘‘symmetric cell division’’ that allows for expansion of the primordial structures (Fig. 2.1A). As the organism develops, the need for ‘‘rapid expansion’’ decreases and new structures begin to form while still allowing for growth of the organism. Therefore, by mid-gestation, a second germinal zone arises, the subventricular zone (SVZ) and cells in this area acquire a modality of cell division characterized by the generation of two different daughter cells (‘‘asymmetric cell division’’): One with the ability to self-renew and the other one with the ability to differentiate into a specific lineage (Temple, 2001). In adult SVZ, the maintenance of homeostasis induces stem cells and multipotential progenitors to divide asymmetrically, unless a need for rapid expansion (e.g. repair after injury) induces the cells to shift to a symmetric modality of division (Fig. 2.1B). Changes in the levels of the extracellular signals, alterations of their receptors or modification of the intracellular signaling molecules regulating proliferation during embryonic development, may result in abnormalities of 1

Department of Neuroscience and Cell Biology, UMDNJ R. Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA 2 Department of Comparative Neurobiology, Instituto Cavanilles de Biodiversidad y Biologia evolutiva, 46980 Paterna, Valencia, Spain 30

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Figure 2.1. Schematic representation of the distinct modalities of cell division. During development (A), the expansion of brain structures is first guaranteed by the rapid and symmetric non-terminal cell division. As new cell types are generated, the pattern of cell division becomes asymmetric. In the adult animal (B) it is likely that stem cells (B cells) in the remaining germinal zones such as the SVZ undergo asymmetric cell division to maintain a specific number of mother and daughter cells. ‘‘Multipotent progenitors (C cells) undergo a similar pattern of asymmetric cell division and generate A cells and oligodendrocyte progenitors (not shown), with the ability to divide following a symmetric division where both daughter cells exit from the cell cycle (Q) and differentiate. Note that upon injury, the need for expansion of the progenitor population leads to symmetric division and expansion of C cells that generate both neurons and oligodendrocytes.’’

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brain structures. Changes or modifications of extracellular or intracellular signals in adult neural stem cells, in contrast, may not affect the histoarchitecture of the brain, but affect the number of multipotential progenitors available for repair after injury. If proliferation is defective, a smaller number of cells will be available for repair, conversely, if proliferation proceeds uncontrolled, larger number of cells may result in hyperplastic foci and eventually lead to neoplastic transformation. Since the responsiveness of neural stem cells and multipotential progenitors to extracellular signals is a dynamic process dependent on the developmental stage and on the regional localization of the cells, it becomes important to recognize that conclusions based on studies on embryonic stem cells may not be translated directly to adult neural stem cells. These temporally and developmentally restricted profiles of responsiveness to mitogenic and anti-mitogenic signals are determined by several parameters, including the bioavailability of extracellular signals, the presence of specific receptors, cross-talks among distinct signaling pathways and modulation of cell cycle regulatory molecules. All of these events can be affected or determined by specific genetic traits, expression of transcription factors and epigenetic modifications of chromatin components resulting in differences of gene expression that modulate the ‘‘context-specific’’ responsiveness of a stem cell. It is worth mentioning that although the steady-state number of neural stem cells at any given stage of development is the result of the equilibrium between proliferation, differentiation, migration and survival of these cells, this chapter will focus only on the experimental evidence on extracellular factors and intracellular molecules affecting proliferation of neural stem cells and multipotent progenitors. This has been a very challenging task and although we have attempted to include several studies in this area, the overwhelming body of available literature has hindered our attempts to be exhaustive.

Extracellular Factors Affecting Proliferation Basic Fibroblast Growth Factor (bFGF) The Fibroblast Growth Factor (FGF) family includes a large number of ligands and receptors initiating signaling cascades that are critical for the early development of the organism (Burke et al., 1998; Klint and ClaessonWelsh, 1999; Reuss and von Bohlen und Halbach, 2003). Of the different members of the FGF family of ligands, for instance, FGF8 is primarily involved in patterning of the midbrain and anterior forebrain (Mason et al., 2000), FGF3 is important for the development of the ear (Represa et al., 1991) and FGF2 is important for proliferation of neural stem cells and neurogenesis both in vitro (Reynolds and Weiss, 1992; Vescovi et al., 1993; Vaccarino et al., 1995; Kuhn et al., 1997) and in vivo (Craig et al., 1996; Tao et al., 1996; Kuhn

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et al., 1997; Wagner et al., 1999). FGF2 (or bFGF) is expressed in the rodent brain at mid-gestation, from E11.5 to E17.5 in mice and from E13.5 to E19.5 in rats (Vaccarino et al., 1999b; Raballo et al., 2000), during a period coincident with active neurogenesis (Bayer and Altman, 1991; Caviness et al., 1995). Targeted deletion of Fgf2 in mice results in a 50% reduction in the number of cortical neurons (Vaccarino et al., 1999b), thus suggesting a critical role for this ligand in neurogenesis. The expression of the bFGF receptor, FGFR1 in the ventricular zone (Fig. 2.2), is observed at E8.5-9.5 (Orr-Urtreger et al., 1991; Wanaka et al., 1991) and it progressively decreases as neuroblasts begin to exit from the cell cycle and start differentiating (Raballo et al., 2000). The phenotypic analysis of mutant mice with targeted deletions in the FgfR1 or FgfR2 receptors supports the idea that FGF signaling mediated by these two receptors, but not by FGFR3 and FGFR4, is critical for regulating proliferation and development of telencephalic structures (Yamaguchi et al., 1994; Deng et al., 1997; Partanen et al., 1998; Xu et al., 1998; Tropepe et al., 1999). The role of FGF receptor signaling in proliferation of neural progenitors and stem cells is also supported by a separate line of investigation on the effect of FGF2 administration at distinct developmental stages (Qian et al., 1997; Kelly et al., 2003). High doses of FGF2 intracerebrally injected during embryogenesis (E14 in mice), result in massive enlargement of the ventricles and aberrant proliferation and differentiation (Ohmiya et al., 2001). However, low doses of recombinant FGF2 in rat embryos (Vaccarino et al., 1999a) or even in neonatal and adult rats (Tao et al., 1996; Wagner et al., 1999) enhance proliferation and neurogenesis.

Epidermal Growth Factor (EGF) Family The epidermal growth factor (EGF) family of polypeptides includes EGF, transforming growth factor-alpha (TGF-alpha), heparin-binding EGF (HBEGF) and related neuregulins. These polypeptides, produced by neurons and glial cells, play an important role in the development of the nervous system, by affecting proliferation, survival, migration and differentiation of neuronal and glial cells (Xian and Zhou, 1999). Neuregulins have been identified during the late embryonic development (Corfas et al., 1995), and their receptors ErbB2 and ErbB4 are expressed in the E12 embryo (Kornblum et al., 2000) as well as in embryonically derived neural stem cells (Calaora et al., 2001). Although neuregulin signaling especially via the ErbB4 receptor is critical for migration of adult neuroblasts and possibly survival and neurogenesis (Calaora et al., 2001; Anton et al., 2004), the available experimental evidence does not support a role of this EGF family member in proliferation. TGF alpha, in contrast, is a potent mitogen and also the predominant form of EGF ligand expressed in the developing brain and in adult SVZ (Kornblum et al., 1997). The importance of this ligand

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in regulating proliferation of adult neural stem cells is supported by the phenotypic characterization of the TGF alpha null mice characterized by decreased number of mitosis exacerbated by senescence (Tropepe et al., 1997). Expression of EGFR in vivo occurs during late embryogenesis (Fig. 2.2) in the developing SVZ and follows the expression of FGFR1 in ventricular zone cells (Burrows et al., 1997). Consistent with this temporal progression, cells isolated from embryonic mouse brain during early development (i.e. E10-E12) are FGFR1þ, while those isolated at later developmental stages are both EGFRþ and FGFR1þ (Ciccolini and Svendsen 1998; Gritti et al., 1999; Lillien and Raphael 2000). This expression pattern is also consistent with the distinct growth factor requirements of early embryonic stem cells for FGF2 and of the late embryonic stem cells for EGF and FGF2 (Tropepe et al., 1999; Martens et al., 2000). The concept of temporal responsiveness to distinct growth factors determined by the sequential expression pattern of distinct receptor subunits is also supported by a comparative phenotypic analysis of the EgfR and FgfR null mice. While the FgfR1 null mice are early embryonic lethal (Deng et al., 1994; Yamaguchi et al., 1994), the phenotype of the EgfR null mice is characterized by reduced cortical size at E18 (Threadgill et al., 1995), and progressive neuronal degeneration during the postnatal period (Sibilia and Wagner, 1995; Sibilia et al., 1998). Despite the similar role as mitogens, FGF2 and EGF have been differentially implicated as modulators of lineage restriction and neurogenesis. It has been proposed that the differential role played by these factors depends on the segregation of the mitogenic effect on distinct cellular populations: EGF preferentially targeting the quiescent stem cells and FGF targeting the more committed neuroblasts (Morshead and van der Kooy, 1992; Morshead et al., 1994). The co-expression of FGFR1 and EGFR within the same cell type at later stages of development, however, argues against this possibility (Gritti et al., 1999). An alternative explanation for the differential effect exerted by these two growth factors is the possibility that through the activation of distinct tyrosine kinase receptors, they may affect distinct intracellular signaling effectors, or the kinetics of activation of specific signaling molecules (Lax et al., 2002; Yamada et al., 2004). The idea that FGF2 and EGF may differentially affect the behavior of multipotent neural progenitors is also based on the evidence that two weeks of intraventricular administration of EGF to adult mice result in decreased neurogenesis and increased generation of astrocytes, while administration of FGF2 results in enhanced generation of neurons (Kuhn et al., 1997). Similar data have been obtained by several other groups with the exception of a study, reporting similar effects of FGF2 or EGF treatment on adult neurogenesis (Craig et al., 1996). The in vivo ultrastructural identification of adult SVZ cells affected by EGF intraventricular infusion, for instance, clearly

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demonstrated the ability of EGF to affect the modality of cell division of the transit amplifying C cell population from asymmetric to symmetric, and to restrict neuroblast formation (Doetsch et al., 2002a). Similarly, intrastriatal infusion of TGF alpha in an animal model of Parkinson’s disease (Cooper and Isacson, 2004) induces the formation of clusters of GFAP=nestinþ cells along the lateral wall of the ventricle, very likely representing an expansion of the transit amplifying C cells (Cooper and Isacson, 2004). Finally, in vitro studies in cultured neurospheres show increased astrocyte generation in response to EGF and enhanced neuronal differentiation in response to FGF2 (Whittemore et al., 1999; Jori et al., 2003). Together, these data identify EGF signaling as permissive for astrocytic but not neuronal differentiation and FGF signaling as permissive for the neurogenic fate in the adult SVZ.

Insulin-Like Growth Factor-1 (IGF1) Insulin growth factor peptides 1 and 2 are members of a family of insulinrelated peptides originally identified by their ability to stimulate growth of chondrocytes (Laron, 2001). IGF1 is secreted by many tissues and its function varies according to the site of secretion and the presence of its receptors. The expression of its receptors is highly conserved throughout evolution (Garofalo and Rosen, 1988). IGF1R, in particular, is expressed in the embryonic brain and co-localizes with the expression of FGFR and EGFR in cells of selective germinal zones (Bondy et al., 1990; Garcia-Segura et al., 1991; Kar et al., 1993). The phenotype of mice with targeted deletion in the Igf1 gene or in the Igf1R gene is severely hypomorphic, with a clear decrease in brain size (Baker et al., 1993; Liu et al., 1993; Beck et al., 1995), thus suggesting IGF1R function as critical for brain development. Conversely, transgenic mice over-expressing IGF1 show a generalized increase in cell number and corresponding increase in brain size (Carson et al., 1993). The role of IGF1 in neurogenesis is still controversial. While in vitro studies on embryonic and adult stem cells suggest a role in neuronal differentiation of EGF-responsive stem cells (Arsenijevic and Weiss 1998; Arsenijevic et al., 2001), other reports underline its role as survival factor for FGF responsive stem cells (Drago et al., 1991) and studies on freshly isolated PSA-NCAMþ cells describe IGF1 as both survival and mitogenic factor for EGF responsive cells (Gage et al., 2003). Its function in oligodendrocytes and myelination is better characterized. In addition, it has been recently suggested that IGF1 also favors the commitment of adult neural stem cells towards the oligodendrocytic phenotype (Hsieh et al., 2004). More studies on the in vivo function of IGF1 will be necessary to decipher the multiple roles played by this factor in the adult SVZ.

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Neurotrophins The neurotrophin family is composed of several trophic factors including the originally discovered founding member nerve growth factor (NGF) (reviewed in Aloe, 2004), and the related brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4 and -5 (NT-3, NT-4, NT-5) (Leibrock et al., 1989; Hohn et al., 1990; Maisonpierre et al., 1990; Berkemeier et al., 1991; Hallbook et al., 1991). Neurotrophin bind to two classes of receptors: tyrosine kinase receptors (Trk A, B and C) and a low affinity neurotrophin receptor called p75 that is structurally related to the TNFR superfamily (Barker, 2004). Each ligand binds with the highest affinity to a specific tyrosine kinase receptor (i.e. TrkA and NGF, TrkB and BDNF, TrkC and NT-3), while all the neurotrophins can bind to the low-affinity p75 (Chao, 2003). The complexity of the system is enhanced by the presence of alternatively spliced isoforms for TrkB and TrkC that may differ in case of the presence of specific catalytic domains (Huang and Reichardt, 2003; Teng and Hempstead, 2004). In the adult SVZ p75 immunoreactivity is confined to proliferating cells. The majority of the p75þ cells are identified also by EGFR immunoreactivity. Few p75þ cells in the SVZ are also nestinþ , but the majority of them does not colabel with GFAP or PSA-NCAM (Giuliani et al., 2004), thus suggesting that p75 is mainly expressed in the fast-proliferating cell population. Interestingly, no TrkA receptor expression is detected in the periventricular region by in situ hybridization (Anderson et al., 1995) or immunohistochemistry (Giuliani et al., 2004; Fiore et al., 2005), while the full-length and truncated form of TrkB receptors are both present. Truncated TrkB is confined to the ependymal cell layer and to choroid plexus, while full length TrkB expression is more widespread (Anderson et al., 1995). Therefore, it is not surprising that BDNF is the primary neurotrophin-affecting neurogenesis in the adult SVZ (Kirschenbaum and Goldman, 1995). In vivo infusion of BDNF in the lateral ventricle of the rat adult brain enhances BrdU incorporation in the SVZ and is associated with an increased number of neurons migrating to the olfactory bulb (Zigova et al., 1998). Since the BrdUþ cells are also p75þ and TrkB (Pencea et al., 2001), it is thought that the proliferative effect of BDNF is mediated by signaling through the low affinity p75 receptor in the fast proliferating cell population. The effect of BDNF on adult neurogenesis (i.e. the generation of neurons from the multipotential stem cells), in contrast, is a TrkB-dependent event, and is likely directed on PSA-NCAMþ neuroblasts. In agreement with this model, treatment of embryonic and adult-derived neurospheres with neurotrophins affects differentiation, but not proliferation of TrkBþ cells (Ahmed et al., 1995; Benoit et al., 2001). BDNF also acts as permissive factor for the maturation and survival of neuroblasts generated from the SVZ (Kirschenbaum and Goldman 1995). A recent study on the effect of BDNF on GABAergic interneurons derived from the SVZ has shown that the sequential activation of p75 and then TrkB signaling pathways is critical for the

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development of the dendritic arbor (Gascon et al., 2005). Together, these studies support a p75-mediated effect in cell proliferation and a TrkBmediated effect in neurogenesis of adult neural stem cells.

Ephrins Ephrins are cell-surface-tethered ligands for Eph receptors, a family of tyrosine kinase receptors. The functional complex ephrin/Eph is involved in several processes, including the formation and guidance of growth cones from differentiating neurons and the induction and maturation of neuronal spines (Palmer and Klein, 2003). There are two subclasses of ephrin ligands: type A (ephrinA) are GPI-linked membrane proteins while type B (ephrinB) are transmembrane proteins (Orioli and Klein, 1997; O’Leary and Wilkinson, 1999). The ephrin family of receptors (Eph) can also be subdivided into class A (EphA) and B (EphB) on the basis of structural similarities in the extracellular domain and on their ability to preferentially bind to specific ligands. Type A ligands (ephrinA) bind to EphA receptors, and type B ligands (ephrinB) bind to EphB receptors, although the EphA4 receptor can bind to both types of ligands (Kullander and Klein, 2002) and ephrinA5 can also bind to the EphB2 receptor (Himanen et al., 2004; Pasquale, 2004). During development, this signaling system modulates attraction/repulsion, cell adhesion and cell migration (Klein, 2004). In addition, a possible direct or indirect role for ephrinB1 in neurogenesis is suggested by its expression in neuroepithelial cells in the VZ at the onset of neocortical neurogenesis and its persistence throughout the neurogenetic period (Stuckmann et al., 2001). It has been suggested that ephrinB1 plays a role in affecting the responsiveness of neuroepithelial cells to other cues and also to favor migration of newly generated neurons towards their targets. In adult SVZ, both ephrin ligands (ephrin-B2,B3 and A5) and ephrin receptors (EphB1-B3, EphA4) are expressed in specific subpopulations of cells (Conover et al., 2000). Intraventricular infusion of ephrin B2 or of the EphB2 ectodomain dramatically disrupts neuroblast migration and increases proliferation in the SVZ, thus resulting in the formation of regions of localized hyperplasia (Conover et al., 2000). Therefore, ephrins/Eph complexes act as environmental cues for migration processes, axonal pathfinding and topographic mapping during development, although they can also modulate proliferation and guided migration of neurons in the adult SVZ.

The Tgfbeta Family, Including Bone Morphogenetic Protein (BMP) Transforming growth factor beta (TGFbeta) signaling controls several intracellular processes including proliferation, apoptosis, differentiation and

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lineage specification. TFGbeta ligands bind to serine-threonin kinase receptors (type I and II) on the cell surface and the signal is mediated by a heterogeneous group of proteins called Smads (Shi and Massague, 2003). The TGFbeta family of cytokines comprises two subfamilies, TGFbeta/ Activin/Nodal subfamily and BMP (Bone Morphogenetic Protein)/GDF (Growth and Differentiation Factor)/MIS (Muellerian Inhibiting Substance) subfamily. TGFbeta cytokines are expressed in the CNS of the developing rodent (Flanders et al., 1991; Millan et al., 1991; Schmid et al., 1991) in regions where neuronal differentiation occurs. In fact, TGFbeta2 in vitro induces cell cycle exit and differentiation of precursor cells (Mahanthappa and Schwarting, 1993; Constam et al., 1994; Kane et al., 1996). The BMP family includes a group of dorsal morphogens whose effect is pleiotropic and ranges from the induction of a dorsal fate in cells of the developing neural tube (Shah et al., 1996; Liem et al., 1997; Panchision et al., 2001), to the suppression of differentiation and maintainance of self-renewal in embryonic stem cells (Ying et al., 2003), from the down-regulation of EGFR expression in embryonic progenitors (Lillien and Raphael, 2000), to the induction of apoptosis (Graham et al., 1996). In addition, BMP signaling has been implicated in neurogenesis (Liem et al., 1995; Reissmann et al., 1996; Li et al., 1998; Panchision and McKay, 2002) as well as in gliogenesis (Gross et al., 1996), and also to favor the commitment to the astrocytic lineage at the expenses of neurogenesis and oligodendrogliogenesis (Grinspan et al., 2000; See et al., 2004). The effect of BMPs on astrogliogenesis is dependent on cross-talks among distinct signaling pathways and involves the activation of critical signaling molecules, including SMADs and STATs (Nakashima et al., 1999a). Since SMADs are downstream of BMP signaling and STATs are downstream of LIF signaling, it is the interaction between these two signaling pathways that appears to be critical for astrogliogenesis. Intriguingly, however, an alternative pathway of activation of STATs by BMP receptor signaling (Rajan et al., 2003) has been suggested. According to this model, STAT activation is mediated by a serine-threonine kinase (called FRAP) that becomes activated upon binding of BMP4 to its receptor (Rajan et al., 2003). Besides their role in development, BMPs favor astrogliogenesis also in the adult animal (Lim et al., 2000; Panchision et al., 2001). Indeed, BMP2 and 4 and cognate receptors are expressed in the adult SVZ where they favor the astrocytic phenotype of adult neural stem cells (Lim et al., 2000) and possibly modulate the cell cycle length of migrating neuroblasts (Coskun and Luskin, 2001). Noggin, a BMP antagonist expressed by the ependymal cells, promotes neurogenesis by counteracting the effect of the BMPs on astrogliogenesis (Lim et al., 2000). Thus, the activation of the TGFbeta signaling modulates both the decision of a cell to exit from the cell cycle and the commitment to an astrocityc fate.

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Ciliary Neurotrophic Factor (CNTF) and Leukemia Inhibitory Factor (LIF) Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are two neuroregulatory cytokines which play a major role in the developing nervous system. They both exhibit broad structural similarities (Bazan, 1991) and share signaling components (Gearing et al., 1991, 1992; Gearing and Bruce, 1992; Ip et al., 1992, 1993) among each other and with other members of the family, including interleukin-6, oncostatin or cardiotrophin 1. CNTF is widely expressed within the nervous system (Ip et al., 1993; Ip and Yancopoulos, 1996; Ip, 1998) and has been implicated in fate choice decision and survival of sensory, sympathetic, ciliary and motor neurons (Sleeman et al., 2000; Turnley and Bartlett 2000). LIF and CNTF share a common receptor, gp130 (Davis et al., 1993; Ip and Yancopoulos, 1996; Nandurkar et al., 1996). Specificity of the signaling response is achieved by selective binding of the ligand with specific receptor components. LIF signaling requires dimerization of the LIF receptor subunit beta (LIFRbeta) with gp130; while CNTF requires trimerization of its cognate receptor subunit (CNTFRalpha) with LIFRbeta and gp130 (Fig. 2.2). CNTFRalpha is expressed in embryonic neural precursor cells (Ip et al., 1993; Lachyankar et al., 1997) and in neurons and astrocytes of the adult central nervous system (Ip et al., 1993; MacLennan et al., 1996; Lee et al., 1997a,b; Kirsch et al., 1998; Dallner et al., 2002), including the subventricular zone (Seniuk-Tatton et al., 1995). In vitro studies on embryonic stem cells suggest a role for CNTF/LIF signaling in maintaining pluripotency (Conover et al., 1993) and preventing differentiation (Pennica et al., 1995) or even promoting survival (De Felici and Dolci, 1991; Pesce et al., 1993). The phenotype of the CNTF null mice, however is relatively normal and exhibits motor neuron losses only later in life, thus arguing against a major role played by this cytokine during development (Masu et al., 1993). The phenotypes of the CNTFR / (DeChiara et al., 1995), of the LIFR / (Li et al., 1995) or the gp130 / (Nakashima et al., 1999a) mice, in contrast, are characterized by a profound motor neuron defect at birth, thus supporting the notion that CNTF is critical for survival and viability of motor neurons (Sendtner et al., 1994; Ip 1998). The neuroprotective effect of CNTF is also observed in vivo, as demonstrated by the intracerebral administration of this cytokine in animal models of Huntington’s disease (Anderson et al., 1996; Emerich et al., 1996) and in injured dopaminergic neurons after transection of the nigrostriatal pathway (Hagg and Varon, 1993). The detection of CNTF receptors during embryonic development and in adult neural germinal zones raises the possibility that CNTF/LIF family members play a role in regulating proliferation and fate choice of neural stem cells. Treatment of neural embryonic progenitors and stem cells with these cytokines suggests a critical role in astroglial differentiation (Bonni et al., 1997;

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Rajan and McKay 1998; Park et al., 1999; Galli et al., 2000; Morrow et al., 2001). Signaling through STAT3, a transcription factor downstream of the LIF/gp130 receptor signaling pathway is critical for the expression of GFAP (Bonni et al., 1997) a fact also supported by the severly perturbed astrogliogenesis in LIFR / mice (Koblar et al., 1998). The role of STAT in astrocytic differentiation has been the subject of several studies. It has been shown that GFAP promoter activation requires the assembly of a complex including SMADs, STATs, the co-activator CBP and the histone acetyl-transferase p300 (Nakashima et al., 1999b). Over-expression of neurogenin, a neuronalspecific basic HLH factor, disrupts this ‘‘gliogenic’’ complex by sequestration of the CBP/p300 component from STATs and thus prevents GFAP promoter activation (Sun et al., 2001). The presence of histone acetyl transferase p300 in the transcriptional complex suggests that activation of the astrocytic program of differentiation necessitates changes in chromatin conformation. Besides acetylation of nucleosomal histones, the GFAP promoter is also regulated by a switch in the methylation of specific lysine residues on nucleosomal histones (Song and Ghosh, 2004). The ‘‘switch’’ from a silencing methylation on lysine 9 to an activating methylation on lysine 4 of histone H3 is affected by the presence of FGF2 and results in an open chromatin conformation in the promoter region, thus facilitating binding of transcriptional activators such as STATs and SMADs (Song and Ghosh 2004). CNTF signaling has also been implicated in oligodendrocytic maturation (Barres et al., 1996; Marmur et al., 1998) and neuronal differentiation (Ernsberger et al., 1989; Saadat et al., 1989; Ip et al., 1994; Rudge et al., 1996; Ezzeddine et al., 1997; Lachyankar et al., 1997). In the adult forebrain, signaling through the CNTFR/LIFR/gp130 complex is responsible for the maintenance of EGF-responsive neural stem cells (Fig. 2.3). CNTF treatment of SVZ-derived cells in vitro, increases self-renewal and expansion (Shimazaki et al., 2001) and in vivo, it enhances proliferation of the EGF-responsive population (Shimazaki et al., 2001; Chojnacki et al., 2003). The effect of CNTF/LIF signaling on proliferation and self-renewal can be explained in terms of receptor cross-talks. Proliferation could be consequent to the effect of CNTF on Notch1 signaling (Chojnacki et al., 2003), while self-renewal could be due to the effect of LIF on differentiation inhibitors, such as the Ids, downstream of BMP receptor signaling (Ying et al., 2003).

Notch1 Notch is a cell-surface receptor activated by contact with a member of the DSL family of ligands (Delta, Serrate, Lag2). Upon ligand activation, the Notch receptor is cleaved and its intracellular domain (Notch ICD) is released into the cytosol, translocates into the nucleus where it activates the transcription of CSL/CBF and induces the expression of HES genes that have been described as basic HLH transcription factors with the ability to

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inhibit neuronal differentiation (Lindsell et al., 1996; Weinmaster, 2000). Therefore, Notch signaling during development has been linked to inhibition of differentiation (Artavanis-Tsakonas et al., 1995). Although the role of Notch in timing of cell fate specification and differentiation is supported by several studies (Yun et al., 2002), the persistence of Notch1 and Jagged 1 expression in the adult SVZ (Stump et al. 2002) suggests that these molecules may also modulate the behavior of pluripotential progenitors and adult stem cells. The depletion of neural stem cells in the Notch1/ mice (Hitoshi et al., 2002) indicates a role for Notch in promoting self-renewal at the expenses of neurogenesis. However, transient Notch activation induced by administration of Notch ligand results in severe decrease of the neurogenic potential paralleled by increased gliogenesis (Morrison et al., 2000b). These apparently contradictory results can be reconciled by evoking the importance of spatial and temporal cues on the responsiveness of progenitor cells to Notch signaling. Indeed, expression of active Notch at midgestation inhibits proliferation and decreases the generation of neurons (Chambers et al., 2001). At later stages, however, Notch ICD promotes proliferation and gliogenesis (Gaiano et al., 2000; Chambers et al., 2001). Thus, similar to what was described for BMP and CNTF, the same signal can result in maintenance of stem-like cells or gliogenesis, depending on the cellular context.

Sonic Hedgehog Sonic hedgehog (Shh), is a very well characterized morphogen expressed at high levels in cells of the ventral telencephalon at embryonic day 11.5 (E11.5) and maintained throughout development (Dahmane and Ruiz-i-Altaba 1999; Wallace 1999; Wechsler-Reya and Scott 1999). Shh has been implicated in several aspects of CNS development such as proliferation (Marti et al., 1995; Roelink et al., 1995; Chiang et al., 1996; Ericson et al., 1996) and cell fate determination (Zhu et al., 1999). It has alo been shown to exert opposing actions to BMP2 in embryonic cortical progenitors (Machold et al., 2003, Viti et al., 2003b). Mice, bearing conditional null alleles of both Shh and its receptor Smoothened, have a dramatic reduction in the number of neural progenitors in the SVZ, possibly resulting from reduced proliferation and increased apoptosis (Machold et al., 2003). Recent studies on the adult SVZ in postnatal and adult mice have identified the Shh responsive SVZ cells as the GFAPþ B cells and the EGF responsive transit amplifying progenitors C cells (Palma et al., 2005). The in vitro data in SVZ cultures treated with Shh do not support a direct effect of this molecule on proliferation, although they do suggest a synergistic effect with EGF (Palma et al., 2005). Similarly, the increased number of neurospheres formed by embryonic stem cells pre-treated with Shh and cultured in the presence of EGF has been ascribed to the up-regulation of EGFR level (Viti et al., 2003b). The lack of proliferation or differentiation in the adult SVZ after intrastriatal injection of a myristoylated form of Shh

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(Charytoniuk et al., 2002) is consistent with the in vitro data. However, the decreased proliferation observed in the SVZ after administration of the Shh antagonist cyclopamine suggests a more complex role for this molecule (Palma et al., 2005). Although the role of Shh in survival of SVZ cells has not been addressed, it is likely to play a role in modulating the responsiveness of neural stem cells to other signaling molecules regulating cell number (Fig. 2.3).

Wnt The behavior of cells in the developing nervous system is tightly regulated by the highly conserved family of Wnt signaling molecules. Wnt proteins can either be secreted or located at the cell surface and may interact with a family of cell surface receptors in the Frizzled family (Ikeya et al., 1997; Yoshikawa et al., 1997; Hall et al., 2000). Binding of the ligand to the

Figure 2.2. Extracellular Receptors in embryonic and adult neural stem cells. Schematic representation of the major subtypes of extracellular receptors observed during embryonic development (panel A) and in the adult SVZ (panel B). Note that during early embryonic development (upper cell in panel A), only FGF and LIF receptors are expressed, but at later stages cells become also responsive to BMPs, Shh and EGF (lower cell in panel A). In the adult SVZ (panel B), a differential pattern of receptor expression is observed. The relatively quiescent B cells are responsive to BMPs, Shh and neurotrophins ephrins, while the transit amplifying progenitors express the receptors for EGF, FGF, CNTF, and Notch (B).

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receptor transduces a signal which involves inactivation of the GSK-3 kinase and the accumulation of the transcriptional regulator beta catenin. Of the several Wnt family members, analysis of the phenotype of mice with targeted deletions in specific genes has revealed the critical importance of Wnt1, Wnt3a and Wnt7a in the developing nervous system (Megason and McMahon, 2002). Cell proliferation is commonly regulated by Wnt signaling and expansion of the CNS fails in Wnt1 mutants (reviewed in Logan and Nusse, 2004) Over-expression of Wnt7a in embryonic stem cells increases proliferation and self-renewal both in vivo and in vitro and further promotes maturation of cortical progenitors by inducing the expression of EGFR (Viti et al., 2003b). Recently, the role of Wnt and its downstream-signaling molecule beta catenin has been explored in neural stem cells. Transgenic mice over-expressing beta catenin have grossly enlarged brains that could not be simply explained in terms of mitogenic effect or decreased apoptosis (Chenn and Walsh, 2002). Rather, it appears that beta catenin affects the decision of progenitors to exit from the cell cycle and this, in turn, results in loss of growth control (Chenn and Walsh, 2002; Zechner et al., 2003). However, in other cellular systems such as embryonic stem cells, beta catenin favors neurogenesis (Otero et al., 2004). This cell context role of b catenin has been linked to the presence of FGF2 (Israsena et al., 2004). In the presence of FGF2, beta catenin contributes to the maintenance of a proliferative state (Viti et al., 2003), while in the absence of FGF2, it enhances neuronal differentiation by forming transcriptionally active complexes on neurogenic promoters (Israsena et al., 2004; Otero et al., 2004; Logan and Nusse, 2004). A better understanding of the role of Wnt pathway in neural stem cell biology will be a very important and critical step for the design of stem cell-based therapies.

Hypoxia-Induced Growth Factors Ischemia and cerebral injury stimulate neurogenesis in neuroproliferative regions of the adult brain, including SVZ and the hippocampal DG (Gould and Tanapat, 1997; Parent et al., 1997; Liu et al., 1998; Takagi et al., 1999; Gu et al., 2000; Magavi et al., 2000; Jin et al., 2001; Yoshimura et al., 2001; Zhang et al., 2001). Concomitantly to ischemic injuries, expression of some factors increases (Kawahara et al., 1999; Marti, 2004): (a) Erythropoietin EPO Erythropoietin (EPO) is a pleiotropic-inducible molecule produced by the kidney and whose function was first described as the regulator of red blood cell production (Carnot and Deflandre, 1906) by promoting erythrocyte survival in the bone marrow (Koury and Bondurant, 1990a; 1990b; Youssoufian et al., 1993; Fisher, 2003). EPO is also a key example of a gene that is regulated in an oxygen-dependent manner and, thus, its expression is

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induced when the oxygen levels are reduced (Wenger, 2002). Recently, EPO and its receptor EPOR have also been detected in the developing CNS, thus suggesting a possible role in neural development (Buemi et al., 2002; Liu et al., 1994; Juul et al., 1998,1999). Indeed, mice with an Epor targeted deletion (Epor/) (Lin et al., 1996), are characterized by the severe reduction in the number of neural progenitor cells and increased apoptosis (Yu et al., 2002). The observation that embryonic precursors in the CNS proliferate and differentiate more in response to lowered oxygen (Morrison et al., 2000a; Studer et al., 2000) suggests that perhaps they could play a similar role in adult neural stem cells. In vitro studies on cultured neural stem cells are consistent with the idea that increased EPO gene expression results in increased adult neurogenesis (Shingo et al., 2001). Furthermore, intraventricular infusion of EPO in mice favors the migration of newly generated neurons to the olfactory bulb and the effect is blocked by anti-EPO antibodies (Shingo et al., 2001). Together these data suggest that EPO can negatively regulate proliferation of stem cells while favoring the differentiation towards the neuronal lineage. (b) Vascular Endothelial Growth Factor VEGF The vascular endothelial growth factor (VEGF) is a hypoxia-inducible secreted protein (Wenger, 2002) that regulates endothelial cell growth and differentiation and is also a survival factor for endothelial cells (Risau, 1997). The loss of a single allele in the mouse results in death during embryogenesis, due to vascular defects (Ferrara et al., 1996). In the nervous system, VEGF is expressed during development (Breier et al., 1992), and is related to the EPO-induced response to hypoxic insults in the brain as a target for the hypoxia inducible transcription factor (HIF-1) (Marti, 2004). VEGF has neurotrophic and neuroprotective effects on distinct types of neurons (Silverman et al., 1999; Sondell et al., 1999, 2000; Jin et al., 2000a, 2000b; ; Matsuzaki et al., 2001) and its receptor VEGFR2/flk-1 is expressed in neural progenitor cells (Yang and Cepko, 1996; Jin et al., 2002b), thus suggesting a possible role in neurogenesis. In the adult murine brain, administration of exogenous VEGF increases proliferation (Fig. 2.3) of neuronal precursors in the SVZ by modulating cell division rather than survival (Jin et al., 2002b). Finally, in cultures from the neonatal SVZ, treatment with FGF2 increases the expression of VEGFR2/flk-1, and in turn, treatment with VEGF enhances the chemotactic response of FGF2-stimulated progenitors, thus suggesting a synergistic effect of these two factors on migration (Zhang et al., 2003). (c) Heparin-Binding HB-EGF Heparin-binding EGF-like growth factor (HB-EGF) is a mitogenic and chemotactic glycoprotein that contains an EGF-like domain and acts

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through several receptors, including ErbB1, ErbB4, and heparin sulfate proteoglycans. Although the targeted deletion of HB-EGF in mice affects mainly heart and skin development (Yamazaki et al., 2003), the expression in neurons and glial cells throughout the brain suggests a role in the CNS (Mishima et al., 1996; Hayase et al., 1998; Nakagawa et al., 1998). As for EPO and VEGF, the expression of HB-EGF in the brain is increased by ischemia and results in neuroprotection (Kawahara et al., 1999). In addition, HB-EGF enhances neurogenesis in vitro, in neonatal cerebellar cultures (Opanashuk and Hauser 1998) and embryonic mouse neurons exposed to hypoxic conditions (Jin et al,. 2002a). In vivo, intraventricular infusion HBEGF enhances neurogenesis in the adult SVZ (Jin et al., 2002a) and restores neurogenesis to young adult levels when administered to aged mice in combination with FGF2 (Jin et al., 2003).

Extracellular Matrix and Cell–Cell Contact It has been proposed that the maintenance of neural stem cells in the adult brain is favored by the presence of extracellular conditions creating a ‘‘niche’’ that favors the preservation of an undifferentiated and proliferative state (Doetsch, 2003; Alvarez-Buyilla and Lim, 2004). The concept of a ‘‘niche’’ including components of the extracellular matrix, is quite attractive and has also been described in the hematopoietic system (Mercier et al., 2002). Remarkably, several components identified in the extracellular matrix of the SVZ (Gates et al., 1995) have been proven effective in modulating the responsiveness to mitogens (i.e. FGF2, EGF) or to morphogens (i.e. Shh, Wnt, BMPs). For instance, ECM molecules such as Tenascin C and chondroitin sulfate proteoglycans, present in the late embryonic SVZ and persist in the adult brain (Garcion et al., 2004), modulate the sensitivity to other extracellular signals at several developmental stages. This effect could be due to indirect binding to other matrix components or to direct interaction with specific cell surface receptors. In the tenascin null mice the responsiveness of embryonic stem cells to FGF2 is dramatically reduced, while the sensitivity to BMP4 is increased (Garcion et al., 2004). Given the previously discussed antagonistic role of BMP and FGF2 on EGFR expression (Lillien and Raphael, 2000), it is not surprising that tenascin loss of function results in decreased proliferation of SVZ cells and delayed EGFR expression. Another component of the ECM, the glycosaminoglycan heparin sulfate, has also been shown to promote the action of FGF2 in embryonically derived cells (Chipperfield et al., 2003), although it inhibits the response to this same factor in cells derived from the adult brain (Leventhal et al., 1999; Shen et al., 2004). These data corroborate and support the idea that the extracellular matrix is a critical component of the niche and that it may affect stem cell behavior by modulating the responsiveness to other extracellular cues and possibly affecting intracellular signals.

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Another essential component of the neural stem cell niche is the vascular compartment. In the developing CNS, the embryonic neural stem cells in the VZ have been shown to produce vascular endothelial growth factor (VEGF) which is known to contribute to the neovascularization of the area. A more direct evidence that endothelial cells enhance proliferation and neurogenesis of embryonic and adult neural stem cells is provided by co-culture experiments (Leventhal et al., 1999). Explants of adult SVZ cultured in the presence of endothelial cells express higher levels of the neurotrophin BDNF (Shen et al., 2004). Time-lapse video recording of dividing clones of neural stem cells, grown in the presence of endothelial cells, indicates that co-culture conditions tend to favor the symmetric modality of cell division (Shen et al., 2004). Therefore, proliferation of the neural stem cells seems to be affected by a wide range of molecular signals, including the production of soluble factors (i.e. VEGF, BDNF), the cross talk with the wnt/beta catenin signaling pathway and/or with the Notch signaling pathway (Temple, 2001, Shen et al., 2004).

Neurotransmitters (a) Dopamine Dopamine is a neurotransmitter produced by neurons in the substantia nigra, ventral tegmental area and preoptic area. It is involved in numerous brain processes and contributes to integration of cortical information underlying motor, limbic and cognitive aspects of behavior (Nieoullon, 2002). Besides its function as neuromodulator, dopamine also plays a role in neurogenesis during development. The D1 and D2-receptors are expressed in the striatal VZ and have been shown to play opposing roles in favoring (D2) or inhibiting (D1) cell cycle progression in the lateral ganglionic eminence (Jung and Bennett, 1996). The effect of D1-receptor activation is dominant over the effect of the D2 receptor and results in an overall reduction of cells entering S-phase (Ohtani et al., 2003). The role of D3 receptor signaling is not well established, although it is expressed in the proliferative neuroepithelium and persists postnatally in the subventricular zone (Diaz et al., 1997). Administration, either in vivo or in vitro of D3-receptor agonists, increases the proliferative rate of neural stem cells and the number of cells expressing neuronal markers (Pilon et al., 1994; Coronas et al., 2004; Van Kampen et al., 2004). This effect is mediated by MAPK activation, a pathway also activated by BDNF to affect neurogenesis (Zigova et al., 1998; Pencea et al., 2001). Given the dual relationship between dopamine receptor activation and BDNF expression (Guillin et al., 2001, 2003; Kuppers and Beyer, 2001; Sokoloff et al., 2002; ), it is likely that they synergize in promoting neurogenesis. (b) Serotonin (5-HT) Serotonin (5-HT) is produced by neurons of the raphe nucleus in the brain stem and modulates sensorimotor control, cognition and mood (Struder and

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Weicker, 2001b, 2001a). In addition to modulating synaptic function in the adult brain, 5-HT also controls important functions in brain development such as neurite outgrowth, cell survival and synaptogenesis (Gaspar et al., 2003). The role of serotonin in neurogenesis is suggested by studies on the class of antidepressants called ‘‘Serotonin Selective Re-uptake Inhibitors (SSRI)’’. Stress is known to inhibit neurogenesis by elevating the levels of gluco-corticoids (Moghaddam et al., 1994; Stein-Behrens et al., 1994). SSRI anti-depressants reverse the effect of stress and increase proliferation and differentiation of newly formed cells into neurons in the hippocampus (Malberg et al., 2000; Santarelli et al., 2003). Since serotonin receptors (5-HT1A and HT2C) are expressed in the SVZ, it is not surprising that systemic administration of various agonists increases proliferation of cells in this brain region (Banasr et al., 2004). Intriguingly, like for dopamine, the effects of serotonin on neurogenesis seem to be related to BDNF signaling, thus suggesting that the effect of the distinct classes of neurotransmitters is possibly linked to the presence of neurotrophins (Mattson et al., 2004). (c) Opioids Opioid peptides are known to act as neurotransmitters or neuromodulators in the adult nervous system. They act through three cognate receptors: m, d, k (Dhawan et al., 1996) that are also expressed in the SVZ (Zagon and McLaughlin, 1986; Stiene-Martin et al., 2001). Blockade of opioid receptors enhances cell proliferation, while their activation induces an antiproliferative effect (Hauser et al., 1996). Although this effect was originally attributed to a fourth opioid receptor z (Zagon et al., 1991), it is likely that the opioid effect on neurogenesis is a m-mediated effect since the m receptor is widely expressed postnatally in neuroproliferative regions (Stiene-Martin et al., 2001).

Hormones (a) Thyroid Hormone T3 constitutes the active ligand of the thyroid hormone (TH). The expression of TH receptors in the brain varies according to the cell type, region and age as it clearly shows a spatial-temporal patterning during development (Bradley et al., 1992) and adulthood (Puymirat et al., 1991). Besides the wellestablished role of TH in maturation of oligodendrocytes (Baas et al., 1997; Baumann and Pham-Dinh, 2001), the presence of its receptors in the adult brain also led to investigate a possible effect in neurogenesis. Indeed, hypothyroid rats showed increased proliferation in the SVZ and olfactory bulb, while hyperthyroid rats showed reduced proliferation and increased tendency to differentiate into oligodendrocytes (Fernandez et al., 2004). Coadministration of thyroid hormone with retinoic acid results in a net increase of proliferation in SVZ and enhanced neurogenesis (Giardino et al., 2000).

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(b) Estrogens The role of sex steroids in neurogenesis has been suggested by the existence of a gender bias in hippocampal-dependent tasks (Roof et al., 1993; Frye et al., 2000; Conrad et al., 2003). At the cellular level, these differences are correlated with the proliferative effects of estrogens in the hippocampus (Tanapat et al., 1999, 2005). Estrogens can bind to two types of receptors, called alpha and beta. Both receptors have been detected in several brain regions throughout development (Shughrue et al., 1990). Both receptors are also present in the ventricular wall of the embryonic neural tube as well as in the adult brain (Brannvall et al., 2002), but the functional role of estrogens at distinct stages of development is quite distinct. While estrogen treatment potentiates the mitogenic effect of EGF in embryonic neural stem cells, it antagonizes the EGF effect in adult neural stem cells, by upregulating the cell cycle inhibitor p21Cip/Waf1 (Brannvall et al., 2002). (c) Prolactin Prolactin is a hormone that increases during pregnancy and at postpartum, signaling lactation. Prolactin stimulates the production of neuronal progenitors in the SVZ (Bridges and Grattan, 2003; Shingo et al., 2003). The increased neurogenesis results in the formation of new neurons in the olfactory bulb (Shingo et al., 2003), and is possibly related to the enhanced olfactory capability of the mother.

Others (a) Amyloid Precursor Protein and Amyloid Peptide The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological functions. Its soluble-secreted form (sAPP), present in normal brain tissue (Palmert et al., 1989), has biological activities resembling a growth factor and increases the in vitro proliferation of embryonic neural stem cells (Ohsawa et al., 1999). The soluble sAPP binds to EGFRþ cells in the adult SVZ and in vitro, EGF induces the secretion of soluble APP (sAPP) by SVZ-derived cells. Intriguingly, sAPP infusions into the lateral ventricle enhances proliferation of the EGF-responsive progenitors and increases the cell number (Caille et al., 2004). In pathological conditions such as Alzheimer’s disease, however, neurons are exposed to the amyloid beta-peptide (Abeta), a self-aggregating neurotoxic protein. This peptide, in contrast to sAPP, has been shown to impair neurogenesis in the SVZ of adult mice and in human cortical neural precursor cells (Fig. 2.3). Amyloid beta peptide treatment suppresses both proliferation and differentiation of neural progenitors and induces apoptosis, associated with a disruption of calcium regulation. The cumulative result of these effects is a severe depletion of neurons possibly contributing to the olfactory and cognitive deficits observed in Alzheimer’s disease (Haughey et al., 2002).

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Figure 2.3. Schematic view of a sagittal section of the adult brain. In red are some of the extracellular signals that inhibit proliferation and favor the exit from the cell cycle. In green are the extracellular signals that promote proliferation and increase neurogenesis.

Intracellular Signals Affecting Proliferation Although it is often assumed that experimental results obtained in stem cells isolated in the developing animal can be extrapolated to the behavior of stem cells in the mature CNS, a large number of studies support the concept of intrinsic differences in distinct neural stem cell populations, depending on their location and birthdate (Temple 2001). The existence of temporally regulated changes intrinsic to the cell is suggested by studies of in vitro time-lapse videos of isolated stem cells. These studies have shown that cells maintained in the same culture conditions can first give rise to neurons and then to glia (Qian et al., 1998, 2000). These ‘‘intrinsic differences’’ may result from genetic differences and epigenetic modifications affecting the pattern of gene expression in a given cell population. Consistent with this interpretation, genetic profiling of embryonic and adult hematopoietic stem cells has identified a relatively small subset of commonly expressed genes and an even smaller number of genes shared with neural stem cells (Ivanova et al., 2002). Changes in gene expression may also result from differences in the extrinsic signaling pathways whose cross talk affects the length of the cell cycle (Tc) and/or the probability of progenitor cells to re-enter the cell cycle or become quiescent (Nowakowski et al., 2002). In this respect, it has been shown that cells in the embryonicVZ undergo a progressive increase in the length of Tc and that an increased proportion of these cells leaves the cell cycle with each cell division (Takahashi et al., 1996). Both these events are likely to be modulated by the expression levels of cell cycle regulatory molecules and other transcription

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factors (Tarui et al., 2005). Thus, progressive changes in the expression of cell cycle genes modify the cycle kinetics and the relative proportion of proliferating cells within each population, depending on the developmental stage and cellular context. Studies on the cell cycle kinetics of progenitors/neural stem cells during embryonic development, for instance, have reported increased cell cycle length with increasing embryonic age (Fig. 2.4) and a switch of cell division from symmetric and rapid (Tc = ~17.6 hr) at E11, to asymmetric and slower (Tc = ~26.5) at E14 (Tropepe et al., 1999). Differences in cell division persist in the adult forebrain subependyma (Fig. 2.4), and at least two distinct populations of proliferating cells have been identified (Morshead et al., 1998). One population, the constitutively proliferating population, has a Tc of 12.7 hr (Morshead and van der Kooy, 1992) and corresponds to the transit amplifying progenitor population (Doetsch et al., 2002a), also called the ‘‘C cell type’’ (Doetsch et al., 1997). The other population has a much longer cell cycle duration (Tc~15 d or more) and corresponds to the quiescent cell population (Morshead et al., 1994, 1998), of adult ‘‘stem cells’’ also called ‘‘B cell type’’ (Garcia-Verdugo et al., 1998). Lengthening of the cell cycle time is thought to be a function of an increase in the duration of G1 as the rest of the cell cycle parameters remain relatively

Figure 2.4. Lengthening of the cell cycle duration in stem cells during development. Note that during the early stages of development (A), the cell cycle duration (Tc) is very fast, possibly allowing for expansion. Around E11 the Tc is 17 hours and the modality of cell division primarily symmetric. As the organism develops and neurogenesis begins (E14) the cell cycle time increases to 26 hours and the modality of division becomes asymmetric. In the adult SVZ (B) two main cell types have been identified. The relatively quiescent B cells has a very long Tc (15 days) and has been proposed to be the precursor of the rapidly expanding population of C cells, characterized by a short cell cycle time (12 hrs) and the ability to give rise to neuroblasts and oligodendrocyte progenitors that become quiescent (Q).

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constant over time (von Waechter and Jaensch, 1972; Caviness et al., 1995). Therefore, it is likely that the expression of cell cycle regulatory molecules in distinct cell populations accounts for differences in cell cycle kinetics. In agreement with this model, studies on cell cycle length in the neonatal rat brain (Schultze and Korr, 1981; Menezes et al., 1995; , 1998; Smith and Luskin, 1998) have indicated that differences in cell cycle kinetics between cells in the neonatal anterior SVZ (that have a fast cell cycle time) and the migratory cells in the RMS with a slower kinetics of cell division (Smith and Luskin, 1998), correlate with the levels of expression of the G1 inhibitor p19INK4d (Coskun and Luskin, 2001). Indeed cell cycle length and the probability to exit from the cell cycle are both affected by cell cycle regulatory molecules and transcription factors whose expression can be modulated by genetic factors, epigeneticmodifications of chromatin and by the integration of extracellular signals.

Cell Cycle Regulatory Molecules In order to discuss intracellular mechanisms of proliferation of CNS progenitors and neural stem cells, it becomes critical to introduce the molecules regulating the progression from G1 into the S phase of the cell cycle. Progression through G1 is regulated by the ordered synthesis, assembly and activation of distinct cyclin-CDK enzymatic complexes (Dyson, 1998; Nevins, 2001). Two main enzymatic activities have been described: CDK4, acting in early-mid G1; and CDK2, acting in late G1, very close to the entry into the S replicative phase (Sherr, 1994; Sherr and Roberts, 1999). These two activities differ in terms of substrate specificity and modality of regulation. CDK4, for instance, is positively regulated by cyclin D and is inhibited by members of the INK4 (INhibitors of CDK4) family. CDK2, in contrast, is positively regulated by cyclin E and negatively regulated by the Kips (Kinase Inhibitory Proteins). The main substrates of cyclinD/CDK4/6 complexes are proteins of the Rb family (including pRb, p107 and p130). INK4 proteins prevent their phosphorylation, thus allowing them to sequester E2F and blocking the transcription of E2F-responsive genes that are responsible for driving the cell into S-phase (Kastan et al., 1992). Besides the role of Rb as growth-inhibitory pathway, another important cell cycle checkpoint acting at the G1 phase is mediated by the p53 tumor-suppressor gene (Paggi et al., 1996; Mundle and Saberwal, 2003). We shall now review literature pertinent to the expression patterns of these cell cycle regulatory molecules in the central nervous system, with a special emphasis on their possible functional role in the SVZ. (a) Rb Family The Retinoblastoma gene family is composed of three members of closely related proteins characterized by a ‘‘pocket’’ domain pRb, p107

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and p130/Rb2 (Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998). These molecules play a critical role in eukaryotic cell cycle progression as negative regulators of proliferation. The retinoblastoma gene product pRb, in its hypophosphorylated state, binds to members of the E2F family of transcription factors, converting them to active transcriptional repressors, by recruiting histone deacetylases (Dyson, 1998). Phosphorylated pRb in contrast, is unable to bind to E2F, the repression is relieved and results in the transcription of genes involved in DNA-replication (Fig.2.5) and nucleotide biosynthesis (Beijersbergen et al., 1994; Ginsberg et al., 1994). Distinct members of the Rb family show association with specific members of the E2F family and pRb preferentially binds to E2F-1, -2 and -3 while p107 and p130 preferentially bind to E2F-4 and -5 (Lees et al., 1992; Li et al., 1993). In addition, p107 and p130 can also bind to cyclin/CDK2 complexes (Gill et al., 1998; Callaghan et al., 1999; Ferguson and Slack, 2001). The expression profile of the ‘‘pocket proteins’’ in the brain has a characteristic cellular and temporal pattern. While pRb is found in both dividing precursor cells and postmitotic neurons during embryogenesis, p107 expression is restricted to the ventricular zone and is rapidly down-regulated at the onset of differentiation (Jiang et al., 1997; Yoshikawa 2000). P130 is expressed mainly in post-mitotic differentiated cells (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1994). Consistent with the temporal pattern of expression, targeted deletions in the Rb locus result in embryonic lethality (Cobrinik et al., 1996; Lee et al., 1996), while mice with deletions in p107 or p130 develop normally (Vanderluit et al., 2004). The expression pattern of

Figure 2.5. Molecular control of cell cycle entry. The G1/S transition of the cell cycle is regulated by the enzymatic activity of cyclin/CDK complexes. The resulting increased phosphorylation of the tumor suppressor gene pRb (or other members of the pocket protein family) induces the release of transcription factors of the family E2F/ DP and allows the transcription of genes involved in S phase entry. The main inhibitors (INK4 and KIP family members) and activators (cyclin D and E) of the cyclin/CDK complexes active at the G1/S transition are shown.

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p107 persists in the adult SVZ, where it is expressed in small clusters of cells around the ventricular wall (Vanderluit et al., 2004). Mice lacking p107 exhibit increased proliferation of the fast proliferating population, but also increased self-renewal of neural stem cells, as indicated by the ability of cells derived from the SVZ of p107 null mice, to generate a larger number of secondary neurospheres than wild type mice (Vanderluit et al., 2004). (b) INK4 Family Members INK4 proteins inhibit S-phase entry by preventing the formation of active cyclin D/CDK4 holoenzymes, due to the formation of binary complexes between the INK inhibitor and the catalytic subunit CDK4 (Quelle et al., 1995, 1997). The Ink4 locus is composed of several genes identified as Ink4a, Ink4b, Ink4c and Ink4d. While each of the Ink4 b-d genes encodes for one protein named on the basis of the molecular weight p15INK4b, p18INK4c, p19INK4d, the Ink4a locus is unusual because its second exon contributes coding sequences to two distinct reading frames resulting in two proteins: p16INK4a and p19ARF (Zindy et al., 1997). In developing mouse embryos, only p18INK4c and p19INK4d have been identified (Zindy et al., 1997). P18INK4c is preferentially localized in neurons as they exited from the cell cycle (Zindy et al., 1999), whereas p19INK4d is mainly detected in post-mitotic neurons and expressed at high levels in the adult brain (Zindy et al., 1999), often together with p27Kip1 (vanLookeren-Campagne and Gill, 1998). In the neonatal rat SVZ, p19INK4d levels are low in proliferating cells at the anterior border of the SVZ and progressively increase in the migratory cells of the rostral migratory stream (Luskin and Coskun, 2002), thus suggesting that this molecule plays a critical role in the induction of cell cycle exit once the migrating cells have reached their final destination (Zindy et al., 1999). Consistent with this interpretation, studies on mice with targeted deletion of two major cell cycle inhibitors, p18INK4c and p27Kip1 continue to proliferate even after the migratory period (Zindy et al., 1997). The results regarding the expression of p16INK4a and its possible role in cell cycle regulation of developing CNS are more controversial. While Northern and Western Blot analysis of extracts from developing mouse embryos (van Lookeren Campagne and Gill 1998) have not detected any p16INK4a signal in the brain, different results have been obtained in the developing rat, where p16INK4a is expressed at high levels in the proliferating cells of the VZ from E16 to E20 (Zindy et al., 1997). Although p16Ink4a expression is apparently down-regulated in the rat brain also, there appears to be a general consensus on the increasing levels of this protein with increasing age of the animal (Zindy et al., 1997). It is important to mention, however, that p16INK4a can be easily detected in vitro, in dissociated primary cultures, thus suggesting that the stress of culturing

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could induce the expression of molecules that may not be present in an in vivo context (Jacobs et al., 1999a, 1999b). As previously mentioned, the p16INK4a represents the alpha transcript of the Ink4a locus and represents an inhibitor of cyclin D/CDK complexes acting on pRb-E2F complexes. The other transcript of the same Ink4a locus is p19ARF (beta transcript) and it originates from a promoter some 15 kb upstream of the alpha transcript resulting in a different reading frame of exon 2 than the alpha transcript (see Fig. 2.6). As a consequence, the beta transcript encodes a protein that has no sequence homology with p16INK4a and that activates p53 rather than the pRb pathway (Fig. 2.6). Given the importance of the Ink4a locus in the transcription of regulatory components for two growth-inhibitory pathways, Rb and p53, it becomes easier to understand the high incidence of deletions or inactivations observed in this locus in patients with brain tumors. The INK4a proteins have not been detected in the developing SVZ, although presumably their expression increases with age. Given the importance of these molecules as modulators of the cell cycle, it becomes critical to understand the mechanisms regulating their expression. In this respect, it has been shown that a member of the polycomb family of chromatin modifiers called Bmi is expressed in the adult SVZ and acts as a potent repressor of the Ink4a locus (Molofsky et al., 2003). Mice with targeted deletions in the Bmi gene have a significant decrease in proliferation of neonatal and adult SVZ cells together with a 20 fold induction of p16INK4a gene product and a 3 fold increase of p19ARF (Molofsky et al., 2003). Besides proliferation, the increased levels of p16INK4a also modulate the ability of the stem cells to self-renew, thus supporting the importance of the Ink4 locus as tumor suppressor. Remarkably, however, spontaneous glial tumors are not observed in the Ink4a/Arf null mutants. Even though both GFAPþ astrocytes and nestinþ cells in these mice have the characteristics of ‘‘immortal’’ cells (Holland et al., 1998a), they still require the delivery of a constitutively active form of the

exon

1beta

beta transcript p19 ARF

p53 pathway

1alpha

2

2

alpha transcript p16INK4a

CDK 4 inhibition (Rb pathway)

Figure 2.6. The INK4a locus. The INK4a locus can generate two transcripts: p19 ARF that regulates p53 function and p16INK4a, that modulates the activity of CDK4 and therefore regulates the Rb pathway.

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EGFR (Bachoo et al., 2002) or of the activated forms of Ras or Akt (Uhrbom et al. 2002; Kamijo et al., 1997) for neoplastic transformation. Finally, it is worth mentioning that mice with selective deletion of p19ARF, with intact p16INK4a, develop spontaneous gliomas (Sherr and Roberts 1995), thus arguing that p19ARF rather than p16INK4a is involved in the neoplastic transformation of SVZ cells. (c) Kip Family Members Inhibitors of the Kip family can bind CDK4/cylinD complexes, although with lower affinity than the INK4 proteins, but this event does not result in efficient functional inhibition of enzymatic activity (Polyak et al., 1994; Toyoshima and Hunter, 1994; Sherr and Roberts, 1999). The ability of the Kips to inhibit S-phase entry is mediated by the formation of ternary complexes with cyclin A or E and CDK2 (Russo et al., 1996). The inhibitory effect of the Kip molecules on cyclin/CDK complexes is two-fold: they prevent substrate binding and rearrange the amino-terminal lobe of CDK2; thus blocking ATP binding (Russo et al., 1996) Three Kip inhibitors have been identified: p27Kip1 (Matsuoka et al., 1995), p21Cip1/Waf1 and p57Kip2 (Sherr and Roberts 1999). The p57Kip2 inhibitor is found in the VZ and SVZ of the developing rat brain at E16 and E18, and higher levels of expression are observed in post-mitotic cells at E20 (van Lookeren Campagne and Gill, 1998). The p21Cip/Waf1 inhibitor is also detected at E16 and E18, but its expression is confined to the ependymal layer of the ventricle and the choroid plexus and dramatically decreases to undetectable levels in the adult brain (van Lookeren Campagne and Gill, 1998). In agreement with this expression pattern p21Cip1/ mice do not show any change in the proliferative ability of cells in the developing or mature brain in physiological conditions (Qiu et al., 2004). Of the three members of the Kip family, p27Kip1 is undoubtedly the most interesting. Its expression is detected in proliferating cells of the VZ at midgestation (van Lookeren Campagne and Gill, 1998) and its levels progressively increase with increasing numbers of cell divisions (Delalle et al., 1999). Given the characteristic pattern of expression during the embryonic neurogenetic period, it has been suggested that p27Kip1 accumulation is part of the mechanism regulating progressive lengthening of the cell cycle and/or increased probability of cell cycle exit (Tarui et al., 2005). Studies on p27Kip1 / mice, however, have shown that the length of the cell cycle (Tc) of cortical embryonic progenitors is not affected by p27Kip1 loss of function (Goto et al., 2004), although there is a definite increase in the probability of the cells to re-enter the cell cycle, and thus an increase of the proliferating population. The expression of p27Kip1, however, persists in cells of the adult SVZ and in the rostral migratory stream, thus suggesting a role for this molecule also in the regulation of the proliferating population in the adult brain (van Lookeren

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Campagne and Gill, 1998). Mice with targeted deletions in the first exon of p27Kip1 show a selective increase in the number of transit amplifying progenitors concomitant with a reduction in the number of neuroblasts and no change in the number of stem cells (Doetsch et al., 2002b). This indicates that cell cycle regulation of SVZ adult progenitors is remarkably cell-type specific with p27Kip1 being a key regulator of cell division in transit amplifying progenitors, but not of the slow proliferating stem cells (Doetsch et al., 2002b). In vitro studies on neurospheres cultured from the neonatal SVZ support this interpretation. The levels of p27Kip1 are low in proliferating neurospheres, they increase during the early stages of differentiation and decrease again with time, in culture, thus indicating a possible role for this protein in regulating the cell cycle of immature, but not stem cells or the more mature neuroblasts (Jori et al., 2003). Together, these data suggest that distinct molecular pathways may be activated in physiological and pathological conditions in order to modulate the number of neural stem cells (Fig. 2.7). (d) p53 Pathway The tumor-suppressor gene p53 is an important checkpoint for mammalian cells in the G1 phase of the cell cycle. Upon genotoxic stress, irradiation, DNA damage, oxidative stress or glucose deprivation, this molecule activates a transcriptional response resulting in either exit from the cell cycle (possibly mediated by up-regulation of p21Cip1/Waf1) or apoptosis. In the developing brain, however, p53 expression is most abundant in proliferating

Figure 2.7. Schematic representation of extracellular signals and intracellular molecules regulating the decision of a cell in the G1 phase of the cell cycle. Although it is not clear whether mitogenic and anti-mitogenic signals affect the same cellular effector molecules in SVZ cells, it is likely that activation of active cyclin/CDK complexes result in proliferation, while their inhibition by Kip and INK family members may result in cell cycle exit.

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cell populations of the embryonic and postnatal rat brain, and is not observed in regions undergoing spontaneous apoptosis (Donehower et al., 1992). At E14, its levels are very high in proliferating cells of the ventricular zone, while from E16 to E20, it is also expressed in the SVZ and the cortical plate. The expression of p53 decreases postnatally, but it remains quite high in the postnatal rostral migratory stream and in the subventricular zone, where it persists together with p27Kip1 (van Lookeren Campagne and Gill, 1998). Interestingly, the pattern of expression of p21Cip1/Waf1, one of the downstream transcriptional targets of p53, is quite different, indicating that the role of p53 in cell cycle regulation of adult neural stem cells is independent of p21Cip/Waf1 expression. Despite the high levels of p53 detected in the VZ and SVZ of the developing rat brain, p53 null mice develop normally, and do not display any major defects in brain histoarchitecture (Donehower et al., 1992). Intriguingly, however, they do display increased susceptibility to the development of glial tumors after transplacental exposure to mutagens (Leonard et al., 2001). Current studies in our laboratory support the hypothesis that the increased susceptibility of these mice to brain tumors is secondary to a specific role of this molecule in modulating the number of adult neural stem cells in vivo (SGP and PCB unpublished). In vitro, the levels of the cell cycle regulator p53 are quite low in proliferating neurospheres generated from neonatal rats and maintained in EGF and its transcript levels are significantly higher in cells differentiated after mitogen withdrawal (Nakamura et al., 2000; Jori et al., 2003). Higher p53 levels correlate with increased apoptotic index in vitro after 3-7 days in culture. Increased protein levels, however, are observed only after 21 days in differentiating conditions, and correlate with the detection of high levels of neuronal and glial markers, thus suggesting a dual role for this molecule in apoptosis and in differentiation or lineage commitment of neural stem cells.

Other Intracellular Signaling Molecules Emx2 Emx2 and the related gene Emx1 are the vertebrate homologues of the Drosophila gene Empty spiracles (ems) involved in cephalic development (Mallamaci et al., 1998). The distinct expression pattern during late embryonic development with Emx2 expression restricted to the VZ, and Emx1 strongly expressed in the subplate and cortical plate (Gulisano et al., 1996), suggests that these two transcription factors play distinct roles in the developing nervous system. Emx2 is involved in proliferation and migration while Emx1 seems to affect neurogenesis (Yoshida et al., 1997). Emx1 null mice, however, do not have the corpus callosum and show only subtle defects in cerebral cortex (Pellegrini et al., 1996), while Emx2 null mice display major alterations of the brain histoarchitecture (Mallamaci et al., 2000; Tole et al., 2000). Recent studies on Emx2 null mice have shown significant enlargement of the proliferative ventricular and subventricular zones (Galli et al., 2002),

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thus suggesting that this molecule acts as a negative regulator of proliferation of neural precursors and adult neural stem cells. Emx2 is expressed in vivo in the adult SVZ (Gangemi et al., 2001; Galli et al., 2002) and in the rostral migratory stream, and in vitro in multipotent neural precursors (Galli et al., 2002). Its expression is significantly decreased when these stem cells differentiate into neurons and glia (Gangemi et al., 2001; Galli et al., 2002). Gain-of-function studies by over-expressing Emx2 decrease the proliferative rate of cells while retaining their differentiative potential (Gangemi et al., 2001; Galli et al., 2002). Based on these in vivo and in vitro studies, it can be concluded that Emx2 acts as a negative regulator of proliferation of adult neural stem cells. Vax 1 The homeobox Vax1 is a homologue of Emx2 and is also strongly expressed in the embryonic and adult SVZ and in the RMS (Soria et al., 2004). In the absence of Vax1, embryonic precursor cells proliferate 100 times more than wild-type controls, in vitro. In addition, the SVZ of Vax1 null mice shows signs of hyperplasia and disorganization (Soria et al., 2004). Together, these data suggested that, like Emx2, the transcription factor Vax1 is an important regulator of proliferation of SVZ cells. PTEN PTEN is a lipid phosphatase originally cloned as a tumor suppressor for glioma (Li et al., 1997; Tamura et al., 1998; Datta et al., 1999). PTEN is a phosphatidylinositol (PIP) phosphatase, responsible for the dephosphorylation of PIP3, thus antagonizing the role of the survival kinases PI3K and Akt and rendering the cells more susceptible to apoptosis (Groszer et al., 2001). In addition, PTEN is responsible for the dephosphorylation of the focal adhesion kinase FAK, resulting in the inhibition of cell migration (Groszer et al., 2001). In the adult brain, PTEN is expressed mainly in neurons and is found both in the nucleus and cytoplasm of cells in the olfactory bulb, in the SVZ and in large projection neurons. Given the importance of this signaling molecule in regulating multiple pathways, several groups have generated conditional knockout mice using the Cre-lox system. The first to be reported is the PTEN deleted by Cre expression in nestinþ cells (Backman et al., 2001; Kwon et al., 2001). These mice show increased proliferation and decreased apoptosis of cells lining the ventricular walls with a dramatic brain enlargement and death immediately after birth (Li et al., 2003). Very different is the phenotype of mice where PTEN is deleted in cells expressing Cre from the GFAP promoter (Recht et al., 2003; Berger et al., 2004). In this case, no change in proliferation or apoptosis has been reported, although the mice displayed an abnormal organization of the cerebellum. These data clearly indicate that the effect of PTEN is cell-context dependent and is affected by the intracellular and extracellular milieu, possibly due to the cross-talk with distinct signaling pathways that are active in different cells at different times.

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Conclusions Although stem cell therapy has been proposed for therapeutic strategies aimed at repairing functions, it is important to realize that as yet, relatively little is known about the behavior of embryonic and adult stem cells in terms of responsiveness to extracellular cues and intracellular signaling molecules. The challenge that awaits ahead is to define possible differences in intracellular signaling molecules between embryonic and adult derived neural stem cells that may underlie the distinctive responsiveness of these different cell types to external signals. A better understanding of the mechanisms regulating proliferation and differentiation of multipotent progenitors into differentiated neurons, astrocyte and oligodendrocytes is, therefore, essential for developing a realistic frame of therapeutic intervention while preventing undesirable - and yet possible-neoplastic transformation of adult neural stem cells. Acknowledgments. The authors are grateful to Dr Aixiao Liu and Siming Shen for critical reading of the text; to Dr Richard Nowakowski and Dr Charles ffrench-Constant for valuable comments and to Ms Bonnefil Valentina for constant support. Dr Casaccia-Bonnefil is supported by funds from NIH-NINDS and from the National Multiple Sclerosis Society. Dr Gil-Perotin is supported by a fellowship from Instituto Salud Carlos III.

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Ying, Q.L., Nichols, J., Chambers, I. and Smith, A. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115(3): 281–292. Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N., Kuratani, S. and Aizawa, S. (1997). Emx1 and Emx2 functions in development of dorsal telencephalon. Development 124(1): 101–111. Yoshikawa, K. (2000). Cell cycle regulators in neural stem cells and postmitotic neurons. Neurosci. Res. 37(1): 1–14. Yoshikawa, Y., Fujimori, T., McMahon, A.P. and Takada, S. (1997). Evidence that absence of Wnt-3a signaling promotes neuralization instead of paraxial mesoderm development in the mouse. Dev. Biol. 183(2): 234–242. Yoshimura, S., Takagi, Y., Harada, J., Teramoto, T., Thomas, S.S., Waeber, C., Bakowska, J.C., Breakefield, X.O. and Moskowitz, M.A. (2001). FGF-2 regulation of neurogenesis in adult hippocampus after brain injury. Proc. Natl. Acad. Sci. U. S. A. 98(10): 5874–5879. Youssoufian, H., Longmore, G., Neumann, D., Yoshimura, A. and Lodish, H.F. (1993). Structure, function, and activation of the erythropoietin receptor. Blood 81(9): 2223–2236. Yu, X., Shacka, J.J., Eells, J.B., Suarez-Quian, C., Przygodzki, R.M., Beleslin-Cokic, B., Lin, C.S., Nikodem, V.M., Hempstead, B., Flanders, K.C., Costantini, F. and Noguchi, C.T. (2002). Erythropoietin receptor signalling is required for normal brain development. Development 129(2): 505–516. Yun, K., Fischman, S., Johnson, J., Hrabe de Angelis, M., Weinmaster, G. and Rubenstein, J.L. (2002). Modulation of the notch signaling by Mash1 and Dlx1/2 regulates sequential specification and differentiation of progenitor cell types in the subcortical telencephalon. Development 129(21): 5029–5040. Zagon, I.S. and McLaughlin, P.J. (1987). Endogenous opioid systems regulate cell proliferation in the developing rat brain. Brain Res. 412(1): 68–72. Zechner, D., Fujita, Y., Hulsken, J., Muller, T., Walther, I., Taketo, M.M., Crenshaw, 3rd, E.B., Birchmeier, W. and Birchmeier, C. (2003). beta-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev. Biol. 258(2): 406–418. Zhang, H., Vutskits, L., Pepper, M.S. and Kiss, J.Z. (2003). VEGF is a chemoattractant for FGF-2-stimulated neural progenitors. J. Cell Biol. 163(6): 1375–1384. Zhang, R., Zhang, L., Zhang, Z., Wang, Y., Lu, M., Lapointe, M. and Chopp, M. (2001). A nitric oxide donor induces neurogenesis and reduces functional deficits after stroke in rats. Ann. Neurol. 50(5): 602–611. Zhu, G., Mehler, M.F., Zhao, J., Yu Yung, S. and Kessler, J.A. (1999). Sonic hedgehog and BMP2 exert opposing actions on proliferation and differentiation of embryonic neural progenitor cells. Dev. Biol. 215(1): 118–129. Zigova, T., Pencea, V., Wiegand, S.J. and Luskin, M.B. (1998). Intraventricular administration of BDNF increases the number of newly generated neurons in the adult olfactory bulb. Mol. Cell Neurosci. 11(4): 234–245. Zindy, F., Cunningham, J.J., Sherr, C.J., Jogal, S., Smeyne, R.J. and Roussel, M.F. (1999). Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases. Proc. Natl. Acad. Sci. U. S. A. 96(23): 13462–3467. Zindy, F., Quelle, D.E., Roussel, M.F. and Sherr, C.J. (1997). Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene 15(2): 203–211.

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Table 1. Extracellular factor FGF2 EGF TGFa IGF-1

BDNF

EPO VEGF HB-EGF Ephrins TNFa BMP

Noggin CNTF/LIF

Shh Wnt (b catenin) Notch Tenascin C Serotonin Dopamine

Opioids Others

Effect " proliferation " neurogenesis " proliferation # neurogenesis " proliferation " migration " proliferation " neurogenesis " survival " proliferation (p75) " neurogenesis (TrkB) " survival " neurogenesis " proliferation " migration " neurogenesis " proliferation # migration " proliferation # proliferation " self-renewal " gliogenesis " neurogenesis " neurogenesis " self-renewal " gliogenesis " neurogenesis " proliferation þFGF2 proliferation FGF2 neurogenesis variable " proliferation " proliferation " neurogenesis " proliferation (Baker et al., 2004) " neurogenesis # proliferation sAPP " proliferation Abeta # proliferation # migration " apoptosis

*shh enhanced the mitogenic effect of EGF

Reference (Wagner et al., 1999) (Wagner et al., 1999) (Kuhn et al., 1997) (Doetsch et al., 2002) (Cooper and Isacson 2004) (Arsenijevic et al., 2001) (Arsenijevic and Weiss 1998) (Gago et al., 2003) (Zigova et al., 1998) (Pencea et al., 2001) (Kirschenbaum and Goldman 1995) (Shingo et al., 2001) (Jin et al., 2002b) (Zhang et al., 2003) (Jin et al., 2002a) (Conover et al., 2000) (Wu et al., 2000) (Coskun and Luskin, 2001) (Ying et al., 2003) (Gross et al., 1996) (Li et al., 1998; Panchison et al., 2001) (Lim et al., 2000) (Shimazaki et al., 2001) (Bonni et al., 1997; Rajian et al., 1998) (Emsley and Hagg 2003) (Charytoniuk et al., 2002; Palma et al., 2005) " (Viti et al., 2003; Israsena et al., 2004) " (Israsena et al., 2004; Otero et al., 2004) (Chambers et al., 2001) (Garcion et al., 2004) (Banasr et al., 2004) (Coronas et al., 2004) (Van Kampen et al., 2004) (Stiene-Martin et al., 2001) (Ohsawa et al., 1999) (Haughey et al., 2002)

Chapter 3 Birth, Migration and Function of SVZ-derived Neurons in the Adult Brain Minoree Kohwi, Rui Pedro GalvA˜o, Arturo Alvarez-Buylla, Ph.D.

Introduction The identification of adult germinal zones that continue generating neurons long after the end of development has generated much excitement and stimulated many questions. In direct opposition to traditional views that germinal potentials are limited to the embryonic developing brain, an increasing number of studies have shown that neurogenesis continues throughout life, albeit in restricted domains. Adult neural stem cell research has led to discoveries that challenge old views on the mechanism of nerve cell generation. Unexpected properties of the neuronal progenitors and new mechanisms for neuronal migration have emerged from these studies. Here, we shall discuss some recent findings that investigate the origin, migration, and function of new neurons derived from the adult SVZ as well as the molecular components that maintain this germinal zone and highlight areas that offer exciting opportunities for future research. Along much of the lateral walls of the lateral ventricles lies the largest germinal zone of the adult mammalian brain, the subventricular zone (SVZ) (Doetsch and Alvarez-Buylla, 1996). In fully adult mammals, new neurons born in the SVZ migrate anteriorly into the olfactory bulb (OB), where they mature into local interneurons (Altman, 1969; Lois and Alvarez-Buylla, University of California, San Francisco, Programs in Neuroscience, E-mail address: [email protected] Instituto Gulbenkian de Cieˆncia, Portugal, Programa Gulbenkian de Biomedicina, [email protected] Department of Neurosurgery, University of California, San Francisco, E-mail address: [email protected], (tel) 415-514-2348; (fax) 415-514-2346, Mailing address: University of California, San Francisco, Programs in Neuroscience, E-mail address: [email protected], 513 Parnassus Avenue, HSW 1201A, San Francisco, CA 94143

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1994; Kornack and Rakic, 2001; Pencea et al., 2001b) (Fig. 3.1A). Cells that possess properties of neural stem cells can be isolated from the SVZ and grown in culture with EGF, FGF or the two combined (Weiss et al., 1996; Temple and Alvarez-Buylla, 1999; Gage, 2000). As such, the SVZ represents an important reservoir of progenitors in the adult brain, perhaps harboring cell populations that could be used for neuroregenerative therapy. Three questions will be discussed in this chapter: (1) Which cells give rise to new neurons in adult brain, and how is the production of these neurons regulated? (2) How do the young neurons move through adult brain and find their way into the OB? (3) What is the function of neuronal replacement in adult OB? Two decades ago, it was difficult to imagine that neurogenesis and neuronal migration could be studied in the adult brain. However, the SVZ-OB system has become an attractive experimental model in which to study neural stem cells, neurogenesis, migration, differentiation and death of the young neurons. Furthermore, the entire process raises basic questions on how neural circuits benefit from a constant exchange of neurons.

Origin of New Neurons The continual production of new neurons in the adult SVZ suggests that neural stem cells persist within this germinal layer. The adult SVZ contains at least four different cell types (A, B, C and ependymal cells) defined by their morphology, ultrastructure, and molecular markers (Doetsch et al., 1997). The young migrating neurons (type A cells) form chains ensheathed by glial fibrillary acidic protein (GFAP)-expressing astrocytes (B cells) (Lois et al., 1996; Peretto et al., 1997). Highly proliferative precursors (C cells) form clusters next to the chains of migrating A cells. Type B cells interact closely with ependymal cells lining the ventricular wall separating the ventricle from the SVZ (Fig. 3.1B). B cells contain a single short cilium that has occasionally been observed to contact the ventricular lumen (Doetsch and Alvarez-Buylla, 1996). This is similar to what has been observed in embryonic neural progenitors.

Identification of Adult SVZ Neural Stem Cells Neural Precursor cells in the adult brain must have the ability to divide. Injections of [3 H]-thymidine reveal that C cells are frequently labeled, indicating fast proliferation, but B cells and A cells also undergo cell division. It has been suggested that multiciliated ependymal cells also divide in vivo (Johansson et al., 1999). However, when labeled cells near the lateral wall of the lateral ventricle are analyzed with electron microscopy, no evidence of ependymal division is found (Spassky et al., 2005). Because A cells also divide (Lois and Alvarez-Buylla, 1994; Luskin, 1998), it was theoretically possible that they simply generate more A cells. However,

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OB NC cc

! LV RMS CB

A

B

LV SVZ

C

LV A

E B

B

C

A

Figure 3.1. (A) The SVZ–OB system. Schematic sagittal view of the adult rodent brain. The SVZ lies along the lateral wall of the lateral ventricle (LV). New neurons are constantly produced throughout the SVZ and become aligned into long chains. These form a complex network of interconnected paths throughout the SVZ (black lines in LV ). Many of the chains in the anterior SVZ connect with the RMS, which leads young neuroblasts (black cells) into the core of the olfactory bulb. Within the olfactory bulb, cells disperse radially (dotted lines) as individual cells and complete their differentiation into granule and periglomerular interneurons. NC, neocortex; cc, corpus callosum; CB, cerebellum. (B) Organization and lineage in the SVZ. Upper Left, Cross section of the anterior rodent brain indicating the location of the SVZ on the lateral wall of the LV. Right, Cellular composition and organization of the SVZ. Chains of young neurons (A cells, black) are surrounded by B cells (squares) that have astrocytic characteristics and form tube-like structures. Clusters of highly proliferative C cells (gray) are associated with the chains of A cells. Ependymal (E) cells form an epithelial layer that separates the SVZ from the ventricle. Lower left, B cells generate transient amplifying C cells that generate the A cells.

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purified A cells in culture do not appear to be self-renewing (Lim and AlvarezBuylla, 1999). In contrast, cultured fractions of B and C cells give rise to large colonies of A cells, suggesting that perhaps B and/or C cells function as the neural stem cell. Neural stem cells can be isolated from embryonic, perinatal, and adult germinal zones and grown in culture as neurospheres, which are self-renewing and multipotent. The ability to form such neurospheres is currently the best in vitro assay to study neural stem cell characteristics (Reynolds and Weiss, 1992; Luskin, 1993; Davis et al., 1994; Tropepe et al., 1999; Geschwind et al., 2001; Svendsen et al., 2001; Capela and Temple, 2002). Both B and C cells can produce neurospheres that can be passaged multiple times, showing self-renewal (Doetsch et al., 2002). These neurospheres can subsequently be differentiated into the three major CNS cell types, astrocytes, neurons, and oligodendrocytes, indicating multipotentiality (Doetsch et al., 2002). These characteristics make B and C cells likely candidates for neural stem cells. There is mounting evidence suggesting that the astrocyte-like, GFAPexpressing cells of adult SVZ, the B cells, are the primary precursors in vivo. Employing a transgenic mouse expressing the receptor for an avian retrovirus under the control of the GFAP promoter, Doetsch et al. (1999) specifically labeled astrocytes of the SVZ by targeting to this region an avian retrovirus carrying a reporter gene and found neurons that migrated to the OB. Furthermore, ablation of C cells and A cells with the anti-metabolite Ara C drugs leaves in the SVZ only astrocytes, which subsequently divide and give rise to new C cells and then to A cells. Thus, under both normal conditions as well as during SVZ regeneration, B cells seem to function as the primary precursors of new neurons. Laywell et al. (2000) observed that mouse GFAP-expressing astrocytes from cortex, cerebellum, and the spinal cord can behave as stem cells in vitro, producing both glia and neurons, but only when isolated up to the first 2 postnatal weeks. In contrast, astrocytes of the forebrain SVZ retain their abilities to behave as stem cells into adulthood. Studies by Imura et al. (2003) are consistent with these findings. The authors used in vitro culture assays and transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) from the mouse GFAP promoter (GFAP-TK) to test the hypothesis that GFAP-expressing cells are neural stem cells. In these mice, dividing GFAP-expressing cells were selectively eliminated by treatment with ganciclovir, an anti-viral agent. They show that very few neurospheres, if any, can be made after the ganciclovir treatment in the adult brain. The authors make an interesting observation that the expression of GFAP in neurosphere progenitors appears during intermediate-late development; progenitors isolated from mouse embryos grew neurospheres even in the presence of ganciclovir, indicating that neural stem cells at this early age do not express GFAP. The above studies have been substantiated by quantitative analyses of new neurons generated in vivo after ganciclovir treatment of the transgenic mouse expressing HSV-TK under the GFAP promoter (Garcia et al., 2004). Here,

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the authors show that new neurons in the OB, identified by their incorporation of BrdU, are depleted after elimination of GFAP-expressing cells. They further use cre-lox mediated recombination to show that essentially all of the new neurons generated in the adult mouse forebrain are derived from GFAPexpressing cells. These studies support the notion that GFAP-expressing cells function as neural stem cells in the adult brain (Doetsch et al., 1999). Are there cells other than those expressing GFAP that can function as adult neural stem cells? Johansson et al. (1999) suggested that ependymal cells lining the lateral wall of the lateral ventricles function as neural stem cells. The authors showed that ependymal cells express Notch1, one of the molecular hallmarks of stem cells in the developing embryonic brain. Furthermore, ependymal cells were able to form neurospheres when isolated either by picking individual cells based on their multiciliated morphologies or by filling the lateral ventricles with DiI and selecting labeled cells. Rietze et al. (2001) similarly used DiI labeling of the cells lining the lateral ventricle to investigate whether ependymal cells had neural stem cell characteristics. The authors took advantage of fluorescence-activated cell sorting (FACS) to find the cell population capable of giving rise to neurospheres and found that although the neural stem cell-enriched population was negative for CD24, an ependymal marker, this population included DiI-labeled cells. These cells also lacked multicilia, suggesting they were not ependymal cells. At the electron microscope level, some astrocytes also touch the ventricle (Doetsch et al., 2002), a characteristic of the adult SVZ that is not detectable at the light microscope level used in the above experiments. It is possible that filling the ventricle with DiI will label ependymal cells as well as a subpopulation of astrocytes, confounding the evidence that ependymal cells have neural stem cells characteristics. Furthermore, Chiasson et al. (1999) and Laywell et al. (2000) observed that although ependymal cells isolated from early postnatal or adult mouse brain were able to give rise to neurosphere-like cell clusters, these structures were neither self-renewing nor multipotent. In fact, adult mouse ependymal cells of the lateral ventricles are born during embryogenesis and do not divide in the adult (Spassky et al., 2005). These experiments indicate that ependymal cells are not multipotent neural stem cells in the adult brain. In another study, Capela and Temple showed with antibody staining that Lewis X (LeX), a carbohydrate in embryonic pluripotent stem cells, is expressed in the adult SVZ (Capela and Temple, 2002). LeX-positive SVZ cells divide in vivo as assayed by the proliferation marker BrdU. By using FACS analyses, the authors demonstrated that the SVZ cells expressing LeX are enriched for cells capable of generating neurospheres, compared to the LeX-negative population, which includes ependymal cells. When ependymal cells were isolated by FACS sorting for CD24, negligible numbers of cells produced neurospheres, in contrast to the CD24-negative fraction, leading to their conclusion that ependymal cells were not neural stem cells. However, their studies revealed that only 5% of LeX-positive cells that incorporated BrdU over a two-week period also expressed GFAP, suggesting that not all

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stem cells are GFAP-positive. In fact, the ciliary margin of the adult mammalian retina does not have GFAP-expressing cells, but also harbors neural stem cells capable of giving rise to multipotent neurospheres in vitro (Tropepe et al., 2000). Interestingly, neurosphere formation by retinal neural stem cells isolated from GFAP-TK mice is not affected in the presence of ganciclovir, suggesting that not all adult forebrain neural stem cells are GFAP-expressing cells (Morshead et al., 2003). Although it remains to be proven whether astrocytes are the only neural stem cells in the adult SVZ, there is evidence that they are not the only cell types capable of generating neurospheres. C cells, the transient-amplifying progenitors generated from B cells in the adult SVZ, also display neurosphere-generating characteristics and are multipotential in vitro (Doetsch et al., 2002). It is not known whether C cells are multipotent in vivo. Possibly, the majority of LeXpositive cells that form neurospheres may be in fact C cells rather than B cells. Therefore, identification of stem cells based on in vitro neurosphere assays may be misleading, as multiple cells within the early lineage in vivo may have this potential. The overall accumulated evidence support the notion that GFAP-expressing cells can function as neural stem cells, both in vitro and in vivo. However, we are still far from fully understanding the cellular and molecular events in the transition between B cells and C cells. Perhaps these issues can be more rigorously addressed once we have better markers to identify primary progenitors in vivo and better assays that closely mimic SVZ germinal activity in vitro.

The Glial Lineage of Neural Stem Cells Several lines of evidence indicate that astrocytes are derived from radial glia during development (Schmechel and Rakic, 1979; Voigt, 1989; Merkle et al., 2004). Radial glia, defined by their expression of RC2, are the only glia during vertebrate CNS development and are found ubiquitously in the embryonic germinal zones. The hallmarks of radial glial cells, as distinguished from a similar but distinct cell type, the neuroepithelial cell, are their astroglial characteristics and appearance around the onset of neurogenesis (reviewed in Campbell and Gotz, 2002). Historically, these cells have been considered to function as scaffold cells for young neurons migrating from the germinal zone into the developing layers of the cortex (Rakic, 1988, 1995). However, recent findings are changing our understanding of radial glial function during brain development (Hartfuss et al., 2001; Noctor et al., 2001; Gotz et al., 2002; Gregg et al., 2002; Noctor et al., 2002; Malatesta et al., 2003). Using in vivo lineage tracing techniques with a GFP-encoded retrovirus, Noctor et al. (2001) labeled radial glia and showed by time-lapse video recording the generation of neurons by radial glia. Malatesta et al. (2003) used a Cre-based fate map analysis and found that the majority of projection neurons of the cortex are derived from radial glia. In some vertebrate species, including the canary, radial glia are

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retained into adult life, serving as the primary precursors of new neurons (Alvarez-Buylla et al., 1990). These data lend credence to a new concept that radial glia function not only as support cells, but can themselves operate as neural precursors during development. The neurogenic potential of cells with astrocytic properties is surprising given the previously unchallenged idea that neuroglia develop from a lineage separate from that of neurons. Furthermore, this phenomenon does not seem to be limited to the SVZ, as there are indications of similar properties in glia from other areas of the CNS. Mu¨ller glia have been identified as neural precursors in the developing retina (Fischer and Reh, 2001). The adult hippocampus, like the SVZ, harbors neural stem cells (Gage et al., 1998). Research on the adult subgranular layer by Seri et al. (2001) indicates that in this germinal region too, astrocytes function as the primary precursors for the new granule neurons in vivo. Taken together, these findings have led to the proposition that neural stem cells lie within the radial gliaastrocyte lineage (Alvarez-Buylla et al., 2001) (Fig. 3.2).

N

N A

N AS

C

RG B

Embryo

Adult

Figure 3.2. Model of neural stem cell development. In the embryo, radial glia (RG) can behave as progenitors, generating neurons (N). Later, radial glia begin to make astrocytes. Most of these astrocytes (As) migrate into the brain parenchyma and no longer behave as progenitors, but some, such as type B astrocytes (B), end up in the SVZ and retain stem cell characteristics throughout adult life. C, type C transit amplifying cell; A, type A neuroblast.

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Recently, a unique ribbon of astrocytes lining the lateral ventricles in the adult human SVZ has been identified (Sanai et al., 2004). Astrocytes within this ribbon were shown to proliferate in vivo and give rise to multipotent neurospheres in culture. Thus, it is likely that stem cells in the human brain are also part of the glial lineage. Interestingly, this study did not find any cells resembling migratory neuroblasts emanating from the astrocytic ribbon or in the pathway towards the OB, but perhaps such cells are rare in humans or they migrate to other areas of the brain instead of the OB. Nonetheless, the study raises many interesting questions regarding the role of adult human neural stem cells and emphasizes the importance of studying stem cells in the human brain. Being able to identify human stem cells will prove vital for their future therapeutic use.

Molecular and Cellular Programs Governing Neurogenesis in the Adult SVZ Very little is known regarding the elements that control neurogenesis in the adult brain and how they are restricted to particular niches. Is the microevironment of the SVZ somehow conducive for neurogenesis? Are the astrocytes of the SVZ inherently different from those elsewhere in the brain, and do these differences allow SVZ astrocytes to produce neurons? Or is it a combination of both factors? We still lack molecular markers that can reliably distinguish terminally differentiated astrocytes from those that can function as stem cells. Most of the signaling molecules that allow neurogenesis to occur in the SVZ also remain to be discovered. However, some progress has been made in understanding the signaling pathways that may be involved in adult SVZ neurogenesis. For adult neurogenesis to occur, it has been hypothesized that an appropriately regulated set of neurogenic signals needs to be present within the stem cells niche (Garcı´a-Verdugo et al., 1998). The notion of a specialized SVZ stem cell niche is supported by experiments in which adult SVZ cells transplanted into the SVZ of another animal can give rise to neurons (Lois and Alvarez-Buylla, 1994; Doetsch and Alvarez-Buylla, 1996), but not when transplanted to non-neurogenic brain regions. Instead, in these latter regions SVZ cells produce mainly astrocytes (Herrera et al., 1999). What are the molecules that form a neurogenic niche in the adult mammalian brain? Notch1 along with its ligand Jagged1 have been found in the adult SVZ (Johansson et al., 1999; Stump et al., 2002), and the expression of the Notch downstream regulator, Hes5, suggests active Notch signaling in this region (Stump et al., 2002). The Notch signaling pathway is highly conserved across species and functions in diverse cell fate decisions during embryonic development both in invertebrates as well as vertebrates (reviewed in ArtavanisTsakonas et al., 1999). Although Notch function in the adult SVZ has not

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been well characterized, important insights can be gained from studies conducted by Gaiano et al. (2000). Here, the authors used retroviral vectors to deliver an active form of Notch to telencephalic progenitors in vivo prior to the onset of neurogenesis. They showed that activation of Notch promoted the infected cells to adopt a radial glial fate, identified by morphology as well as markers against radial glia. When infected embryos were allowed to develop to adulthood, the infected cells became astrocytes, many of which were located in the SVZ (Gaiano et al., 2000; Chambers et al., 2001). When activated Notch was stereotaxically introduced to the postnatal SVZ, the infected cells remained quiescent and did not give rise to any progeny. In contrast, infected embryonic cortical progenitors gave rise to glia at the expense of neurons (Chambers et al., 2001), perhaps suggesting that Notch activation inhibits differentiation in the adult SVZ. Hitoshi et al. (2002) demonstrated that in Notch1 mutant mice, neural stem cells are depleted from the embryonic brain, as measured by a decrease in neurosphere-forming cells. The authors also showed that neural stem cells are generated independently of RBP-Jkappa (the mammalian homologue of Suppressor of Hairless), a key signaling molecule in the Notch pathway, suggesting that Notch is not required for the generation of neural stem cells but rather, their maintenance. These data are consistent with the notion that Notch signaling inhibits neuronal differentiation by promoting a progenitor fate, perhaps accomplished by adopting a glial identity. One can imagine how such functions of Notch signaling would be important in the adult SVZ where inhibition of differentiation would maintain a lifelong pool of progenitors. Similar to Notch function, bone morphogenetic proteins (BMPs) are also known to inhibit neuronal differentiation, and recent experiments suggest signaling cross-talk between the two pathways (Takizawa et al., 2003). BMPs and their antagonistic modulator Noggin play crucial roles in neurulation and dorso-ventral patterning of the neural tube in the developing vertebrate embryo (for review, Hogan, 1996; Mehler et al., 1997). Increasing evidence reveals that Noggin and BMPs are expressed in SVZ cells, and neutralization of BMP signaling by Noggin is critical for the production of neurons in the adult SVZ (Lim et al., 2000; Liu et al., 2004; Peretto et al., 2004). BMP signaling in SVZ precursors directs glial differentiation at the expense of neurogenesis, and high Noggin expression by ependymal cells promotes neuronal differentiation (Lim et al., 2000). However, Noggin is also expressed in adult brain areas where neurogenesis has not been observed. It is likely that no one signaling pathway is sufficient for neurogenesis to occur as in the SVZ. Even within the SVZ-OB system Noggin and BMP signaling appears to elicit different effects on different populations of cells. Recent studies suggest that Noggin and BMPs are expressed not only in the SVZ, but also in the RMS into the OB (Liu et al., 2004; Peretto et al., 2004). Liu et al. (2004) propose that BMP4 induces cell cycle exit and facilitates differentiation into astrocytes in the SVZ and enhances differentiation into neurons in the RMS and OB.

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The Sonic Hedgehog (Shh) signaling pathway has also been implicated in the maintenance of the adult stem cell niche (Lai et al., 2003; Machold et al., 2003). Shh, a signaling molecule known for its involvement in cell differentiation and patterning in the neural tube and limb bud (reviewed in McMahon et al., 2003), is required for various events during CNS development including precursor proliferation (Rowitch et al., 1999), but its functions in the adult brain have been elusive. Studies by Lai et al. (2003) and Machold et al. (2003) indicate that Shh plays a role in proliferation in adult neurogenesis. Lai et al. showed that progenitors in the adult hippocampus, a neurogenic germinal zone, respond to Shh exposure by upregulating proliferation both in vitro and in vivo. Similar to the hippocampus, Shh appears to regulate proliferation in the adult SVZ (Machold et al., 2003). The authors used genetic approaches to target loss-of-function of Shh to later stages of embryogenesis and specifically to neural progenitors in order to circumvent the problem of perinatal lethality of Shh mutants. They observed that much of telencephalic patterning was preserved, yet these animals had a marked decrease in the number of neural progenitors in the postnatal SVZ as well as the hippocampus. Recent studies further indicate that some GFAP-expressing astrocytes of the SVZ express Gli 1, a downstream transcriptional target, and may respond directly to Shh signaling (Palma et al., 2005). These data suggest that Shh plays a continued role in adult germinal zones maintaining progenitor pools. Transcription factors of both the basic helix-loop-helix and homeobox family are historically known for their functions in regulating various aspects of neurogenesis, ranging from proliferation to cell fate specification (Schuurmans and Guillemot, 2002). These factors likely play important roles in adult neurogenesis. The homeobox transcription factor Vax1 appears to be important in maintaining SVZ cytoarchitecture, proliferation of precursors, and generation of migrating neuroblasts (Soria et al., 2004). Likewise, the homeobox transcription factor Sox2 and basic helix-loop-helix transcription factor Mash1 appear to play a role in SVZ neurogenesis by affecting the number of dividing precursors (Ferri et al., 2004; Parras et al., 2004). The absence of the homeobox transcription factors Dlx1 and Dlx2 result in the loss of most GABAergic OB neurons (Bulfone et al., 1998). In contrast, Pax6, a member of the paired-homeobox transcription factor family, is required for the production of specific subtypes of OB interneurons (Hack et al., 2005; Kohwi et al., 2005). We are beginning to identify the molecular players involved in adult SVZ neurogenesis and the mechanisms that specify the particular neuronal phenotypes generated. This information will undoubtedly prove critical in future therapeutic applications for neurodegenerative diseases and brain tissue damage due to stroke and trauma. Growth factors probably also play an important role in the regulation of proliferation in the SVZ. Epidermal growth factor (EGF), transforming growth factor a (TGFa) and basic fibroblast growth factor (bFGF) can stimulate the proliferation of SVZ cells in vivo (Morshead et al., 1994; Craig et al., 1996; Kuhn et al., 1997; Tropepe et al., 1997), but of these, only bFGF

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is able to increase the number of newly born OB neurons after two weeks of intracerebroventricular administration (Kuhn et al., 1997). In contrast, EGF infusion results in a decrease of OB neurogenesis concomitant with the dramatic expansion of dividing cells in the SVZ (Craig et al., 1996; Kuhn et al., 1997; Fallon et al., 2000), suggesting that these growth factors display a differential effect on adult neurogenesis in vivo (Kuhn et al., 1997). As indicated above, multipotent EGF-responsive neural stem cells can be isolated from the SVZ and grown as neurospheres (Reynolds and Weiss, 1992; Morshead et al., 1994). C cells expressing the EGF receptor (EGF-R) have been detected in vivo in the SVZ (Morshead et al., 1994; Seroogy et al., 1995; Weickert et al., 2000; Doetsch et al., 2002). Signaling through this receptor appears to play a role in vivo, as proliferation in the SVZ is reduced in mice null for TGFa; an endogenous ligand of the EGF-R (Tropepe et al., 1997). The in vivo role of growth factors and their receptors is, however, poorly understood. Consequently, the identity of the EGF-responsive cells in vivo is controversial. Whereas earlier work suggested that the EGFresponsive cells are relatively quiescent (Morshead et al., 1994), experiments in the adult brain show that the majority of EGF-responsive cells in the adult SVZ correspond to the rapidly dividing transit-amplifying C cells (Doetsch et al., 2002). These data suggest that transit-amplifying cells retain stem cell characteristics when induced to continually proliferate by the addition of exogenous growth factors. Interestingly, growth factor-amplified SVZ cells appear to acquire the ability to give rise to differentiated cells of other organs such as blood and muscle cells (Vescori, et al., 2001) . Together with classical growth factors like FGF and TGFa other molecules may also regulate proliferation in the SVZ. SVZ astrocytes express ligands for EphB2, and infusion of the soluble forms of either this receptor or ephrinB2 into the brain increases proliferation of SVZ cells and induces many B cells to contact the ventricle (Conover et al., 2000). These effects are also observed after the infusion of EGF (Doetsch et al., 2002). The regulation of proliferation in the SVZ is a particularly interesting problem not only at the molecular level, but also at the cell biological level. The symmetry of B cell division remains unknown and is a crucial piece of information needed to understand one of the key features of stem cells, selfrenewal. Transient amplifying C cell proliferation is likely to play an important role in the regulation of cell generation in the SVZ. However, the number of times C cells divide prior to A cell production is still unclear. The cell cycle of the different cell types in the SVZ needs to be determined. Previous estimates of cell cycle duration in the SVZ assumed that the SVZ is composed of a homogeneous population of non-migratory dividing cells (Morshead et al., 1994). These assumptions are not met in the SVZ, particularly given the rapid tangential migration of dividing A cells. We are still far from understanding how the SVZ regulates the number of new neurons and glial cells generated. This information is important to determine the dynamics of

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cell generation and draw a precise lineage from the dividing SVZ stem cells to the differentitated cells. More accurate methods are required to establish the cell cycle duration for the specific cell types in the SVZ.

Migration of Newly Generated Neurons Once neuroblasts are born within the SVZ, they migrate a long distance to their final destination in the OB. In adult mice, the SVZ is millimeters away from the OB, quite a journey for a cell that is just 10–30 mm long. The problem is further compounded if we consider that A cells originate throughout most of the lateral wall of the lateral ventricle and traverse a complex network of interconnected paths before joining the rostral migratory stream (RMS) (Doetsch and AlvarezBuylla, 1996) (Fig. 3.1). A similar migration has now been described in much larger brains including those of primates (Kornack and Rakic, 2001; Pencea et al., 2001b). It has been suggested that this may also occur in the infant human brain (Weickert et al., 2000). In these large brains the migration of SVZ derived cells is impressively long. How do these cells move and orient themselves over such long distances and traverse the dense parenchyma of the adult brain?

How SVZ Neuroblasts Move In adult rodent SVZ and RMS, A cells move along each other forming chains (Lois et al., 1996; Peretto et al., 1997) which are surrounded by processes from type B astrocytes (Fig. 3.1B). A cells have elongated morphologies with a prominent leading process tipped by a growth cone (Kishi, 1987; Lois and Alvarez-Buylla, 1994; Wichterle et al., 1997). In chains reconstituted in vitro, neuroblasts move in incremental steps. Constantly exploring their surrounding environment with their leading process, the cells appear to respond to unidentified signals that trigger the cell body to move. This type of migration can be measured at average speeds of approximately 120 mm=h (Wichterle et al., 1997). The molecular machinery that propels young neurons at such high speeds is not understood. Studies of cortical development have shown that the microtubule network plays a key role in translocation of neuronal cell bodies during migration (nucleokinesis) (Lambert de Rouvroit and Goffinet, 2001). Doublecortin (DCX), a microtubule associated protein important for neuronal migration in development (Francis et al., 1999) is expressed by cells in chains of the RMS (Gleeson et al., 1999), suggesting that migration to the OB in the adult shares key molecular players with migration in the embryo. It is likely that microtubule polymerization and depolymerization play an important role in the exploratory behavior and net translocation that occurs during the stepwise migration of A cells. The prominent growth cone and the active extension and retraction of the leading process of A cells (Wichterle et al., 1997) suggest that these cells may use some of the locomotory mechanisms employed by growing axons.

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Accordingly, collapsin response mediator protein 4 (CRMP-4), a molecule involved in axonal guidance, is present in the migrating A cells (Nacher et al., 2000). What role molecules like DCX and CRMP-4 may play in migration of A cells is not known. How the A cell cytoskeleton functions to decide between process elongation and cellular migration and what types of signals trigger the cell to advance a step are interesting problems for future research.

Importance of Glial Tubes In mature brain, chains of migrating A cells become ensheathed by type B astrocytes (Fig. 3.1B) (Jankovski and Sotelo, 1996; Lois et al., 1996; Peretto et al., 1997). These cells cannot be found in the RMS of the early postnatal brain when neuroblast migration is at its peak (Kishi et al., 1990; Law et al., 1999), and chain migration in vitro can occur in the absence of astrocytes (Wichterle et al., 1997). Therefore, it seems that in the immature brain as well as in vitro, astrocytes are not essential for chain migration, but they may become necessary as the brain matures. The function of these glial tubes is not known, but they are likely to play important roles, perhaps preventing A cells from prematurely escaping normal migratory routes. Olfactory ensheathing glia enwrap bundles of axons of olfactory receptor neurons, enabling the processes of this constantly regenerating cell population to enter the differentiated CNS (reviewed in Raisman, 2001). Similarly, RMS glial tubes may facilitate the migration of type A cells through the differentiated neural tissue of the adult brain. In fact, GABA is present in the SVZ and RMS and reduces the migration speed of neuroblasts by an unknown mechanism (Bolteus and Bordey, 2004). It has been discovered that the glial tube-forming astrocytes express GAT-4, a GABA transporter, allowing them to absorb GABA near the chains of neuroblasts (Bolteus and Bordey, 2004). This, in turn, may help create an environment more permissive to migration within the glial tubes. Furthermore, factors secreted by astrocytes such as MIA (Migration-Inducing Activity), appear to enhance the migration of SVZ neuroblasts (Mason et al., 2001). Additionally, these ensheathing astrocytes may support the survival of and/or provide directional information to the A cells.

Importance of Cell Adhesion Interactions Interactions between neuroblast cell surface adhesion molecules and the RMS extracellular matrix seem to be important, providing favorable conditions for cell migration. Research has begun to identify cell surface adhesion molecules important for chain migration. Polysialic acid-neural cell adhesion molecule (PSA-NCAM) is expressed along the RMS in migrating A cells (Bonfanti and Theodosis, 1994; Rousselot et al., 1995). The PSA moiety of PSA-NCAM reduces the adhesive properties of NCAM (Hoffman and Edelman, 1983; Sadoul et al., 1983; Rutishauser et al., 1985; Johnson et al., 2005) and may help in keeping A cells from establishing tight contacts with

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cells that may slow them down in their long journey. Initial observations had suggested that (PSA-NCAM) was essential for chain migration (Hu et al., 1996) because rostral migration of A cells is disrupted in animals lacking NCAM or PSA (Tomasiewicz et al., 1993; Cremer et al., 1994; Ono et al., 1994). More recent work, however, indicates that A cells lacking PSA or NCAM are less effective in their migration but still capable of forming chains and moving along the RMS (Chazal et al., 2000). This study also shows that, although PSA-NCAM is not expressed by B cells, its presence on neuroblasts is critically important for the organization of B cells as glial tubes around the migrating progenitors in the RMS and suggests that disruption of glia-neuron interaction may hinder the migration of A cells. The immature A cells may depend on PSA-NCAM in other ways: enzymatic removal of the PSA moiety causes premature differentiation of SVZ neuroblasts (Petridis et al., 2004), possibly due to an increase in their cell-cell interactions. A variety of integrins are also expressed in the RMS and appear to be required for chain migration of type A cells (Jacques et al., 1998; Murase and Horwitz, 2002). Ganglioside 9-O-acetyl GD3 (9-O-acGD3), a glycolipid present in migrating A cells (Miyakoshi et al., 2001), has also been shown to be important. Immunoblocking this molecule in vitro drastically reduces A cell migration (Hedin-Pereira et al., 2001). The extracellular matrix also contributes to the migratory environment of the RMS. Tenascin-C and chondroitin sulfate proteoglycans, molecules involved in boundary formation, have been shown to be present in the RMS (Thomas et al., 1996). They may prevent A cells from straying from the RMS pathway. Furthermore, Tenascin-C acts as a ligand for several integrins (Yokosaki et al., 1996) as well as for 9-O-acGD3 (Probstmeier and Pesheva, 1999), although little is known of the consequences of such interactions. Further work is needed to understand the interplay between surface molecules and the extracellular matrix during chain migration. It seems that molecules such as PSA-NCAM and integrins endow A cells with surface properties necessary for motility, whereas other types of molecules are necessary for guiding their migration.

Directionality of A Cell Migration The rostral migration of the neuroblasts over the long distance between the SVZ and the OB is perhaps one of the most fascinating problems in this system. It has been suggested that chemo-repulsion mediated by Slit-Robo signaling may be involved in the migration of young neurons in the RMS. Slits are expressed by the septum and choroid plexus (Hu, 1999; Li et al., 1999; Nguyen-Ba-Charvet et al., 1999) and are capable of repelling SVZ neuroblast migration in vitro (Hu, 1999; Wu et al., 1999). This activity is probably mediated through Slit receptors Robo-2 and Robo-3, which are expressed in the SVZ and RMS (Nguyen-Ba-Charvet et al., 2004). In Slit-1 /

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brains, PSA-NCAMþ neuroblasts can be found ectopically, extending into the corpus callosum dorsally and caudally (Nguyen-Ba-Charvet et al., 2004). Slits may, therefore, serve as general inhibitors of migration (Mason et al., 2001), perhaps preventing SVZ neuroblasts from migrating into certain regions of the brain. These ideas await confirmation with in vivo data. Slit activity in vitro can occur only within the distance of 1mm (Wu et al., 1999), and both septum and choroid plexus lie at a considerable distance from the RMS. Therefore, although Slits may play a role in regulating the initial steps of A cell migration, it is difficult to imagine how stable gradients of this repulsive agent can be established over such a long and complex migratory route. However, it has recently been discovered that Slit-1 is expressed by type C cells and the migrating type A neuroblasts throughout the SVZ and RMS. It was proposed that Slit-1 acts in an auto/paracrine manner in this case, affecting the neuroblasts’ ability to migrate (Nguyen-Ba-Charvet et al., 2004). Nonetheless, the activity of Slit proteins alone seems insufficient to explain the directionality of neuroblast migration. Other guidance cues must exist, acting in combination with Slits, to not only guide A cells away from the SVZ but also towards the OB. Laminin, which can be found in the RMS (Murase and Horwitz, 2002), may contribute to the directionality of A cell migration by acting as a chemoattractant for A cells. Although laminin has been traditionally regarded as a substrate for neuronal migration, ectopic infusion of a laminin peptide appears to also influence the direction of neuroblast migration (Emsley and Hagg, 2003). Recent work have suggested that the OB is the source of attractants important for tangential migration of A cells (Liu and Rao, 2003; Ng et al., 2005). Secreted molecule Prokineticin-2 can act as a chemoattractant for A cells and is expressed by the OB (Ng et al., 2005). Netrin-1 has similarly been implicated in attracting A cells to the OB (Astic et al., 2002; Murase and Horwitz, 2002). However, some studies report either no activity or even repulsion of SVZ neuroblasts by this molecule (Mason et al., 2001; Liu and Rao, 2003). Other studies have failed to detect an attractive activity of the OB on migrating cells (Hu and Rutishauser, 1996), and it has been shown that surgical disconnection and/or removal of the OB does not prevent rostral migration of SVZ neuroblasts (Jankovski et al., 1998; Kirschenbaum et al., 1999). It is likely that the OB plays a role in attracting migrating neuroblasts rostrally. However, such activity is not essential, and the molecular mechanisms by which it would be achieved have not been identified. The mechanism of directional migration in the SVZ and RMS remains, therefore, enigmatic. Understanding this process may yield novel mechanisms of neuronal orientation.

Migration Within the OB Once the cells reach the core of the OB, they separate from the chains, migrate radially to more superficial layers and differentiate into granule

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and periglomerular neurons. What regulates this change in migratory behavior? Reelin is a protein crucial for the laminar organization of the cortex by regulating the final steps in radial migration of cortical neurons (Ogawa et al., 1995). Reelin also seems to be involved in OB radial migration. This molecule is expressed in mitral cells, and one study indicates that it is necessary for the tangential to radial change in A cell migration (Hack et al., 2002). However, Reelin does not appear to have an attractive effect on A cells, affecting only their separation from the chains of cells in the core of the OB. Therefore, once the cells detach from the RMS, other signals must steer their migration to their final destination. In development, radial glia are the main substrate for neuronal radial migration (Rakic, 1972, 1990). Interestingly, these cells are no longer present in the adult CNS, raising the question of what guides these young neurons from the OB core to more superficial layers. Recent studies have identified Tenascin-R and Prokineticin-z as extracellular molecules important for neuroblast radial migration (Saghatelyan et al., 2004; Ng et al., 2005). Not only do these molecules induce detachment of neuroblasts from the RMS chains, but they also seem to reorient neuroblast migration towards the OB layers in which they are expressed. Interestingly, Tenascin-R expression in the OB is regulated by neuronal activity, providing a possible link between activity patterns and new neuron recruitment in the OB circuitry. In summary, many molecules regulating neuroblast migration have been identified. Most of these are cell surface adhesion molecules and extracellular matrix molecules that create a permissive environment for A cell migration from the SVZ to the OB. We are also beginning to identify molecules responsible for the directionality of A cell migration (Fig. 3.3). Much proSVZ/RMS Slit-1,2 (septum, cp) Slit-1 (A cells) DCX (A cells) CRMP-4 (A cells) 9-O-acGD3 (A cells) PSA-NCAM (A cells) Integrins (A cells) GABA (A cells) MIA (B cells) Tenascin-C (ECM) Chondroitin sulfate proteoglycans (ECM) Laminin (ECM)

OB Netrin-1 (MCL, ONL) Reelin (MCL, EPL, ONL) Tenascin-R (GCL, MCL)

Figure 3.3. Molecules involved in SVZ neuroblast migration. A number of different molecules have been identified that play a role in chain migration of SVZ neuroblasts towards the OB (see text for details). Many more are probably still to be discovered. cp, choroid plexus; ECM, extracellular matrix; MCL, mitral cell layer; EPL, external plexiform layer; ONL, olfactory nerve layer.

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gress is expected in elucidating the mechanisms underlying these processes as novel techniques such as microarray analysis begin to provide a plethora of candidate genes (Pennartz et al., 2004).

Function of Continuous Neuronal Replacement in the OB Every day, over 50,000 new cells migrate into the OB of an adult mouse. This process is even more conspicuous in the early postnatal brain where the RMS is massive (Luskin, 1993). Why are these new neurons incorporated into the olfactory system during postnatal life? What is their role and how is their incorporation into an adult circuitry regulated?

Circuitry of the OB The OB, a highly laminated structure (Fig. 3.4), receives input directly from the olfactory epithelium and is the first processing stage of olfactory information (reviewed in Shepherd, 1972; Shepherd and Greer, 1998). It is composed of two main cell types: the principal, or output, neurons and the local interneurons. The output neurons are the mitral and tufted cells, functionally similar and collectively called mitral/tufted (M/T) cells. Both cells send dendrites to the surface of the OB, where they contact the axons of olfactory receptor neurons (ORNs) from the olfactory epithelium. These connections form a spherical neuropil structure called a glomerulus. Each glomerulus receives axons from a single type of ORN, and each M/T cell contacts only one glomerulus. This organization is responsible for most of the odor specificity in the activation of M/T cells. M/T cells then project their axons to the pyriform and entorhinal (main and accessory olfactory) cortex, where a second level of information processing occurs. The OB circuitry and processing capability is, however, not limited to the relay of neuronal excitation between ORNs and M/T cells. The majority of neurons in the OB are inhibitory interneurons: the periglomerular and granular cells. Unlike most neurons in the adult CNS, these cell populations undergo continuous cell death (Kaplan et al., 1985). It is exactly these neurons that are produced in the distant SVZ and replaced throughout life. Periglomerular cells are located around glomeruli and establish contacts between different glomeruli and ORN axon terminals. Granular cells, by far the most numerous, are in the deepest layer of the OB (the granular cell layer) and project dendrites into the external plexiform layer. There they establish bidirectional, dendro-dendritic synapses with several M/T cells (Rall et al., 1966; Nicoll, 1969; Shepherd, 1972; Woolf et al., 1991). Activity in M/T cells leads to their release of glutamate onto the dendritic spines of granular cells. The granular cells then in turn release GABA back onto the M/T cell that activated it as well as other neighboring M/T cells (Isaacson and Strowbridge, 1998; Chen et al.,

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on

onl orn

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Figure 3.4. Olfactory bulb circuit diagram. Upper panel, Olfactory bulb interneurons (black) are derived from the SVZ. Granule cells make dendrodendritic synapses between mitral cells (light gray) connected to different glomeruli. Periglomerular cells make synaptic contacts between different glomeruli. on, olfactory nerve; onl, olfactory nerve layer; orn, olfactory receptor neurons; glom, glomeruli; gl, glomerular layer; epl, external plexiform layer; mcl, mitral cell layer; gcl, granule cell layer; lot, lateral olfactory tract. Lower panel, schematic diagram of a sagittal section of the OB showing the laminated organization of the different cells.

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2000). Thus, because of OB interneurons, activation of M/T cells results in feedback inhibition as well as lateral inhibition of neighboring M/T cells (Rall and Shepherd, 1968; Nicoll, 1969; Yokoi et al., 1995).

Role of OB Interneurons in Information Processing One cannot understand the meaning of interneuron replacement in the OB without understanding the function of these cells in the OB circuitry. Important progress has been made in that direction. As aforementioned, OB interneurons modulate the activation of M/T cells. The apparent function of this modulation is to maximize the differences between the patterns of neuronal activation elicited by different odorants (which can differ by as little as chirality of the molecule) before this information is sent to the olfactory cortex for further processing. Exactly how that is achieved is not certain. Akin to what occurs in the visual or auditory systems, local inhibitory interneurons may sharpen sensory responses, silencing neighboring neurons that are less strongly activated by the afferent stimulus (Yokoi et al., 1995; Mori et al., 1999). Other models have been proposed. Any odor leads to the activation of a variety of ORNs, which is translated in the OB into activation of multiple glomeruli often located all over the OB’s surface. Closely related odors evoke activation patterns that are initially very similar. However, due to the interactions mediated by interneurons between M/T cells, these patterns evolve over ~800ms, becoming increasingly dissimilar. It is believed that this change in patterns is used by the OB to sharpen odor discrimination (Friedrich and Laurent, 2001; Laurent et al., 2001). Another mechanism used by the OB to enhance odor detection and perhaps identification is an oscillatory synchronization of M/T cell firing. These oscillations are believed to synchronize M/T cells responding to the same odor to render transmission of information to the olfactory cortex more efficient (Stopfer et al., 1997; MacLeod et al., 1998; Schoppa and Westbrook, 1999). OB interneurons are thought to be essential for all these mechanisms.

Proposed Function of Interneuron Replacement in OB What then could be the advantage of a continuous removal and addition of OB interneurons for the organism’s sense of smell? Several studies have suggested that adult neurogenesis can enhance olfactory discrimination. Neuroblasts are produced in excess by the SVZ and migrate to the OB, where they arrive 3 to 5 days later. Over the course of the next 10 to 25 days, they migrate to their appropriate positions in the OB, mature and become fully connected to M/T cells. However, only a fraction of the cells that initially arrived in the OB survive. Petreanu and Alvarez-Buylla (2002) showed that between 15 and 45 days after they are born, 50% of the new neurons are eliminated. Interestingly, they found that this process seems to be activity-dependent, whereas production, migration and maturation seem to be activity-independent.

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After 45 days, neuronal death occurs at a slower pace, perhaps related to the overall rate of neuronal replacement. Based on this information and on the knowledge of the general pattern of OB connectivity, a mathematical model was constructed to study how adult neurogenesis could affect olfactory processing (Cecchi et al., 2001). In this model, granular cells are added randomly to the network and selected based on activity: cells with higher levels of activity are kept and those with lower levels are eliminated. After a number of iterations (or ‘‘trainings’’), the system becomes capable of optimally discriminating the 10 odors for which it was trained and the network is stable (no more granular cells are added or lost). However, if the odors are changed, the network cannot adapt to achieve maximal performance again. To do so, an additional rule must be added to the model. There must be not only an initial activity-dependent selection of incoming granular cells but also a random, slow elimination of pre-existing granular cells from the circuit. In this model, this elimination creates vacancies that allow for the incorporation of new neurons. With these rules, the system can now readjust itself to accommodate new odors. Therefore, according to this model, a continual replacement of these interneurons is necessary for maximal discrimination of odors in an olfactory environment that is constantly changing. A series of experimental observations are consistent with the predictions of this model. Mice subjected to an odor enriched environment show marked increases in the number of new OB interneurons correlating with enhanced olfactory performance (Rochefort et al., 2002). Conversely, reduced survival of granular cells caused by olfactory deprivation disrupts the representation of odors by M/T cells (Guthrie et al., 1990). NCAM mutant mice, in which migration of neuroblasts to the OB is reduced, have greater difficulty in discriminating different odors (Gheusi et al., 2000), suggesting that production of new neurons is necessary to maintain a functional olfactory system. However, this study does not directly address the role of neuronal replacement in odor discrimination, as these animals only have a deficit in the addition of new neurons to the OB. What is unique about adult neurogenesis is that it provides the capability to continuously modify the inhibitory circuitry by combining the addition of new interneurons with the removal of older ones. The model described predicts that this process allows animals to have plasticity during changes in the olfactory environment. Therefore, it would be important to study the consequences on olfactory performance of simultaneously blocking the arrival of new cells and preventing the death of older cells in the OB. Unfortunately, none of the aforementioned studies has been able to directly manipulate the replacement of OB interneurons. This problem remains to be investigated. Despite limited experimental evidence, a role for neurogenesis in olfaction can already be hypothesized. Neurogenesis might continue throughout life in brain regions that receive inputs with a high statistical variability that preclude the predetermined assembly of a unique optimal circuit. As the olfactory environment changes, it may become necessary to change the

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wiring of OB interneurons to different combinations of M/T cells. This type of change may be too drastic to be accommodated by synaptic plasticity alone. The elimination of old neurons and incorporation of new ones would thus, provide a means by which this type of plasticity could be attained (Nottebohm, 2002a, b). Additionally, young neurons may have an enhanced potential for synaptic plasticity (Schmidt-Hieber et al., 2004). The incorporation of new neurons in the OB would then enable the brain to accommodate changes in the environment. Hippocampal neurogenesis may serve a similar function (Kempermann, 2002).

Hormonal Control of Neuronal Replacement In birds as in mammals, it is becoming apparent that neurogenesis can be modulated seasonally and/or by ‘‘life events.’’ To date, most examples are related to reproductive behaviors such as singing. Seasonal fluctuations of testosterone are well correlated with new neuron addition to the avian High Vocal Center (HVC) (Nottebohm, 2002b), where there is a continuous death and incorporation of interneurons and projection neurons. Testosterone and estradiol can increase the number of new neurons in HVC, apparently by regulating their survival rather than their production rate (Rasika et al., 1994; Burek et al., 1995; Hidalgo et al., 1995; Rasika et al., 1999). In the adult mammalian hippocampus, estrogen appears to increase the numbers of dividing cells in the subgranular layer of the dentate gyrus (Tanapat et al., 1999; Ormerod et al., 2003; Perez-Martin et al., 2003). SVZ neurogenesis seems to be influenced by similar mechanisms. This may provide the mammalian olfactory system with the capacity to change perception of odors for specific purposes. Female prairie voles are induced into estrous by olfactory cues. Interestingly, in these rodents, there is a dramatic increase in the number of dividing cells in the RMS during estrous (Smith et al., 2001). In sheep, parturition leads to a drastic remapping of odor representation in the mother’s OB, a phenomenon associated with recognition of its lamb’s odor (Kendrick et al., 1992). It has recently been shown that the production of neuronal progenitors in the SVZ and recruitment of new OB neurons increases in the mother at specific days during pregnancy and after parturition, a process mediated by the hormone prolactin (Shingo et al., 2003). It is possible that neurogenesis in the SVZ/ OB system is regulated to accommodate changes in OB performance that may be necessary for specific tasks such as parturition and young rearing. New interneurons are also added to the accessory OB (Bonfanti et al., 1997), a region known to process olfactory information related to sexual behavior, but whether this process is regulated by hormones remains unknown. In birds, the neurogenic effects of testosterone seem to be mediated by BDNF (Rasika et al., 1999; Louissaint et al., 2002) and in mice, BDNF

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administration increases the number of neurons produced by the SVZ (Benraiss et al., 2001; Pencea et al., 2001a). Many other growth factors are known to act as stimulants to SVZ neurogenesis (see above). However, the role of these other factors under physiological conditions and their possible link to changes in olfactory behavior are not known.

The Matter of Numbers The adult SVZ constantly generates large numbers of neuroblasts destined for the OB. Why produce such an excess of neuroblasts in the SVZ if many probably die during migration and 50% or more are lost 15 to 45 days after they are born? The strategy may be to first produce neurons in excess and select them later in the OB, perhaps based on activity patterns (as discussed above). A common strategy during development is to produce neurons in excess of what is needed. Later, these numbers are adjusted by competition for target-derived growth factors and unnecessary neurons are eliminated by apoptosis (Levi-Montalcini, 1987). In the case of the SVZ/ OB, it is possible that the incorporation of new interneurons and the connections they establish with M/T cells is random. Many of these connections would be innapropriate and/or unnecessary and would subsequently be eliminated. It is also possible that the SVZ simultaneously produces several subtypes of interneurons, and the OB selects which survive according to their relevance to the environment at that moment. This would allow the modification of the type of neuron selected as conditions change over time. The regulation of adult neurogenesis may occur in two stages. The SVZ may regulate the overall production of neurons (influenced by hormonal and growth factor levels), whereas the OB may control their selection according to the needs of the animal. Such random production followed by specific selection may be a more favorable strategy than the OB instructing the SVZ to produce certain types of cells, due to the length of time it takes for new neurons to migrate to and integrate in the OB circuitry. By then, changes in the olfactory environment may have rendered it useless. Many questions on the role of mammalian SVZ neurogenesis remain unanswered. It is known that activity can regulate the survival of OB interneurons (Frazier-Cierpial and Brunjes, 1989; Corotto et al., 1994; Petreanu and Alvarez-Buylla, 2002), but is it merely a matter of activity versus inactivity, or is there a more specific pattern of activity that is necessary for a cell to be selected? What induces older interneurons to die? Do new neurons stimulate old ones to die, or do old dying neurons stimulate incorporation of new neurons? Can activity patterns determine both survival of new neurons and death of older cells, or do OB interneurons have a predetermined life span to continuously make space for new ones? It has been argued that incorporation of new neurons to an already functioning network may unacceptably disrupt activity (Rakic, 1985). It is,

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therefore, interesting that incorporation of new neurons in the adult OB, although similar in many ways to what occurs during development, is different in at least one aspect. In the adult OB, neuronal excitability is acquired late during maturation of new neurons (Carleton et al., 2003). This is in contrast to what happens during development, when neurons start firing action potentials when they are relatively immature. The late activation of newly born cells in the OB may allow addition of neurons to old circuits of the OB without perturbation of essential activity. This is just the beginning of our understanding of what is perhaps the most intriguing problem: the role of the new neurons in the OB. More sensitive behavioral models to test olfactory discrimination are needed. Animal models in which only a subpopulation of granular cells born postnatally can be ablated are also required so that developmental history would not interfere with interpretation of behavioral effects. In addition, we need a better understanding of the contribution of the new neurons to electrophysiological changes in OB function. The SVZ-olfactory system offers a unique opportunity to link molecular and cellular understanding of neurogenesis with function and plasticity in neural circuits.

References Altman, J. (1969). Autoradiographic and Histological Studies of Postnatal Neurogenesis. IV. Cell Proliferation and Migration in The Anterior Forebrain., With Special Reference to Persisting Neurogenesis in The Olfactory Bulb. J. Comp. Neurol. 137: 433–458. Alvarez–Buylla, A., Theelen, M. and Nottebohm, F. (1990). Proliferation ‘‘hot spots’’ in adult avian ventricular zone reveal radial cell division. Neuron 5: 101– 109. Alvarez–Buylla, A., Garcia–Verdugo, J.M. and Tramontin, A.D. (2001). A unified hypothesis on the lineage of neural stem cells. Nat. Rev. Neurosci. 2(2): 287–293. Artavanis–Tsakonas S., Rand, M.D. and Lake, R.J. (1999). Notch signaling: Cell fate control and signal integration in development. Science 284: 770–776. Astic, L., Pellier–Monnin, V., Saucier, D., Charrier, C. and Mehlen, P. (2002). Expression of netrin–1 and netrin–1 receptor., DCC., in the rat olfactory nerve pathway during development and axonal regeneration. Neuroscience 109: 643– 656. Benraiss, A., Chmielnicki, E., Lerner, K., Roh, D. and Goldman, S.A. (2001). Adenoviral brain–derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment from endogenous progenitor cells in the adult forebrain. J. Neurosci. 21: 6718–6731. Bolteus, A.J. and Bordey, A. (2004). GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone. J. Neurosci. 24: 7623–7631.

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Chapter 4 Contributions of the Neocortical Svz to Human Brain Development Nada Zecevic, Sonja Rakic*, Igor Jakovcevski, Radmila Filipovic

Introduction One of the main characteristics of the developing CNS is that all neurons and a majority of the macroglia originate in the proliferative layer situated near the lumen of the cerebral ventricles and the central canal in the brain and spinal cord, respectively, and then migrate to their final destinations. In the developing forebrain, this proliferative layer can be subdivided into the ventricular (VZ) and subventricular (SVZ) zones. The SVZ reaches a very large size in the human fetal forebrain where it was originally discovered. This transient zone can be considered a secondary proliferative compartment, which has been mitotically active in the human for several gestational months, serving as a major source of cortical interneurons and glial cells. Besides the SVZ of the dorsal forebrain, two additional secondary proliferative zones in the brain are the ganglionic eminence (GE) at the floor of the lateral ventricle, and the rhombic lip (RL) in the lateral recess of the IVth ventricle. The GE-SVZ produces interneurons and glial cells that migrate tangentially to become incorporated into the structures already containing neurons that have arrived radially from the cortical VZ/SVZs. The enlargements of the secondary proliferative zones in humans can be considered as an evolutionary adaptation to increase selective areas of the central nervous system, and thus have great importance for the expansion and cellular organization of the human cerebral cortex. This chapter focuses on the human fetal neocortical SVZ, which will be referred to as the SVZ or neocortical SVZ throughout the text. Department of Neuroscience, University of Connecticut School of Medicine, Farmington, CT 06030, USA, Tel: þ1-860-679-1768, Fax: þ1-860-679-8766, Email: [email protected] *Present address: Sonja Rakic, MD, Department of Anatomy and Developmental Biology, University College London, Gower Street London WC1E 6BT, Tel: þ4420-7679-3289, Fax: þ44-20-7679-7349, E-mail: [email protected]

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Emergence of the SVZ in the Human Fetal Brain Construction of the human brain undergoes protracted development, with basic developmental processes lasting considerably longer than in commonly studied non-primate species. The lengthy development of 40 gestational weeks (g.w.) in humans provides a better time resolution of individual developmental processes than in animal models. It is important to stress that during this period, the human brain grows in size and continues to grow well into the postnatal period, until the adult size is achieved during adolescence (Blinkov and Glazer, 1968; Badsberg Samuelsen et al., 2003). Equally important, however, is that the human brain achieves a cellular and regional diversity and complexity unmatched in any other species. The overall medical significance of understanding the developmental period for normal functioning of the adult brain has inspired scientists to study it since the time of His (1904), Cajal (1911), and Hochstetter (1919), who provided the first major principles of the brain’s organization, including the fact that neurons are produced near ventricles and migrate to the outer regions of the brain. The unique significance of the SVZ for the development of the human cerebral cortex is highlighted by its much larger size in the human forebrain than in the forebrains of any other examined species. Perhaps this is why it was initially described in the human fetus (Rakic and Sidman, 1968). The incubation of a fresh tissue block of the fetal brain in a medium containing, 3 H-thymidine, which is incorporated into dividing cells only, has revealed that, in addition to progenitor cells situated near the ventricular surface, cells located further away were also labeled (Rakic and Sidman, 1968) (Fig. 4.1). This observation, made perhaps on the first use of slice preparation for the study of developmental events, concluded that these progenitors form a separate mitotic compartment. The Boulder Committee (1970) recommended that this proliferative layer be termed the subventricular zone, and this name has now been in use for over thirty years (Fig. 4.2). During these three decades, we have learned more details about the generative capacity and the role played by the SVZ in the development of the cerebral cortex. We now know that in humans this zone contributes all major cell types to the fetal forebrain over several months of intrauterine development. Simultaneous proliferation, migration and differentiation of various cells within a relatively confined space of the fetal SVZ provide ample opportunity for these cells to influence each other in ways still not fully understood. Thus, it is likely that an insult affecting one cell type during prenatal development will affect other cell types as well. As a result, specific pathologies of neonates, often accompanied by motor deficit and mental retardation, are closely related to this region (Volpe, 1997, 2001; Back et al., 2001). In preterm infants, an intracerebral hemorrhage is predominantly located in the ganglionic eminence (GE), whereas the periventricular leukomalacia (PVL), the hypoxic-ischemic insult, is most often observed in the neocortical

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Figure 4.1. Tissue slice of the cerebral wall of the of 18.5 week human fetus immersed in a medium containing 3 H-Thymidine 25 minutes after clinical death. The cells continue to synthesize DNA supravitally and thus mark the dividing cells. The drawing in the left upper corner indicates the position of the microscopic fields, from the lateral ventricles to the medial cerebral surface. (A) Ventricular zone (VZ) and (B) wide subventricular zone (SVZ) contain many labeled cells (arrows). (C) The intermediate zone contains some labeled cells. GE-ganglionic eminence, Th-thalamus. (Modified from Rakic and Sidman, 1968.)

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Figure 4.2. Schematic drawing of five stages (A-E) in the development of the vertebrate central nervous system, which delineates the transient embryonic cellular zones recommended by the Boulder’s Committee and based on the P. Rakic drawing. Abbreviations: CP, cortical plate; I, intermediate zone; M, marginal zone; S subventricular zone; V, ventricular zone. (From Boulder Committee, Anat. Rec. 1970.)

SVZ and subcortical white matter close to the lateral ventricles. It is believed that this characteristic localization is due to the immaturity of blood vessels in this region and the unique vulnerability of immature oligodendrocyte progenitor cells (OPCs) to oxidative stress and glutamate (Volpe, 2001, Back et al., 2001). Since these lesions typically involve cortical SVZ and developing white matter, various cell groups present at these sites could be damaged. Hypoxic-ischemic injury of the periventricular white matter in premature infants seems to specifically target oligodendrocyte progenitors, which results in abnormal myelination in the forebrain (Back et al., 2001, 2002). A similar observation was made in neonatal rats (Levison et al., 2001). Other cells targeted by hypoxia/ischemia are the subplate neurons, generated in the cortical SVZ (Kostovic and Rakic, 1990; Kostovic et al., 2002; Smart et al., 2002). The subplate layer becomes the most prominent in the human cortical forebrain (Kostovic and Rakic, 1990; Kostovic et al., 2002), and although transient, this layer is very important for normal organization of thalamocortical connections and normal cortical function (Kostovic and Rakic, 1990; Shatz et al., 1988; Ghosh et al., 1990). Furthermore, this layer has an essential role in structural plasticity after periventricular lesions in premature infants (Kostovic et al., 1989; Volpe, 1997; Kostovic and Judas, 2002). Specific damage of the subplate cell progenitors in the SVZ may be one of the causes of various impairments of cortical functions, as recently described in a mouse model of periventricular leukomalacia (McQuillen et al., 2003). Similar damage is the likely contributor to

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cognitive disorders and cortical visual loss encountered in children with PVL (Piecuch et al., 1997; Cioni et al., 1997). In addition, cortical interneurons in the human brain, in contrast to other mammals, are mainly generated in the cortical SVZ (Letinic et al., 2002; Rakic and Zecevic, 2003b). Their elimination or disrupted migration can result in various cortical pathologies that range from lissencephaly (smooth brain) to cortical ectopias (displaced cells) with epilepsy (Gleeson and Walsh, 2000). Cortical interneurons seem to play a role even in some very complex psychiatric disorders, such as in schizophrenia (Akbarian et al., 1995; Lewis, 2000) or in bipolar disorder (Knable, 1999). On the other hand, uncontrolled proliferation in the SVZ or its postnatal remains, the subependimal zone, could lead to formation of brain tumors usually found in childhood (Rydberg, 1932; Kershman, 1938; Lewis, 1968).

Changing Appearance of the SVZ At the beginning of CNS development, in humans, similar to all other mammals, neural stem and progenitor cells are aligned around the ventricular system, where they rapidly divide and generate other progenitors. When the cortical plate emerges in the middle of the cell sparse primordial plexiform layer (PPL), at 7–8 g.w., a small but distinct proliferative zone (SVZ) can be recognized between the highly proliferative ventricular zone and the non-proliferative intermediate zone (Figs. 4.2 and 4.3) (Boulder Committee, 1970; Sidman and Rakic, 1973, 1982; Marin-Padilla, 1988; Zecevic, 1993; Meyer et al., 2000; Chan et al., 2002; Zecevic et al., in press). Similar to humans, in monkeys too, the SVZ appears at the time when the cortical plate forms, around E43-E55 (e.g., Rakic, 1972; Smart et al., 2002). From this period onwards, this zone increases in width and cellular complexity in all mammals (rev. Brazel et al., 2003), but achieves its maximum size and complexity over subsequent months of intrauterine development in primates, and especially in humans (e.g., Sidman and Rakic, 1982; Kendler and Golden, 1996; Smart et al., 2002; Zecevic et al., in press). On Nissl-stained sections of the human fetal brain, it is apparent that the histological organization of the SVZ changes considerably during the course of intrauterine development (Fig. 4.3). Early during development, at 9 g.w., the SVZ consists of radially oriented cells that at its lower border appear to be a continuation of radially arranged VZ cells. At its upper border, cells close to the intermediate zone (IZ), are more loosely organized (Fig. 4.3A, B). From 17 g.w. on, many fiber bundles are crossing tangentially through the SVZ, changing its initial compact appearance (Fig. 4.3C). The appearance of the SVZ also varies in respect to its position in the forebrain. Its width differs from the rostral to caudal pole, as well as from medial to dorso-lateral regions of the forebrain (Zecevic et al., in press). The anterior or rostral SVZ is located around the frontal pole of the lateral

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Figure 4.3. The SVZ in human fetal brain. (A) SVZ cells are radially aligned in continuation with the VZ in this Nissl-stained section of 9 g.w. fetus. (B) Higher magnification of this section reveals mitotic figures in the SVZ (arrows). (C) At 17 g.w. in the visual cortex, two parts of the SVZ are seen; the inner (ISVZ) is cell dense, whereas the outer (OSVZ) contains numerous tangentially traversing fibers (IFLinner fiber layer). (D) Numerous cell bands streaming from the medial SVZ towards the subplate layer of the cerebral cortex; Nissl stained section from the rostral pole at 20 g.w. (E) Golgi impregnated section of 19 g.w. fetus reveals bipolar morphology and either horizontal or radial orientation of SVZ cells. Arrows point to the direction of the leading processes. CP-cortical plate Scale bars: A = 50 mm, B,C = 10 mm, E = 20 mm.

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Figure 4.4. Composite drawing of coronally sectioned right hemisphere in 22 g.w. fetus, starting from the rostral (frontal) to caudal (occipital) end, M-medial, D-dorsal, Th-thalamus, GE-ganglionic eminence, LV-lateral ventricle, lateral (lSVZ) and medial subventricular zone (mSVZ). Note difference in thickness of the lateral and medial SVZ.

ventricle and apparently contributes cells to the cerebral cortex, GE, cingulum, and septum. On coronal sections, the SVZ around the frontal pole looks asymmetric, with the lateral part being three times as thick as the medial part (Fig. 4.4). On the medial side, facing the interhemispheric fissure, one of the main characteristics of the human SVZ is the presence of numerous cell bands that spread towards the overlaying cortical plate (Fig. 4.3D). Although these cell bands can be seen in the lateral SVZ, they are more pronounced in the medial SVZ, stretching over 2000 mm to the subplate layer (Zecevic et al., in press). These bands consist of closely linked bipolar cells, and resemble the rostral migratory stream (RMS) described in rodents (Altman, 1969; Luskin, 1993; Lois and Alvarez-Buylla, 1994). Similar to the RMS, cells in these SVZ bands express the astroglial markers GFAP (glial fibrillary acidic protein) and vimentin, but also a large number of other markers, such as lectins for microglia/macrophage, PDGFRa for oligodendrocyte progenitors, ventral forebrain transcription factor Nkx2.1, and neuronal marker MAP2 (Rakic and Zecevic, 2003b). Thus, it seems that

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neurons, microglia and oligodendrocyte progenitor cells could all use this route to migrate out of the SVZ (Rakic and Zecevic, 2003b; Jakovcevski and Zecevic, 2005). In contrast to the adult mouse brain, where the network of cell chains in the lateral wall of the lateral ventricle is labeled with PSANCAM (polysialylated form of neuronal cell adhesion molecule) (Doetsch and Alvarez- Buylla, 1996), cell chains of the fetal human SVZ are not labeled with PSA-NCAM antibody, indicating different adhesion properties of these cells. Cells in the bands are often dividing as revealed by their PCNA (proliferating cell nuclear antigen) labeling (Rakic and Zecevic, 2003b). Division of cells during migration from the SVZ was also described in rodents (Chapter I). In adult rodents, the rostral SVZ has been described to produce olfactory neurons, oligodendrocytes and astrocytes that migrate through the rostral migratory stream (RMS) (Altman, 1969; Luskin, 1993; Lois and AlvarezBuylla, 1994). A smaller RMS as a restricted pathway of migrating cells has also been described in the adult monkey brain (Kornack and Rakic, 2001). In the human fetal brain, in situ hybridization with ventral transcription factors, Dlx2 and Nkx2.1, revealed a strong signal that connects the ganglionic eminence and cortical SVZ with the olfactory bulb. This result suggests that, similar to other mammals, in developing human brain the rostral SVZ may be a source of olfactory bulb neurons (Fig. 4.5, Rakic and Zecevic, 2003b). In contrast to the more compact medial SVZ, the dorso-lateral SVZ along the whole rostro-caudal length of the lateral ventricle consists of two sublayers of cells intercepted with fiber bundles. This is best observed in the occipital pole (Fig. 4.6). A similar organization of the SVZ was recently described in the occipital cortex of the fetal monkey brain (Smart et al., 2002). This complex organization of the SVZ develops through a histogenetic program, which seems to be a unique feature of the primate brain (Smart et al., 2002; Zecevic et al., in press). The outer fiber layer (OFL in the occipital cortex) is formed by an early arrival of optic radiation, and has a distinct palisade appearance. It consists of radially aligned migrating neurons, intercepted by fiber bundles. The OFL does not have an equivalent counterpart in mouse (Smart et al., 2002). As the SVZ increases in depth it becomes divided into an inner SVZ (ISVZ) and an outer SVZ (OSVZ) by the inner fiber layer (IFL) (Fig. 4.3C). The formation of radially aligned cells in the OSVZ coincides with the decrease of proliferation in the VZ, which occurs prior to major neuronal production, suggesting that in primates, the OSVZ is the main source of later-born cortical neurons (Smart et al., 2002). The ISVZ consists of randomly organized subventricular cells close to the VZ, and resembles the SVZ described in rodents (Smart et al., 2002). In monkey, the OSVZ contains nuclei of proliferating radial glia and neurons, which results in the increased size of the forebrain and an enlarged cortical surface area (Levitt et al., 1981; Smart et al., 2002). Similarly, in the human fetal forebrain, both the OSVZ and ISVZ contain many dividing

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Figure 4.5. Expression of Dlx2 and Nkx2.1 mRNAs in human developing forebrain during midgestation as seen by radioactive in situ hybridization. (A) In the frontal sections through both hemispheres of 17 g.w. fetus, a strong Dlx2 mRNA signal in the GE-SVZ and weaker signal in the cortical VZ and in the cerebral cortex could be observed; (B) On sagittally cut section at 25 g.w. a strong Dlx2 mRNA signal connects the GE and the olfactory region and in the hippocampus. The frontal pole of the brain is to the left of the field; occipital pole to the right; (C,D) The expression of Nkx2.1 mRNA is revealed in two sagittal sections through the rostral part of the forebrain at 22 g.w.; (C) in the more medial section, Nkx2.1 mRNA is expressed in the neocortical SVZ, the cerebral cortex and in the GE, spreading

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nuclei, which attest to their highly proliferative activity. In the VZ, proliferation, however has been greatly reduced at this time and cannot account for the enlargement of the cortex. One possibility is that radial glia are relocated to the OSVZ where they continue to proliferate and possibly produce neurons (Smart et al., 2002). Proliferating radial glia cells have been indeed demonstrated in the human SVZ at midgestation (Howard et al., 2005). This is an interesting finding, particularly in the light of the potential of radial glia to make neurons (Malatesta et al., 2000, 2003; Noctor et al., 2001; Campbell and Gotz, 2002). The outer SVZ contains a striking mesh of tangentially and radially oriented fibers and migrating cells, as observed in developing monkey (Zecevic and Rakic, 2001) and human forebrains (Rakic and Zecevic, 2003b). Tangential axonal fibers crossing through the SVZ belong mainly to the cortico-cortical fiber system that crosses through the corpus callosum, but also the cortico-thalamic and thalamo-cortical fibers which are passing through the outer SVZ (Molnar and Blakemore, 1995; Miller et al., 1993). In the occipital cortex of the fetal monkey (E86), geniculostriate fibers travel through the lower part of the outer fiber layer (Smart et al., 2002), similar to what has been reported in human fetal brains (Kostovic and Judas, 2002). Numerous monoaminergic fibers from the brain stem are also observed in the human fetal forebrain at midgestation, but these fiber bundles are positioned superficial to the SVZ, at the border between the IZ and the subplate layer (Zecevic and Verney, 1995). Transversally crossing fibers may provide a substrate for tangential migration of interneurons and oligodendrocyte progenitors through the SVZ (Zerlin et al., 1995; Parnavelas, 2000). It has been described using retrovirally labeled cells that migration of SVZ cells is approximately 10 times faster than migration from the VZ (Kakita and Goldman, 1999). In the neonatal rat brain, these cells proceed tangentially through the SVZ, and then radially into the cerebral cortex, following vimentin-labeled radial glia fibers (Zerlin et al., 1995). These authors suggested that many migrating cells represent glial cells, consistent with our finding in human fetal SVZ (Jakovcevski and Zecevic, 2005). Cortical interneurons coming from the GE also use this pattern of migration through the SVZ, which will be described later.

towards the olfactory region, whereas (D) in the more lateral section, a signal is also observed in the proliferative zones of the GE and in the basal ganglia. Frontal pole of the brain is to the left. VZ – ventricular zone; SVZ – subventricular zone; Cx – cortex; GE – ganglionic eminence; NC – nucleus caudatus; P – putamen; IC – internal capsule; LV – lateral ventricle; Th – thalamus; Hipp – hippocampus; OR – olfactory region; GP – globus pallidus. Scale bars = 5mm (A-D). (From Rakic and Zecevic, 2003b.)

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Figure 4.6. The coronal section of the occipital pole at midgestation stained with aniline dye shows several cell and fiber sublayers in the lateral SVZ. From the VZ up, the inner cell layer (ISVZ), the inner fiber layer (IFL), the outer cellular layer (OSVZ) and the outer fiber layer (OFL). Note: radial alignment of cells in the OFL. VZ-ventricular zone, CP-cortical plate, MZ-marginal zone. Scale bar = 100 mm

A caudal SVZ is located around the ventro-posterior part of the lateral ventricle. This region has not attracted a lot of attention probably because of its relatively smaller size in rodents compared to primates. In the human fetal brain, the posterior SVZ is well developed in the second half of gestation. A stream of cells extends from the posterior SVZ to the white matter of the temporal lobe, consistent with the described role of the posterior SVZ in production of various glia cell types, and perhaps also neurons in the adult human brain (Bernier et al., 2000).

Cell Proliferation in the Human Fetal SVZ The enlargement of the human brain is a result of cell proliferation that occurs through the entire intrauterine period and in some regions after birth. Human brain growth can be divided into a rapid phase of exponential growth from 13 to 20 weeks of gestation, and a slower phase with a linear

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increase of cell number from 22 weeks to term (Badsberg Samuelsen et al., 2003). The unique expansion of the SVZ in the human brain is due mainly to continuous proliferation of the progenitor cell pool, which results in culmination of the size of the SVZ after midgestation (20–35 g.w.). At the same time, the proliferative activity in the VZ decreases, as the VZ becomes reduced to a one-cell layer -the ependyma (Simonati et al., 1999; Rakic and Zecevic, 2000; Chan et al., 2002). Although significantly reduced in size, the SVZ remains in rudimentary form in the adult human brain as the subependymal zone. Cells in the human fetal SVZ actively proliferate for a period of several months, as confirmed by H3 -thymidine autoradiography (Rakic and Sidman, 1968), immunoreaction to PCNA (Møllgard and Schumacher, 1993; Simonati et al., 1999; Rakic and Zecevic, 2000, 2003b; Chan et al., 2002), and labeling with propidium iodide (Letinic et al., 2002), or BrdU (Filipovic and Zecevic, 2004; Zecevic et al., in press). Cell proliferation in the cortical SVZ differs from that in the VZ in several aspects. First, cells proliferate in situ and, thus, lack the nuclear translocation that is typical for the VZ (Sidman and Rakic, 1973). Second, cells that divide in the SVZ can at the same time migrate, as shown in the rodent brain (Menezes et al., 1995; Zerlin et al., 1995; Kakita and Goldman, 1999) and human fetal brains (Letinic et al., 2002; Rakic and Zecevic, 2003b). Finally, whereas the VZ mainly supplies projection neurons to the cerebral cortex (Sidman and Rakic, 1973, 1982), the cortical SVZ has a much larger repertoire. In primates, this zone contributes to interneurons, oligodendrocyte and astrocyte cell populations. It is, however, difficult to distinguish between the proliferation of progenitor cells that arrived from the ganglionic eminence and proliferation of cells that have neocortical SVZ origin (Letinic et al., 2002; Rakic and Zecevic, 2003b). The increased number of progenitor cells in the proliferative zone affects the gross morphology of the forebrain. This has been best shown in experiments with transgenic mice, where either cell death was reduced (Kuida et al., 1998; Haydar et al., 1999) or cell proliferation was increased (Chenn and Walsh, 2002, 2003). These last authors generated a transgenic mouse with a stabilized form of b-catenin in neural precursors, which further resulted in an increased number of progenitor cells, larger brains with enlarged cortical surface and gyration pattern reminiscent of higher mammals. These results indicate that the enlarged human forebrain with pronounced gyri and sulci is probably related to the size of the progenitor pool residing in the VZ/SVZ region. The control of cell proliferation in the SVZ is not well understood, either in non-primate or primate brains. In rodents, several extracellular factors influence cell proliferation in the forebrain. For example, the basic fibroblast growth factor (bFGF) increases the number of cortical neurons when added to cell culture or injected into embryonic rat brains (Vaccarino et al., 1995, 1999). The adult mouse forebrain contains stem-like cells in the rostral SVZ

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that in vitro proliferate in response to either bFGF or EGF (epidermal growth factor) (Reynolds and Weiss, 1992; Gritti et al., 2002). Furthermore, when cycling glia progenitors are isolated from the adult rat SVZ, they proliferate in response to PDGF-AA, bFGF and IGF-1 (Mason and Goldman, 2002). Chemokines such as Gro-a increase proliferation of OPCs in the human fetal SVZ slice culture (Filipovic and Zecevic, unpublished results). In addition, both neurotransmitters GABA and glutamate affect the proliferating cells in the VZ and SVZ in rat (LoTurco et al., 1995; Haydar et al., 2000), but this effect is specific to each zone. GABA and glutamate increase proliferation of progenitors in the VZ, but decrease their proliferation in the SVZ (Haydar et al., 2000). It is worth noting that GABA is expressed in human forebrain at 5–6 g.w., before the SVZ is established and, therefore, this neurotransmitter is available to regulate cell proliferation from the early stages of development (Zecevic and Milosevic, 1997).

Cell Death in the Human Fetal SVZ Programmed cell death (PCD) is a universal feature of the embryonic and postnatal central nervous system, and has been described in detail in several animal species (Cowan et al., 1984; Oppenheim, 1991; Ferrer et al., 1990; Blaschke et al., 1996; Thomaidou et al., 1997; Spreafico et al., 1999). PCD is genetically regulated and most often occurs in the form of apoptosis, characterized by particular cell morphology, lack of inflammation, and end-point removal by macrophages or by adjacent glial cells (e.g., Burek and Oppenheim, 1996). There are two types of developmental PCD: early, target-independent, that control the size of progenitor pools; and late, target-dependent, which is related to synapse formation (Oppenheim, 1991; Blaschke et al., 1996). At the initial stages of development when progenitor cells are multiplying, programmed cell death can critically regulate the size of the adult brain by decreasing the number of progenitor units (Rakic, 1988). Concurrent with cell proliferation, many cells in the human fetal SVZ are dying, as seen with the TUNEL in situ method (Fig. 4.7, Rakic and Zecevic, 2000). In the human fetal brain, the highest number of TUNEL (þ) nuclei per volume of brain tissue is demonstrated in the proliferative VZ/SVZ (Rakic and Zecevic, 2000); apoptoic cells appear during the embryonic period and last through the entire fetal period (Chan and Yew, 1998; Spreafico et al., 1999; Rakic and Zecevic, 2000; Simonati et al., 1999; Chan et al., 2002). Their peak is achieved at 17 g.w. in the rostral SVZ, as shown by comparing the three levels, rostral, medial and caudal, of the fetal brains (Fig. 4.7). The somewhat unexpected finding of cell death in proliferative zones prompted several speculations. It is suggested that errors may occur during cell division or differentiation, or that the specific phenotype of some cells may cause their death (Voyvodic, 1996). In addition, cell death may be influenced by trophic

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Figure 4.7. Neurolucida drawings illustrating the distribution of TUNEL (þ) nuclei (dots) in three coronal sections through the forebrain at 17 g.w. Sections A-rostral, B-middle, C-caudal, were made at the level presented on the small drawing of the brain. Layer I and cortical plate are presented as stripped area, while the proliferative zones, VZ, SVZ and GE, are dark gray and marked with asterisks TUNEL (þ) nuclei were concentrated in the proliferative zones (VZ, SVZ, GE) and basal ganglia (NC-nucleus caudatus, PU-putamen, CL- claustrum, GP-globus pallidus), whereas they were less abundant in the surrounding subcortical white matter. The concentration of TUNEL (þ) nuclei was also observed in the corpus callosum (arrowheads) and cingulum (arrows). Scale bars in A, B, C = 3 mm. IC-internal capsule; GE-ganglionic eminence. (From Rakic and Zecevic, 2000.)

factors from surrounding cells or regulated by intrinsic mechanisms within the cell (Blaschke et al., 1996; Thomaidou et al., 1997; Voyvodic, 1996). The simultaneous use of BrdU and TUNEL method revealed that 71% of TUNEL-labeled cells just exited the S phase of mitosis (Thomaidou et al., 1997), consistent with the idea that mitosis may trigger apoptosis (Freeman et al., 1994). Similar mechanisms may be activated in the SVZ where cell proliferation is actively going on during the protracted developmental period (Blaschke et al., 1996; Simonati et al., 1999; Rakic and Zecevic, 2000). A regulatory gene bcl-2 and its product, Bcl-2 oncoprotein, which represses apoptosis, have an inverse correlation with TUNEL-labeled cells in human

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cortical layers (Chan and Yew, 1998). Either increased apoptosis as a result of targeted mutations of death-effector genes, or decreased apoptosis as a result of disturbed caspase cascade, significantly change cortical size and shape, as was discussed earlier (Kuida et al., 1998; Haydar et al., 1999; Chan et al., 2002). It is generally believed that neurons are the main target of programmed cell death, but other cell types die as well. It has been shown that up to 50% of oligodendrocytes in the rat optic nerve (Barres et al., 1992), and 50% astrocytes in rat neonatal brain (Soriano et al., 1993) die during development. In addition, the number of microglia could also be regulated by apoptosis in the developing brain (Bonetti et al., 1997). However, it is hard to resolve this issue by double labeling immunocytochemistry, since shrunken nuclei of dying cells are difficult to label with other cell-specific markers.

Various Cell Populations are Present in the Human Fetal SVZ One of the fascinating characteristics of the SVZ is its cellular diversity. Virtually all cell types that are present in the brain can be observed in the SVZ during the several months of intrauterine development. The cellular population in the SVZ consists of cells that originate in situ as well as cells that are migrating through this region. The shape of SVZ cells is best revealed by the Golgi impregnation method, where they appear mainly as bipolar cells, with leading processes directed either radially, or horizontally (Fig. 4.3E). This shape is consistent with the radial and tangential migration described both in non-primate and primate brains. In addition, some cells seem to be migrating in an opposite direction, towards the VZ, as suggested by the orientation of their leading process (Rakic and Zecevic, 2003b). This bi-directional migration was also described in rodents, using time-lapse microscopy on live brain slices (Nadajarah et al., 2002, 2003; Ang et al., 2003). A vast majority of human SVZ cells at midgestation look immature, both on the Nissl-stained sections and at the ultrastructural level (Zecevic et al., in press). Although they share an immature look, certain morphological differences between these cells can be noticed. On Nissl-stained sections, one cell class has regularly shaped, pale and larger nuclei, whereas the other cell class has irregular, darker and smaller nuclei. The third class, lying on the ventricular surface, is either mitotic or displays tight junctions and villi that protrude into the ventricle (Figs. 4.3, 4.8). On the ultrastructural level, cells with smooth and elongated nuclei have a small amount of dark cytoplasm with numerous ribosomes, similar to type A cells (neurons) described in the adult mouse SVZ (Doetsch et al., 1997). The other cell type has irregular nuclei with clumps of chromatin, and more cytoplasm, which may

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Figure 4.8. Cell morphology in the SVZ. (A) At 18 g.w. on 1 mm thin Toluidin blue stained section, cells with light (a) and dark (b) nuclei and various amounts of cytoplasm are observed in the SVZ. Mitotic figures in the VZ (arrow) and in the SVZ (arrowheads). (B) At 22 g.w. on the ultrastructural level cells with smooth nuclei and evenly distributed chromatine are intermingled with cells with irregular nuclei with clumps of chromatine. Scale bar on A-10 mm. B-direct Mg. 2000X. (Modified from Zecevic et al., in press)

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correspond to type B cells (astrocytes). In human fetal brain, it is difficult to distinguish cells subclasses on the basis of cytoplasm density, since such cells are still developing. Only after immunocytochemical reactions can one fully appreciate a great variety of cell types present in the SVZ. Different neuronal subtypes, oligodendrocyte progenitors, astrocytes, and microglia/macrophages, are all present in the human fetal SVZ and can be labeled with appropriate markers. In addition, several subpopulations of multipotent progenitors labeled with various markers of immature cells, or neural and non-neural stem cells, are also located in the human fetal SVZ.

Neurons in the Cortical SVZ Neurons in the cortical SVZ represent either the cells that originate in the local SVZ, or cross through the cortical SVZ from another site of origin, such as the ganglionic eminence (GE). Subpopulations of cortical interneurons and subplate neurons originate in the SVZ, whereas pyramidal (projection) cells and remaining cortical interneurons are trespassing through the SVZ. These two cell classes, projection and interneurons, express different transmitters, and have different migratory routes. Projection (pyramidal) neurons contain glutamate, and radially migrate from the cortical VZ towards the cortical plate (Sidman and Rakic, 1973; Rakic, 1974, 1988), while cortical interneurons contain GABA, calretinin, calbindin and neuropeptides, probably have dual origin, and migrate following complex migratory routes through the SVZ (Yan et al., 1992; Zecevic and Milosevic, 1997; Tan et al., 1998; Zecevic et al., 1999, in press; Letinic et al., 2002; Rakic and Zecevic, 2003b). In non-primate mammals the majority, if not all, cortical interneurons originate in the GE and then migrate tangentially through the cortical marginal zone, subplate layer, and cortical SVZ on their way to their final position in the cerebral cortex (rev. Marin and Rubenstein, 2001). Tangential migration is probably facilitated by numerous corticofugal fibers that are developing at the same time (Parnavelas, 2000). However, in contrast, the majority of cortical interneurons in the primate brain originate in the cortical SVZ (Letinic et al., 2002; Smart et al., 2002; Rakic and Zecevic, 2003b). Several observations speak in favor of this conclusion. Using shortterm organotypic human fetal slice cultures, and labeling cells with Dlx1,2, Mash1 and GABA, it has been possible to show that two-thirds of the cortical GABAergic interneurons are actively produced in the cortical VZ and SVZ, whereas only 35% migrate from the ganglionic eminence (Letinic et al., 2002). Immunocytochemical and in situ hybridization studies using the ventral transcription factor Dlx2 also support a dual origin of cortical interneurons in the human forebrain (Rakic and Zecevic, 2003b). In the early human embryonic stages, sub-populations of interneurons that express ventral transcription factors (Nkx2.1 and Dlx2) are observed to be radially

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oriented in the cortical wall, consistent with their radial migration from the VZ (Rakic and Zecevic, 2003b). An alternative explanation is that Dlx2 and Nkx2.1 cells first migrate tangentially from the ganglionic eminence to the cortical VZ, and then radially through the SVZ and the cortical wall, as described in rodents (De Carlos et al., 1996; Anderson et al., 1997, 1999; Lavdas et al., 1999; revs. Parnavelas, 2000; Marin and Rubenstein, 2001) and human (Letinic et al., 2002; Rakic and Zecevic, 2003b). This, however, might not be the case for all cortical interneurons. Tangentially oriented Nkx2.1 and Dlx2þ cells are not observed in the cortical SVZ, as one would expect if these cells tangentially migrate from the ganglionic eminence. At the same time, in situ hybridization and immunolabeling show dorsal expansion of these transcription factors in human cortical VZ/SVZ (Fig. 4.5 Rakic and Zecevic, 2003b). It is, however, difficult to rule out the possibility that precursor from the ganglionic eminence seed the SVZ and continue proliferating there. Nevertheless, combined results obtained on the human fetal brain strongly suggest that, in contrast to rodents, Nkx2.1 and Dlx2þ cells present in the human cerebral cortex do not arise exclusively from the GE, but rather a significant subpopulation of these cells originate in the cortical VZ/SVZ. This is consistent with the idea that the much larger primate brain needs various sources of cortical interneurons that include cortical VZ/SVZ and the GE. These interneurons are probably destined to deeper cortical layers, and use both neuronal fibers and radial glia to migrate to their final destinations. In addition, upper cortical layers might be supplied by interneurons from the subpial granular layer in both monkey (Zecevic and Rakic, 2001) and human brains (Rakic and Zecevic, 2003b).

Oligodendrocyte Progenitors in the Cortical SVZ Oligodendrocyte progenitor cells (OPCs) can be labeled in the human fetal forebrain with the same specific markers previously described in rodents: early OPCs are labeled with antibodies to PDGFRa (platelet-derived growth factor receptor alpha) and NG2 (chondroitin sulfate proteoglycan), late OPCs are labeled with antibody O4, whereas mature oligodendrocytes express myelin proteins, MBP (myelin basic protein) and PLP (proteolipid protein) (Pringle et al., 1992; Pfeiffer et al., 1993; Nishiyama et al., 1996). The cortical SVZ has been described as a site of origin for OPCs, both in rat (LeVine and Goldman, 1988; Levison and Goldman, 1993; Levison et al., 1993; Nery et al., 2002; Kakita and Goldman, 1999) and in the human forebrain (Back et al., 2001; Rakic and Zecevic, 2003a). In addition, some OPCs originate in the GE. For example, it has been shown that in prenatal mice, a fraction of NG2 positive cortical OPCs are double-labeled with Dlx2, a marker for GE-originated cells (He et al., 2001). Furthermore, in transgenic mice that express Dlx2/lacZ, it was possible to follow these cells to

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Figure 4.9. Specific distribution and possible migratory pathways of oligodendrocyte progenitors (OPCs) in the human forebrain at midgestation. Early OPCs (PDGFRaþ , Olig1þ , NG2þ ), DLX2þ cells, CD68þ microglia and nestinþ or CD34þ stem cells (‘rainbow’ cells) are present in the distinct area of the GE-SVZ in the lateral forebrain and in anterior cortical SVZ (red asterisk, left). From the cortical proliferative zone these multipotent cells take two routes, one towards the prospective white matter (IZ) and another in the direction of the medial forebrain, first to the SVZ (red asterisk, right) and then the IZ. Arrows mark the possible migratory pathways of the multipotent cells. Late OPCs, labeled with O4 antibody (white cells), are not confined to the telencephalic proliferative zones only and have a dense appearance in the subplate (SP) layer of the cerebral cortex. Early OPCs, expressing PDGFRa only (green cells), are present within and outside the stream representing a population of OLs that apparently originate in the cortical SVZ. A small population of PDGFRa cells expresses NKX2.1 (yellow nucleus). In the amygdala, early OPCs are present (green and blue cells), but do not express DLX2. In the same region DLX2 is expressed in neuronal nuclei (red). GE – ganglionic eminence; Cx – cortex; SVZ – subventricular zone; IZ – intermediate zone; SP – subplate zone; LV – lateral ventricle; BG – basal ganglia; A – amygdala; Hipp – hippocampus; CC – corpus callosum; OR – olfactory region. (From Rakic and Zecevic, 2003a.)

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the cortex where they differentiated in either GFAPþ astrocytes, or CNPþ oligodendrocytes (Marshall and Goldman, 2002). Similarly, in the human fetal brain, cells in both GE and cortical SVZ can be co-labeled with early OPC markers and ventral transcription factor Dlx2, suggesting that these Dlx2 labeled oligodendrocyte progenitors originate in the GE and migrate to the cortical SVZ (Rakic and Zecevic, 2003a). Supporting their GE origin, a narrow stream of double-labeled cells (Dlx2 and OPC antibodies) connect the GE-SVZ and the cortical SVZ in human fetal forebrain at mid-gestation (Fig. 4.9 -Rakic and Zecevic, 2003a). Extensive double-labeling studies confirmed that these immature-looking bipolar cells with round soma could be labeled with additional markers that include lectins, CD68, and PSA-NCAM. It has been proposed that these cells represent multipotential progenitors based on their immature morphology, specific location and expression of multiple proteins that characterize progenitor cells. In the same sections, however, cells expressing markers of more differentiated cell types, such as neuronal markers (MAP2, b-III-tubulin, calretinin, calbindin and GABA), astrocytes (GFAP), late OPCs (O4), or mature oligodendrocytes (MBP), are not localized in the stream (Fig. 4.9, Rakic and Zecevic, 2003a). The observation of migrating OPCs restricted to a well-delineated pathway suggests that environmental cues channel these cells towards their final destinations. Factors described to have either a chemorepulsive role, such as Slit1 (Zhu et al., 1999) and Semaphorins (Marin et al., 2001), or that facilitate migration of cortical interneurons, such as TAG1 (Denaxa et al., 2001) and hepatocyte growth factor/scattered factor (Powell et al., 2001), might affect forebrain oligodendrocyte progenitors in a similar way. Indeed, class 3 semaphorins and netrin-1 regulate migration of oligodendrocyte progenitors in the rat optic nerve (Spassky et al., 2002). From the stream, OPCs appear to migrate tangentially either directly to the IZ, or first to the rostral SVZ, and then radially to the neocortex. It seems that from all OPCs, only the ones that are migrating express Dlx2, which indicates that this transcription factor might be required for their tangential migration to the cerebral cortex (Rakic and Zecevic, 2003a). Sonic hedgehog (Shh) was shown to be necessary for the expression of ventral transcription factors Dlx2 and Nkx2, and essential for the initial specification of oligodendrocyte lineage in the rodent brain (Lu et al., 2000; Nery et al., 2001; Tekki-Kessaris et al., 2001). Mice lacking Nkx2.1, do not express Shh in the medial GE, and have a reduced number of oligodendrocytes in the forebrain (Nery et al., 2001; Tekki-Kessaris et al., 2001). Interestingly, the role of Shh in the specification of oligodendrocytes is yet to be assessed in the human brain. For later radial migration of OPCs to neocortex radial glia processes are important, as was determined in studies that used retrovirus labeling and in vivo time-lapse microscopy (Zerlin et al., 1995; Kakita and Goldman,

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Figure 4.10. Chemokine GRO-a and CXCR2 in the fetal human SVZ at midgestation. (A) Drawing of the coronal section through the rostral pole of the fetal forebrain. Boxed areas correspond to the immunocytochemical results presented in B, C and D. A strong expression of (B) GRO-a and (D) receptor CXCR2, is observed in the medial SVZ, with individual labeled cells spreading through cell bands to the upper levels. (C) In the lateral SVZ, two parallel bands of CXCR2 immunoreactivity (asterisks), one at the border of the VZ and SVZ and the other in the outer SVZ, are separated by a non-immunoreactive fiber bundle. LV-lateral ventricle. Scale bar: A = 3 mm, C = 100 mm, B,D = 50 mm. (From Filipovic et al., 2003.)

1999). In the human fetal brain, close contacts between vimentin-labeled radial glia fibers and early OPCs were demonstrated (Jakovcevski and Zecevic, 2005). At the same time (mid-gestation), the highest density of early OPCs is observed in the SVZ, decreasing towards the upper levels of the cortex. There are roughly ten times more PDGFRa cells in the SVZ than in the overlying white matter, and only sporadic PDGFRa cells are found in the cortical plate at midgestation (Jakovcevski and Zecevic, 2002, 2005). This gradient, as well as the shape of the labeled cells, suggests that early OPCs migrate from the SVZ to a dorsal position in the developing white matter and the neocortex, where they differentiate into mature oligodendrocyte that express MBP and PLP.

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A fraction of OPCs in the human fetal forebrain originate in the cortical SVZ. Support for this notion derives from the finding that numerous PDGFRaþ cells are observed in sections of the forebrain, double-labeled with PDGFRa and Dlx2. Single-labeled PDGFRa cells are present both in the cortex and in the GE, consistent with their origin in the cortical SVZ, and the subsequent migration in the opposite direction, from the cortical SVZ to the GE (Rakic and Zecevic, 2003a). This finding is in accord with previously published results in rodents, where the postnatal cortical SVZ has been described as a source for both oligodendrocytes and astrocytes (see Chapter I; Levison et al., 1993; Levison and Goldman, 1997). During several months of human intrauterine development, early OPCs differentiate into late progenitors that can be labeled with the O4 antibody. In rat O4þ progenitors have been described as non-migratory cells (Pfeiffer et al., 1993). In the human fetal brain, late O4þ OPCs are present in the SVZ and overlying white matter (Back et al., 2001; Rakic and Zecevic, 2003a, Jakovcevski and Zecevic, 2005). In contrast to early progenitors (PDGFRa and NG2), O4þ cells are not confined to the stream but, instead, are evenly distributed in the cortical and GE VZ/SVZ. This cell distribution indicates that environmental factors affect early and late OPCs differentially (Rakic and Zecevic, 2003a). The premyelinating oligodendrocytes, which express myelin proteins, MBP or PLP, can be observed in the SVZ around midgestation (Back et al., 2001; Jakovcevski and Zecevic, 2005). However, MBPþ cells are relatively sparse in the human fetal SVZ, whereas they are more numerous in the emerging white matter and the corpus callosum. Transcription factors Olig 1, 2 are also shown to play an important role in oligodendrocyte lineage specification in mice (Lu et al., 2000; Zhou and Anderson, 2002). Both factors are expressed in the human cortical SVZ at mid-gestation by a heterogeneous populations of neuronal, oligodendroglial and stem cells (Jakovcevski and Zecevic, unpublished). Thus, the oligodendrocyte population in the human fetal cortical SVZ is heterogeneous, characterized by the expression of different markers that indicate different sites of cell origin: either from GE, in which case they express ventral transcription factors, Dlx2 and Nkx2.1, or from the cortical SVZ, in which case they do not express ventral transcription factors. It is, however, hard to exclude the possibility that some Dlx2þ OPCs might originate in the cortical SVZ, since the Dlx transcription factor spreads dorsally in the human brain, as was previously discussed in relation to the origin of cortical interneurons (Rakic and Zecevic, 2003b). Additional oligodendrocytes may arise from nestinþ or vimentinþ stem cells present in the cortical SVZ, or even from CD34þ hematopoietic stem cells (Rakic and Zecevic, 2003a).

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Astrocytes/Radial Glia in the Cortical SVZ In the human fetal brain, radial glia (RG) fibers cross through the SVZ during the early stages of development. Antibodies to the intermediate filament proteins, vimentin and GFAP (glial fibrillary acidic protein) are most often used to label RG in primates (Rakic, 1971; Levitt and Rakic, 1980; Levitt et al., 1981; Choi and Lapham, 1978; Choi, 1986; Sasaki et al., 1988; Stagaard and Mollgard, 1989; Gould et al., 1990; Honig et al., 1996; Sarnat, 1992; Zecevic et al., 1999; Ulfig et al., 1999; Rezaie et al., 2003). Studies in primates revealed differences in the molecular and biochemical characteristics of RG as well as in their final cell fate, as compared to various other animal species, which is important to take into consideration when comparing results from different laboratories. For example, in contrast to rodents and other mammals, where GFAP appears later in development, in primates, GFAP is expressed in the radial glia/astrocyte lineage early on (Antanitus et al., 1976; Choi and Lapham, 1978; Levitt et al., 1981; Choi, 1986; Zecevic, 2004; Howard et al., in press). In addition, while both vimentin and GFAP persist together in radial glia cells throughout at least two-thirds of gestation in humans, in other mammals, GFAP replaces vimentin as development proceeds and radial glia mature (Rickmann et al., 1987; Voigt, 1989). RG are a transient cell type in mammalian brains, and after the end of neuronal migration, the majority of these cells transform into astrocytes. This idea was first suggested by Cajal (1911) and later confirmed using different neuroanatomical methods (Schmechel and Rakic, 1979; Voigt, 1989; Marin-Padilla, 1995; deAzevedo et al., 2003). In the human brain, this transformation is gradual, starting at 12 g.w in the frontal pole and lasting to term (Choi and Lapham, 1978; Kadhim et al., 1988; MarinPadilla, 1995; deAzevedo et al., 2003). Using carbocyanin dyes (DiI/DiA), it has been described that RG transformation is dependant on their position in the cerebral wall (deAzevedo et al., 2003). In the SVZ transition profiles, monopolar cells with pyriform soma were seen at 18 g.w., while appearing later on in more superficial layers. Astrocytes, however, can also originate from SVZ progenitor cells, as mentioned before, when they diverge into specified astroglia or oligodendroglia lineages (Levison et al., 1993; Levison and Goldman, 1993; Luskin and McDermott, 1994; Brazel, 2003; Marshal and Goldman, 2003). The number of astrocytes increases in subsequent weeks of development. They seem to be concentrated in the subplate layer and the marginal zone, leaving the cortical plate more or less devoid of astrocytes (Wilkinson et al., 1990; Marin-Padilla, 1995; Ulfig et al., 1999; deAzevedo et al., 2003, Rezaie et al., 2003). At mid-gestation, vimentinþ and GFAPþ fibers, abundant in all cortical layers, are especially dense in the SVZ from where they radiate towards the upper levels of the cerebral cortex. Interestingly, in addition to radial fibers, a subset of vimentinþ fibers is tangentially oriented in the upper

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SVZ and lower intermediate zone (Zecevic et al., in press). These fibers might guide tangential migration of interneurons and glia cells, or may have a role in guiding callosal axons (Norris and Kalil, 1991; Ulfig et al., 1999).

Stem Cells/Multipotential Progenitors in the Cortical SVZ At mid-gestation, human SVZ contains cells that can be immunostained simultaneously for various stem cell markers such as nestin, NG2, microglial markers, and markers of oligodendrocyte progenitors. This immunostaining pattern suggests the existence of multipotent progenitor cells in the SVZ (Rakic and Zecevic, 2003a; Jakovcevski and Zecevic, 2002), consistent with previously reported multipotent stem cells observed in the human fetal forebrain (Flax et al., 1998; Vescovi et al., 1999; Ourednik et al., 2001, Suslov et al., 2002). When these progenitors are isolated from the human fetal SVZ, cultured, and then implanted into the lateral ventricle of the monkey fetal brain, they differentiate into olfactory neurons, astrocytes and oligodendrocytes, with a fraction remaining undifferentiated in the form of stem cells in the SVZ near the lateral ventricle (Ourednik et al., 2001). However, determined neuronal precursors are also present in the human fetal SVZ (Zecevic, 2004; Howard et al., in press). Experiments on living slice preparations from human fetal SVZ, where neuronal stem cell progenitors were labeled by retroviral gene transfer, showed that labeled cells divided several times before migrating to the cerebral cortex as cortical neurons (Letinic et al., 2002). Furthermore, neuronal stem cells isolated from human fetal SVZ generate neurospheres in vitro. These neurospheres are heterogeneous and contain neuronal stem cells, but also neuronal and glial progenitors (Suslov et al., 2002). These observations are consistent with the notion that during development, multipotential progenitors become gradually more restricted to bipotential and lineage restricted progenitors of either neurons or glia, and finally specified towards one type of glia, giving rise to only astrocytes or only oligodendrocytes (Grove et al., 1993; Luskin et al., 1993; Luskin and McDermott, 1994; Levison and Goldman, 1997). Neuronal progenitors also can be restricted to produce only one subtype of neurons, or even neurons for a particular cortical layer (McConnell, 1995). It is still not clear at what point during development these restrictions happen, and when two separate progenitor cell lines, one for glia and the other for neurons, can be observed for the first time in human proliferative zones. One of the obstacles in research in this field is the lack of a specific marker that can distinguish between stem cells and multipotent progenitors, or even between stem cells and more differentiated cell types. The markers used so far are not sufficiently specific (e.g. Steindler and Laywell, 2003). For example, nestin, often used as marker of neural stem cells, is primarily expressed by radial glia progenitor cells (Hockfield and McKay, 1985). But nestin also labels oligodendrocyte progenitors, microglia and type 2 astro-

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cytes (Almazan et al., 2001; Rakic and Zecevic, 2003a). Similarly, NG2 antibody labels a major population of proliferating cells in the postnatal and adult rodent brain, and so, it, is used as a stem-like cell marker. However, this antibody also labels early oligodendrocyte progenitors (e.g., Nishiyama et al., 1996; Dawson et al., 2000; Levison et al., 1999; Mallon et al., 2002; Filipovic et al., 2002), human microglia cells (Pouly et al., 1999; Filipovic et al., 2002), and various cell types after brain injury (Moon et al., 2000). A clinically relevant discovery is that neural stem cells contribute to cell populations not only through development, but also in the adult brain. Neuronal stem cells labeled with nestin are believed to remain in the subependymal zone in the periventricular region of the adult brain, where they contribute to the replenishment of different cell types, including neurons (e.g., Reynolds and Weiss, 1992; Morshead et al., 1994; Young and Levison, 1996; Alvarez-Buylla et al., 2001; Bernier et al., 2000, 2002). These stem cells may be radial glial descendents that have persisted in the subependymal zone (Alvarez-Buylla et al., 2001; Alves et al., 2002). In adult rat brain, both astrocytes and oligodendrocyte originate from the progenitors located in the SVZ, as suggested by classical methods (Altman, 1966; Gressens et al., 1992), and confirmed in neonatal rats by retroviral labeling studies (Levison and Goldman, 1993; Levison et al., 1993, 1999, Dawson et al., 2000; Levine et al., 2001). The human subependymal zone also seems to be a source of progenitors that, when isolated and cultured with growth factors, generate both glial cells and neurons (Kirshenbaum et al., 1994). Thus, various stemlike cells observed in the human fetal SVZ may remain in adult brains and represent a source of new cells throughout a lifetime. Non-neuronal stem cells should also be considered as possible contributors to the developing forebrain. Hematopoietic stem cells (HSC) are an example of non-neural stem cells that might be particularly interesting in this context. In the adult, HSC from the bone marrow are mainly responsible for the constant renewal of blood and immune cells (Morshead and van der Kooy, 1990). However, in the human fetal SVZ, HSC labeled with marker CD34 are present at midgestation. Moreover, a fraction of these CD34þ cells can be double labeled with transcription factor DLX, indicating that the DLX family of transcription factors might have a role in stem cell development (Rakic and Zecevic, 2003a). CD34/DLX2 double-labeled cells are often in close contact with the fetal brain blood vessels, which suggests their entry from the circulation, as has been shown for microglia precursors. In support of their role in forebrain development, both experiments in the mouse (Bonilla et al., 2002), and our preliminary experiments into human fetal SVZ, indicate that hematopoietic stem cells transplanted in the fetal SVZ, can differentiate into oligodendrocyte progenitors (Jakovcevski et al., 2003). In the latter example, it seems that the SVZ environment favors oligodendrocytic differentiation of transplanted HSCs. However, fusion of bone marrow cells could be an alternative explanation of these results

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(Terada et al., 2002; Alvarez-Dolado et al., 2003) and more studies are necessary to explore whether HSCs are a source of oligodendrocytes in normal development.

Microglia in the Cortical SVZ During early development, monocytes from the bone marrow cross the blood brain barrier and enter the CNS to become microglia, a macrophage population of the CNS (Ling and Wong, 1993; Andjelkovic et al., 1998). The observation that microglia accumulate in the SVZ was made by del Rio Hortega who described ‘‘microglia fountains’’ (1932) and by several laboratories, including ours, using various histochemical markers (Hutchins et al., 1990; Esiri et al., 1991; Andjelkovic et al., 1998; Zecevic et al., 1998; Rezaie and Male, 1999). In the fetal brain, at mid-gestation, aggregates of microglia labeled with lectins (tomato lectin, TL and Ricinus Communis Aglutinin-1, RCA-1) or human macrophage antigen (CD68) have been consistently observed in the cortical SVZ and in cell bands radiating from the SVZ (Rakic and Zecevic, 2000). Similar to oligodendrocyte progenitors, the density of microglial cells shows a gradient from the SVZ to the upper levels of the cerebral cortex (Rezaie and Male, 1999; Rakic and Zecevic, 2000). The reasons for microglial accumulation in the SVZ are not well understood, but several possibilities can be considered. For example, microglial cells are necessary to clear cell debris left after significant cell death shown in this highly cellular region (Rakic and Zecevic, 2000). Furthermore, the SVZ may be a portal for microglial colonization of the brain, as suggested by del Rio Hortega (1932), before they migrate to more dorsal regions of the brain (Rezaie and Male, 1999). The importance of microglial accumulation in the SVZ is underlined by the fact that these cells secreate various growth factors and chemokines. In this way, microglia may influence the other cell types that are simultaneously differentiating in the SVZ. It is important to understand the role of microglia both during normal brain development and during disease, because of significant medical implications for the human brain. For example, normal myelination as well as on the observed re-myelination in multiple sclerosis (Prineas et al., 1993; Raine, 1997; Franklin, 2003) may depend, among other factors, on the activation of microglia and their influence on oligodendrocyte progenitors. Here, only the effect of microglia on oligodendrocyte differentiation will be described, although it is likely that neurons and astrocytes could be affected in a similar way. Several lines of evidence indicate that microglia actively influence oligodendrocyte development and myelination in the SVZ and white matter. First, it has been observed that numerous microglia are located in the developing fiber tracts prior to myelinogenesis (Ling et al., 1982; Innocenti et al., 1983). Second, in vitro studies have shown

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that microglia can stimulate the synthesis of the myelin- specific proteins, MBP and PLP (Hamilton and Rome, 1994). Third, both microglia and astrocytes produce growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-b), neurotrophins (NT-3, NGF), insulin-like growth factor (IGF-1) and basic fibroblast growth factor (bFGF) while, receptors for these growth factors have been found on oligodendrocyte progenitors (Gogate et al., 1994; Presta et al., 1995; Bansal et al., 1996). PDGF influences proliferation and differentiation of early, uncommitted progenitor cells (O-2A) into oligodendrocytes (Noble et al., 1988; Satoh and Kim, 1994). Recently, it has been shown that microglia, acting through the PDGFR pathway, influence the survival and maturation of oligodendrocyte progenitors (Nicholas et al., 2001). bFGF, acting alone or with PDGF, induces early oligodendrocyte progenitors to proliferate but also stops their differentiation (Noble et al., 1988). Moreover, bFGF, expressed by radial glia, microglia and astrocytes in the SVZ, is crucial for maintaining the number of progenitor cells, as observed in experiments with bFGF knockout mice (Zheng and Vaccarino, 2002). IGF-1, similar to bFGF, acts primarily on more mature O4þ progenitors and influences their differentiation into oligodendrocytes (Satoh and Kim, 1994). Microglia can also act through astrocytes, as has been shown in demyelination models, where microglia induce astrocytes to produce IGF-1, which further results in oligodendrocyte proliferation and limited remyelination of axons (Gehrmann et al., 1994). NT-3 has been described to influence proliferation of oligodendrocyte progenitors in vivo and in vitro, suggesting a microglial role in remyelination of axons and survival of neurons (Hamilton and Rome, 1994; Loughlin et al., 1997; Cuzner, 1997). Various chemokines, a group of small chemo-attracting cytokines, are also present in the human fetal SVZ at mid-gestation (Rezaie et al., 2002; Filipovic et al., 2003). All major cell types, neurons, microglia and macroglia, produce chemokines and express their receptors, creating a signaling network in the CNS that is still not well understood (Westmoreland et al., 2002; Rezaie et al., 2002; Tsai et al., 2002). Whereas the role of chemokines is often associated with response to injury or infection of the CNS, specific chemokines are also involved in normal CNS development where they can influence cell proliferation and migration, cell survival and synaptic action (rev., Asensio & Cambell 1999; Bolin et al., 1998; Hesselgesser et al., 1997). In rodents, one of the chemokines, GRO-a (growth-related oncogene-a), acting through its receptor CXCR2, promotes oligodendrocyte genesis while inhibiting their migration (e.g., Robinson et al., 1998; Wu et al., 2000; Tsai et al., 2002). Both GRO-a and its receptor, CXCR2 are prominent in the human fetal SVZ, while they are less abundant in the adjacent ventricular zone and forming white matter (Fig. 4.10; Filipovic et al., 2003). Interestingly, GRO-a/CXCR2 are preferentially expressed by early PDGFRaþ and late O4þ oligodendrocyte progenitors present in the SVZ

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and the white matter, while they are less prominent in mature MBPþ oligodendrocytes. This distribution suggests that chemokine signaling in the human brain might have a role in the early stages of oligodendrocyte differentiation and their migration. Finding of numerous microglia/macrophages immunolabeled with antibodies to GRO-a and CXCR2 in the fetal SVZ (Filipovic et al., 2003), is consistent with the expression of other chemokines and their receptors in human fetal microglia (Rezaie et al., 2002). It is possible that in rat GRO-a secreted by early OPCs and surrounding ameboid microglia, interacts through CXCR2 receptor present on O4þ cells to inhibit their migration and enhance their differentiation into myelinating MBPþ oligodendrocytes (Tsai et al., 2002). Our preliminary results on human fetal slice culture are in favor of that mechanism since it has been demonstrated that GRO-a enhances proliferation and differentiation of oligodendrocyte progenitors through its receptor, CXCR2 (Filipovic and Zecevic, 2003). Further studies are necessary for a detailed understanding of the role of chemokines, including GRO-a, in migration of OPCs from the SVZ to the white matter in the human fetal brain.

Conclusion The transient proliferative SVZ increases in size during evolution and achieves its peak in the human fetal brain. Mainly due to intensive proliferation in this zone, the human brain grows during the second half of gestation linearly, with the cell number increasing by a factor of 2.9 from mid-gestation to birth (Badsberg Samuelsen et al., 2003). In this period of development, proliferation in the VZ has diminished or stopped, and the cortical and GE-SVZ are the only sources of new cells in the forebrain. The importance of the SVZ as a secondary proliferative zone is highlighted by the fact that all major cell types are generated in this zone. However, one important difference between human and experimental animal brains should be stressed. In contrast to animal models, where the majority of cortical interneurons have a subcortical origin, in the human brain these cells seem to mainly originate in the neocortical SVZ. In addition, transient subplate neurons, as well as macroglia (oligodendrocytes and astrocyte) originate in the SVZ during the extended period of corticogenesis in human. Furthermore, stem cells and multipotential progenitors reside in the fetal SVZ and later on the subependymal zone of the adult forebrain. In addition, microglia use this zone as an entrance port to colonize the brain. Thus, a better understanding of the human SVZ, including its cellular and molecular characteristics as well as its contribution to the overall development of the forebrain, is extremely important for understanding brain organization and function. Although we have learned a lot about this zone in the last decade alone, still many questions remain. Maybe one of the most interesting ones

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is: how many different subtypes of stem cells are present in the human SVZ? What are the mechanisms that initiate their differentiation to specialized cell types, in embryonic and in adult life? What factors influence the migration of cells through the SVZ? Do different subtypes of cortical interneurons in human forebrain have different progenitors, including the radial glia cells? Are astrocytes derived solely from radial glia transformation, or also from a common progenitor with oligodendrocytes? Do all oligodendrocytes originate in the GE, or some originate in the neocortical SVZ? An important consequence of the protracted intrauterine development in humans is that external or internal factors can interfere with developmental processes for a longer period of time. Insults that occur during the prenatal period could interrupt the proliferation and migration of cells in the SVZ, and cause irreparable damage to the normal function of the brain. Therefore, a more detailed understanding of cellular and molecular characteristics of the human SVZ may provide new options for treating common neonatal pathologies, such as periventricular leukomalacia or intracerebral hemorrhage. The discovery of stem cells in the adult subependymal zone, the residue of the fetal SVZ, emphasizes the importance of the SVZ not only in development, but also later in life. More subtle brain damage could deplete stem cells from the fetal SVZ and remain unnoticed by neurological or even sophisticate imaging diagnostics, but such injuries could compromise repair processes later in life. For all of these reasons, a better understanding of the human SVZ is both needed and important. Acknowledgements. We are thankful to Dr. Steve Levison for helpful suggestions on the manuscript. These studies were supported by grants from NIH-NS 41 489 and the National Multiple Sclerosis Society, RG- 3083-A-2.

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Abbreviations: bFGF – basic fibroblast growth factor BrdU – bromodeoxyuridine CD – clusters of differentiation CNP – 2’,3’- cyclic nucleotid 3’-phosphodiesterase CNS – central nervous system E – embryonic day GABA – gamma amino butyric acid GE – ganglionic eminence GFAP – glial fibrillary acidic protein GRO-a – growth-related oncogene alpha g.w. – gestational weeks HSC – hematopoietic stem cell IFL – inner fiber layer IGF – insulin-like growth factor IL – interleukin ISVZ – inner subventricular zone IZ – intermediate zone MAP2 – microtubule associated protein 2 MBP – myelin basic protein NT – neurotrophin OFL – outer fiber layer OPC – oligodendrocyte progenitor cell OSVZ – outer subventricular zone PCD – programmed cell death PCNA – proliferating cell nuclear antigen PDGFRa -platelet-derived growth factor a PLP – proteolipid protein PPL – primordial plexiform layer PSA-NCAM – polysialylated neuronal cell adhesion molecule PVL – periventricular leukomalacia RCA-1 – Ricinus Communis Aglutinin 1 RMS – rostral migratory stream RG – radial glia RL – rhombic lip TUNEL – TdT-mediated dUTP-biotin nick-end labeling Shh – sonic hedgehog SVZ – subventricular zone TL – tomato lectin TNF – tumor necrosis factor VZ – ventricular zone

Chapter 5 Responses of the Adult SVZ to Neuronal Death and Injury Jason G. Emsley and Jeffrey D. Macklis

Introduction The central nervous system (CNS) is constantly developing. For some time, the cellular plasticity of the nervous system in response to its internal and external environment has been recognized as a hallmark of the brain’s adaptability. Our understanding of these exciting phenomena has been considerably expanded upon with recent demonstrations that neural precursors or ‘‘stem cells’’ reside in the adult CNS, and that these unique cells are capable of generating new neurons in the adult brain. These findings question long-standing dogmas about the CNS and its capacity for change, while challenging many fundamental concepts about neuronal development and degeneration. In addition, and especially important for the discussion here, such an understanding brings with it the promise of novel therapeutic approaches to brain repair for a broad spectrum of neurodegenerative diseases, and acquired CNS injury. The subventricular zone (SVZ) may play a particular role in responding to various forms of neurodegeneration. Indeed, it has been suggested that the process of adult neurogenesis may play a key role in compensating for a variety of types of neural damage or neurodegeneration (Kuhn et al., 2001). This chapter aims to describe the present understanding of the response of the SVZ to a variety of neurodegenerative conditions. A brief overview of the SVZ as a neurogenic region is followed by a description of the various forms of responses seen in the SVZ undergoing neurodegeneration, and lastly there is a brief overview of the factors that influence the proliferation, differentiation, and migration of SVZ-derived cells in the adult CNS. The SVZ is a commonly used source for the isolation and preparation of neural precursor cells, which themselves are a commonly used source for neural transplantation paradigms into a variety of diseases or models of disease. An understanding of the response of the SVZ to neurodegenerative MGH-HMS Center for Nervous System Repair, Departments of Neurosurgery and Neurology, Program in Neuroscience; and Harvard Stem Cell Institute, Harvard Medical School, Massachusetts General Hospital, Edwards 4 (EDR 410), 50 Blossom Street, Boston MA, 02114, USA, Telephone: 617-726-5776, Fax: 617-726-2310, Email: [email protected] 159

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conditions must, therefore, also include a discussion of the nature of the diseased environment as it responds to cells derived from the SVZ. It is also clear that studying how host environments change during neurodegenerative disorders will have a bearing on our understanding of the discrete steps through which SVZ-derived cells pass as they participate, or fail to participate, in reconstructing damaged host circuitry. A broad definition of neurodegenerative conditions is presented which will allow for a more thorough examination of the array of responses of the SVZ. Indeed, a neurodegenerative condition is defined here as one in which any form of degeneration occurs in the CNS, and this may result from normal aging, specific discrete disease states such as Parkinson’s or Huntington’s Disease, as well as for more diffuse conditions such as Alzheimer’s disease. The chapter continues with a brief summary of some of the known responses of the SVZ in a variety of other neurological conditions, such as the neurodevelopmental disorders ataxia telangiectasia and tuberous sclerosis, along with other neurodegenerative diseases, including those caused by viruses and other pathogens, as well as epileptiform disorders and some psychiatric conditions. Ischemic injury, as well as the demyelinating disorder, multiple sclerosis, could also be considered to be forms of neurodegeneration; these two specific topics are discussed in detail elsewhere in this volume. The chapter concludes with an examination of a specific model of neurodegeneration. This model, from our lab, uses targeted apoptosis of cortical projection neurons to alter the micro-environment for the study of neuronal repopulation and repair. This system will be reviewed in detail, and discussed as an approach by which one can systematically study both the molecular and cellular responses of the SVZ in a controlled model of neurodegeneration of a complex region of the CNS.

Factors Influencing the Proliferation, Differentiation, and Migration of SVZ-Derived Cells in the Adult CNS In order to better understand the responses of the SVZ to the spectrum of neurodegenerative disorders, it is necessary to briefly consider the factors known to affect proliferation, differentiation, and migration of cells within or derived from the SVZ. An extensive range of factors are capable of regulating the proliferation of SVZ-derived cells and their progeny, both in vitro and in vivo. These factors include epidermal growth factor (EGF) (Reynolds et al., 1992; Gritti et al., 1995; Craig et al., 1996; Reynolds and Weiss, 1996), basic fibroblast growth factor (or fibroblast growth factor-2 (FGF-2)) (Gage et al., 1995; Gritti et al., 1995, Vicario-Abejon et al., 1995;Gritti et al., 1996), brain-derived neurotrophic factor (BDNF) (Pencea et al., 2001), as well as thyroid hormone (T3) (Ben-Hur et al., 1998), and transforming growth factor a (TGFa) (Fallon et al., 2000). Ciliary

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neurotrophic factor (CNTF), insulin-like growth factor (IGF-1), and estrogen all appear to be involved in increasing cellular proliferation (Tanapat et al., 1998; Aberg et al., 2000; O’Kusky et al., 2000; Trejo et al., 2001; Emsley and Hagg, 2003). Inflammation, either by mechanical intervention or induced by lipopolysaccharide (LPS), also enhances SVZ cellular proliferation in vivo, as demonstrated by increases in PSA-NCAM labeling (Szele and Chesselet, 1996) or BrdU incorporation (Emsley and Hagg, unpublished observations). A variety of growth factors, neurotrophins, cytokines, signaling molecules, and the in vivo environment itself can influence differentiation of SVZ-derived cells. These factors include BDNF (Ahmed et al., 1995; Kirschenbaum and Goldman, 1995), CNTF (Piquet-Pellorce et al., 1994; Wolf et al., 1994; Ip, 1998; Whittemore et al., 1999), ascorbic acid (Yan et al., 2001), or the homeodomain transcription factor Phox2 (Lo et al., 1999). Bone morphogenetic proteins (BMPs) potently inhibit neurogenesis both in vitro and in vivo, but the protein Noggin can promote neurogenesis by inhibiting BMPs (Lim et al., 2000). The protein Notch appears to limit neuronal differentiation by maintaining a cell’s proliferative capacity (Faux et al., 2001; Solecki et al., 2001). Sonic hedgehog (SHH) is also involved in patterning and growth in a variety of systems (Smith, 1994). Along with proliferation and differentiation, migration is a key feature of the progeny of SVZ-derived cells. Type A cells are migrating neuroblasts that are PSA-NCAM-, TuJ1-, and nestin-positive (Doetsch et al., 1997). The normal migration pattern of these cells is reviewed in Luskin (1998). Briefly, these neuroblasts migrate along a ‘‘rostral migratory stream’’ (RMS) from the anterior portion of the subventricular zone (SVZa) to the olfactory bulb (Luskin, 1993; Lois and Alvarez-Buylla, 1994; Lois et al., 1996) as chains of closely apposed elongated cells (Lois and Alvarez-Buylla, 1994; Wichterle et al., 1997).

SVZ-Derived Cells and Transplantation in Neurodegenerative Conditions The environment into which SVZ-derived neural precursors/‘‘stem cells’’ and their progeny are transplanted also exerts a strong influence on these cells’ differentiation. Neural progenitors transplanted into the striatum are reported to preferentially take on a GABAergic or dopaminergic neuronal phenotype (Zigova et al., 1998b), or express DARPP-32 (Armstrong et al., 2000). The yield of dopaminergic neurons after transplantation to the striatum appears to be dramatically enhanced when these cells are transplanted into a dopamine-depleted rather than a normal striatum (Nishino et al., 2000). Finally, when partially fate-restricted neuronal precursors are

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transplanted into the cortex under specific cellular and molecular microenvironmental conditions, they not only take on the appropriate morphological and synaptic phenotype, but a subset of the transplanted neurons are also capable of making appropriate contralateral connections (Shin et al., 2000). In some cases, however, neuronal restricted precursors maintain their default phenotype regardless of the environment into which they have been transplanted. For example, spinal cord-derived neuronal precursors continue to express choline acetyltransferase (ChAT) when grafted into the forebrain SVZ (Yang et al., 2000).

The SVZ in Various Neurodegenerative Disorders Aging Aging is often thought to include a progressive loss of neural cells, accompanied by a reduction in dendritic processes (reviewed in Uylings and de Brabander, 2002), and an age-related decline in brain weight and volume (Peress et al., 1973; Dekaban, 1978; Miller et al., 1980). Although there continue to be questions regarding whether neurodegenerative diseases such as Alzheimer’s might simply be extreme examples of normal aging, increasingly, specific neuronal pathology has been identified in each distinct disease. Nonetheless, some studies suggest that normal aging does not simply involve non-specific loss of cells, brain volume, or dendritic complexity or volume, but that degeneration is localized to specific areas (Uylings and de Brabander, 2002). For example, most of the degeneration seen with aging occurs more in the cortical laminae, specifically the frontal cortex (Raz et al., 1997), possibly in the parahippocampal cortex (Uylings and de Brabander, 2002) and in specific areas of the hypothalamus (Zhou and Swaab, 1999). Several studies have examined changes in the response of the SVZ during the normal aging process. It has been established that cells in the SVZ can normally respond to growth factors such as EGF (Craig et al., 1996; Yamada et al., 1997). Indeed, EGF and NGF can increase the number of proliferating cells in the SVZ of adult mice (Fiore et al., 2002; Tirassa et al., 2003). In addition, EGF and NGF, when delivered into the lateral ventricles, can increase choline acetyltransferase (ChAT) mRNA, as well as the number of ChAT-positive cells in an area of the SVZ in aged mice (greater than 15 months old) (Tirassa et al., 2003). These changes are seen concomitant with an increase in BDNF mRNA and protein (Tirassa et al., 2003). These results indicate that cells within the SVZ retain the capacity to proliferate and respond to growth factor treatment. In contrast to the studies described above, it has also been shown that cells in the SVZ of aged animals have a reduced ability to respond to lysolecithin (LPC) induced demyelination of the overlying corpus callosum compared with the response in younger animals (Decker et al., 2002a). This reduced

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ability to respond includes a decreased ability to proliferate with age, and a decreased ability to respond to the lesion, compared to the ability with which cells in the SVZ of younger animals respond to a similar lesion (NaitOumesmar et al., 1999; Decker et al., 2002a, b). However, this reduced ability to repopulate the corpus callosum may not be due to decreased migrational capacity, because cells from neural precursors derived from young and older adult mice do not appear to differ with respect to migrational capacity in vitro (Decker et al., 2002a). These results suggest that the lack of ability to repopulate corpus callosum cells in LPC-induced demyelination may be due to a loss of the intrinsic capacity of SVZ-derived cells to respond to growth factor treatment (Decker et al., 2002a). Furthermore, SVZ cells in older (18–20 month old) mice also have reduced proliferative response to FGF-2 or TGFa compared to those in younger mice (Decker et al., 2002a). The cell adhesion molecule PSA-NCAM is the highly polysialylated form of the neural cell adhesion molecule (NCAM), and is associated with a migratory immature neuronal phenotype (Hu et al., 1996). There are suggestions that the normal expression of PSA-NCAM differs between young and aged rats, with PSA-NCAM expression in dorsal and ventral parts of the SVZ in aged brains, while expression is seen only in the dorsal SVZ of younger brains (Sato et al., 2001). In addition, after transient middle cerebral artery occlusion (tMCAO), SVZ PSA-NCAM levels peak in younger rats at one day, but do not peak until three days post MCAO in older rats (Sato et al., 2001).

Parkinson’s Disease Parkinson’s Disease (PD) is the second most common neurodegenerative disorder, after Alzheimer’s disease. While neurodegeneration can be more widespread in many cases, the primary neurodegeneration of PD is a progressive cell death of a particular population of dopaminergic nigro-striatal projection neurons in the substantia nigra pars compacta, leading to the cardinal clinical manifestations: bradykinesia, cogwheel rigidity, and tremor (reviewed in Calne et al., 1992; Calne, 1992; Uitti and Calne, 1993; Hoehn and Yahr, 1967; Nussbaum and Ellis, 2003). While the exact etiology and pathogenesis of most sporadic forms of Parkinson’s disease have not been completely elucidated, there is increasing evidence that neuronal death results from intraneuronal protein aggregation. In addition to a genetic etiology for a small subset of patients (Nussbaum and Ellis, 2003), numerous environmental factors have been implicated, including pesticide exposure (Olanow, 1996; Lozano et al., 1998; Sherer et al., 2002). The course of the disease, such as the rate at which dopaminergic neurons die, is not clear. It is not yet certain, for example, whether PD involves a linear neuronal loss, or if there is a massive neuronal loss immediately prior to or long before the onset of clinically detectable symptoms (Olanow and Tatton, 1999). Aside from a

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loss of substantia nigra neurons, PD is characterized by the presence of Lewy bodies and Lewy neurites, which comprise ubiquinated protein deposits in the neuronal cytoplasm (Kuzuhara et al., 1988; Pollanen et al., 1993; Nussbaum and Ellis, 2003), and aggregated a-synuclein has been reported in Lewy bodies and Lewy neurites in PD (Spillantini et al., 1997; Mezey et al., 1998). Very little is known about the response of cells within the SVZ during PD, and there are few reports of the response of cells in the SVZ within animal models of PD. Much more is known about the role of SVZ-derived neural cells following transplantation in various models of PD than is known about the endogenous response of the SVZ and its progeny. However, a few studies have looked at the response of the SVZ in situ following a model of PD, using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to selectively kill dopaminergic neurons in the substantia nigra. Nestin immunoreactivity has been reported to increase in the neostriatum and adjacent SVZ under these simulated neurodegenerative conditions (Chen et al., 2002). It has also been suggested that dopamine, or the integrity of the nigrostriatal projections themselves, might influence precursor cell proliferation in the SVZ (Baker et al., 2004; Hoglinger et al., 2004). Finally, a recent report has suggested that a constitutive neurogenesis of dopaminergic neurons in the substantia nigra, as well as an increased generation of substantia nigra cells in response to experimentally induced neuronal death (Zhao et al., 2003). However, these results have come into question based on reports by other groups (Frielingsdorf et al., 2004; Kay and Blum, 2000; Lie et al., 2002). SVZ-derived neural precursors have been used in a specific 6-hydroyxdopamine (6-OHDA) model of PD (Lundberg et al., 1997), and it has also been shown that such SVZ-derived cells can migrate and differentiate in the normal striatum (Zhang et al., 2003). Finally, using infusions of TGFa in combination with 6-OHDA induced substantia nigra neuronal degeneration, Fallon et al. report that cells within the adult SVZ are capable of proliferating and then migrating towards the growth factor infusion site (Fallon et al., 2000).

Huntington’s Disease Huntington’s Disease (HD) is a fatal neurodegenerative disease characterized by disruptions in the extrapyramidal motor system, choreic movements, as well as psychiatric manifestation and progressive dementia (Huntington, 1872, cited in Davies and Ramsden, 2001). The basal ganglia and cortex are primarily affected in HD, and the main neurodegeneration in HD includes loss of GABAergic medium spiny neurons in the striatum, as well as corticostriatal projection neurons in Layer 6 and, to a lesser extent, in Layers 3 and 5 of the cerebral cortex, resulting in a marked atrophy of both striatum and cortex (Sharp and Ross, 1996; Martin, 1999; Reddy et al., 1999 Ho et al.,

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2001). In addition, neuronal loss has also been reported in other brain regions, including the substantia nigra, hypothalamus, and cerebellum (Davies and Ramsden, 2001; Ho et al., 2001; McMurray, 2001). The molecular basis for HD is an over-expansion of CAG repeats within the huntingtin gene coding sequence, along with expression of the mutant huntingin protein (The Huntington’s Disease Collaborative Research Group, 1993) (Davies and Ramsden, 2001; Ho et al., 2001; McMurray, 2001). The pathology of HD has also been thought to involve glutamate excitotoxicity, and may also involve a variety of other factors, such as oxidative stress (Tarnopolsky and Beal, 2001) and impaired energy metabolism (Jenkins et al., 1993). Very little is known about the response of the SVZ to HD. There is a larger body of literature focusing on the behavior of SVZ-derived precursors following transplantation into models of HD (Armstrong et al., 2000; Mundt-Petersen et al., 2000; Watts and Dunnett, 2000; McMurray, 2001). With respect to in vivo developmental studies, huntingtin protein is known to be involved in neurogenesis. For example, mice homozygous for reduced levels of CAG repeats show abnormal brain development; however, those mice with expanded human CAG repeats do not appear to have abnormal neural development (White et al., 1997). Recently there have been reports of increased cellular proliferation in the SVZ of human Huntington’s diseased brains (Curtis et al., 2003). Finally, it has also been suggested that in a quinolinic acid lesion model of HD, there may be an increase in proliferation in the SVZ, and that this increased proliferation includes increased production of Dcx-positive migrating neuroblasts (Tattersfield et al., 2004).

Alzheimer’s Disease Alzheimer’s Disease (AD) is the most common neurodegenerative disorder, the prevalence of which increases with age, centrally affecting memory, language, cognition, and behavior (McKhann et al., 1984; Citron, 2002a; Cummings and Cole, 2002; Dugu et al., 2003; Nussbaum and Ellis, 2003). Neuropathological hallmarks of AD include widespread cortical and hippocampal amyloid plaques and neurofibrillary tangles. The plaques comprise deposits of aggregated amyloid proteins, and the latter are helical filaments of abnormally phosphorylated tau protein in the cell body and/or dendrites (Cummings and Cole, 2002; Nussbaum and Ellis, 2003). In addition to plaques and tangles, AD is characterized by a reduction in neuronal number and a decline in synaptic density. The predominant cause of sporadic AD is not clear, as genetic mutations in the amyloid precursor protein (APP), or presenilins 1 and 2, account for less than 5% of reported cases. The accumulation of APP over a period of time is believed to be directly responsible for cell death (Cummings and Cole, 2002) via a number of potential mechanisms, including lipid oxidation, membrane disruption, inflammation,

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secondary tangle formation, or initiation of specific cell death pathways (Moechars et al., 1996; Citron, 2002b; Cummings and Cole, 2002; Nussbaum and Ellis, 2003). A connection between the SVZ and AD is found both in examples of neural development as well as in in vitro and in vivo studies seeking to understand AD-specific proteins. Developmentally, Presenilin 1, mutations of which are a major cause of familial AD, is critical for normal neural development (Kim et al., 1997; Moreno-Flores et al., 1999; Tanimukai et al., 1999; Kostyszyn et al., 2001), at least in part because it affects cleavage of Notch, and release of the active Notch intracellular domain. Specifically, Presenilin 1 is expressed developmentally, including in the human embryonic CNS. PS1 plays a significant role in cell fate decisions during neural development (Moreno-Flores et al., 1999; Handler et al., 2000). Its specific role in cell fate choice within the SVZ, or in SVZ-derived neural precursors, is yet to be elucidated. However, it has been suggested that in PS1 -/-mice, neural cell proliferation and apoptosis are unaltered, but there may be a significant decrease in the number of adult neural progenitors in these mice. It has been suggested that these differences may be due to premature differentiation of these progenitors, and PS1 may exert its effects via down regulation of Notch signaling during normal embryonic neurogenesis (Handler et al., 2000). Another example of a developmental connection with AD comes from studies of the Cdk5 and Gsk3beta proteins, which are critical for the phosphorylation of tau proteins (Maccioni et al., 2001a). It has been shown, for example, that the protein kinase Cdk5 is critical during developmental neurogenesis (Maccioni et al., 2001b); its specific role in adult neurogenesis in the SVZ requires further exploration. Amyloid precursor protein (APP) has been detected in ependymal cells lining the lateral ventricle, and has also been reported to be expressed in glial cells of the SVZ and RMS (Yasuoka et al., 2004). APP has also been shown to alter neurogenesis in SVZ-derived cells in vitro. For example, it has been reported that, in cultures of SVZ-derived cells from APP mutant mice, or in cultures that have been taken from normal mice receiving infusion of amyloid beta, amyloid beta suppresses both proliferation and differentiation of SVZ-derived cells. In addition, the amyloid beta protein can induce apoptosis in these cells, in part via disruption of calcium regulation (Haughey et al., 2002a, b). However, it has also been shown that APP does not impair the rate of neurogenesis in neural stem cells in vitro, but it does enhance the number of cells that differentiate into neurons, suggesting both positive and negative influences of APP in neurogenesis (Lopez-Toledano and Shelanski, 2004). Furthermore, the in vivo proliferation of cells within the SVZ of adult rodents is impaired in amyloid beta mutant mice, as well as in normal mice infused with amyloid beta peptide (Haughey et al., 2002a). Finally, it has recently been reported that transgenic mice with mutated forms of APP have increased neurogenesis in both the dentate gyrus and SVZ (Jin et al., 2004).

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Other Neurodegenerative Disorders There are a variety of other neurological disorders for which some limited information about the response of the SVZ is known, either in specific animal models or from the clinical literature. Certainly, a better understanding of neural stem cells, and the SVZ itself, will be critically important in the characterization and treatment of a variety of diffuse neurodegenerative disorders (Zoghbi et al., 2000) such as tuberous sclerosis/cortical dysplasia, other forms of developmental disorders such as ataxia telangiectasia, as well as multiple sclerosis. Other conditions involving neuronal injury having an influence on the SVZ include those caused by viruses and other pathogens, epileptiform disorders, as well as a select number of psychiatric diseases. Tuberous sclerosis and cortical dysplasia are developmental disorders that fall into a category of neurological abnormalities involving developmental disorders of neurogenesis and neuronal migration (Curatolo et al., 2002). Tuberous sclerosis is characterized by the presence of Dcx-positive giant cells, at a time when the expression of Dcx should be in decline (Mizuguchi et al., 2002). This disease is characterized by ventricular subependymal nodules, the presence of which allows for some prediction on the severity of cerebral dysfunction in tuberous sclerosis (Hosoya et al., 1999). In addition to the morphological abnormalities and abnormalities in Dcx expression seen in tuberous sclerosis, there are reports of abnormalities in the SVZ during lissencephaly and pachygyria. There have been reports in these disorders of the formation of nodules within the developing subependymal zone (Sarnat et al., 1993). Ataxia telangiectasia (AT) is a childhood neurodegenerative syndrome. It has been shown that the mutated protein (AT mutated, or ATM) is important in early neurogenesis, and may act via elimination of damaged postmitotic neural cells (Lee et al., 2001). The role of ATM in regulation of cell number and survival in the normal adult SVZ requires further examination. Finally, multiple sclerosis is a neurodegenerative, primary demyelinating disorder thought to result from autoimmune challenges to the CNS (Hanson and Cafruny, 2002). This disease will be discussed in greater detail elsewhere in this volume. A few other disorders involving damage to the nervous system, such as those involving viruses or other pathogens, as well as some epileptiform and psychiatric conditions, have been reported to elicit responses in the SVZ. For example, the serum and antibodies from patients with paraneoplastic encephalomyelitis strongly stain immature cells in the SVZ in rat (Antoine et al., 1993). With respect to epileptiform disorders and the SVZ, models of status epilepticus (SE) in rats have shown that one to two weeks after SE induction, there is an increase in BrdU labeling in the SVZ, along with increased levels of Nissl staining in this region (Parent and Lowenstein,

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2002; Parent et al., 2002). Furthermore, by two to three weeks, there is a greater density of SVZ-derived neuroblasts in the RMS, as well as an increase in Dcx-, PSA-NCAM-, and Tuj1-positive cells in the RMS (Parent and Lowenstein, 2002; Parent et al., 2002). Finally, while there is very little direct information regarding links between psychiatric disorders and the SVZ, recently, several interesting links and hypotheses have been put forward. For example, it has been shown that depletion of serotonin, which can act both as a neurotransmitter and as a developmental signal (Lauder, 1990; Whitaker-Azmitia, 1991), can influence the SVZ. Specifically, serotonin depletion has been shown to decrease developmental neurogenesis (Lauder, 1990). More importantly, depletion of serotonin via injection of the neurotoxin 5,7-dihydroxytryptamine (5,7DHT) results in a decrease in the proliferation in the SVZ (Brezun and Daszuta, 1999). Although not directly related to the SVZ, a connection between hippocampal neurogenesis and depression has been postulated (Gage, 2000; Gould et al., 2000). It has also been suggested that the effects of anti-depressants may be mediated by hippocampal neurogenesis (Santarelli et al., 2003). It is an intriguing possibility that similar effects could also play a role in the response of the SVZ.

The SVZ in Models of Neurodegeneration Various animal models exist that aim either to mimic or model discrete aspects of neurodegenerative conditions. Some of these models can be used to dissect discrete cellular or molecular mechanisms underlying the behavior of the CNS in general, and adult neurogenic zones such as the SVZ in particular, in response to region-specific or phenotype-specific forms of cell death. And although animal models seek to characterize the response of the adult SVZ to various forms of neurodegenerative injury, it should be emphasized that the adult human SVZ may have a structure and complexity that differs from other species (Doetsch et al., 1997; Sanai et al., 2004). These models include experimental allergic encephalomyelitis (EAE) as a model of multiple sclerosis, a number of models of ischemic injury, models using brief pulses of ionizing irradiation, and a model used by our lab, which involves in situ induction of targeted apoptotic neuronal death of specific populations of projection neurons in the adult CNS. Models of ischemia, multiple sclerosis, and irradiation will be discussed in other chapters. Therefore, this section will focus in greater detail on the response of SVZ-derived cells, and the SVZ itself, in a model of targeted apoptotic neuronal death.

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Targeted Apoptotic Cell Death of Cortical Projection Neurons Our lab has developed a biophysical approach to induction of synchronous, cell-type-specific neurodegeneration of targeted populations of projection neurons in the adult cerebral cortex and other regions of the vertebrate CNS (Macklis and Madison, 1985; Macklis, 1993; Sheen and Macklis, 1994, 1995; Hernit-Grant and Macklis, 1996; Snyder et al., 1997; Wang et al., 1998; Sheen et al., 1999; Leavitt et al., 1999; Magavi et al., 2000; Scharff et al., 2000; Shin et al., 2000; Fricker-Gates et al., 2002; Eyding et al., 2003). Studies employing this approach have investigated the capacity of either transplanted or endogenous cells to integrate into complex cortical circuitry, under specifically manipulated cellular and molecular micro-environmental conditions. Most importantly, for the purposes of this discussion, this approach provides a selective model of neurodegeneration in which one can study the response and characterize the role of the SVZ under conditions of highly specific and controlled neurodegeneration. The following section describes this model in greater detail, outlines its prior use in studies of cellular circuit repair via neural precursor and neuroblast transplantation, and includes a discussion of the induction of neurogenesis from endogenous neural precursors/‘‘stem cells’’ following targeted neuronal death of cortical projection neurons. Throughout this discussion, the response of the SVZ in repopulating the manipulated cortex will be emphasized. This biophysical model of targeted apoptotic neurodegeneration leads to highly specific and relatively synchronous cell death of targeted projection neurons in defined regions of the neocortex. Importantly, other cells in the regions surrounding those targeted for cell death (such as astroglia, interneurons, and other projection neurons) are not injured (Macklis, 1993; Madison and Macklis, 1993). To induce this specific cell death, latex nanospheres are conjugated with the photoactive chromophore chlorin e6 , and these nanospheres are injected into the terminal fields of the projection neurons to be targeted (Macklis, 1993; Sheen and Macklis, 1994, 1995). These conjugated nanospheres are transported retrogradely to the somata of the projection neurons, where they are stored within neuronal lysosomes. The projection neurons are then non-invasively exposed to long wavelength light (similar to that used in multi-photon confocal microscopy), and it is only by photoactivation that the labeled cells are induced to die via apoptosis. Specifically, cell death occurs via production of singlet oxygen from the chlorin e6 , which leads to apoptosis of the projection neuron. This long wavelength light is not absorbed by the surrounding neural tissue, and there is no non-specific damage associated with heating or radiation. Because neither the chlorin e6 nor the long wavelength light alone induces cell death, the combination can be used to offer two-fold specificity of axon

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target field and location in cortex to lead to specific cell death of a select population of cortical projection neurons. This approach of targeted apoptosis has been used in a variety of mammalian and avian systems (Macklis, 1993; Sheen and Macklis, 1995; Magavi et al., 2000; Scharff et al., 2000; Eyding et al., 2003). In the neocortex of adult mammals, this system has been used to induce selective neurodegeneration in Layer 2/3 and Layer 5 callosal projection neurons, or in Layer 6 cortico-thalamic neurons (Macklis, 1993; Madison and Macklis, 1993; Sheen and Macklis, 1995; Magavi et al., 2000). These targeted neurons undergo a distinct program of cell death, which includes DNA fragmentation, heterochromatin condensation, and the formation of apoptotic bodies (Sheen et al., 1992; Sheen and Macklis, 1994). In addition, such cells are TUNEL-positive, and no inflammation is associated with their death. Based initially upon results using transplanted neural precursors and immature neurons, our lab has also investigated the potential for these same molecular factors to manipulate endogenous precursors in the SVZ and/or cortex itself following targeted degeneration of deep layer corticosubcortical projection neurons fairly close to the SVZ. Specifically, in the first demonstration of induction of neurogenesis, synchronous apoptotic degeneration was induced in Layer 6 cortico-thalamic projection neurons in the anterior cortex, and the fate of dividing cells was examined in the cortex, corpus callosum, and underlying SVZ (Magavi et al., 2000). Using BrdU as well as phenotype specific markers of progressive neuronal differentiation, it was found that newborn cells progressively developed expression of the mature neuronal marker NeuN, and survived to at least 28 weeks. Initially, a subset of BrdU-positive precursors expressed Dcx (Francis et al., 1999; Gleeson et al., 1999) and displayed migratory morphology; they also expressed the early neuronal marker Hu (Marusich et al., 1994; Barami et al., 1995). The newly generated neurons developed distinct pyramidal neuron morphology, with 10-15 mm diameter somata and apical processes (Magavi et al., 2000). A subset of these newly generated mature neurons were also able to form appropriate long-distance thalamic projections, as demonstrated with the retrograde label FluoroGold. From where do these new neurons arise? These experiments (Magavi et al., 2000) suggested two potentially co-existing sources (Fig. 5.1). A small population of the newborn cells appeared to originate from precursors resident within the cortex itself. However, the main population appeared to originate in or within the vicinity of the SVZ. Using BrdU combined with Dcx labeling, it was found that there were early, postmitotic, immature neurons with migratory morphologies. These neurons were found to be apparently migrating from the underlying SVZ, through the corpus callosum, and into the degenerating cortical layer (Layer 6). That the cells took on a more mature phenotypic morphology only once they reached the appropriate cortical layer suggests that they underwent progressive differentiation on their way from the SVZ itself to their final position within Layer 6 of the

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Figure 5.1. Induction of neurogenesis in the neocortex of adult mice. Cartoon showing targeted apoptosis of cortico-thalamic projection neurons and subsequent recruitment of new neurons from endogenous neural precursors, without transplantation, in adult mouse neocortex. (a) In intact adult mouse neocortex, endogenous precursors exist in the cortex itself, and in the underlying subventricular zone (SVZ). Normally, these precursors produce only glia in cortex. Neurons produced in the anterior SVZ migrate along the rostral migratory stream to the olfactory bulb (not shown). (b) When cortico-thalamic projection neurons are induced to undergo synchronous targeted apoptosis, new migratory neuroblasts are born from endogenous precursors. These cells migrate into cortex, differentiate progressively into fully mature neurons, and a subset send long-distance projections to the thalamus, the appropriate original target of the neurons being replaced. The new neurons appeared to be recruited from SVZ precursors and potentially also from precursors resident in cortex itself. Adapted from Bjorklund and Lindvall, Nature (news and views), 405: 892-4, 2000, re: Magavi et al., ibid, 405: 951–955, 2000. Reprinted by permission from Nature copyright 2000 Macmillan Magazines Ltd.

cortex. Further, although it seems clear that at least a large subset of the newly generated neurons which replace dying neurons in the cortex are derived from SVZ precursors, it is possible or even likely that precursors in other regions (e.g., the cortical parenchyma) also give rise to new neurons (Magavi et al., 2000). More detailed studies of the exact location of these

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precursors, as well as the relative contributions of precursors from different areas, will need to be performed in the near future. Finally, it should also be noted that the exact means by which these newly generated cells reach their correct position has not yet been fully elucidated. Indeed, this migration may occur via homotypic chain migration, such as that seen in the rostral migratory stream, or else there may be some form of glial guidance mechanism, assisting the newly generated cells as they migrate from the SVZ to their correct cortical layer. It has been shown, for example, in studies involving transplantation of precursors into cortex undergoing targeted apoptosis, that mature astrocytes are capable of transforming into transitional radial glia (Leavitt et al., 1999). More recently, prior work in the cortico-thalamic system to induce neurogenesis has been modified to induce neurogenesis of cortico-spinal motor neurons in Layer 5 of motor cortex in adult mice (Chen, et al.,2004). The fates of dividing cells were examined within the nearby SVZ and overlying cortex, again using BrdU and markers of progressive neuronal differentiation. Within the first two weeks, subsets of BrdU-positive precursors in the SVZ express Dcx, and then migrate through the corpus callosum into overlying Layers 6 and 5 of cortex. Later, between approximately two and four weeks, BrdU-positive newborn cells express the early-expressed neuronal marker Hu, and NeuN, a mature neuronal marker, only in regions of cortex undergoing targeted neuronal death. Newborn neurons survive at least 56 weeks, and retrograde labeling from cervical spinal cord demonstrates that they can form long-distance cortico-spinal connections from 12 weeks onward, but not earlier. Together, these results demonstrate that endogenous neural precursors in or near the SVZ can be induced in situ to differentiate into cortico-spinal neurons that can survive for more than one year and, most critically, a subset of them form appropriate long-distance projections to cervical target regions in the adult spinal cord. This system of targeted apoptotic degeneration of cortical projection neurons provides a unique opportunity to examine the cellular and molecular controls underlying the response of the SVZ to discrete neurodegenerative pathology. It is well established that a variety of growth factors can influence the proliferation, differentiation, and survival of SVZ-derived cells, both in vitro and in vivo. These factors include EGF (Craig et al., 1996; Kuhn et al., 1997; Whittemore et al., 1999; Doetsch et al., 2002), FGF-2 (Kuhn et al., 1997), platelet-derived growth factor (PDGF) (Whittemore et al., 1999), ciliary neurotrophic factor (CNTF) (Whittemore et al., 1999; Emsley and Hagg, 2003), and BDNF (Zigova et al., 1998a; Pencea et al., 2001). Therefore, we have performed work aimed at expanding the endogenous precursor pool, the differentiation and survival of neurons derived from these precursors, and characterizing the responsiveness of cells in the SVZ during ongoing, targeted neurodegeneration. We have performed pilot experiments based on the literature and studies conducted in our lab (Fricker-Gates et al., 2002) and have unpublished observations suggesting that at least some subcortical SVZ pre-

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cursors express receptors for and respond with increased proliferation to the critical growth factors EGF, FGF, and PDGF. We investigated if we could increase the available ‘‘pool’’ of endogenous precursors in vivo by sustained infusion of growth factors. These pilot experiments were performed either with or without targeted neuronal death. We infused EGF, FGF, PDGF, or control vehicle (artificial cerebrospinal fluid with mouse serum albumin) into the lateral ventricles of adult mice for two weeks, using unilaterally implanted, specially-designed cannulae and Alzet osmotic minipumps (n = 36 in one set of experiments, and n = 20 in an additional set of experiments). We treated the same for two weeks with BrdU (given in the drinking water) and analyzed the subcortical SVZ and deep layers of cortex primarily, in addition to assessing the rostral migratory stream and hippocampus. In response to both EGF and FGF, we observed both: (1) previously described dramatic proliferation of cells in the anterior SVZ (Craig et al., 1996; Kuhn et al., 1997); and (2) an even more substantial increase in proliferation in more caudal SVZ ventral to sensorimotor cortex. There were fewer BrdU-positive cells overall caudally than in the anterior SVZ. Many of the expanded population of newborn cells entered Layers 4-6 of overlying cortex. More recently, we completed a pilot investigation of the effect of intraventricular EGF infusion on recruitment of newborn cortico-thalamic projection neurons in adult neocortex undergoing induced neurogenesis (Magavi, Emsley, and Macklis, unpublished observations). We observed 50–60/mm3 BrdU/Dcx-positive newborn migratory neuroblasts in experimental cortex one week after induction of cortico-thalamic neuron apoptosis, versus 0 in controls. Previous work in our laboratory has also shown that adult cortex undergoing synchronous, targeted apoptotic degeneration of projection neurons forms an instructive environment, which serves to guide the subsequent differentiation of transplanted immature neurons or neural precursors. These cells, when transplanted into degenerating adult mouse neocortex, can migrate selectively to the appropriate cortical layer (Macklis, 1993; Sheen and Macklis, 1995; Hernit-Grant and Macklis, 1996), differentiate into projection neurons (Macklis, 1993; Sheen and Macklis, 1995; HernitGrant and Macklis, 1996; Snyder et al., 1997; Leavitt et al., 1999; Shin et al., 2000; Fricker-Gates et al., 2002), receive afferent synapses (Macklis, 1993; Snyder et al., 1997; Shin et al., 2000; Fricker-Gates et al., 2002), express appropriate neurotransmitters and receptors (Shin et al., 2000; FrickerGates et al., 2002), and re-form appropriate long-distance connections to the original contra-lateral targets of the degenerating neurons (Hernit-Grant and Macklis, 1996; Shin et al., 2000; Fricker-Gates et al., 2002). Altered gene expression by cell populations local to the dying neurons exert effects over approximately 300-500 mm, via at least some soluble factors (Wang et al., 1998). These prolonged alterations in gene expression, induced by relatively synchronous initiation of neuronal apoptosis, may differ from that which would occur as a result of the relatively sporadic cell death that occurs in neurodegenerative conditions (Wang et al., 1998).

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In summary, it has been established by our lab that under specific conditions of targeted neuronal degeneration, endogenous precursors are capable of proliferating, migrating to the correct cortical layer, differentiating and, in many cases, assuming a progressively more mature neuronal phenotype with long-distance projections, even in the adult mammalian neocortex. A predominant subset of these cells is a product of the adult SVZ. In addition, the response of the SVZ during these defined circumstances of neurodegeneration provides a unique opportunity to study the molecular and cellular controls underlying the behavior of this distinct region during neurodegenerative conditions. Current work in this system is focusing on the role of a select panel of growth factors in enhancing the proliferation, differentiation, and survival of SVZ-derived cells, and is also examining changes in gene expression following targeted cortical apoptosis. These experiments, coupled with a greater understanding of the cellular and molecular characteristics of the SVZ, will provide further insight into how this unique brain region is capable of responding to neurodegenerative conditions.

Summary Neurodegenerative disorders can take many forms, from discrete neurological insults to diffuse degenerative conditions; from those associated with developmental disorders to those associated with ischemic injury or aging. Just as there is a broad range of neurodegenerative disorders, there are a variety of responses of the SVZ to these disorders. These responses include, in general, increased cellular proliferation, changes in the overall morphology of the SVZ, changes in the differentiation or production of certain cell types, altered migration of SVZ-derived cells, and changes in gene expression. It is not sufficient to view or characterize the SVZ as merely responding to an injury or a degenerative condition. As the SVZ is a developmental source that continues to proliferate in the adult CNS, there is a dynamic interplay between the role of the SVZ in development and its response to neurodegenerative conditions. To take such an argument further, it has been suggested that events in the SVZ itself may be the cause of disease—via either lack of appropriate SVZ cell generation in the adult, or via the unchecked proliferation and differentiation of immature cells seen in some forms of cancer. It is clear that there is much more work that needs to be done on understanding the response of the SVZ to neurodegenerative disorders. This increase in understanding must come not only from further cellular pathological studies of disease, but from continued, rigorous analyses of the development of the SVZ, its cellular architecture, and the molecular controls underlying its behavior in both normal and pathological conditions. Such elucidation of normal and pathological responses of the SVZ, and of controls over pre-

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cursors/’stem cells’ resident in the SVZ, may allow for directed differentiation and manipulation of SVZ-derived cells toward functional repair of the CNS. Acknowledgments. This work was partially supported by grants to J.D.M. from the NIH (NS41590, NS45523, HD28478, MRRC HD18655), the Alzheimer’s Association, the Human Frontiers Science Program, the National Science Foundation, the Christopher Reeve Paralysis Foundation, and the ALS Association. J.G.E. is partially supported by a Heart and Stroke Foundation of Canada Fellowship, and by a grant from the Children’s Neurobiological Solutions Foundation to J.D.M. Some confocal imaging was performed in facilities supported by the Harvard Center for Neurodegeneration and Repair. A portion of this chapter was updated and modified from a review article for different readership (Arlotta et al., 2003).

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Chapter 6 Responses of the SVZ to Radiation and Chemotherapy Ami M. Karkar1, Radoslaw Rola1 and John R. Fike1,2,3

Introduction The largest germinal zone in the adult mammalian brain, the subventricular zone (SVZ), has been meticulously described with respect to its component cells and their morphologic orientations to one another (Doetsch et al., 1997). The SVZ is mitotically active throughout the life span of a living being and the products of this extensive proliferation include neurons (Lois and AlvarezBuylla, 1993; Luskin, 1993; Doetsch and Alvarez-Buylla, 1996; Lois et al., 1996 Luskin, 1998) as well as glia (Levison and Goldman, 1993; Lois and Alvarez-Buylla, 1993; Luskin et al., 1993; Luskin and McDermott, 1994). In vitro analyses show that multipotent stem cells reside within the SVZ (reviewed in Alvarez-Buylla and Lois, 1995; Weiss et al., 1996; Temple and AlvarezBuylla, 1999; Gage, 2000), a finding recently verified in vivo (Doetsch et al., 1999b; Johansson et al., 1999). Cells produced in the SVZ are mobile, migrating long distances before differentiating into mature phenotypes. In adult rodents (Lois et al., 1996) and primates (Kornack and Rakic, 2001), the primary route of cell migration is to the olfactory bulb where migrating neuroblasts differentiate into granule neurons. This tangential migration presumably occurs along the entire rostrocaudal extent of the lateral ventricular walls (Lois and Alvarez-Buylla, 1994; Doetsch and Alvarez-Buylla, 1996), ultimately coalescing into the rostral migratory stream, which terminates in the olfactory bulb. While this pattern of cell movement is the most prevalent, radial cell movement away from the SVZ is also seen, particularly in the young brain (Suzuki and Goldman, 2003). Furthermore, in conditions of injury, disease, Departments of Neurological Surgery1 and Radiation Oncology2, University of California, San Francisco, San Francisco, CA 94143–0521 3 Mailing address: Brain Tumor Research Center, Department of Neurological Surgery, 533 Parnassus, Room U378, University of California, San Francisco, San Francisco, CA 94143–0520, Tel.: 415 206-3383, Fax: 415 206-3373, E-mail: [email protected] 185

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or infusions of growth factors, SVZ precursor cells are mobilized and move into nearby areas where they can differentiate into astrocytes and oligodendrocytes (Craig et al., 1996; Kuhn et al., 1997; Nait-Oumesmar et al., 1999; Wagner et al., 1999; Fallon et al., 2000; Zhang et al., 2001; Decker et al., 2002a, 2002b; Picard-Riera et al., 2002). Given the migratory activity of cells produced in the SVZ and their ability to differentiate into divergent cells types, it is possible that the SVZ may play a role in response to brain injury. In fact, it has been suggested that the SVZ may act as a reserve population of undifferentiated cells that could be recruited after tissue damage (Morshead and van der Kooy, 1992; Luskin and McDermott, 1994; Doetsch et al., 1997; Alvarez-Buylla and Garcia-Verdugo, 2002). Furthermore, recently it has been suggested that it might be possible, using specific growth factors, to reprogram specific transit amplifying cells to allow them to function as multipotent precursor cells, perhaps expanding their potential to act in repair or recovery (Doetsch et al., 2002). Brain cells can be injured or lost under a wide variety of circumstances including disease, injury, toxic substances or normal aging, and replacement of those cells, perhaps by stimulating normal neurogenic processes, could potentially ameliorate or change the evolution of damage within the brain. While such capabilities have not yet been realized, the idea of manipulating new cell production to affect recovery or repair seems possible given the proliferative, migratory and differentiation potential of cells in or from the SVZ. Understanding the regulation of cell proliferation, migration and differentiation under normal and pathologic conditions will not only enhance our overall understanding of neurogenesis, but also may provide keys to overcoming or ameliorating the adverse effects of injury or disease on the SVZ. This chapter will focus on how the SVZ responds to two insults associated with the treatment of certain types of cancer: ionizing irradiation and chemotherapeutic drugs.

Ionizing Irradiation The brain is exposed to ionizing irradiation during the management of specific disease states, particularly cancer. In the case of brain tumors, the amount or dose of irradiation that can be given is dictated to a large extent by the tolerance of normal tissues surrounding the tumor (Sheline et al., 1980). Radiation injury is manifold in character, involving multiple regions and cell/tissue types and involves a variety of structural and functional consequences. A large number of physical and biologic factors influence the expression and extent of radiation injury (Hopewell, 1998; Tofilon and Fike, 2000). Generally, radiation injury is localized to the white matter and sophisticated imaging studies have shown that in many cases, imaging abnormalities can be seen in the periventricular area (Tsuruda et al., 1987; Stylopoulos et al., 1988; Valk and Dillon, 1991). In fact, the presence of neurological complications associated with irradiation were reported to be

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correlated with periventricular and subcortical white matter changes (Tomio et al., 1998). Although the morphologic consequences of irradiation are well documented (Caveness et al., 1968; Burger et al., 1979; Hopewell, 1979; Calvo et al., 1988; Fike and Gobbel, 1991; van der Kogel, 1991; Tofilon and Fike, 2000), the pathogenesis of these changes remains uncertain. It has been suggested that the cellular events leading to radiation injury should be explained in terms of a loss in the reproductive capacity of cells regarded as targets within the CNS (Hopewell, 1979). In the past, those targets were primarily restricted to oligodendrocytes and vascular endothelial cells, two cell types in the mature brain capable of division (Hopewell, 1979; van der Kogel, 1986, 1991). Based on clinical and laboratory data, there is considerable support for both of these targets as critical players in the development of radiation injury. However, it seems unlikely that radiation injury can be attributed to only a single cell target (Tofilon and Fike, 2000). In fact, current approaches to the problem of radiation effects in the brain involve the assessment of factors not specifically associated with assays of clonogenic cell survival (Rubin et al., 1995; Tofilon and Fike, 2000). Regardless, an understanding of the response of potential target cell populations within the CNS still forms an important strategy for studying the development and possible treatment of radiation injury. Over thirty years ago, it was hypothesized that cell depletion within the mitotically active SVZ (referred to then as the subependymal plate) could play a role in radiation-induced white matter injury (Hopewell and Wright, 1970; Cavanagh and Hopewell, 1972; Hopewell and Cavanagh, 1972). The authors of those studies suggested that if the restorative response observed in the subependymal plate should fail, a gradual decline in the number of glial cells could ultimately lead to white matter necrosis, a characteristic of latedeveloping radiation brain injury. Citing that newly born cells move away and differentiate into neuroglia (Smart, 1961; Lewis, 1969), and that a ‘stem cell’ population in the subependymal plate could be a significant factor in the pathogenesis of white matter damage, investigators gave single x-ray doses of 2, 8, 20 and 40 Gy to rats, and assessed proliferative activity using mitotic cell counts (Cavanagh and Hopewell, 1972; Hopewell and Cavanagh, 1972). The Gray (Gy) represents the ‘amount’ or dose of irradiation, and is defined as the energy absorbed/unit mass (joule/kg). In earlier radiation studies, absorbed dose was defined by the ‘rad’ which represents 100 ergs/gm; one Gy ¼ 100 rads. Whole brain doses of 2–40 Gy have very different biological consequences with doses 20 Gy inducing gross changes in the vasculature following a latent period of > 12 months (Reinhold and Hopewell, 1980), while higher doses (> 22.5 Gy) result in glial cell depletion/white matter necrosis after a shorter latent period (Calvo et al., 1988). The results from studies of the SVZ showed that one day after irradiation, there was a dosedependent loss of mitotic activity ranging from 75% after 2 Gy to 100% after 40 Gy, and numerous pyknotic nuclei were noted in conjunction with the acute cell loss. This result was similar to what another investigator had

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reported in the mouse brain after a large dose of 23.7 Gy (Hassler, 1966). In the rat brain (Cavanagh and Hopewell, 1972; Hopewell and Cavanagh, 1972), over time (week to months, depending on the dose), mitotic cell counts returned to normal levels after all doses except 40 Gy; that dose ultimately resulted in extensive white matter necrosis. One remarkable feature reported by the authors was that chromosomal abnormalities were visible within the subependymal plate weeks to months after irradiation, suggesting either that the cell cycle time in many cells was considerably longer than the 18 hours reported earlier (Lewis, 1968), or that some cells in the subependymal plate were unusually sensitive to radiation-induced mitotic delay. Hopewell and Cavanagh concluded from their rat studies (Cavanagh and Hopewell, 1972; Hopewell and Cavanagh, 1972) that white matter necrosis might be due to a suppression of proliferation in the subependymal plate along with a gradual neuroglial cell loss in situ due to a slow natural cell turnover. Using the subependymal plate model described above, Chauser et al. (1977) assessed the difference in response between x-rays, as described above, and fast neutrons, a type of radiation that is more damaging to the CNS. While the changes induced in the subependymal plate after x-rays were similar to those reported earlier (Hopewell and Cavanagh, 1972), after neutron irradiation there was no obvious threshold dose above which there was recovery in terms of numbers of subependymal cells. Furthermore, those authors (Chauser et al., 1977) described the response of ‘light’ and ‘dark’ cells (Smart, 1961), the functions and proliferation characteristics of which were not known. Regardless, that study (Chauser et al., 1977) showed that the dark cells disappeared after irradiation while the light cells did not, leading the authors to speculate that early responses might be dictated by one cell type (i.e. dark cells), while later-developing and more clinically significant effects (e.g. necrosis) might be due to the response of the light cells. Subsequently, to determine what cell fraction within the subependymal plate was affected by irradiation, and also to determine if cell kinetics were affected, investigators used tritiated thymidine (3 H TdR) to label cell proliferation in the subependymal plate after a single 8 Gy dose of x-rays (Hubbard and Hopewell, 1980). The identification of specific subpopulations of cells was based on work by Lewis (Lewis, 1968) and Smart and Leblond (Smart, 1961) and included three morphologically distinct cells types: (a) cells with small dark-staining (SD) nuclei which were mitotically active and which the authors felt represented ‘stem cells’; (b) cells with small light-staining (SL) nuclei which were considered to be neuroglial precursors; and (c) cells with large, light-staining (LL) nuclei which were either neuroglial precursors or astrocytes (Hubbard and Hopewell, 1980). The numbers of SD, SL and LL nuclei decreased with age as well as with radiation dose. Those studies showed that cells having SD nuclei were the most sensitive to irradiation, and that about half of the SD nuclei were gone 1–2 weeks after irradiation. Normal cell counts were re-established 12 weeks later, followed

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by an ‘overshoot’ above age-matched controls at 26 weeks, and a subsequent decline back to control levels by 39 weeks post-irradiation. Changes in mitotic cell counts showed this same response both in terms of time and magnitude of change. The numbers of SL nuclei remained constant for the first two weeks after irradiation but then declined in concert with an increase in LL nuclei. The techniques used in that study allowed the investigators to estimate cell cycle time and length, which were shown to be not different between control and irradiated rats although the absolute number of labeled cells was less in irradiated brains. The sharp decline in both cell density and mitotic activity in the subependymal plate was interpreted by the authors to represent a change but not a cessation of function of the subependymal plate (Hubbard and Hopewell, 1980). Furthermore, the authors concluded that their study provided evidence of cell transformation and cell migration, noting that SD nuclei transformed into SL nuclei and that losses of SL nuclei were accompanied by a corresponding rise in LL nuclei. Consequently, a model was proposed to represent the production and differentiation of cells in the subependymal plate (Fig. 6.1), starting with the SD proliferative ‘stem cell’ population. Given what we now know about the various populations of cells within the mammalian SVZ, the ‘stem cells’ reported by Hopewell’s group likely do not represent the relatively quiescent stem cells reported by Morshead et al. (1994) almost 15 years later. Regardless, one of the main conclusions of these early radiation studies was that the absence of white matter necrosis after modest radiation doses was due to the repair capacity of the subependymal plate, allowing a gradual restoration of the supply of neuroglia to the brain. (Hubbard and Hopewell, 1980). Lacking corroborative cause and effect data, this conclusion remains speculative. It is of interest that the model put forth by Hubbard and Hopewell (1980) (Fig. 6.1) was not really expanded upon until Doestch et al. (1997) provided their elegant description of the SVZ. Using more sophisticated methods such as electron microscopy and cell-specific immunohistochemical labels, those investigators were able to provide a topographical model for the adult SVZ. Cells with SD nuclei, “stem” cells

Proliferation

Cells with SL nuclei, neuroglial precursors

Cells with LL nuclei, neuroglial precursors or astrocytes

Migration

Migration

Figure 6.1. A model representing the production and differentiation of cells in the subependymal plate of the adult rat. SD, small dark; SL, small light; LL, large light. Modified from Hubbard and Hopewell, 1980.

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In the more comprehensive description offered by Doestch et al. (1997), neuroblasts or Type A cells corresponded to the SD nuclei visible with the light microscope. The type B cells, which had astrocytic properties, corresponded to the previously described LL staining nuclei. Lastly, Type C cells, which likely correspond to the SL cells described by Hopewell’s group, were described as putative precursors. Interestingly, recent work has suggested that SVZ stem cells have astrocytic properties (B cells) (Doetsch et al., 1999b) The early radiation studies of Hopewell and his colleagues (Cavanagh and Hopewell, 1972; Hopewell and Cavanagh, 1972; Hubbard and Hopewell, 1980) were similarly not followed up for over 2 decades. However, with new information available regarding the potential role of 0–2A glial cell progenitors and radiation injury to the spinal cord (van der Maazen et al., 1991, 1992, 1993), along with provocative in vitro and in vivo studies relating to the presence of multipotential stem cells in the adult SVZ (Reynolds and Weiss, 1992; Lois and Alvarez-Buylla, 1993; Okano et al., 1993; Morshead et al., 1994), new studies were initiated to further investigate the radiation response of the SVZ. Bellinzona et al. (1996) showed that the early cell loss observed by Hopewell and his colleagues, was due to a programmed cell death, or apoptosis. Using a distinct morphologic pattern of nuclear fragmentation combined with TdT-mediated dUTP-biotin nick end labeling (TUNEL) (Fig. 6.2), which specifically labels the 3’-hydroxyl termini of DNA strand breaks (Gavrieli et al., 1992), Bellinzona et al. (1996) showed that in the rat SVZ, apoptosis was maximum 6 hours after irradiation. While only 2 doses of irradiation were used, 5 and 30 Gy, there were more apoptotic nuclei seen after the higher dose. This study was the first to show that acute cell loss in the subependymal area after irradiation could be attributed to a specific type of cell death. However, no information was provided regarding which cell type(s) in the SVZ were affected. Citing that there were ways to reduce or inhibit apoptosis (Ferrer, 1992; Langley et al., 1993), Bellinzona and his coworkers suggested that it might be possible to modulate the extent of later radiation injury if that injury was mediated in part by an apoptotic process. Shinohara et al. (1997) expanded on the work of Bellinzona et al. (1996) by assessing SVZ radiation response after single and fractionated doses of x-rays. Using single doses ranging from 1 to 30 Gy, the authors showed a steep increase in apoptosis after low doses of 1–2 Gy, followed by a plateau after doses above 2 Gy (Fig. 6.3). The presence of a plateau has been shown to represent the fraction of the total cell population that is susceptible to apoptosis (Dewey et al., 1995), and at 6 hours after irradiation, that population accounted for about 30% of the cells in the region. However, due to the dynamic nature of apoptosis and the fact that the measurement was done only at one time after irradiation, this value probably underestimated the actual fraction of sensitive SVZ cells. The extreme sensitivity of SVZ cells to relatively low doses of x-rays (1–2 Gy) raised the question of how the SVZ

Figure 6.2. Photomicrographs of un-irradiated mouse brain (bottom) and mouse brain irradiated with a single dose of 2 Gy (top), showing apoptotic cells in the dorsolateral portion of the SVZ. Apoptotic cells can be seen as either brown-staining TUNEL-positive cells (arrows) or darkly stained fragmented/pyknotic nuclei (arrowhead). Few apoptotic nuclei are observed in un-irradiated tissues, while substantial numbers of apoptotic nuclei are seen after irradiation. cc: corpus callosum; cpu: caudate putamen; v: lateral ventricle. Mark ¼ 100 mm.

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Figure 6.3. Apoptosis in the SVZ of young adult F344 rats after whole brain irradiation. Apoptotic index (percentage of total cells showing characteristics of apoptosis) was determined using TUNEL and distinct nuclear morphology (nuclear fragmentation, pyknosis) consistent with apoptosis. These data show that there are very radiosensitive cells in the SVZ and that no additional apoptosis is observed above doses of 2 Gy. Each datum point represents a mean of 3–6 rats; error bars are standard error of the mean (SEM). Modified from Shinohara et al., 1997.

might respond to a more clinically relevant radiation paradigm, i.e. multiple low dose radiation fractions, and if there was recovery or repair when radiation was given in small doses. When 1.5 Gy fractions were administered over a 1–7-day period, Shinohara et al. (1997) showed a steep decrease in apoptotic index after 1, 2, and 3 fractions, followed by a plateau after 4–7 fractions. In terms of SVZ cellularity within their specified region of interest, Shinohara et al., found that the first 4 fractions appeared to be equally effective in terms of decrease the cell population within the SVZ. There was no additional cell loss after fractions 5–7. On the basis of these data, it appeared that there was no apparent recovery between 1.5 Gy fractions, and similar to what was inferred from the single dose response data, i.e. there was a finite population of cells within the SVZ zone that was exquisitely sensitive to x-irradiation. Consideration of the time course of SVZ apoptosis along with data from the fractionation study led Shinohara et al. (1997) to estimate that the sensitive cells constitute at least 40% or more of the cells in the SVZ. It is of interest that this value is very similar to the total proliferating population (43 + 1.5%) determined using multiple injections of the thymidine analog 5-bromo-2’-deoxyuridine (BrdU) (Shinohara et al., 1997). This suggests that cells apoptosing, after irradiation, largely represent the population described as constitutively proliferating cells of the SVZ (Morshead

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et al., 1994). This was confirmed by irradiating rats that had previously received injections of BrdU to label actively proliferating cells. At the time of peak apoptosis, a significant proportion of cells showing apoptotic characteristics (TUNEL staining or nuclear fragmentation) were shown to be BrdU-positive (Shinohara et al., 1997). Shinohara et al. (1997) showed that in response to the loss of cells after a single dose of x-rays, the BrdU-labeling index in the SVZ increased 24–72 hours after exposure. That was interpreted to mean that radiation-induced cell loss in some way stimulated proliferation in surviving SVZ cells. It was further suggested that such a phenomenon might reflect an attempt to regenerate the SVZ. This finding is similar to results seen in mouse brain irradiated using 3 HTdR (Morshead et al., 1994). While machine-produced xrays are delivered from outside the animal, 3 HTdR is an isotopic source that produces low energy beta particles of short range. The main advantage of 3 HTdR is that it is taken up during the S-phase of the cell cycle and thus localizes the radiation exposure exclusively to proliferating cells. A disadvantage of an isotopic source is that the cellular dose is difficult to determine in vivo, and because proliferating cells are not synchronized, not all cells will receive the same dose and perhaps express the same biological damage. Nonetheless, 3 HTdR is a reasonable way to selectively irradiate most of the constitutively proliferating cells in the SVZ without affecting other, nonproliferating cells. In the study by Morshead et al. (1994), not only was the apparent stimulation of cell proliferation shown, but subsequent 3 HTdR injections during the accelerated proliferation showed that there was a relatively short time window (days) during which active cell proliferation occurred after the initial cell depletion. This short time window is not unlike that seen in the hematopoietic system, using a similar two-challenge paradigm, where stem cells stimulated to divide are more susceptible to a second insult (Harrison and Lerner, 1991). The 3 HTdR results described above led Morshead et al. (1994) to conclude that under normal conditions, there is a relatively quiescent stem cell population residing in the SVZ that is less mitotically active than the constitutively proliferating population. The studies above provided important insights into the presence and proliferation characteristics of multipotent stem cells in the rodent SVZ. Stem cells are found in a variety of normal tissues, and radiation studies of those tissues show that in general, such cells are able to respond after irradiation and repopulate the damaged tissues (Paulus et al., 1992; Do¨rr, 1997). Based on that information, the early works by Hopewell and his co-workers (Cavanagh and Hopewell, 1972; Hopewell and Cavanagh, 1972; Hubbard and Hopewell, 1980) and on the studies by Morshead et al. (1994), Tada et al. (1999) hypothesized that x-rays would impact the SVZ in a dose-dependent fashion and that repopulation would occur as a function of time after irradiation. This study differed from that of Morshead et al. (1994) in a number of important ways, not the least of which was the use of external beam x-irradiation. As discussed earlier, irradiation from an

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isotopic source like 3 HTdR is localized only to proliferating cells and at least under normal conditions, should not affect the quiescent stem cell population. External beam x-rays, on the other hand, will not only affect proliferating cells, but also any non-cycling stem or progenitor cells. Due to the stochastic nature of x-irradiation, the probability of inducing damage in any given cell will increase with dose, so determining the numbers of surviving SVZ cells should provide an indirect measure of the sensitivity of the stem cells. Therefore, assuming that the SVZ stem cells are responsible for maintaining the normal cellularity/mitotic character of the SVZ (Morshead et al., 1994, 1998; Doetsch et al., 1997), it is possible to estimate the extent of stem cell death by quantifying the cellular composition of the SVZ as a function of radiation dose and time after irradiation. Tada et al. (1999) irradiated the brains of young adult rats with doses of 2–15 Gy, and at various times thereafter, quantified specific cellular components within a representative area (0:05 mm2 ) of the SVZ. How this area was chosen, and the studies done to standardize its use have been described by Shinohara et al. (1997). In terms of biological effectiveness, the doses used in the study by Tada et al. (1999) are considered low to moderate, and below the threshold for inducing obvious tissue changes such as demyelination or necrosis (Calvo et al., 1988). After such doses to the brain, and using appropriate statistical analyses (e.g. Dunnett’s many to one t test), Tada et al. (1999) showed that 24 hours after irradiation, there were significant (p < 0.05) reductions in total cell number, number of proliferating cells and immature neurons in the SVZ (Table 6.1). Furthermore, within the designated region of interest, the calculated areas of nestin and vimentin immunoreactivity decreased at 24 hours, supporting the contention that most of the cells being lost early after irradiation were undifferentiated proliferating cells. No acute changes were noted with respect to the area of GFAP immunoreactivity. The acute cell loss seen by Tada et al. (1999) ranged from about 50% after 2 Gy to 95–98% after higher doses, and was comparable to that seen by others (Bellinzona et al., 1996; Shinohara et al., 1997). Following up on the post-irradiation proliferation studies by Shinohara et al. (1997) and Morshead et al. (1994), Tada et al. (1999) also investigated the Table 6.1. Changes in cellular composition in the SVZ 24 hours after X-irradiationa Endpoint Total cells P34cdc2 L.I.d (%) BrdU L.I.d (%) TuJ1 L.I.d (%) a

Shamb

2 Gyc

5 Gyc

7.5 Gyc

628 + 51.9 403.5 + 22.9 302.3 + 20.9 303.5 + 20.9 38.9 + 1.0 8.8 + 0.9 3.2 + 0.22 1.5 + 0.5 15.1 + 0.62 4.4 + 0.7 0.2 + 0.2 0.2 + 0.1 27.3 + 2.5 0.7 + 0.4 0.4 + 0.1 0.4 + 0.2

Modified from Tada et al., 1999. Sham represents rats that were anesthetized but not irradiated. c Each value represents the mean 4 rats + standard error of the mean. d L.I., labeling index (percent of labeled cells). b

10 Gyc

15 Gyc

304 + 8.5 1.3 + 0.3 0.1 + 0.1 1.1 + 0.4

319 + 14.4 1.0 + 0.3 0.6 + 0.1 0.1 + 0.1

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stimulation of SVZ cell proliferation after varying doses of x-rays using an antibody against p34cdc2 . p34cdc2 is a protein expressed in proliferating cells (Hayes et al., 1991) and is involved in driving those cells into mitosis (Nurse, 1990; Hayes et al., 1991). After 2–15 Gy of x-rays, the p34cdc2 labeling index (percent of total cells counted expressing p34cdc2 ) increased between 1 and 7 days after irradiation and there was an apparent delay in the onset of proliferation with increasing radiation dose (Fig. 6.4). Seven days after irradiation, there was a clear dose response in terms of p34cdc2 and Class III b-tubulin (TuJ1) labeling index, indicating that while cell proliferation and numbers of immature neurons rebounded after the acute cell loss, their ultimate numbers varied with radiation dose. Assuming that SVZ repopulation is due in part to the stimulation of relatively quiescent stem cells (Morshead et al., 1994), this result is consistent with the idea that more stem cells are killed with increased radiation dose. To address the longer-term ability of surviving stem cells to repopulate the SVZ, Tada and his co-workers gave 2–15 Gy of x-rays and quantified cellular changes up to 6 months later. After peaking at 7 or 14 days post irradiation, numbers of p34cdc2 -positive cells then decreased to levels that were maintained for the entire study (Fig. 6.5). Similar responses were seen in terms of total cell number within a defined region of the SVZ. At all the post-irradiation times studied

Figure 6.4. An early effect of x-rays on proliferating cells in the rat SVZ. Cell proliferation was detected using an antibody against p34cdc2 , a cyclin-dependent kinase. The p34cdc2 labeling index (percent of total cells positive for p34cdc2 ) for all treatment groups fell at 24 hours due to apoptosis of proliferating cells. The increase in cell proliferation from days 1–7 likely represents the stimulation of relatively quiescent stem cells. Each datum point represents a mean of 4 rats; error bars are SEM. Modified from Tada et al., 1999.

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Figure 6.5. The number of proliferating SVZ cells in young adult F344 rats stays relatively constant from 30 to 180 days after irradiation. Prior to irradiation, the number of proliferating cells in a standardized region of interest was 2245.5 + 25.6 (mean + SEM). Twenty-four hours after all doses, significant reductions were seen in the numbers of proliferating cells. The magnitude of subsequent changes was dosedependent. Each datum point represents a mean of 4 rats; error bars are SEM. Modified from Tada et al., 1999.

(7, 14, 30, 60, 120 and 180 days), the percentages of p34cdc2 -positive cells were plotted versus dose and analyzed using a general linear modeling procedure (Tada et al., 1999). The slopes of the individual dose response curves were not statistically different (p ¼ 0.85, Fig. 6.6), which was consistent with a dose-dependent decrease in the number of SVZ stem cells. Those data also showed a lack appreciable repopulation of the SVZ for at least 6 months following irradiation. As described above, numbers of proliferating cells and immature neurons seen 7 days after irradiation are transient, rising initially and falling at later times to levels that are maintained for up to 180 days (Fig. 6.5). Such transient changes have also been seen in other renewing tissues such as the gut (Paulus et al., 1992), and may reflect a variety of factors including abortive cell divisions, changes in proliferation rate or a change from asymmetrical to symmetrical division within the stem cell population (Do¨rr, 1997). Although Tada et al. (1999) did not specifically address the mode of cell division after irradiation, the apparent lack of re-establishment of total cellularity, numbers of proliferating cells and numbers of immature neurons suggested symmetrical divisions did not occur, at least to the extent that would result in a normal-appearing SVZ. This conclusion is in agreement with the work of other investigators (Morshead et al., 1998), who concluded that SVZ stem cells self-renewed in an asymmetric mode even during

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Figure 6.6. Radiation dose response for proliferating SVZ cells. At all time points studied, the fraction of p34cdc2 -positive cells was reduced in a dose-dependent fashion. Each datum point represents a mean of 4 rats; error bars are SEM. Modified from Tada et al., 1999.

repopulation after irradiation with 3 HTdR. Tada et al. (1999), therefore, suggested that under conditions where stem cells were damaged or lost, there was no compensatory change to symmetrical division, and that the doserelated loss of SVZ stem cells may be permanent. Given that SVZ stem cells apparently have the ability to undergo symmetrical divisions when exposed to the right conditions in vitro (Gritti et al., 1996; Reynolds and Weiss, 1996), or in vivo (Craig et al., 1996), the data from Tada et al. (1999) and others (Morshead et al., 1998) suggest that the surviving stem cells may not have received the ‘proper’ signal(s) to initiate a change in mode of division. Alternatively, inhibitory factors might exist in vivo to limit symmetric self-renewal (Morshead et al., 1998), or perhaps stem cell recovery may be dependent in part on surrounding supportive elements such as glia. Data are becoming available showing that a variety of factors/conditions stimulate cell proliferation, migration and differentiation of cells in the normal SVZ (Craig et al., 1996; Cameron et al., 1998; Smith et al., 2001; Decker et al., 2002b; Jin et al., 2003). How or if these or other factors may be involved in the radiation response of the SVZ is not yet clear. However, recent data regarding hippocampal neural precursor survival and differentiation after irradiation (Monje et al., 2002; Mizumatsu et al., 2003) show that local microenvironmental factors such as vascular integrity and/or inflammation may also play inhibitory roles in post-irradiation stem cell function/recovery.

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Astrocytes are critical components to the integrity of the SVZ, and may provide directional cues or trophic support for migrating neuroblasts, or may provide a partition for isolating migrating cells from surrounding parenchyma (Lois et al., 1996; Garcia-Verdugo et al., 1998). In addition, recent data indicate that an astrocytic subpopulation may actually constitute the stem cell component within the SVZ (Doetsch et al., 1999b). Tada et al. (1999), using a quantitative morphometric analysis, showed a subtle increase in astrocytic area within the SVZ shortly after the radiation-induced loss of undifferentiated cells, nestin-positive cells, Tuj1-positive neuroblasts and proliferating cells, but the data did not specifically address if the slight change in GFAP area represented increased stem cell activity. However, 180 days after irradiation, there was a clear dose-related decrease in GFAP area within the SVZ (Fig. 6.7), giving more indirect support to the idea that the astrocytic component changed as a function of radiation dose. Concomitantly, there was a dose-related decrease in the production of TuJ1-positive neuroblasts (Fig. 6.7), again supporting the contention that stem cells were diminished in a dose-dependent manner. While the data from Tada et al. (1999) suggest a possible link between radiation-induced depletion of astrocytes and the lack of SVZ repopulation, that relationship is not yet well understood. While some uncertainties still remain regarding precisely how radiation injury develops in and around the SVZ, clearly this structure is sensitive to irradiation. Therefore, based on the older data from Hopewell’s group (Fig. 6.1) as well as more recent radiation and neuroscience studies, a simple model can be constructed documenting how the SVZ responds to irradiation (Fig. 6.8). While the response of the component cells of the SVZ is relatively easy to detect and quantify, the identification of the cellular and molecular mechanisms responsible for regeneration or rescue of the this area after acute cell loss need to be determined. Studying the radiation response of the SVZ cells has a clear therapeutic relevance inasmuch as it has long been speculated that the depletion of such cells may underlie radiation-induced late effects. However, the results to date show that significant depletions of the cells of the SVZ occur after doses that are not associated with such late effects, and functional consequences of radiation-induced SVZ damage have not been assessed. While the available data do not yet prove that the SVZ is or is not involved in the development of late radiation injury, the fact that SVZ cells are proliferative, able to migrate and can differentiate into multiple cell types makes this structure and its cellular components of particular interest, especially if the various characteristics associated with neurogenesis can be manipulated. While the responses of the SVZ after irradiation remain incompletely characterized, and the effects of age, gender, genetic factors, pre-existing disease/injury, etc on those responses have not been specifically addressed, the use of ionizing irradiation as a tool to study the vulnerability and plasticity of the SVZ is not in question. Selective ablation of proliferating cells with 3 HTdR provides

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Figure 6.7. Loss of astrocytes (top) and immature neurons (bottom) observed 180 days after x-irradiation were dose dependent. Total numbers of cells or percent area were determined for a standardized region of interest. Numbers of immature neurons were detected using an antibody against Class III b-tubulin (TuJ1), and the percent area measurements for astrocytes represent the relative area immunoreactive for GFAP. Each datum point represents a mean of 4 rats; error bars are SEM. Modified from Tada et al., 1999.

a means by which subsequent recovery effects due to the stimulation of quiescent stem cells can be analyzed. On the other hand, external irradiation provides the possibility of actually damaging the stem cells and assessing their subsequent ability to repopulate. One potential problem of the external

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Figure 6.8. Representation of radiation-induced cell loss within the rodent SVZ after either a low (2 Gy) or moderate (10 Gy) dose of x-rays. The diagram is based on the model of Doetsch et al. (1999b) which designates migrating neuroblasts as Type A cells, surrounding astrocytes as Type B cells and actively dividing cells (e.g. putative precursors) as Type C cells. A low dose affects the most sensitive cells, Type A and C, while a higher dose affects those cells as well as the surrounding astrocytes, some fraction of which may represent SVZ stem cells.

beam approach is that all cells in the area of interest will be exposed, and the damage/recovery of those cells could impact the stem cell response that is being investigated. Recent data concerning the radiation response of the dentate subgranular zone clearly elucidates the complex microenvironmental interactions that can occur in irradiated tissues, and which can affect the function of stem cells or their progeny (Monje et al., 2002). In conclusion, irradiation is known to significantly affect the brain, and after doses that do not cause widespread tissue damage, there are substantial effects on the cells of the SVZ. These effects involve the constitutively proliferating cells as well as the stem cells that give rise to them. While uncertainty still exists with respect to the function of the SVZ, the study of radiation effects provides a means to study the neurobiology of this interesting structure and perhaps provide insight into its functional role under normal conditions and after injury or disease.

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Chemotherapeutic Agents Chemotherapeutic agents are currently used in conjunction with local brain irradiation in the management of a number of neoplastic diseases. While the use of adjuvant chemotherapy has decreased mortality rates of a number of conditions such as acute lymphoblastic leukemia, complications associated with the CNS have been reported (Lovblad et al., 1998). The character of such changes can be variable, and includes vascular and parenchymal changes. In addition, imaging studies have shown diffuse periventricular and white matter hypodensities (Lovblad et al., 1998); similar changes have been seen after brain irradiation as well (reviewed in Valk and Dillon, 1991). As described earlier, some effort has been made to try to understand the response of the periventricular region after irradiation. However, few data are available regarding how chemotherapeutic agents affect the SVZ, and most studies that address have the topic used mitotic inhibitors to study the neurobiology of the region. The work of Hopewell and his colleagues represents one of the only studies to specifically address how a chemotherapeutic agent affects the SVZ or as they called it, the subependymal plate (Morris and Hopewell, 1983). In that particular study, the effects of methotrexate (MTX) alone or combined with radiation was evaluated in the SVZ relative to radiation alone. Intrathecal MTX was administered to rats as a single dose (4 mg/ kg) and cellular changes were assessed using light microscopy. Using cellular parameters described in previous radiation studies (Hubbard and Hopewell, 1980), Morris and Hopewell (1983) chronicled changes over a 3–9 week period. Cells with small dark (SD) nuclei were most sensitive to x-ray and combined treatment, and those authors suggested that the losses in SD nucleated cells accounted for the fall in mitotic count. Given more recent data describing the SVZ, this result likely reflects, in part, the sensitivity of the constitutively proliferating cells. While the mechanism of action of MTX on the radiation response of cells in the SVZ was not clarified by this study, Morris and Hopewell (1983), nonetheless, implied that the actions of these two cytotoxic agents were additive. Based on this information, they concluded that if MTX is to be used with irradiation, the radiation dose should be reduced to avoid exceeding the tolerance of the normal brain to these insults. A decade later, Morris et al. (1995) revisited the effects of MTX on the SVZ, comparing them to the effects of another compound, the bioreductive drug misonidazole. Arguing that the SVZ may be relative hypoxic under normal conditions, those authors concluded that this brain region might be particularly susceptible to misonidazole, which is toxic to hypoxic cells (Hall and Biaglow, 1977). Maximum tolerated doses of intraventricular MTX or orally administered misonidazole were given and the cellular changes in the SVZ were examined days to weeks after treatment. Total nuclear density in the rostral extension of the SVZ declined to about 70% of control values

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rapidly (1–2 days) after MTX treatment, reflecting the loss of mitotic cells and cells characterized by SD or small light (SL) nuclei; numbers of large light (LL) cells initially increased relative to controls then decreased to baseline levels. These early cell losses were paralleled by an increase in the number of pyknotic cells. Significant reductions (i.e. 50%) in the numbers of mitotic figures, total nuclear counts and numbers of SD nuclei were seen at 12 weeks following MTX treatment, but at 24þ weeks, the total number of cells had returned to control levels. The numbers of SL and LL nuclei were increased relative to age-matched controls at 12 and 48 weeks following MTX treatment. In comparison to what was seen after MTX treatment, declines in total nuclear counts and numbers of SD nuclei were less marked (i.e. 30%) after treatment with misonidazole. However, changes in mitotic counts were similar between the two compounds, emphasizing the extreme sensitivity of proliferating cells in the SVZ to cytotoxic agents. Small but significant reductions in SL nuclei were observed on day 2 after misonidazole treatment while no changes were observed in LL nuclei. Taken together, the data from this study suggest that the SD nuclei are the most sensitive cells in the SVZ, and the authors suggest that the chosen chemotherapeutic drugs have a differential toxic effect on the stem cell population of the SVZ. Given the current knowledge regarding the cellular make-up of the SVZ and the response of the various cell types to radiation, the sensitive cells described by Morris et al. (1995) are not likely to be the stem cells themselves, but rather their progeny. With the exception of the work described above, little information is available describing the effects of chemotherapeutic agents on the morphologic/functional integrity of the SVZ. Given the widespread use of chemotherapeutic agents in cancer treatment and the potential neurotoxicity of many of these compounds, it is surprising that a sensitive cell population such as that seen in the SVZ has not been a subject of study. However, some research groups have used the extreme sensitivity of SVZ cells to cytotoxic drugs as a tool to study the neurobiology of the SVZ. The lab of AlvarezBuylla in particular has been interested in depleting the proliferating population in the SVZ in order to identify and understand the biology of cells in the SVZ. In the studies by Doetsch et al. (1999a), the antimitotic drug cytosine-b-d-arabinoside (Ara-C) was administered to specifically eliminate all the immature precursor cells (type C cells). The drug was infused onto the brain surface for a total of 6 days, which resulted in a complete elimination of all migrating neuroblasts as well as all type A cells. During the next 30 days, there was a complete regeneration of the SVZ and the migrating chains of neuroblasts. This was considered an unexpected plasticity within the mammalian brain and suggested to the authors that the stem cells in the SVZ were not killed by the Ara-C and were able to completely restore the population of neuroblasts. Using this same methodology Doetsch et al. (1999b), were able to show that the surviving stem cells share features with astrocytes. Furthermore, in a recent study, after Ara-C depletion of type C

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cells and neuroblasts, these investigators wanted to determine if the number of neurospheres that could be generated from the SVZ changed at different stages of regeneration (Doetsch et al., 2002). The results from that study showed that neurospheres generated at various time points after Ara-C had the same properties as neurospheres produced from non-treated animals, indicating that SVZ stem cells were not affected by the drug treatment. According to those authors, the findings related to the use of Ara-C may have clinical implications, because if longer drug treatments are used it may be possible to eventually deplete the stem cells as well as their progeny. While the numbers of studies directed toward understanding the effects of chemotherapeutic agents on the SVZ are very few, the data from those studies have provided important information regarding the biology of this structure. The early studies of Hopewell and his colleagues (Morris and Hopewell, 1983; Morris et al., 1995) identified the most sensitive cellular elements in the SVZ to drug therapy, while the studies of Doetsch et al. (1999a, 1999b, 2002) exploited that sensitivity to elicit critical details about the identity of the SVZ stem cell and its ability to regenerate the SVZ.

Conclusions It is now evident that new cells are produced in the adult SVZ throughout life, and numerous studies have shown that multipotent stem cells residing in the SVZ are capable of producing neurons and glia. While considerable data exist showing that stem cell proliferation is upregulated under a variety of circumstances, the potential of SVZ stem cells to regenerate or repopulate damaged neurons and glia and to rescue damaged areas of the brain is not yet clear. Radiation and chemotherapy are commonly used in the management of cancer, and exposure of brain to damaging doses of radiation or drugs is not uncommon. It is tempting to speculate that neurogenesis in the SVZ may provide a source of undifferentiated cells that may be able to play some role in the response to injury. However, the available data show that while the SVZ is extremely sensitive to irradiation, the resulting cell depletion occurs after doses not generally associated with late developing radiation injury. While this may imply that SVZ neurogenesis may not play a role in the development of late injury, it still may be possible to induce reparative processes using either exogenous or endogenous factors (e.g. cytokines, growth factors). Much more work is necessary to determine exactly if/how neurogenesis can be manipulated to increase the recovery potential of the SVZ. Given the apparent plasticity of the SVZ, such manipulations seem conceivable. While there is still uncertainty regarding the role of the SVZ after brain exposure to radiation or drug, the sensitivity of this structure to adverse stimuli offers a unique opportunity to study neurobiology. Given the dynamics of the cell populations in the SVZ and the availability of approaches to selectively deplete various populations, it is possible to specifically address

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the role of stem cells as well as their progeny under normal and pathologic conditions. To date, such manipulations have helped identify an astrocytelike cell as the stem cell component of the SVZ and have shown that at least after irradiation, those cells have a limited regenerative capacity. Having this information, we are poised to ask more mechanistic questions regarding exactly why regeneration is so affected by radiation and if/how such effects can be overcome.

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Chapter 7 The Subventricular Zone Responds Dynamically to Mechanical Brain Injuries Maria L.V. Dizon and Francis G. Szele

Introduction Brain injury is a common yet relatively untreatable problem (Thurman, 1999). Of the 500,000 cases of traumatic brain injury annually in the United States, greater than 90,000 result in disability (Valadka, 2000). Mechanical injuries often occur to external regions of the brain: the cerebral cortex and other parts of the telencephalon. They can produce a wide variety of longterm and devastating symptoms and pathologies due to neuronal death. The underlying biological predicament in recovery from brain injury is that the adult central nervous system is generally incapable of replacing dead neurons. This concept has been challenged by the discovery of neurogenic adult stem cells in the subventricular zone (SVZ) (Alvarez-Buylla et al., 2000). It has been estimated that the SVZ replaces tens of thousands of olfactory bulb (OB) interneurons per day in rodents. Behavioral studies suggest that the constant turnover of OB neurons allows olfactory discrimination (Gheusi et al., 2000). Adult humans and other primates have also been shown to possess neurogenic SVZ cells with many of the features delineated in rodents (Eriksson et al., 1998; Bernier et al., 2000; Weickert et al., 2000; Kornack and Rakic, 2001; Pencea et al., 2001b). Granted, the sense of smell is not of vital importance for humans, unless one is a sommelier. Yet, since the SVZ is in close proximity to the cerebral cortex and other functionally important forebrain nuclei, hope has risen that the great neurogenic and migratory potential of adult stem cells may be co-opted for repair. This optimism is bolstered by the fact that adult SVZ cells are descendants of the proliferative neuroepithelia which, during development, give rise to the myriad neural cells in the telencephalon. 2300 Children’s Plaza, No. 209, CMIER Neurobiology Program, Children’s Memorial Hospital, Department of Pediatrics, Feinberg School of Medicine, Northwestern University Chicago, IL 60614-3394, Tel.: (773) 755-6356, Fax: (773) 755-6344, E-mail: [email protected]

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Two general strategies for cell replacement therapy have been conceived. In one scenario, SVZ cells would be molecularly stimulated in situ to migrate to injured regions and then differentiate appropriately. The other strategy would rely on the capacity of SVZ cells, even from humans, to be cultured (Lois and Alvarez-Buylla, 1993; Kirschenbaum et al., 1994; Kukekov et al., 1999) and genetically manipulated in vitro (Ostenfeld et al., 2002). This property suggests a scenario whereby a small portion of a patient’s SVZ would be excised, expanded and genetically manipulated in vitro, and then transplanted into the site of injury. It is crucial that we understand how SVZ cells respond to brain injuries before attempting genetic manipulation of SVZ cells for either approach. An enormous amount of work needs to be done to answer fundamental questions of how SVZ cells behave in response to specific modes of injury. Does the SVZ change its rates of proliferation after mechanical trauma to the brain? Why don’t SVZ cells normally move into non-olfactory bulb regions in the adult? If some SVZ cells do migrate into injured areas, how long can they survive, and do they differentiate? What are the genes that inhibit or promote these processes? Do lesions that directly damage the SVZ elicit different responses from those that are restricted to surrounding regions? A few studies have addressed these issues. In this chapter we shall review them, attempting to identify major gaps in our knowledge, and proposing potentially fruitful approaches. We focus on the adult, since there is a paucity of literature on SVZ responses to injury in the immature brain, and because the biology of the SVZ changes through development. A common sequence of events occurs after brain trauma in a time course ranging from seconds to months. It starts with a rapid mechanical jolt (microseconds to seconds), followed by hemorrhage (minutes to hours), edema (hours to days), and gliosis (days to months). In all likelihood, each of these events is associated with identifiable molecular changes that have distinct effects on the SVZ. Thus, it is important to determine the time course of molecular changes in the SVZ and correlate it with phases of neural reaction to injury. As described below, some very specific ‘‘schedules’’ of SVZ cellular responses to injury in rodents have been discovered, suggesting that they are tightly regulated at the molecular level. These time courses should provide clues as to when to look for the molecular signals regulating SVZ responses to injury. In addition to molecular and cellular responses, most head injuries bring about physical forces that may impact the SVZ. One of the main functions of the lateral ventricles (LV) is to provide an internal cushion for changes in intracranial pressure. During the initial trauma, which often includes a rapid acceleration and deceleration of the cranium, intracranial pressure increases transiently and causes compression of the lateral ventricles, forcing CSF into more caudal regions. Subsequently, edema causes more long duration increases in intracranial pressure, sometimes resulting in obstruction of CSF circulation (obstructive hydrocephalus). Later, death of brain tissue can result in decreased pressure and increased LV volume (hydrocephalus ex

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vacuo) (Volpe, 1995; Reider et al., 2002). Mechanical stress causes multiple molecular effects on a variety of other cellular systems (Ingber, 1997; Grodzinsky et al., 2000; Ingber, 2002; Keller et al., 2003), so it is probable that compression or distension of the LV also influences the SVZ. The molecular consequences of ventricular volume changes on SVZ and ependymal cells are unknown. Recently, mechanical forces were shown to regulate developmental genes by causing b-catenin movement to the nucleus in Drosophila (Farge, 2003). Since b-catenin inhibits cell cycle exit of neuronal precursor cells, thereby increasing cerebral cortex size (Chenn and Walsh, 2002), it would be interesting to examine its regulation by mechanical forces in the developing and adult SVZ. A few studies have shown that hydrocephalus in humans is characterized by ependymal tears and gliosis (Bruni et al., 1985; Sarnat, 1995). Ependymal cells, which line the LV, are interconnected by specialized junctions that provide mechanical force (Mitro and Palkovits, 1981). Likewise, SVZ cells are interconnected by specialized junctions (Lois et al., 1996), and are embedded in an ultrathin extracellular matrix and basal lamina that are molecularly different from the surrounding neuropil (Miragall et al., 1990; Jankovski and Sotelo, 1996; Mercier et al., 2002). Thus, the mechanical integrity of the SVZ probably differs from adjacent tissue. It remains unexplored if changes in levels of SVZ adhesion molecules, extracellular matrix, or basal lamina following brain injury (see below) decrease the stiffness of the walls of the LV and contribute to the ventricular enlargement characteristically observed after head injury (Reider et al., 2002; Chen et al., 2003b). As ventricular enlargement after brain injury is well simulated in rat (Iwamoto et al., 1997; Bramlett and Dietrich, 2002), the answers to some of these questions are tractable and deserve further study. Brain injuries are exceedingly difficult to model because they are highly variable and also because they elicit a wide variety of cellular and molecular responses. Variability in the types of lesions and species used in studies of brain injury leads to problems in interpreting results (Table 7.1). For example, lesions which directly affect the SVZ or the rostral migratory stream (RMS) may initiate a different cascade of effects than those that only injure surrounding areas. Whereas, percussion and aspiration lesions do not directly impinge upon the SVZ (Szele and Chesselet, 1996; Holmin et al., 1997; Chirumamilla et al., 2002; Goings et al., 2002, 2004; Chen et al., 2003b), they probably do cause sudden pressure changes that may affect the SVZ, and complicate interpretation. Differences in the way lesions of the same brain regions are made can cause variable attempts at repair. We along with others examined whether deafferentation of the striatum by cortical lesions induced sprouting of remaining axon terminals (Szele et al., 1995; Napieralski et al., 1996; Kartje et al., 1999), a form of plasticity which can restore function in other brain areas (Deller and Frotscher, 1997; Caroni, 1998). We found that aspiration lesions (Fig. 7.1), but not focal ischemic lesions of the somatosensory cortex induced sprouting in the striatum (Szele et al., 1995; Napier-

Table 7.1 Responses of the SVZ to mechanical brain injuries Proliferation

Migration

Differentiation

Ref.

rat

Increased number of cells, mitosis ND

ND

ND

Willis, 1976

Knife cut through cerebral cortex, corpus callosum, and fimbria fornix

rat

Number of cells ND, increased LI

ND

Newborn cells in SVZ did not express markers of neurons, or glia

Weinstein, 1996

Knife cut through RMS and knife cut through cerebral cortex (separate rats)

rat

Number of cells ND, increased number of BrdUþ cells

Rostral migration continued; emigration into cerebral cortex and striatum

PSA-NCAMþ cells in wound and striatum expressed GABA and TH

Alonso, 1999

Stab wound in cerebral cortex

rat

Number of cells ND, nonsignificant transient increase in number of BrdUþ cells

ND

ND

Tzeng, 1999

Weight percussion injury of cerebral cortex

rat

ND

Emigration of nestinþ/ GFAPþ cells towards lesion in cortex

SVZ cells co-expressed nestin and GFAP

Holmin, 1997

Fluid percussion injury

rat

Number of cells ND, increased number of BrdUþ cells

ND

Newborn cells in SVZ did not express markers of neurons, or glia

Chirumamilla, 2002

Fluid percussion injury

rat

Number of cells ND, increased number of Ki67þ and PCNAþ cells at long survival times

ND

Increased numbers of neurofilamentþ and GFAPþ cells one year after injury

Chen, 2003

Aspiration lesion of somatosensory cortex

rat

Increased total number of cells, but no change in number of BrdUþ cells

No evidence of emigration to adjacent areas despite appearance of radial glia like fibers

Increased numbers of PSA-NCAMþ cells but no expression of mature markers in SVZ

Szele, 1996

213

Species

Knife cut through cerebral cortex, corpus callosum, and striatum

7. The Subventricular Zone Responds Dynamically to Mechanical

Type of Injury

(Continued )

214

Table 7.1. (Continues) Species

Proliferation

Migration

Differentiation

Ref.

Aspiration lesion of somatosensory cortex

mouse

Number of cells not changed, decreased number of BrdUþ cells

ND

ND

Goings, 2002

Aspiration lesion of somatosensory cortex

mouse

Number of cells ND, decreased number of retrovirally-labeled cells

Emigration into corpus callosum, and injured cortex

Differentiation into oligodendrocytes in corpus callosum, and astrocytes in lesioned cortex

Goings, 2003

Aspiration lesion of frontal cortex and olfactory peduncle

mouse

Increased total number of cells and size of RMS, decreased number of BrdUþ cells

Rostral migration continued; emigration into anterior olfactory nucleus, frontal cortex

Increased number of calretininþ cells indicating increased neuronal differentiation

Jankovski, 1998

Olfactory bulbectomy

mouse

Number of cells ND, but increased size of RMS, decreased LI

Rostral migration continued, no emigration noted

ND

Kirschenbaum, 1999

BrdU ¼ bromodeoxyuridine; GFAP ¼ glial fibrillary acidic protein; LI ¼ labeling index (the percentage of cells going through the cell cycle); ND ¼ not determined; PCNA ¼ proliferating cell nuclear antigen; PSA-NCAM ¼ polysialylated neural cell adhesion molecule; RMS ¼ rostral migratory stream; SVZ ¼ subventricular zone; TH ¼ tyrosine hydroxylase.

Maria L.V. Dizon and Francis G. Szele

Type of Injury

7. The Subventricular Zone Responds Dynamically to Mechanical

215

Figure 7.1. Aspiration lesion of the senosorimotor cortex. Note that the lesion does not include the corpus callosum (arrowhead) and that it is close to the dorsolateral SVZ (arrow). Lesions in the same location were made in mouse and rat (Szele and Chesselet, 1996; Goings et al., 2002, 2004).

alski et al., 1996; Kartje et al., 1999). This discrepancy underscores the need to examine SVZ responses to multiple defined models of injury within a given species, as well as across different species (Szele and Chesselet, 1996; Goings et al., 2002, 2004).

Changes in SVZ Proliferation After Brain Injury Differs Among Species Changes in cell proliferation have been examined more than any other response of the SVZ to brain injury (Table 7.1). Investigators most commonly use pulse studies to examine the rates of BrdU incorporation at various time points after injury. While very useful for determining the

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number of cells going through S-phase at a given time, this method reveals only part of the story. For example, it is important to correlate the number of cells going through S-phase with the total number of cells in the SVZ. This allows determination of the percentage of cycling cells; the ‘‘labeling index’’. The absolute number of cells in the SVZ is ultimately regulated by an interplay of the labeling index, cell cycle length, migration away from the SVZ, and cell death. Thus, increased BrdU incorporation in the SVZ does not necessarily mean that the population as a whole has increased in number. If increased rates of mitosis are accompanied by a corresponding increased emigration from the SVZ or by increased cell death, then the total number of cells in the SVZ may remain constant, or even decrease. Relatively few studies have attempted to determine how these different SVZ processes change in relation to each other, making comparison of the different experiments difficult. Nonetheless, it seems that SVZ proliferation, and possibly total cell numbers, increase in response to a variety of injuries in the rat. In contrast, the few studies conducted in mouse suggest that proliferation decreases in the murine SVZ after injury (Table 7.1). It is impossible to know at this point which rodent may better model the human. But if the rat versus mouse difference is born out, it will strengthen the argument for histopathological investigations of human SVZ after brain injury, in order to help choose the best preclinical model. Because of the high frequency of injuries to the cerebral cortex and its functional importance in humans, many labs have studied cortical injuries. Two of the best-studied models are unilateral aspiration lesion of the somatosensory cortex (Fig. 7.1) and fluid or weight percussion injury of the cerebral cortex. A series of studies examined the success of grafting fetal tissue into cortical aspiration lesions in rats (Castro et al., 1987, 1988; Girman and Golovina, 1988; Kolb et al., 1988; Sorensen et al., 1990). Grafting into the lesion cavity has often resulted in survival and development of new connections by implanted tissue, suggesting that if SVZ cells can migrate to the lesion, then perhaps they would also survive and differentiate. Aspiration lesions of somatosensory cortex have been shown to affect motor behavior (Shipley et al., 1980; Glassman, 1994; Zepeda et al., 1999), amygdaloid kindling (Corcoran et al., 1975), object discrimination (Deacon and Rawlins, 1996), and fear potentiated startle (Rosen et al., 1992). This extensive body of knowledge on the behavioral effects of aspiration cortical lesions provides an ideal framework within which to interpret the SVZ’s response to such lesions. The time course of the proliferative response of the SVZ to unilateral aspiration cortical lesions has been studied in rat and mouse (Szele, 1994; Szele and Chesselet, 1996; Goings et al., 2002). The two species exhibit markedly different results to identical lesions (Fig. 7.2). In rats, the total number of cells in the SVZ increases after one week of survival (Fig. 7.2A, B). In order to determine the topographic heterogeneity of the SVZ’s response, three subregions of the SVZ were quantified: the dorsolateral ‘‘DL’’

Rats

Total cell numbers

*

500

Nissl

*

400

cc

300

sl

BrdU

lv

str 200

str

100

control

0 0 Hr

A

Day 1

Day 7

Day 12

Day 16

Day 7

B

Days after lesion

control

Day 14

Days after lesion

Mice

C

BrdU 120

1000

Number of BrdU-positive cells/section in SVZ

Number of cells/section in SVZ

Total cell numbers 900 800 700 600 500 400 300 200

100

*

80

**

*

**

Day 1

Day 2

60

*

*

40 20

100 0

0 Controls

6 hours

Day 1

Day 2

Day 3

Day 6

Day 15

Days after lesion

Day 25

Control

Day 35

D

6 hours

Day 3

Day 6

Day 15

Day 25

Day 35

Days after lesion

217

Figure 7.2. The SVZ of rats (A, B) and mice (C, D) respond differently to the same type of cortical lesion. A: number of cells in the dorsolateral SVZ increased after aspiration cortical lesions in rats. B: left panels show the increase in total cell numbers at 7 days after cortical injury, right panels the increase in survival of BrdU labelled cells. BrdU was given 10 days afer lesion and rats survived for 4 days. Scale bars ¼ 100 mm. In contrast to rat, total numbers of cells did not change significantly in mouse SVZ after the same type of lesion (C). D, the number of cells going through S-phase actually decreased biphasically in mouse SVZ after cortical injury. A-B: Szele PhD thesis, 1994; Szele and Chesselet, 1996; C-D: Goings et al., 2002.

7. The Subventricular Zone Responds Dynamically to Mechanical

Number of cells/section in SVZ

600

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Maria L.V. Dizon and Francis G. Szele

SVZ (SVZ cells extending from the dorsolateral angle of the lateral ventricles), the striatal SVZ, and the septal SVZ. In control rats, the DL SVZ has the largest number of proliferating cells. In lesioned rats, the DL SVZ is physically proximate to potential molecular changes associated with the lesion (Fig. 7.1). Yet, all three subregions of the SVZ exhibit similar and statistically significant increases in the total number of cells between one and two weeks after injury (Szele and Chesselet, 1996). Cell numbers in SVZ contralateral to the lesion also increase. This contralateral effect emphasizes the importance of using separate animals as non-lesion controls instead of relying on the contralateral SVZ. The long-distance responses of the contralateral SVZ leads to the hypothesis that a mitogen for SVZ cells diffuses through the CSF and causes increased proliferation. However, the number of cells going through S-phase, labelled with pulse injections of BrdU, does not change significantly at various time points after injury (Szele and Chesselet, 1996). Although the number of cells proliferating at any given time after cortical lesion does not change, multiple BrdU injections followed by sacrifice after several days suggests that SVZ cells survive longer after cortical injury (Fig. 7.2B), (Szele, 1994), possibly accounting for the larger cell numbers. Recently, using stereological techniques, the Chesselet lab demonstrated that the total SVZ cell number and numbers of BrdU-incorporating SVZ cells increase after focal ischemic lesions of the cortex in rats (Gotts and Chesselet, 2002), suggesting that the increased cell numbers are at least partly due to increased proliferation. The number of cells going through mitosis in the SVZ may be differentially regulated in the context of thermocoagulatory versus aspiration lesions, or alternatively the differences may reflect variations in counting methodologies (Szele and Chesselet, 1996; Gotts and Chesselet, 2002). Subsequent studies have examined the effects of the same unilateral aspiration cortical lesions in mice (Goings et al., 2002, 2004). In contrast to rats, the total number of cells in the murine striatal and DL SVZ does not change significantly (Fig. 7.2C) (Goings et al., 2002). The relatively constant cell numbers may have been caused by increased migration coupled with increased proliferation. Therefore, the rate of BrdU incorporation was determined with pulse studies. In fact, the number of cells going through S-phase in mice significantly decreases in a biphasic manner after cortical aspiration lesions (Fig. 7.2D). The decreases are rapid (six hours) and are seen as late as 35 days (Goings et al., 2002). Theoretically, decreases in the number of cells going through S-phase should be associated with concommitant decreases in cell number. However, the decreased proliferation is probably offset by decreases in rostral migration (see below) resulting in unchanged total cell number (Goings et al., 2004). The decreased number of SVZ cells incorporating BrdU has been corroborated by retroviral studies demonstrating that, four days after aspiration lesions of the somatosensory cortex, half as many SVZ cells were labelled, suggesting decreased numbers of cells going through mitosis (Goings et al., 2004). Thus unilateral aspiration cortical lesions cause:

7. The Subventricular Zone Responds Dynamically to Mechanical

219

(1) increases in total SVZ cell number in rats but no change in total SVZ cell number in mice; and (2) increases in the number of BrdUþ cells in rats, but decreases in the number of BrdUþ cells in mice. As we shall see, probably not all, but at least a few other types of lesions differentially affect proliferation and cell numbers in the SVZ of these two species. The following experiments studied whether lesions that involve the RMS affect proliferation and cell numbers in the SVZ. In a study using rats, Alonso et al. (1999) made knife cuts through the RMS and found increased numbers of BrdUþ cells in the SVZ caudal to the lesion at 14 and 52 days after surgery, suggesting that rates of proliferation had increased. Because BrdU was administered seven days before sacrifice, the increased numbers of BrdUþ cells may also reflect increased survival (Alonso et al., 1999). In contrast to the study in rats, two other studies suggest decreased proliferation after interruption of the RMS in mice (Jankovski et al., 1998; Kirschenbaum et al., 1999). Jankovski et al. (1998) made aspiration lesions of the frontal cortex and olfactory peduncle in mice, effectively interrupting the stream of cells migrating to the OB. The number of cells caudal to the lesion increased as did the size of the RMS. However, the number of BrdUþ cells in the SVZ/RMS caudal to the lesion significantly decreased from 2 days to 7 weeks compared to non-lesioned controls (Jankovski et al., 1998). Although the lesions were unilateral, the response was bilateral, similar to the changes in rat SVZ cell numbers after aspiration lesions of the somatosensory cortex (Szele and Chesselet, 1996). Also, the rapidity and duration of decreases in BrdU incorporation is reminiscent of that observed after aspiration lesions in the mouse (Goings et al., 2002). In other experiments that interrupted the RMS, Kirschenbaum and colleagues performed unilateral olfactory bulbectomies in mice also finding that the size of the RMS increased (Fig. 7.3) (Kirschenbaum et al., 1999). The density of cells in the RMS was not changed after bulbectomy, suggesting that there were increases in total cell numbers (Kirschenbaum et al., 1999). Interestingly, the largest changes observed with lateral views of wholemounts were in the DL SVZ (arrows Fig. 7.3C), whereas the striatal SVZ seemed to be less affected. Unlike most migratory cells which are postmitotic during neural development, SVZ cells continue to divide as they migrate in the RMS to the OB (Lois and AlvarezBuylla, 1994). Therefore, another possible explanation for the increased number of BrdUþ cells or total number of cells seen after interruption of the RMS is that rostral migration decreases, causing mitotic cells to accumulate (see below). Kirschenbaum and his co-workers measured the percentage of cells going through S-phase and found that it was similar to controls at three days and three weeks, but was decreased at three months after bulbectomy. This result differs from the studies of Jankovski in which decreased BrdU incorporation was observed as early as two days after lesion. Another difference was that Jankovski found bilateral effects, whereas Kirscenbaum did not. The former made lesions that included the frontal cortex, while the latter’s surgeries were restricted to the olfactory

40 OBX

30 25

15

N=6 N=3

10

N=1

N=3

N=5 N=5

N=6

5 0

A

N=1

20

B

CONT OBX 3 Days

CONT OBX 3 Weeks

CONT OBX 3 Months

CONT SHAM 3 Months

Percentage of BrdU-labelled Cells (RMS)

Control

OBX

C

9 8 7 6 5

N=4 N=3

N=4

N=4

N=3

4 3

N=4

2 1 0 CONT OBX 3 Days

CONT OBX 3 Weeks

CONT OBX 3 Months

D

Figure 7.3. Olfactory bulbectomy induced changes in the SVZ/RMS. (A) Unilateral olfactory bulb removal (OBX) caused increased numbers of cells to accumulate in the RMS. (B) The volume of the RMS ipsilateral to the lesion (dark bars) increased with time, and since the cell density did not significantly change, it suggests that the number of cells increased in the SVZ/RMS. (C) Sagittal views of PSANCAM immunohistochemistry show that the majority of the accumulation of cells in the dorsolateral SVZ after OBX (arrows). (D) the labeling index was decreased at long survival times after surgery. From Kirschenbaum et al., 1999.

Maria L.V. Dizon and Francis G. Szele

CONTROL

220

Volume RMS ( mm33103)

35

7. The Subventricular Zone Responds Dynamically to Mechanical

221

bulb, suggesting that perhaps surgical and not species differences gave the different effects (Fig. 7.3A). It would be informative to perform Jankovski’s and Kirschenbaum’s methods in rats and Alonso’s methods in mice to test the hypothesis that the former would increase, and the later decrease, proliferation. Thus, similar to cortical lesions, lesions that interrupted the RMS generally cause the rate of proliferation to increase in rats but decrease in mice. Another model of traumatic brain injury that has been used extensively is the fluid percussion model in which a controlled impact is made on the surface of the cerebral cortex (McIntosh et al., 1987; Schallert et al., 2000; Chen et al., 2003a, 2003b; Prins and Hovda, 2003). One advantage of this model is that the surface area, location, intensity of impact, and extent of tissue loss can be tightly controlled. It is also thought to be one of the best animal models for human non-penetrating brain injuries. The level of BrdU-incorporation in SVZ cells increased two and four days after fluid percussion injury in rat (Chirumamilla et al., 2002). However, in this study, it was not determined whether the increases in the number of cells going through S-phase were associated with increased total number of cells in the SVZ. Another group examined the long term effects of such injuries on the expression of cell cycle markers in the SVZ (Chen et al., 2003b). They allowed rats to survive for up to one year and found that, while control rats had significant age-related declines in the numbers of PCNA-positive and Ki67-positive cells in the SVZ, lesioned animals did not (Chen et al., 2003b). The majority of fluid percussion injury studies have been performed in rat, but this model can also be used in mouse (Carbonell et al., 1998), and we have found that numbers of BrdU labelled cells increase in mouse after a percussion injury of the cerebral cortex (Ramaswamy et al., 2005). Two studies have examined the reaction of the SVZ to penetrating brain injuries that do not impact, or only minimally impact, the SVZ. They are interesting to consider because they are similar to human brain surgery and the effects of human brain surgery on the SVZ are completely unknown. Stab wounds of the cerebral cortex (needle sticks) in rat do not change the number of BrdU-incorporating cells in the SVZ compared to the contralateral side between twentyfour and seventytwo hours (Tzeng and Wu, 1999). This suggests that, compared to larger lesions, relatively discrete injuries do not affect proliferation in the SVZ. In another experiment, the effects of a one mm long sagittal cut through the cerebral cortex and the fimbria were examined on the rat SVZ (Weinstein et al., 1996). This injury does not directly cut through the SVZ (except for the thin layer of subcallosal SVZ cells); it interrupts the septo-hippocampal cholinergic axons and causes death of hippocampal cells and of cholinergic cells in the medial septum (Hefti, 1986). Proliferation in the SVZ was determined with tritiated thymidine. Ten days after fimbrial lesions, a two-fold increase in the percentage of SVZ cells going through S-phase was detected (Weinstein et al., 1996). As

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Maria L.V. Dizon and Francis G. Szele

with the fluid percussion model, the total number of cells was not determined in either of these studies, therefore, it remains uncertain whether the increases in labeling index observed by Weinstein et al. (1996) is associated with an expansion of the SVZ. In rats, aspiration cortical lesions, fluid percussion injuries of the cerebral cortex, and knife cuts all cause increases in total SVZ cell numbers and/or in the number of cells going through S-phase. Conversely, in mice, aspiration lesions of the cerebral cortex as well as lesions that remove the olfactory bulbs cause decreases in total and/or BrdUþ SVZ cell numbers. These results strongly suggest that the two species differ markedly in their response to some brain injuries. It will be interesting to determine whether the proliferative response of human SVZ to brain injury is better modeled in the rat or the mouse. Attempts to tease apart molecular differences in the SVZ between the two species may shed light on mechanisms that control SVZ proliferation. Also, xenotransplantation experiments may reveal whether these potential molecular differences are intrinsic or extrinsic to the SVZ cells.

Cells can Migrate from the SVZ/RMS to Various Brain Regions After Injury In thinking about the biology of the SVZ after brain injury, and of the potential use of SVZ cells for replacement therapy, the question of whether SVZ cells migrate to the area of injury is of paramount importance. Whereas cells born in the ventricular zone and SVZ of the developing forebrain migrate to many nuclei along a variety of pathways, neuroblasts in the intact adult SVZ migrate primarily to the OB in the RMS. A number of studies indicate that brain injury can induce, or allow, SVZ cells to migrate to nonOB areas, especially towards and into injured areas (Table 7.1). It is essential to use methodologies which allow the distinction of SVZ cells from local astrocytes, microglia and macrophages, cells which also migrate towards and into lesions. Since systemic BrdU labels all mitotic cells in the brain, and lesions increase the proliferation of glial cells, it is often difficult to interpret whether BrdUþ cells near the injury are intrinsic glial cells or cells which have migrated from the SVZ. Similarly, systemic BrdU injections label dividing cells in the bone marrow which can enter the brain through the disrupted blood brain barrier and be mistaken for in situ neural cells. Injections of BrdU, viral vectors, DiI, fluorescent microspheres, or tagged cells directly into the lateral ventricle or the SVZ can help circumvent this problem, though each of these techniques has its own inherent problems, not the least of which is that the injection, by definition, is a small lesion. Once it is definitively shown that cells have migrated from the SVZ, it becomes important to discriminate whether neuroblasts that normally migrate to

7. The Subventricular Zone Responds Dynamically to Mechanical

223

the OB redirect their pathways out of the SVZ/RMS, or whether the RMS is unaffected and the emigrating cells comprise a separate population. The studies described below address some of these issues and set the stage for more detailed experiments designed to ascertain the molecular regulation of SVZ cell migration. During brain development, migration occurs along radial glia (radial migration) and across radial glia (tangential migration). In the adult SVZ, neuroblasts move through a meshwork of astrocyte-like cells (Jankovski and Sotelo, 1996; Lois et al., 1996), but are not dependant on them for migration, at least not in vitro (Wichterle et al., 1997; Jacques et al., 1998). It is unclear whether the loss of radial glia after development accounts for the lack of SVZ cell migration into adjacent nuclei such as the striatum. Several converging experiments suggest that it may be possible for the radial glial phenotype to be expressed by astrocytes in the adult and for these cells to serve as substrates for neuroblast migration. The reversibility of the adult astrocyte morphology was first shown by exposing adult astrocytes to embryonic cell-conditioned media, whereupon these cells re-expressed a radial glial phenotype in vitro (Hunter and Hatten, 1995). Adult astrocytes in vivo have also been shown to revert to a radial glia-like phenotype in a model of cortical apoptosis that induces neurogenesis. Videomicroscopy was used to show that the newborn neurons migrate along the radial glia-like processes (Leavitt et al., 1999). Radial glia-like cells with processes perpendicular to the LV were observed concomitantly with decreases in the density of normal astrocytes in the medial striatum of rats that had received aspiration cortical lesions (Fig. 7.4), (Szele and Chesselet, 1996). These observations suggested that the processes may provide a substrate for migration from the SVZ into the striatum after aspiration cortical lesions. This hypothesis was strengthened by the fact that PSA-NCAM, which is permissive for SVZ migration, increases in the medial striatum after cortical injury (Poltorak et al., 1993; Ono et al., 1994; Szele, 1994; Szele et al., 1994; Szele and Chesselet, 1996). In contrast to PSA-NCAM, tenascin, which inhibits migration of a variety of cells (Joester and Faissner, 2001; ChiquetEhrismann and Chiquet, 2003), decreases after cortical lesion (Fig. 7.5 C,D). When we examined migration of SVZ cells into the striatum after cortical injury by injecting BrdU and allowing rats to survive for four to sixteen days after the injections, no evidence of SVZ migration into the striatum was found (Szele and Chesselet, 1996). The formal possibility exists that only postmitotic cells can emigrate from the SVZ after aspiration cortical lesions, but it is unlikely since others have found that many emigrated SVZ cells were BrdUþ (Alonso et al., 1999). Alternatively, it may be that SVZ cells were only induced to migrate out of the SVZ before the time points examined. In contrast to rat, in mice, aspiration cortical lesions did not result in the appearance of GFAPþ radial glia-like fibers in the medial striatum (SundholmPeters et al., 2004). Nevertheless, cortical lesions in mice are associated with changes in migration patterns from the SVZ (Goings et al., 2004). We

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Maria L.V. Dizon and Francis G. Szele

Figure 7.4. Twelve days after cortical lesions in rat, radial glia like fibers appeared to emanate from the SVZ into the medial striatum. From Szele and Chesselet, 1996).

injected a library of retroviral vectors (Golden et al., 1995) into the lateral ventricles and examined migration to the OB and the area of injury. Four days after injury, fewer cells migrate to the OB, and a greater number of cells migrate toward the injury (Goings et al., 2003). In particular, SVZ cells emigrate to the corpus callosum and the cerebral cortex in the area of the injury. Thus, even lesions that do not directly damage the SVZ can affect migration within the SVZ/RMS. Our results are also consistent with a scenario in which migration from the SVZ to the OB is redirected towards the area of injury, as opposed to a new population of migrating cells being generated. As described in the previous section, experiments that interrupt the RMS result in SVZ cells accumulating caudal to the lesion (despite decreased proliferation in mice) (Jankovski et al., 1998; Alonso et al., 1999; Kirschenbaum et al., 1999). Since removal of the OB does not abolish the rostral movement of cells in the SVZ, the OB may not be necessary for migration in the RMS (Jankovski et al., 1998; Kirschenbaum et al., 1999), although other studies suggest that the rostral OB does express chemoattractants (Liu and Rao, 2003). In one of the lesion studies, endogenous SVZ cells, as well as homotopically and homochronically engrafted b-gal expressing cells, migrated into rostral forebrain nuclei. The cells did not migrate abnormally in all directions but instead were primarily found dorsal to the RMS in the frontal cortex and the anterior olfactory nucleus. No abnormally emigrated

7. The Subventricular Zone Responds Dynamically to Mechanical

225

Figure 7.5. Adhesion molecule expression in the SVZ and striatum after lesions of the cerebral cortex. The strong tenascin immunoreactivity seen in control SVZ (A)

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cells appeared in the striatum and only a few were found in the nucleus accumbens (Jankovski et al., 1998). This study showed that in the context of injury in the adult, SVZ cells are capable of migrating into adjacent nuclei other than the OB. Interestingly, emigration only occurred into specific nuclei suggesting that it may may be informative to compare the molecular composition of these nuclei with regions into which migration does not occur. In another study, knife cuts through the RMS of rat were made and cells migrated out of the RMS into the lesioned area of the cortex and striatum as well as caudal to the lesion. The cells frequently moved to the edge of the lesion and spread from the SVZ dorsally to the pial surface of the cerebral cortex and ventrally through the striatum to the anterior commissure (Fig. 7.6) (Alonso et al., 1999). The migrating cells were PSA-NCAM positive and stayed in their normal chain-like configuration as they migrated from the SVZ, suggesting that they were the type A neuroblasts described by Alvarez-Buylla and colleagues (Lois et al., 1996; Doetsch et al., 1997). Although PSA-NCAM can also be expressed by reactive astrocytes after lesion (Le Gal La Salle et al., 1992), the majority of the PSA-NCAM cells that had migrated were positive for neuronal markers and possessed neuronal ultrastructural morphology (Alonso et al., 1999). This fact signified that the increased PSA-NCAM immunoreactivity (Fig. 7.4) was not merely due to astrocytosis. This study demonstrates that lesions involving the SVZ/ RMS, similar to those of Jankovski, can induce adult SVZ cells to migrate out of the RMS into adjacent structures and differentiate into neurons. It is important to note that in the studies mentioned thus far, where SVZ emigration was observed, the cells moved toward the injured area (Jankovski et al., 1998; Alonso et al., 1999; Goings et al., 2003). Similarly, in a model of weight percussion injury of the cerebral cortex in rat, cells seemed to migrate towards the injured cortex through the corpus callosum (Holmin et al., 1997). In contrast, after a mild form of needle stab injury to the cerebral cortex, BrdUþ SVZ cells migrated into the dorsolateral septum (Tzeng and Wu, 1999), thus directing migration towards the injury may not always occur. The results indicate that emigration from the SVZ can be caused by a variety of injuries in both rats and mice. Two major question that remain are what molecules induce migration out of the SVZ in the context of injuries, and conversely, what molecules inhibit migration into adjacent brain areas was decreased 7 days after lesion (B). Tenascin was also decreased in the medial striatum 12 days after lesion (D) compared to controls (C). Quantification showed statistically significant decreases in the SVZ (E) but not in the striatum (F). CSPG immunoreactivity in the SVZ was not appreciably different from controls (G) 5 days after cortical lesion (H); although here it appears higher in the medial striatum, this effect was inconsistent and therefore not quantified. Scale bars A, B ¼ 50; C, D ¼ 150; G, H ¼ 20 microns. From Szele and Chesselet, 1996 and Szele PhD thesis, 1994.

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Figure 7.6. PSA-NCAMþ SVZ neuroblasts migrate into the lesioned area and the striatum after surgical knife cuts (location shown by vertical arrow in schematic). Boxed areas in schematic show locations of A and B. (A) Arrowheads point to PSA-NCAM positive cells that appear to have migrated from the SVZ into the lesion, which passes through the corpus callosum (cc) and the striatum (15 days post lesion). (B) Arrowheads indicate the rostral limit of the surgical cut. Note that numerous PSA-NCAM positive cells have migrated caudally into the striatum. From Alonso et al., 1999.

in normal animals. It is not understood why cells stop migrating in the coronal plane (into the striatum, corpus callosum, and cerebral cortex) after the second postnatal week of life. In the adult, structures adjacent to the SVZ may establish a cellular barrier or they may lose molecules that stimulate coronal migration during earlier phases of development. Alternatively, the absence of radial glia in the adult may be responsible for the limited capacity for migration out of the RMS. Since there are several models of injury which induce migration to non-OB areas in the adult, we can begin to test these different possibilities. Comparison of molecular expression patterns in development and in an adult model of injury which induces migration would be expected to identify candidate genes which regulate migration out of the SVZ. The roles of these genes could then be tested with gain or loss of function approaches.

Survival and Differentiation of SVZ Cells is Altered by Brain Injury Neurons born during embryogenesis can survive for the entirety of one’s life (if the sommelier’s selections are consumed with temperance). In contrast, the majority of neurons born in the SVZ are thought to only live for a few weeks in the OB (Kaplan et al., 1985). How long do neurons that migrate out

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of the SVZ to injured areas survive? If they do not survive for long, will the SVZ continue to replenish them? If they do live for appreciable lengths of time, will they differentiate appropriately? Another set of related questions concerns survival and differentiation of cells that remain within the SVZ and the OB; how are they affected by brain injury? It has been known for some time that head injury in humans can be associated with olfactory dysfunctions, in particular olfactory discrimination (Eslinger et al., 1982; Yousem et al., 1996; Green et al., 2003). These human data are very intriguing because animal studies hint that olfactory discrimination is one of the main functions of the newborn interneurons of the OB (Gheusi et al., 2000). Also, survival and differentiation of SVZ cells that remain in their normal locations can be altered in animal models of injury (Table 7.1). In at least some head injuries, olfactory receptor neuron axons are sheared as they pass through the cribriform plate (Kern et al., 2000). It will be important to discriminate this possibility from lesions that affect some combination of proliferation, migration, survival, or differentiation of SVZ/OB neurons. Some clues are beginning to emerge that SVZ cells may survive for weeks and may actually acquire mature neural cell phenotypes in non-OB areas after brain injury. In the studies of aspiration lesions of the somatosensory cortex, survival of SVZ cells was examined. Three injections of BrdU were given ten days after cortical lesion in rats and survival of BrdUþ cells examined after the last injection. A larger number of cells survived in the SVZ 14 days after injury (Fig. 7.2B) (Szele, 1994). Quantification at 22 and 26 days after lesion showed similar increases in both the dorsolateral and striatal SVZ, and the increases were statistically significant at the latter time point (Szele, 1994). These data strongly suggest that SVZ cells survive for longer periods of time in the rat SVZ after aspiration cortical lesions. In the mouse, retroviral vector studies showed increased numbers of labelled cells around the lesion and in the OB 21 days after cortical lesions (Goings et al., 2003). Although proliferation and total cell numbers are regulated in opposite directions in rats and mice following cortical lesions, both species seem to respond with increased survival in the SVZ. Analysis of differentiation in the brain can be quite difficult and deserves comment. The majority of investigators, including ourselves, often attempt identification of phenotype with cell subtype specific markers. As exemplified below, this can be fraught with problems of specificity, since most individual molecules are expressed by more than one cell subtype and because after injury, specificity further decreases. In particular, reactive astrocytes can express a panoply of ‘‘neuron-specific’’ proteins. Most neural cells go through a complex series of morphological transitions making identification of immature cell subtypes based on morphology difficult. Mature neural cells have distinctive shapes and sizes which often allow incontrovertible phenotypic identification. Molecular markers usually only correspond to the translation of a single gene, but the elaboration and

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maintenance of mature morphology probably requires the coordinated regulation of dozens of genes. Reliable identification of differentiated neural cells ultimately depends on a combination of markers, morphology, and function; the latter has seldom been attempted in vivo in the context of injury induced changes of the SVZ. We will first consider the differentiation of cells that remain in the SVZ. Although other cell subtypes of the SVZ lack specific markers, migrating neuroblasts are the only cells that express class III b-tubulin and PSANCAM (Rousselot et al., 1995; Doetsch et al., 1997). After aspiration lesions of the somatosensory cortex, there is a dramatic increase in the number of PSA-NCAM positive cells in the SVZ (Szele, 1994; Szele and Chesselet, 1996). The number of PSA-NCAMþ cells is increased in all three subdivisions of the SVZ, suggesting that neurogenesis increases in the SVZ even at relatively far distances from the injury. In fact, the increases in the septal SVZ (24-fold) were greater than the increases in the dorsolateral SVZ (threefold) and the striatal SVZ (two-fold). As mentioned before, PSA-NCAM has been detected on reactive astrocytes (Le Gal La Salle et al., 1992) and on oligodendrocyte progenitor cells in vitro (Grinspan and Franceschini, 1995). This caused us to consider whether it could also be expressed on SVZ cells other than neuroblasts after brain injury. Two markers of the astrocyte-like type B cells (vimentin and GFAP) were not changed, suggesting that there was no astrogliosis in the SVZ after cortical injury (Szele and Chesselet, 1996). This was in contrast to the strong glial reaction in the area of the injury and in the denervated dorsolateral striatum (Szele et al., 1995). The expression of carbonic anhydrase, which labels oligodendrocytes (Cammer and Zhang, 1992), was also examined but was absent in the SVZ before and after lesion (Szele, 1994; Szele and Chesselet, 1996). Since some oligodendrocytes are born in the SVZ quite late in development (Levison et al., 1993; Levison and Goldman, 1993), and since the SVZ makes them after demyelination in the adult (Nait-Oumesmar et al., 1999; Picard-Riera et al., 2002), it may be informative to reexamine the rat SVZ after aspiration cortical lesions with markers of immature oligodendrocytes. The data suggested that cortical lesions increase survival of cells in the SVZ, so it was logical to ask whether the neuroblasts would have sufficient time to mature into neurons. However, there was no expression of markers of mature neurons (synaptophysin, neuron specific enolase, and MAP-2), in the SVZ before or after injury, suggesting that neuroblasts were not differentiating into mature neurons (Szele, 1994; Szele and Chesselet, 1996). The most parsimonius explanation is that increased PSA-NCAM immunoreactivity after cortical lesion in rats reflects an increase in the number of SVZ neuroblasts, that these neuroblasts do not differentiate prematurely in the SVZ, and that the proportion of other cell types remains the same (Szele and Chesselet, 1996). In studies of mouse examining differentiation of SVZ cells after cortical aspiration lesions, we have not observed expression of markers of mature neural phenotypes (Szele lab, unpublished observations). Others have also

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examined markers of astrocytes, microglia, and neurons in the SVZ after different types of injury; newly born 3 H-thymidineþ SVZ cells do not express any of the glial or neuronal markers examined (Weinstein et al., 1996; Chirumamilla et al., 2002). The sum of the data show that as long as cells stay in the SVZ, they do not acquire a mature phenotype, suggesting that there may be extrinsic factors specific to the SVZ that inhibit differentiation and that these factors are not changed after cortical injury. Now we shall examine how cells that have migrated away from the SVZ into adjacent tissues differentiate. Late in development, the SVZ is the site of origin of astrocytes and oligodendrocytes that migrate to a variety of forebrain regions (Levison and Goldman, 1993). The majority of reactive astrocytes that appear after injury are probably born in situ, yet some of them may originate from the SVZ versus the parenchyma. In one study, weight percussion injury to the cerebral cortex transiently increased the number of double-labeled nestinþ/GFAPþ cells in the SVZ at two days after injury (Holmin et al., 1997). The number of double-labeled cells in the corpus callosum (between the SVZ and the injury), and near the injury gradually increased thereafter, and the nestinþ/GFAPþ astrocytes that were located between the SVZ and the lesion had a migratory morphology (Holmin et al., 1997). The authors postulated that SVZ-derived cells were differentiating into astrocytes and migrating towards the injury (Holmin et al., 1997), although the astrocyte like cells may have been born in situ in the corpus callosum. Other studies have also suggested that the SVZ can produce daughter cells that migrate out of the SVZ toward the injury and differentiate into glia. Studies in mice were carried out using a retroviral vector that contains human placental alkaline phosphatase (Golden et al., 1995), a membrane protein allowing detailed morphological identification of mature cell types. These experiments suggest that cells which migrate from the SVZ to the corpus callosum and towards the lesion differentiate into oligodendrocytes, and those that reach the lesioned cortex differentiate into astrocytes (Goings et al., 2004). This makes it tempting to surmise that white matter provides an environment that pushes SVZ-derived cells towards an oligodendrocyte fate whereas gray matter induces an astrocyte fate. Overall these results strongly imply that certain brain injuries in the adult can induce cells to migrate out of the SVZ and then differentiate into glia. A few studies have indicated that cells emigrating into adjacent brain regions from the SVZ after brain injury can differentiate into neurons. Aspiration lesions of the frontal cortex and olfactory peduncle in rats result in emigrated cells expressing PSA-NCAM and calretinin; the latter is also expressed by newly-generated olfactory bulb neurons derived from the SVZ. These cells gradually down-regulate their level of PSA-NCAM as they became morphologicaly more complex, exhibiting dendritic and axonal processes (Jankovski et al., 1998). This is consistent with the notion that neurons lose PSA-NCAM after migration and as they differentiate. By injecting BrdU a month after injury, Jankovski and colleagues confirmed

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that these neurons were newly born. Interestingly, SVZ-derived cells migrated into surrounding nuclei, differentiated into calretininþ neurons and survived for up to two months after injury (Jankovski et al., 1998). However, not all the BrdUþ cells were calretininþ raising the question of their phenotype. Knife cuts through the RMS in mice caused an enormous number of PSA-NCAM positive cells which were oriented in chains to migrate caudal to the lesion and into the striatum (Alonso et al., 1999). A subset of these cells expressed neuronal markers such as class III b-tubulin, TH and GABA up to one and a half months after the surgery (Alonso et al., 1999). The literature suggests that SVZ cells that remain in the SVZ or RMS do not change but remain relatively undifferentiated, as they do in controls. Although they do not differentiate in the SVZ after brain injury they may survive longer than in controls. SVZ cells that have migrated out in response to lesions, can both survive and differentiate into a variety of phenotypes. It remains to be determined if this response is common to many different types of injury and to what extent, if any, these cells extend axons and synapse in appropriate regions.

Molecular Regulation of SVZ Response to Brain Injury A tremendous volume of information on the molecular regulation of brain development has been generated in the past two decades. Spatiotemporal expression of large gene families are often reciprocally coordinated and regulate tissue patterning as well as cell proliferation, migration, survival, and differentiation. It is vital to examine the expression of these genes in the context of brain injury to learn if developmental programs are recapitulated in the adult. To what extent does ‘‘repair recapitulate ontogeny’’? Since many cardinal features of development (presence of stem cells, proliferation, migration, neurogenesis) persist in the SVZ, it is especially likely that genetic regulation of SVZ cell response to injury will be predictable from development. Neurogenesis during development is in large part regulated by growth and trophic factors. Injury to the central nervous system frequently results in upregulation of growth factors and in fact, aspiration lesions of the cortex cause increased expression of the trophic nerve growth factor receptor p75 (van Eden and Rinkens, 1994) and of FGF2 in the spared cortex (GomezPinilla and Cotman, 1992; Szele, 1994). Injuries to the brain result in astrocytosis in and around the lesion, and one of the most characteristic features of these cells is the production of growth factors (Ridet et al., 1997; Chen and Swanson, 2003). It is probable that many of the responses of the SVZ to brain injuries are due to activated astrocyte-derived growth and trophic factors (Ishikawa et al., 1991). This is all the more likely since EGF, FGF2, and astrocyte-conditoned media induce proliferation of SVZ cells in vitro (Reynolds and Weiss, 1992; Richards et al., 1992).

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It remains relatively unclear to what extent endogenous growth factors induced by brain injury alter SVZ biology, but a number of studies have examined the effects of exogenous growth factors (see chapter 2) (Craig et al., 1996; Kuhn et al., 1997; Benraiss et al., 2001; Pencea et al., 2001a). Increasing the levels of growth and trophic factors in the SVZ increases the numbers of SVZ cells and of new cells in adjacent structures. This suggests that growth factors can induce emigration from the SVZ and neuronal differentiation in adjacent nuclei. Two recent studies provide especially convincing evidence that BDNF stimulates SVZ cells to migrate into adjacent structures (Benraiss et al., 2001; Pencea et al., 2001a). Benraiss and colleagues infected the ependymal cells lining the lateral ventricles with an adenoviral vector expressing BDNF. Subsequently, newborn neurons appeared in the striatum, strongly suggesting that they had emigrated from the SVZ. These neurons expressed striatum-specific neuronal markers and survived for several weeks. Pencea administered BDNF and BrdU into the lateral ventricles for twelve days and then examined the presence of newborn neurons twentyeight days after the beginning of the injection. The number of cells found in the walls of the ventricles increased dramatically and neurons were found not only in the striatum, but also in the septum and diencephalic regions (Pencea et al., 2001a). EGF and FGF2 have also been injected into the lateral ventricles and both growth factors caused increased proliferation of SVZ progenitor cells (Craig et al., 1996; Kuhn et al., 1997). EGF injections, and to a lesser extent FGF2, were associated with an increase in newly born striatal cells although they did not appear to be neuronal (Kuhn et al., 1997). Interestingly, EGF pushed differentiation of SVZ cells towards an astrocytic lineage showing that SVZ cells retain the potential to generate astrocytes into adulthood (Kuhn et al., 1997). In another study, EGF caused SVZ cell emigration to other brain regions such as the cerebral cortex and septum and subsequent differentiation into primarily astrocytes (Craig et al., 1996). In Craig’s study, TGF-a also caused emigration of SVZ cells into the striatum (Craig et al., 1996). Whereas the appearance of newborn cells in the striatum and other regions is most easily explained in all of these studies by migration from the SVZ, it may also be possible that growth factors induced neurogenesis in a latent population of stem cells in the neural parenchyma (Palmer et al., 1999). In addition to understanding how the SVZ responds to exogenously administered growth factors, we must also learn which growth factors are induced by specific types of lesions and how the SVZ responds to endogenous growth factors. The combination of these two data sets may lead to more refined strategies for pharmacologic and /or genetic modulation of growth factor signalling as an adjunct to using SVZ cells for repair of injured brains. Because unilateral aspiration cortical lesions increased the numbers of cells in the rat SVZ, the hypothesis was generated that upregulated EGF and/or FGF2 were causing the effect (Szele and Chesselet, 1996). Although the expression of these growth factors was increased in reactive astrocytes in

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the striatum and cerebral cortex, they were not increased in the SVZ (Szele and Chesselet, 1996), suggesting that if they were responsible for the expanded SVZ, they must have diffused into it from surrounding regions. In addition to growth factors, adhesion molecules and extracellular matrix molecules are key players in a variety of developmental processes such as migration. The SVZ retains adhesion molecules and extracellular matrix molecules from development that are lost from the majority of the CNS (Miragall et al., 1990; Szele et al., 1994; Gates et al., 1995; Thomas et al., 1996). The astrocytic response to brain injury is characterized by the modulation of these molecules, which are thought to alternatively promote or inhibit axonal outgrowth depending on the circumstances (Ridet et al., 1997). Similarly, astrocyte-derived cell surface or matrix molecules may also regulate migration (Husmann et al., 1992). We examined the expression of PSA-NCAM (as described above), tenascin, and chondroitin sulfate proteoglycan (CSPG) in the SVZ after unilateral aspiration cortical lesion (Szele and Chesselet, 1996). Tenascin was expressed at high levels in the control dorsolateral SVZ (Fig. 7.5A) but decreased significantly one and seven days after unilateral aspiration cortical lesions (Fig. 7.5B, E) (Szele, 1994). The distribution of tenascin in the medial striatum was also decreased compared to controls although this effect did not reach significance (Fig. 7.5C, D, F) (Szele, 1994). Interestingly, decreases in tenascin in the medial striatum have also been observed in the mouse after cortical lesions (Poltorak et al., 1993). CSPGs have been implicated in the regulation of cell migration and actually do so in part by interacting with NCAM and tenascin (Grumet et al., 1996). In contrast to PSA-NCAM and tenascin, CSPG expression in the SVZ and medial striatum was not changed after unilateral aspiration cortical lesion (Fig. 7.5) (Szele, 1994). The sum of these studies suggests that adhesion and extracellular matrix molecules are regulated in complex patterns and that they contribute to the SVZ’s response to brain injury.

Conclusion Compared with other aspects of SVZ biology, relatively few studies have examined the SVZ’s reaction to mechanical brain injury. Nevertheless, some general patterns are beginning to emerge from the experiments described here, and they should help steer the course of future studies. The good news is that almost all studies of lesions near, or including, the SVZ result in changes in some combination of proliferation, migration, survival, or differentiation. Thus, we may be able to eventually control and harness these responses for therapy. However, it remains to be determined, how distant the lesion can be from the SVZ to elicit an effect. This and other questions raised in this chapter can only be accurately assessed with well characterized and controlled models of injury. Yet even with well characterized models,

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generalizations of SVZ response should be avoided since diverse species can respond differently to very similar lesions. Dissimilarities in cellular responses to given stimuli may help unravel regulatory genes. Ultimately, the mouse may be better for examining the genetic regulation of SVZ function in response to injury, whereas the rat may be a better model for SVZ mediated behavioral recovery. The response of the primate SVZ to brain injury remains very poorly characterized, but it is likely that simple histologic analyses of postmortem human brains would reveal changes in the SVZ after brain injury. One recent study has shown that proliferation increases in the SVZ of humans with Huntington’s disease (Curtis et al., 2003). Similar examination of the human SVZ after brain injury should be correlated with MRI data on the time course of tissue loss, edema, and ventricular expansion since biological parameters within the SVZ can change within hours and last for months. Furthermore, there may be topographical heterogeneities in the SVZ’s response to injury dependant on the distance from the lesion or inherent biological differences within subregions of the SVZ. The temporal and spatial variability in SVZ cell changes suggest clues of when and where to look after injury for molecules known to regulate normal and developmental processes. The tremendous suffering caused by brain injury, the inadequacy of current treatments, the tremendous potential of using SVZ cells therapeutically, and their inherent biological complexity combine to make an irrefutable case for increasing research in this field. Acknowledgments. F.G.S. supported by NIH RO1 NS/AG42253-01. The authors thank Martha Bohn, Shekhar Mayanil, and Jeff Gotts for their helpful comments on the chapter.

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Chapter 8 Responses of the SVZ to Hypoxia and Hypoxia/Ischemia Ryan J. Felling1, H. VanGuider1, Michael J. Romanko1 and Steven W. Levison1,2

Introduction Stroke is a devastating injury caused by interruption of the blood supply to the brain. Each year, about 700,000 people in the United States suffer a stroke incurring healthcare costs of more than $50 billion (Heart Disease and Stroke Statistics - 2003 Update, 2002). While advances in the acute treatment of stroke have improved the survival rates, little success has been realized in decreasing the associated morbidity. Stroke survivors suffer a wide range of neurological deficits depending on the location in the brain where the stroke occurs. These effects include, but are not limited to, paralysis, sensory deficits, memory loss and personality changes. Additionally, hypoxic/ischemic (H/I) injury is the single most important cause of brain damage resulting from complications during birth, leading to permanent neurological deficits. Every year, perinatal H/I afflicts approximately 1-2 per 1000 term births and roughly half of surviving preterm infants. Many of these infants suffer long-term handicaps that include learning disabilities, mental retardation, epilepsy and cerebral palsy (Volpe, 2001). For more than 20 years, experimental animal models have been widely used to study the effects of ischemic brain injury. The most common model is transient middle cerebral artery occlusion (MCAo) used primarily in adult animals to generate unilateral focal cerebral infarcts (Longa et al., 1989). 1

Department of Neural and Behavioral Sciences, Pennsylvania State University College of Medicine, Hershey, PA 17033 2 Department of Neurology and Neuroscience, UMDNJ-New Jersey Medical School, Newark, NJ 07103 Mailing Address: Steven W. Levison, Ph.D., Department of Neurology and Neuroscience, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, H-506, Newark, NJ 07101-170, Email: [email protected]

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Bilateral common carotid artery clamping also simulates global ischemia in both perinatal and adult animals, causing infarcts in both hemispheres (Smith et al., 1984). The standard perinatal model involves permanent unilateral common carotid artery ligation followed by systemic hypoxia (Rice et al., 1981; Vannucci et al., 1996). More recently, methods of inducing brain damage in utero have garnered attention for modeling prenatal H/I (Cai et al., 1995). The experimental models described above have allowed investigators to extensively characterize these insults at the gross, cellular and molecular levels. Recently, the subventricular zone (SVZ) has become a focus of investigation in these injury models. Evolving data suggests that the precursors in this region are capable of responding to hypoxic and ischemic insults, findings that bolster evidence against the long-held tenet that the brain is a nonregenerating organ. Both the perinatal and the mature brain harbor neural stem/progenitor cells (NSPs) that persist throughout life in the SVZ. The central location of the SVZ within the brain, paired with constitutive neurogenesis and gliogenesis that support physiological cell turnover in the olfactory bulb and white matter (see Chapters 1, 3 and 9), renders this region a potential target for treatment following both adult and perinatal stroke. In this chapter we review findings on the vulnerability and responses of the SVZ cells to hypoxic/ischemic insults as well as to the effects of hypoxia alone.

Damage in the SVZ Following H/I Injury Overview of SVZ Damage after Perinatal H/I In premature infants, the damage subsequent to H/I is generally limited to periventricular structures. As the infant matures, the damaged area expands radially from the ventricles towards the pial surface. Thus, in premature infants, the prominent histopathological features of perinatal H/I are germinal matrix infarction, periventricular hemorrhage and intraventricular hemorrhage (possibly stemming from stenosis of the deep venous drainage system). Additionally, due to poor vascularization of the white matter and the specific vulnerability of the late oligodendrocyte progenitors in the white matter, there is frequently focal as well as diffuse damage to the white matter (Towbin, 1998; du Plessis and Volpe, 2002). A substantial structure in the immature developing brain is the germinal matrix. This general term is applied to the histologically defined region that contains the precursors in both the ventricular and subventricular zones. In the human brain of 29–34 weeks of gestation, the germinal matrix largely comprises cells of the SVZ. Studies have characterized the perinatal SVZ as a mosaic of immature cell types that include multipotential, bipotential, and unipotential stem cells and progenitors at different stages of lineage restriction (See Chapter 1). During perinatal development, the SVZ is the major source of myelinating oligoendrocytes in the forebrain (Levison et al.,

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1993; Levison et al., 1999). Both perinatal H/I of prematurity and term birth asphyxia occur prior to the peak of myelination, exerting their effects precisely when SVZ cells are generating oligodendrocyte precursors. It is not surprising then that inclusive of obvious neurodegeneration, there is also oligodendrocyte progenitor depletion and subsequent dysmyelination (Back et al., 2001; Levison et al., 2001; Ness et al., 2001; Back et al., 2002). In the Vannucci model of perinatal H/I, P6 rat pups (when the day of birth is P0) are exposed to various durations of 8% oxygen following unilateral ligation of the right common carotid artery (Rice et al., 1981). Ninety minutes of 8% oxygen exposure result in severe unilateral brain injury characterized by columnar damage of the cortex, mild white matter damage, and up to 50% cell death in the striatum. Furthermore, this injury drastically affects the cells of the ipsilateral SVZ. By four hours following the insult, the ipsilateral SVZ suffers greater than a 20% reduction in cellularity. Necrotic death predominates during this early stage of recovery in both the choroid plexus and subependyma, and the resulting edema leads to a 10% enlargement of cells in the ipsilateral hemisphere. At 12 hours of recovery, numerous pyknotic cells, indicative of apoptotic cell death, are present throughout the SVZ. This correlates to a further reduction in cellularity of 25% by 48 hours of recovery. Notably, the progression of damage persists through 3 weeks of recovery, markedly reducing the size of the SVZ and inducing extensive astrogliosis. A loss of axons, dysmyelination and thinning of the periventricular and subcortical white matter as well as infiltration of hypertrophied astrocytes into areas once occupied by myelinating oligodendrocytes are also characteristic of this late time point of recovery. The enrichment of astrocytes likely contributes to the glial scarring and restructuring of the brain (Levison et al., 2001).

Sequence, Sources, and Nature of Cell Death Following Perinatal H/I The nature of damage in the SVZ following perinatal H/I evolves during the recovery period (Fig. 8.1). Electron microscopy reveals a range of cell death morphologies, and these are indicative of the type of cell death. The predominant type of cell death during the first 4 hours of recovery is necrosis, defined by condensation and marginalization of chromatin, swelling of organelles and loss of cell membrane integrity. The next 48 hours are characterized by cells exhibiting characteristics of both necrosis and apoptosis with pure apoptotic figures observed at later intervals (Levison et al., 2001). At the peak of cell death within the SVZ, it appears that the majority of the dying cells in this region exhibit the features of hybrid cell death, also characteristic of excitotoxic cell death (Martin et al., 1998). Hypoxia/ischemia causes increased release of excitatory amino acid (EAA) neurotransmitters that open ionotropic channels, allowing an influx

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Figure 8.1. Hypoxia/ischemia causes necrotic, hybrid and apoptotic deaths within the subventricular zone. Electron microscopy at 4 h recovery demonstrates that dying cells appear necrotic (B) compared to their well preserved counterparts in the contralateral hemisphere (A). At 12 h recovery, necrotic, hybrid, and apoptotic cells are seen interspersed among normal and dividing cells within the SVZ (C). At 24 h the affected SVZ contains apoptotic profiles and reduced cellularity (D). Mag. bar represents 3 mm in A,C,D, and 1:5 mm in B. From Levison et al. (2001). Copyright (2001) Karger, reprinted with permission.

of calcium, water, and cations into receptive cells (Puka-Sundvall et al., 1997, 2000). The influx of excessive calcium will activate cell death pathways, especially the activation of m-calpain, a calcium-dependent cysteine protease that has been implicated in the demise of brain cells after H/I (Blomgren et al., 2001). In addition, numerous studies have highlighted the importance of caspases as major death effectors in perinatal H/I brain damage (Hu et al., 2000; Zhu et al., 2000; Northington et al., 2001; Wang et al., 2001; Gill et al., 2002). Our recent studies indicate that calpains and caspases are activated subsequent to perinatal H/I and that caspase 3 is downstream of m-calpain. Both m-calpain and caspase-3 can cleave a common substrate, fodrin, with distinct cleavage products; therefore, we used fodrin cleavage to determine the

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levels of both m-calpain and caspase-3 activity in the SVZ following perinatal H/I. Activation of m-calpain is detectable by Western blot as early as 3 hours following the injury and its activity remains sustained through 48 hours of recovery (Romanko, 2004). Immunostaining for fodrin’s unique m-calpain cleavage site reveals that after 14 hours of recovery 4.5% of the SVZ cells are positive for calpain cleaved fodrin and calpain cleaved fodrin postive cells remain elevated above control levels through 72 hours of recovery. Separate counts taken from the distinct regions of the SVZ reveal regional variation in m-calpain activity. Most of the calpain activity is evident in the lateral regions of the SVZ while the medial regions exhibit little activity (Romanko, 2004). Caspase-3 proteolytic activity is delayed compared to m-calpain activity, as we are unable to detect the caspase cleaved fragment of fodrin by Western blot until 24 hours of recovery (Romanko, 2004). However, caspase-3 begins to become activated by 12 hours of recovery following H/I, when approximately 3% of the cells in the ipsilateral SVZ exhibit strong cytoplasmic active caspase-3 staining compared to 1% positive cells in the contralateral SVZ. These active caspase positive cells represent approximately 1/3 of the total dying cells at this timepoint, suggesting a major role for other (as yet unidentified) death effectors (Fig. 8.2). Caspase-3 staining also exhibits regional variation similar to m-calpain activity, with the majority of dying cells situated in the mediolateral and lateral regions of the SVZ. These regions in the ipsilateral hemisphere showed a 5-fold increase in the percentage of caspase-3þ cells relative to contralateral regions, whereas no difference appeared between the medial regions of the 2 hemispheres (Romanko et al., 2004). An important correlate of these studies is that others have reported neuroprotection afforded by intraventricular infusion of the pancaspase inhibitor BAF following a perinatal H/I insult, suggesting that a therapeutic window may exist during which the administration of antiapoptotic agents will prevent cell death and the subsequent loss of tissue subsequent to perinatal H/I (Cheng et al., 1998).

Distribution and Identity of Dying Cells in the SVZ Cell death within the SVZ following perinatal H/I does not occur in a uniform pattern. The majority of dying cells reside in the mediolateral and lateral tail regions. Data demonstrate that many of these vulnerable cells are restricted progenitors. PSA-NCAM, a cell adhesion molecule, plays a critical role in the morphogenesis of the nervous system and is expressed in a subset of migrating progenitors that give rise to both neurons and glial cells. Restricted neuronal progenitors express PSA-NCAM, whereas NSPs, sometimes referred to as Type B cells or SVZ astrocytes, do not (Doetsch et al., 1997). Furthermore, cells in the mediolateral and lateral SVZ, where the majority of the SVZ cell death occurs, strongly express PSA-NCAM (Szele et al., 1994). Following perinatal H/I, active caspase-3þ =PSA-NCAMþ cells

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Figure 8.2. Progenitors, rather than stem cells are vulnerable to perinatal H/I. Panels A,B depict the medial SVZ, whereas panels C,D depict the lateral SVZ at 4 h of recovery of the contralateral (A,C) and ipsilateral (B,D) hemispheres. Cells in the ipsilateral medial SVZ (B) swell at this early recovery time point; however, no detectable cell death is present. Conversely, both hybrid (arrows) and apoptotic (arrowheads) figures are present in the lateral region of the ipsilateral SVZ (D). Panel E depicts the regional distribution of cell death measured by TUNEL labeling, the occurrence of pyknotic nuclei and active caspase-3 staining at 12 h of recovery. There is extensive cell death in the lateral regions of the SVZ compared to the medial fields. Caspase-3þ cells comprise a subset of the dying cells. Panel F shows an optical slice through a caspase-3þ (Cy3) / PSA-NCAMþ (Alexa 488) progenitor cell in the ipsilateral (lateral region) of the SVZ. Panel G shows the lack of nestinþ (Alexa 488)/Caspase 3þ cells (Cy3) in the medial region of the ipsilateral SVZ. Caspase-3þ cells in the medial region are nestin- (inset). Scale bar represents 15 mm in panels A-D and 4 mm in panels F, G. Modified from (Romanko, 2004; Rothstein and Levison, 2002). Copyright (2004) Lippincott Williams and Wilkins (2002), Karger, respectively, with permission.

have been found in the lateral SVZ, suggesting that these cells are vulnerable to the insult (Romanko et al., 2004). Neural progenitors are not the only cell types to suffer as a consequence of perinatal H/I. Myelin basic protein (MBP) gene expression in the corpus callosum is reduced by 3 hours of recovery, and this is accompanied by degeneration of SVZ cells that express the proteolipid protein (PLP) gene, indicating that oligodendrocyte progenitors within the SVZ also are vulnerable to the insult (Skoff et al., 2001). The demonstration that cells positive

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for the oligodendrocyte progenitor markers Yp, NG2 and Gst-p die in the periventricular white matter, reaching a peak at 12 hours following the insult, support this idea and the depletion of these cells likely contributes to the resulting progressive dysmyelination (Levison et al., 2001; Ness et al., 2001). This feature is consistent with the clinical presentation of periventricular leukomalacia (PVL), the most prominent pathaphysiological form of perinatal H/I in humans (du Plessis and Volpe, 2002) which contribute to the cognitive and motor dysfunctions that are typical sequelae of perinatal brain insults. While cell death is extensive in the lateral regions of the SVZ, the medial region including the ependyma and immediate subependyma is relatively spared. Evidence demonstrates that the medial SVZ provides a niche that harbors the NSPs in the postnatal brain (Doetsch et al., 1997; GarciaVerdugo et al., 1998; Chiasson et al., 1999). Cells in this region exhibit swelling as early as 4 hours of recovery; however, indicators of cell death including TUNEL or ISEL labeling, apoptotic or necrotic morphology, and caspase-3 activation are rare, even at the peak of cell death (Fig. 8.2). In fact, caspase-3þ activation in the most medial cells of the ipsilateral SVZ is equivalent to the baseline levels of activation observed in the contralateral medial SVZ (Rothstein and Levison, 2002; Romanko et al. 2004). Double immunofluorescence studies demonstrate an absence of caspase-3 activation in cells positive for nestin, an intermediate filament used to identify neural precursors (Fig. 8.2) (Romanko et al., 2004). These data indicate that although restricted progenitors are vulnerable to perinatal H/I, the putative NSPs residing in the medial SVZ are resistant to the injury. The infrequency of dying cells within the medial SVZ suggests that these cells may be intrinsically resilient to the deadly effects of H/I insults. Glycogen granules have been found within ependymal cells and SVZ cells which may provide an energy reserve to protect the cells from injury (Blakemore, 1969). Ependymal and SVZ cells also rely greatly upon anaerobic respiration, which renders them naturally resistant to hypoxia (Shimizu, 1957; Cammermeyer, 1965). Additionally, large blood vessels are prominent in the medial regions of the human germinal matrix and the rodent SVZ, affording rapid reperfusion during recovery (Towbin, 1998). In addition to metabolic and structural mechanisms rendering these cells resistant to hypoxia and ischemia, NSPs have higher levels of anti-apoptotic Bcl-2 and Bcl-XL , which may endow them with resistance to stimuli causing cell death (Brazel, 2003). It is important to note that, despite increased survival when compared to other cell populations, the cells situated in the most medial regions of the SVZ are affected by H/I, becoming significantly swollen in the early stages of recovery following the insult (Rothstein and Levison, 2002).

Damage to the Adult SVZ following Ischemia The SVZ has only recently garnered attention in models of adult ischemia, primarily because of its potential to provide precursors necessary for

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regeneration. The bulk of research regarding cell death following ischemia has focused on the nearby striatum, where near complete loss of NeuNþ cells has been documented in the lateral and caudal regions of the ipsilateral striatum with sparing of the most medial areas in MCAo models (Arvidsson et al., 2002). Whereas damage to the perinatal SVZ has been well documented, future research will need to further detail whether the adult SVZ sustains damage after ischemia.

Regenerative Potential of the SVZ Following H/I Injury The potential of the SVZ to participate in regeneration following H/I injury has made it an exciting target for study. Recent studies have demonstrated that new cells are generated in both the adult and perinatal SVZ following stroke, rebutting the long held tenet that the brain was a non-regenerating organ (Felling and Levison, 2003). Much of the research in this area has focused on the genesis of neurons, but the presence of putative NSPs in the SVZ offers the promise of replacing multiple cell types. The following sections summarize the regenerative opportunities that are possible as a consequence of activating cells in the SVZ.

Proliferation of Neural Stem Cells following H/I injury Within the last decade, many investigators have demonstrated that new cells are generated from proliferative regions of the brain following stroke. Most of these data indicate that proliferation in the ipsilateral SVZ following MCAo peaks at 7 days and persists to at least 14 days of recovery (Fig. 8.3) (Zhang et al., 2001; Li et al., 2002). Cumulative BrdU labeling has been used to define the total number of proliferating cells during the first 2-weeks after MCAo. This study demonstrates that approximately 37% more cells proliferate in the ipsilateral SVZ compared to both the contralateral SVZ and nonischemic controls. No difference is observed at four weeks of recovery (Zhang et al., 2001). As the investigators only injected BrdU once daily during the 2 week recovery interval, the results of this analysis underestimate the absolute level of proliferation due to a lack of constantly available BrdU and rapidly cycling cells. Similar experiments that also observe increased BrdU labeling in the ipsilateral SVZ find BrdUþ cells in the olfactory bulb, indicating that the increased number of BrdUþ cells seen is not simply a failure of migration resulting in an accumulation of progenitors within the SVZ (Parent et al., 2002). One concern with using BrdU to examine cell proliferation is that DNA repair can lead to BrdU incorporation. One study failed to show overlap of BrdU incorporation with labeling for PCNA, a cell-cycle dependent protein marker of cell proliferation, in the SVZ (Jin et al., 2001). The failure to confirm the BrdU results with PCNA may be a technical artifact, as two

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4000 BrdU-positive cells/mm2 (±SE)

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Figure 8.3. Quantitative analysis from a pulse labeling paradigm reveals that the number of BrdU immunoreactive cells in the ipsilateral SVZ significantly increases 2, 7 and 14 days after MCA occlusion compared with that in the contralateral SVZ (E, F). ( p < 0:05; p < 0:01). (Adapted from Zhang et al., 2001). Copyright (2001) Elsevier, with permission.

studies have demonstrated that administration of Ara-C, a toxin for dividing cells, combined with BrdU administration eliminates the observed increases in the number of BrdUþ cells (Arvidsson et al., 2002), supporting the interpretation that the increases in BrdU labeling are primarily due to cell division following H/I. The SVZ is an amalgamation of putative NSPs and other lineagerestricted progenitors, and direct evidence indicates that progenitor cells comprise the majority of the proliferating population at 1 week following H/I injury. Doublecortin (DCX) is a marker of early neuronal progenitors, and many BrdUþ cells in the SVZ and striatum also label for DCX 2 weeks after MCAo (Fig. 8.4) (Arvidsson et al., 2002; Parent et al., 2002). Other precursor markers, including Pax6, Emx2, and Mash1 are elevated in the posterior SVZ following transient global ischemia in adult rats (Nakatomi et al., 2002). Further evidence that these proliferating cells are precursors comes from studies showing that BrdUþ cells in the rat SVZ do not label with NeuN, a marker of mature neurons, after 1 week of recovery from MCAo and that these BrdUþ cells do not express another neuronal marker, Hu, providing additional support for the hypothesis that progenitor cells rather than mature cells proliferate in response to ischemic injury and that BrdU is not being incorporated into dying neurons (Jin et al., 2001). Current evidence indicates that an expansion of the neural stem/progenitor (NSP) cell population may provide the basis for the burst in progenitor cell proliferation described above. Presently, there are no definitive markers to distinguish NSPs; hence, it is difficult to prospectively analyze them.

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Figure 8.4. Male Wistar rats were injected with BrdU IP twice daily on days 1–14 following MCA occlusion and sacrificed directly thereafter. Sections were processed for immunohistochemistry using antibodies against BrdU (red) and Dcx (green), a marker for migrating neuroblasts. a and b, Overview of Dcx and BrdU immunoreactivity in the dorsomedial striatum contralateral (a) and ipsilateral (b) to the insult at 2 week after MCAO. On the contralateral side, Dcx immunoreactivity is restricted to the SVZ and isolated cells in the striatum (a), whereas abundant Dcxþ cells are present in the ipsilateral striatum (b), distributed in a density gradient from the SVZ, bordered dorsally by the corpus callosum (CC) and medially by the lateral ventricle (LV). c–f, Confocal microscopic images showing the boxed areas in a and b. c, A Dcxþ =BrdU cell with the morphology of a mature neuron located in the striatum contralateral to MCAO. d, Densely clustered Dcxþ cells in the SVZ, showing extensive colocalization with BrdU immunoreactivity. A migrating Dcx =BrdUþ cell can be seen in the CC dorsal to the SVZ (arrow). e, A Dcxþ =BrdUþ cell with the morphology of a migrating neuroblast. f, Multiple Dcx-labeled cells with migratory or mature morphologies. Some of the cells are double-labeled with BrdU (arrows). Scale bar is 200 mm for a, b; 40 mm for c–f. (Adapted from Arvidsson et al., 2002). Copyright (2002) Nature Publishing Group, with permission.

Retrospectively, however, primary and secondary neurosphere assays have been used to demonstrate that there is an increase in the abundance of NSPs between 2 and 3 days after H/I in the perinatal rat brain (Felling et al., submitted). Recent data demonstrate that the adult rat SVZ also generates higher numbers of neurospheres 7 days following ischemia (Zhang et al., 2004). In both cases, the neurospheres derived from the ischemic brain selfrenew and differentiate into neurons and glia, suggesting that the increase in neurospheres represents an expansion of the NSP population. Consistent with this hypothesis, the level of nestin, an intermediate neurofilament protein expressed by neural precursors both during development and in adults, is significantly elevated in the region surrounding the infarct as early as one day following MCAo in adult rats (Li and Chopp, 1999).

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Furthermore, we have observed an increase in the number of cells co-expressing PNCA, a marker of cell proliferation, and nestin in cells residing in the medial SVZ. The increase in the numbers of primitive neural progenitors may result from a shift from asymmetric cell divisions to symmetric, expansionary divisions as suggested by secondary neurosphere data (Felling, 2005). These data suggest that H/I initially triggers proliferation in the NSP compartment of the SVZ to allow for a subsequent burst in committed progenitor cell proliferation.

Migration of Newly Generated SVZ Cells to Sites of Injury The data reviewed above provides evidence of a significant proliferative response of SVZ progenitors and NSPs to ischemic injury. This response, however, is only advantageous if the new cells ultimately repopulate brain regions depleted of cells as a consequence of stroke. Indirect evidence suggests that newly generated cells migrate away from the SVZ toward sites of injury. For instance, BrdUþ =DCXþ cells with the morphology of migrating neurons stream from the SVZ to the striatum in the ipsilateral hemisphere after MCAo in adult rats. A gradient of migrating profiles is seen, with the greatest number of spindle-shaped DCXþ cells located up to 0.5 mm lateral to the SVZ but with scattered cells located as far as 2 mm from the SVZ. In the contralateral hemisphere, DCX immunoreactivity is restricted to the SVZ and to rare single cells of the striatum (Fig. 8.4) (Arvidsson et al., 2002; Parent et al., 2002). Although indirect, these observations suggest that newly generated neuroblasts are migrating into damaged areas of the brain. Analysis of PSA-NCAM, a cell adhesion molecule important in cell migration, provides further evidence indicating increased migration of SVZ cells after H/I. Many of the BrdUþ cells that appear to be migrating away from the SVZ also express PSA-NCAM. While PSA-NCAM is also highly expressed by reactive astrocytes, there is no colocalization of PSA-NCAM and vimentin or GFAP, astrocytic markers (Sato et al., 2001; Parent et al., 2002). It is currently unknown whether cells detected in damaged regions following H/I insults represent proliferating cells resident in the parenchyma or cells that have migrated from proliferative regions. While the observation of DCXþ cells with spindle-shaped profiles hundreds of microns from the SVZ is consistent with migration, fate-mapping studies using stereotacticallyinjected retroviruses or time-lapse video microscopy studies are needed to define where the cells that proliferate and migrate from the SVZ early after an insult ultimately reside. These types of data will help to define the migratory patterns of progenitors after ischemic injury to verify that progenitor cells repopulate areas where they are needed.

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Differentiation of Newly Generated SVZ Precursors into Functionally Mature Cells Several studies have addressed the long-term fates of cells that proliferate early after an ischemic insult in adult stroke models. Injections of BrdU given during the first week after MCAo in adult rats reveals a 31-fold increase newborn neurons in the ipsilateral striatum compared to the contralateral striatum and sham-operated controls when analyzed 4 weeks following the injury (Arvidsson et al., 2002). The difference in cell densities is even more prominent than that of absolute cell numbers because parts of the damaged striatum disintegrate as a result of ischemia. The density of BrdUþ =NeuNþ cells is nearly 10 times greater at 4 weeks of recovery than at 2 weeks, indicating that progenitor cells continue to mature over the 1 month recovery period. Consistent with the interpretation that newly generated neurons are maturing within the damaged striatum, BrdUþ =DCXþ cells stain for Meis2 and Pbx, two markers that are expressed by developing striatal cells. Furthermore, at 5 weeks of recovery, 42% of the BrdUþ =NeuNþ cells are also positive for DARPP-32, a marker of mature medium-spiny neurons characteristic of the striatum (Arvidsson et al., 2002). These data suggest that a significant proportion of the newly generated cells in the striatum develop in a regionally appropriate manner. After an ischemic insult, some newly generated cells differentiate into a mature neuronal phenotype, but survival has not been assessed at longer recovery intervals. Although 4 weeks may be enough recovery time for cells to mature, any therapeutic role for these cells requires that they maintain a functional presence in the local networks for extended periods of time if not permanently. While current literature focuses on the generation of neurons, an involvement of NSPs in the physiological responses to ischemia suggests that other cell types may be involved in the recovery process. The astrocytic response has been thoroughly investigated (Stoll et al., 1998), but less is known about oligodendrocyte production following stroke. One group demonstrates an accumulation of oligodendrocytes in both the border and core of the infarct after an initial loss of PLP mRNA expression (Mandai et al., 1997). Another group shows a significant increase in the number of NG2þ oligodendrocyte progenitors surrounding the infarct within two weeks after MCAo (Tanaka et al., 2001), but the fate of these cells has not yet been determined. From a therapeutic perspective, the ability to direct the differentiation of these newly generated cells following brain injury would be of the utmost importance. Recent experiments have identified a myriad of different factors that support neurogenesis. Some of these, including heparin-binding EGFlike growth factor (HB-EGF), basic fibroblast growth factor (FGF-2), stem cell factor (SCF), and erythropoietin (EPO), have been shown to increase after ischemic injury, indicating that they may play a restorative role in cell proliferation and differentiation following H/I insults (Shingo et al., 2001;

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Jin et al., 2002a, 2002b). Infusion of EGF and FGF-2 into the lateral ventricles following stroke in adult rats dramatically improves neuronal production and incorporation into the hippocampus as well as performance on behavioral tests (Nakatomi et al., 2002). Harnessing these factors and processes may allow future studies and therapies that will enhance recovery following the H/I insult associated with stroke by focusing on the neural stem cells and progenitors present in the SVZ.

Effects of Hypoxia on the SVZ In contrast to H/I, cerebral hypoxia occurs when the brain receives an inadequate oxygen supply despite normal blood flow. Hypoxia can result from drowning, carbon monoxide poisoning, and suffocation as well as from prolonged exposure to high altitudes. Symptoms, including inattentiveness, dizziness, loss of motor coordination, and memory impairment, are usually mild and may proceed undetected. Cerebral hypoxia is a common component of severe brain insults, including perinatal asphyxia and stroke, both of which produce more severe symptoms and can also cause neuronal death and brain lesions. Although hypoxia poses a significant clinical threat, little research has been conducted to facilitate prevention and treatment following hypoxic insult, and only a few laboratories have studied the effects of hypoxia on the SVZ. The immature brain is sensitive to hypoxic stress. In experimental animals, chronically low levels of O2 decrease the volume of the cerebral cortex and subcortical white matter (Ment et al., 1998). In light of these results, it is surprising that rat pups raised in 9.5% oxygen from postnatal day 3 to postnatal day 33 have a slightly higher number of neurons than normoxic controls (the sea level atmosphere contains 21% oxygen) (Stewart et al., 1997). While this increased number of neurons is attributed to decreased programmed cell death, this result also may be due to compensatory neurogenesis, as it has been shown that maintaining NSPs in hypoxic conditions in vitro stimulates their proliferation and differentiation into neurons (Studer et al., 2000). In addition to a slight increase in neuronal numbers, neuronal density also is increased by approximately 50% as a consequence of chronic hypoxia; however, this effect is due to a reduced cortical volume as a consequence of decreased neuronal arborization and smaller soma volumes. The molecular bases for some of these changes are now being identified. Using a chronic hypoxia model, Ganat et al. (2002) have established that levels of the known growth/trophic factors FGF-1 and FGF-2 increase in rat pups exposed to 3 weeks of hypoxia. By contrast, levels of the neurotrophins BDNF and NT-3 are unchanged. Interestingly, increased numbers of radiallike glial cells expressing vimentin and Brain Lipid Binding Protein are observed in the SVZ during chronic hypoxia and a subset of these cells express higher levels of the type 1 FGF receptor. As dividing radial glial cells can produce new neurons and serve as a scaffold for cell migration,

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these observations support the hypothesis that there may be enhanced neurogenesis during chronic hypoxia.

Conclusion It is now evident that the cells in the SVZ that are essential for the development of the brain continue to produce new neurons and glia throughout life. While uncertainties remain regarding how injury develops in and around the SVZ, clearly this structure is sensitive to, and responds to, damage. Our studies reveal that progenitors are more vulnerable than more primitive precursors, which in part appears to be due to intrinsic differences in the levels of pro- and anti-apoptotic molecules. That progenitors within the fetal brain are depleted as a consequence of H/I has important implications, as a single SVZ cell can generated over 100 neocortical progeny (Levison et al., 1999). Thus, these findings provide new insights into why developmental brain insults impair congnitive and motor function. Whereas progenitors are depleted from the SVZ after H/I, the resilience of the more primitive cells provides opportunities to enhance the modest regeneration of the brain that appears to occur. Major inroads on the molecular regulation of the stem cell state have been made in the recent past. Therefore, it will be vital to continue to identify those factors that will increase the numbers of primitive neural progenitors in SVZ cells so that these regulators can be used to amplify the numbers of these neuroregenerative cells. A number of studies have injected growth factors into the uninjured brain to examine their effects on proliferation and survival of SVZ cells. These studies suggest that growth and trophic factors can induce emigration from the SVZ and neuronal differentiation in adjacent nuclei. Based on these studies, growth factors have been applied after ischemia to stimulate neurogenesis, and electrophysiological and behavioral analyses of these growth factor-treated animals suggest a recovery of brain function following treatment. Thus, the existing data are promising and offer hope that with a further appreciation of which growth factors regulate the proliferation and survival of neural precursors we will obtain practical information on restoring neurologic function after a stroke. However, based on a century of research, we know that without manipulation, there appears to be limited directed cell migration and cell replacement from the SVZ. Moreover, there is significant glial scarring after stroke, with the likelihood that the majority of newly generated cells become astrocytes rather than new neurons or oligodendrocytes. Therefore, strategies aimed at stimulating cell replacement from endogenous precursors (as well as studies providing exogenous progenitors) will not only have to identify strateties to maintain the pools of primitive progenitors while they are dividing, but they will also need to establish the means to direct newly

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generated cells away from astrocytic fates. Given the plasticity of the SVZ, and the encouraging results of interventions applied to animal models of stroke, we are optimistic that manipulating the responses of SVZ cells is not only conceivable for eventually treating human disease, but likely.

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Stoll, G., Jander, S., and Schroeter, M. (1998). Inflammation and glial responses in ischemic brain lesions. Prog. Neurobiol. 56: 149–171. Studer, L., Csete, M., Lee, S.H., Kabbani, N., Walikonis, J., Wold, B., and McKay, R. (2000). Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J. Neurosci. 20: 7377–7383. Szele, F.G., Dowling, J.J., Gonzales, C., Theveniau, M., Rougon, G., and Chesselet, M.F. (1994). Pattern of expression of highly polysialylated neural cell adhesion molecule in the developing and adult rat striatum. Neuroscience 60: 133–144. Tanaka, K., Nogawa, S., Ito, D., Suzuki, S., Dembo, T., Kosakai, A., and Fukuuchi, Y. (2001). Activation of NG2-positive oligodendrocyte progenitor cells during post-ischemic reperfusion in the rat brain. Neuroreport 12: 2169–2174. Towbin, A. (1998). Brain Damage in the Newborn and its Neurologic Sequels: Pathological and Clinical Correlation. PRM Publishing Company, Inc., Danvers, MA. Vannucci, S.J., Seaman, L.B., and Vannucci, R.C. (1996). Effects of hypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature rat brain. J. Cereb. Blood Flow and Met. 16: 77–81. Volpe, J.J. (2001). Neurobiology of periventricular leukomalacia in the premature infant. Pediatr. Res. 50: 553–562. Wang, X., Karlsson, J.O., Zhu, C., Bahr, B.A., Hagberg, H., and Blomgren, K., 2001. Caspase-3 activation after neonatal rat cerebral hypoxia-ischemia. Biol. Neonate. 79: 172–179. Zhang, R., Zhang, Z., Wang, L., Wang, Y., Gousev, A., Zhang, L., Ho, K.L., Morshead, C., and Chopp, M. (2004). Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats. J. Cereb. Blood Flow Metab. 24: 441–448. Zhang, R.L., Zhang, Z.G., Zhang, L., and Chopp, M. (2001). Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia. Neuroscience 105: 33–41. Zhu, C., Wang, X., Hagberg, H., and Blomgren, K. (2000). Correlation between caspase-3 activation and three different markers of DNA damage in neonatal cerebral hypoxia-ischemia. J. Neurochem. 75: 819–829.

Chapter 9 Responses of the SVZ to Demyelinating Diseases B. Nait-Oumesmar, L. Decker, N. Picard-Riera and A. Baron-Van Evercooren

Introduction The mammalian adult central nervous system (CNS) has an inherent ability to generate neurons and glia. The dogma that the adult CNS remains incapable of regeneration came from the observation that most neurons and glia are post-mitotic cells. However, this idea is now widely challenged by the demonstration that indeed mitotic cells are detected in restricted areas of the adult CNS. Among these regions, the subventricular zone (SVZ) of the forebrain is the largest germinative zone of the adult brain. It is also one of the most characterized germinative area at the cellular and molecular level. While most studies highlight the neurogenic potential of the SVZ, we will review its gliogenic potential in normal and demyelinating conditions.

The Adult Subventricular Zone: Structure and Cellular Composition The presence of mitotic cells in the adult SVZ was reported nearly hundred years ago (Allen, 1912). Later studies in rodents and human demonstrated the presence of undifferentiated dividing cells in the adult subependymal layer lining the lateral ventricles (Altman, 1969), suggesting that the SVZ persists in the adulthood (Privat and Leblond, 1972; Tramontin et al., 2003). These cells were first characterized by electron microscopy. Many cells within the subependymal layer possessed dark-stained nuclei, had smaller round cell bodies and showed a high rate of proliferation as evidenced by tritiated thymidine incorporation (Smart et al., 1961), while other cells more distant from the ventricle wall had light nuclei. More recently, AlvarezByulla and colleagues proposed a classification of the adult SVZ based on INSERM U.546, Hopital Pitie´-Salpeˆtrie`re, Paris, France. 260

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three cell types. These cells were classified as type A, B and C cells on the basis of immunological and ultrastructural features. The type B cells, the presumed neural stem cells, give rise to primary precursors the transit-amplifying type C cells, which in turn generate type A cells (neuroblasts). Type B cells, divide slowly and share characteristics with astrocytes and radial glia (Doetsch and Alvarez-Buylla, 1996; Doetsch, 2003). While these cells are well characterized at the ultrastructural level, specific immunological markers are still missing. Type B cells express GFAP and LexA/SSEA-1 (stage specific embryonic antigen-1), a cell membrane carbohydrate on embryonic and other stem cells (Capela and Temple, 2002). They are closely associated with the ependymal cells and often bear a single cilium that contacts the lateral ventricle (Tramontin et al., 2003). The function of this short single cilium is as yet unknown but the contact of type B cells with the ventricle fluid may be required for the proliferation, survival or differentiation of this quiescent cells. Type C cells are rapidly proliferating, have an elongated morphology and a dark-stained nucleus. They are characterized by the expression of the homeodomain transcription factor Dlx2, Epidermal Growth Factor Receptor (EGFR) and do not express the embryonic form of NCAM (Doetsch and Alvarez-Buylla, 1996; Doetsch, 2003). Type A cells migrate as homotypic chains along the rostral migratory stream to the olfactory bulb. They express PSA-NCAM, the intermediate filament nestin and the mCD24 antigen.

Ontogenesis of the SVZ and its Relationship to Oligodendrogliogenesis During early embryogenesis in rodents (E9-E12.5), numerous studies demonstrated that the first telencephalic oligodendrocyte precursors originate from ventral ganglionic eminences and entopedoncular area (Ross et al., 2003; Spassky et al., 2001; Pringle and Richardson, 1993). This assumption is based on the expression pattern of early oligodendrocyte precursor markers such PLP/DM20 and PDGF-receptor alpha and more recently of the basic-helix-loop-helix transcription factors Olig1 and Olig2 (Lu et al., 2002; Zhou and Anderson, 2002). Oligodendrocyte precursors of these restricted regions are specified by gradient concentrations Shh and BMPs, two antagonist morphogens that play pivotal roles in oligodendrocyte specification. Gradient concentrations of Shh induce the expression of genes, such as Olig2, that are required for the specification of the neural progenitors to the oligodendroglial fate within the LGE/MGE and AEP). The adult SVZ is a remnant structure of the embryonic lateral and medial ganglionic eminences (LGE and MGE) (Fig. 9.1). Progenitors cells of the LGE and MGE take tangential migration routes and several pathways were identified (Marin and Rubenstein, 2001; Corbin et al., 2001). For instance, some progenitor cells of the LGE migrate dorsally to the presumptive

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Figure 9.1. Ontogenesis of the SVZ in rodent. Schematic illustration of coronal sections of mouse telencephalon, illustrating the formation of the SVZ from E12 to P0 (A). The SVZ, shown in green is a remanent structure of lateral ganglionic eminence (LGE). Tangential migration pathways from the MGE to the LGE and cortex are indicated in red. (B): Localisation of the adult SVZ and its cellular composition. The SVZ is composed of three cell types (A, B and C). The type B cells are considered as the neural stem cells, which give rise to the transient-amplifying type C cells and then to the type A cells (neuroblasts).

dorso-lateral SVZ, where they take up residency. Some of these progenitors express the ventral forebrain marker Dlx1/2 and contribute to the expansion of the dorsolateral SVZ. While most of the Dlx1/2-expressing progenitors that migrate dorsally give rise to interneurons of the cortex, the cell fate of the entire Dlx1/2 population of the SVZ remains unknown. Based on the expression of Zebrin II (a brain specific aldolase C) two populations of cells that comprise most of the dorsolateral perinatal SVZ were identified (Marshall et al., 2003). The first population is composed of large cells that express Zebrin II and located at the border of the lateral ventricle. The second population comprise Zebrin II negative cells and are presumed to originate from ventral regions of the forebrain and migrate dorsally into the SVZ. This cell population was stained with the pan-Dlx antibody (Marshall and Goldman, 2002). Recent cell-fate mapping using the Dlx2/tauLacZ knock-in mouse identified the Zebrin II-cells as the progeny of the Dlx2 expressing cells. During the formation of the SVZ, Dlx2 þ cells intermix with the Zebrin expressing neuroepithelial cells at the corticostriatal sulcus. Then at post-natal stages, Dlx2þ progenitors of the SVZ migrate to the cerebral cortex, striatum and white matter, where they give rise to astrocytes and oligodendrocytes (Marshall and Goldman, 2002). In summary, these studies

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highlight the cellular diversity of progenitor cells of the SVZ, a concept that should undoubtedly help our molecular and cellular understanding of the adult SVZ. In vitro analysis indicate that progenitors of the LGE and early postnatal SVZ are multipotent and generate the three major cell types of the CNS. However, in vivo, many of the post-natal SVZ cells become committed to a neuronal lineage and take a tangential migratory pathway, the rostral migratory stream (RMS), to the olfactory bulb where they differentiate into granular and periglomerular neurons (Lois and Alvarez-Buylla, 1994; Luskin, 1993). Others become specified as astrocytes and oligodendrocyte precursors (Levison and Goldman, 1993; Parnavelas, 1999) or as yet remain uncommitted until their migration outside the SVZ into the corpus callosum, cerebral cortex and striatum, where they differentiate in astrocytes and oligodendrocytes. Early studies with the immunological markers of oligodendrocyte precursors, such as GD3 and A2B5 (Levine et al., 1993; Levison and Goldman, 1997) have suggested that the SVZ is a major source of the telencephalic oligodendrocyte precursors during early postnatal development (from P0 until around P15). These data were secondly confirmed with retroviral tracing of SVZ progenitors (Levison and Goldman, 1993; Luskin and Boone, 1994). From these studies it clearly appears that SVZ cells generate clones of astrocytes and oligodendrocytes in vivo. Moreover, they indicate that SVZ cells are not irrevocably specified to oligodendrocyte or astrocyte fate and their commitment may take place after their migration outside the SVZ. While the genesis of the oligodendrocytes from the adult SVZ in normal conditions is still at debate, this site account for most of the oligodendrocyte precursors during embryogenesis. However, it does not explain the emergence of the oligodendrocyte during the postnatal weeks. One possible explanation for the emergence of oligodendrocytes from the postnatal SVZ is the following one. Oligodendrocyte precursors are specified ventrally and then migrate into the forming SVZ and do not yet express common markers of the oligodendrocytes, such PLP/DM20 and PDGF-aR.

The Adult SVZ Progenitors Does Not Contribute to Oligodendrogenesis in Normal Adult Brain but are Multipotent In Vitro During adulthood, one of the major roles of the adult SVZ is to generate interneurons and periglomerular neurons within the olfactory bulb. The generation of olfactory bulb interneurons is not unique to rodents since rabbits and primates also generate neurons in the olfactory bulb (Luzzati et al., 2003; Kornack and Rakic, 1999; Bernier et al., 2000). While the SVZ is a source of oligodendrocytes during postnatal development, this activity mostly ceases in the adult. But why does the adult SVZ cease to give rise to glia? One possible explanation is that the molecular determinants that are required for glial specification are no longer expressed in this structure. As oligodendrocyte determination is controlled by morphogen signal such Shh,

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one could predict that the micro-environment of the neural stem cells in the adult SVZ are lacking this extrinsic signals or that the molecular niche created by ependymal cells are not in favor of oligodendrocyte specification. Accordingly, the Shh receptor patched (Charytoniuk et al., 2002), BMPs and their cognate receptors (Lim et al., 2000) as well as the BMP antagonist Noggin are expressed in the adult SVZ. One of the effects of BMPs signalling in the SVZ is to inhibit neurogenesis and direct glial differentiation. However, this glialpromoting effect is blocked by the antagonist Noggin secreted by ependymal cells, thereby preventing glial differentiation of the SVZ neural stem cells. Thus Noggin creates a molecular niche for adult neurogenesis (Lim et al., 2000; Doetsch, 2003). In vivo, the adult SVZ appears to be mainly neurogenic in normal condition (Lois et al., 1996). However, the possibility that oligodendrocyte progenitors may be generated from adult SVZ neural stem cells in some particular CNS insults should not be disregarded (Paterson et al., 1973). In vitro, cells from the adult SVZ are capable to generate astrocytes and oligodendrocytes indicating that progenitor cells within this germinative area are competent to differentiate along a glial pathway (Weiss et al., 1996). It was then demonstrated that the transit amplifying type C cells respond to EGF and FGF2 and constitute the main source of adult SVZderived neurospheres (Doetsch et al., 1999). EGF treatment converts Type C cell into multipotential neural stem cells, thus indicating that this transient population of cells is not totally committed to a neuronal lineage and may still bear features of multipotential stem cells. For instance, intra-ventricular injection of the anti-mitotic drug cytosine beta–D-arabinofuranoside (AraC) in the lateral ventricles leads to the elimination of type C cells and results in decreased formation of neurospheres derived from the adult SVZ (Doetsch et al., 1999). This ability of type C cells to form neurospheres is explained by the expression of the EGFR. Various levels of the RMS are sources of neurospheres capable of differentiating between astrocytes, oligodendrocytes and neurons. However, the most rostral tip of the RMS is more prone to generate oligodendrocytes than the other sites (Gritti et al., 2002). These observations indicate that environmental cues restrict gliogenesis in vivo or that there may be a subset of unidentified precursors which are more committed than the remaining population to become oligodendrocytes. In this regard, future studies on the expression of early markers of the oligodendrocytes such as Olig1 and Olig2 may help to further define the cellular composition of the adult SVZ/RMS system and provide a definitive answer on the oligodendrogliogenic potential of the intact adult SVZ.

The Adult SVZ as a Cellular Source for Cell Therapy of Myelin Lesions Cell therapy is a prominent area of investigation for treatment of neurodegenerative and demyelinating disorders and the adult SVZ is considered as a

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potential source for cell therapy (reviewed in Galvin and Jones, 2002). Indeed, adult-derived neural ‘‘stem cells’’ propagated in vitro as neurospheres in the presence of FGF and EGF retain their full plasticity in terms of their ability to generate a variety of cell types of the CNS or even to revert to immature cells which have the ability to form other tissues (Bjornson et al., 1999). Epigenic stimulation gives the ability to derive neurospheres in one particular cell type. This strategy was used to force adult neurospheres to become ‘‘oligospheres’’ enriched in oligodendrocyte precursors. Their transplantation into md rat pups lead then in extensive myelin formation with myelin repair potential similar to neonatal oligospheres (Zhang et al., 1999; Avellana-Adalid et al., 1996). We found that adult SVZ fragments containing neural stem cells and immature neural progenitors also form myelin when transplanted into the shiverer and wildtype neonate brain (Lachapelle et al., 2002). Although this capacity was limited, priming SVZ donors with growth factors such as FGF-2 and PDGF-A enhanced the amount of myelin formed by the transplanted cells. In this regard, FGF-2 was more potent than PDGF-A, and EGF treatment induced the formation of tumors. Since the adult brain is less prone to plasticity than the neonate, we also studied the effect of growth factor treatment on the adult SVZ grafted in the adult demyelinated CNS. We found that the potential of these cells to remyelinate lesions of the adult CNS was strikingly reduced. In fact, we found myelin in the adult brain only when donors were primed with FGF-2 and the number of brains in which SVZ fragments generated new myelin was 4 fold less than in the newborns subjected to the same treatment. While these experiments show that the adult SVZ is able to generate functional oligodendrocytes, they also stress the influence of the environment on the ability of competent cells to participate in repair events. The behavior of adult neural progenitors was also studied in the context of inflammatory demyelination. They were introduced by intrathecal delivery in the brain of mice affected with EAE. The grafted cells were able to penetrate the CNS parenchyma, the clinical profile was improved and the extent of demyelination was reduced (Pluchino et al., 2003). Moreover, there was a significant reduction of astrogliosis and axonal loss. However, a direct relationship between the clinical recovery and the amount of CNS remyelination performed by the grafted cells could not be found. Since neural precursors express a series of trophic factors and their transplantation results in upregulation of growth factor transcripts, the authors demonstrated that the effect of neural stem cells on EAE arose mainly through an immunomodulatory mechanism thus stressing a novel way for neural stem cells to cooperate to myelin repair (Pluchino et al., 2005). Multipotential neural stem cells were also derived from the adult human SVZ and expanded in vitro as neurospheres. However, little is known on their ability to restore myelin in the diseased brain. One experiment indicates that grafting adult human neurospheres in chemically induced chronic lesions in rodents leads to the formation of myelin, which

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surprisingly has characteristics of PNS myelin (Akiyama et al., 2001). Whether they have the ability to generate large quantities of oligodendrocytes or provide trophic support for myelin repair remains an open issue.

The Adult SVZ as an Endogenous Source of Cells for Myelin Repair The adult SVZ appears to be also of great interest to promote auto-repair of the diseased brain, since this structure contains multipotential neural stem cells, which are able to self-renew, migrate extensively and differentiate into the three major CNS cell types. In fact, proliferation and ectopic mobilization of neural precursors has been widely reported in experimental rodent models of seizure, ischemia, trauma and selective targeting of striatal neurons. In most cases, the cells mobilized by the lesion site generated new neurons or astrocytes according to the disease type reviewed in (Picard-Riera et al., 2004). The generation of newly born oligodendrocytes was rarely investigated. Until recently, the ability of SVZ progenitor cells to generate oligodendrocytes for myelin repair has not been assessed. To address whether neural stem cells or progenitors of the adult SVZ are able to respond to a demyelinating lesions, we investigated the effects of demyelination on the proliferation, migration and differentiation of the SVZ-RMS system. We used several demyelination models of the adult rodent CNS. In a first attempt, we used intra-parenchymal injections of lysophosphatidyl-choline (LPC) in the adult corps callosum distant from the lateral ventricle of the adult mouse CNS. As previously described, the injection of this gliotoxin in the corpus callosum, rostral to the SVZ induced focal demyelination, characterized by a depletion in myelin basic protein immunostaining and the presence of numerous naked axons at the injection site. In this model, demyelination is fully accomplished 2 days after LPC injection and remyelination onsets at the end of the first week. It was nearly accomplished after the third week of injection. We followed the reactivity of the SVZ-derived neural progenitors using PSA-NCAM immunohistochemistry. Interestingly, neural progenitors in the SVZ and RMS were expanded as early as 2 days after LPC injection with a peak of amplification at two weeks after demyelination. Moreover, tangential migration of PSA-NCAMþ cells seemed to be disturbed by the lesion since PSA-NCAM expressing cells emerged in the demyelinating corpus callosum as small migrating chains leaving the SVZ and the RMS. This phenomenon was specific of demyelination since it occurred in injured mice but not in controls (Nait-Oumesmar et al., 1999). These PSA-NCAM chains were often oriented radially and

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were found in close contact with astrocyte processes oriented perpendicularly to the SVZ thus indicating that the migration pattern of the SVZ cells changes in response to subcortical demyelination. Using tritiated thymidine incorporation combined with immunological markers for glial cells, we demonstrated the fact that SVZ progenitors were able to reach the lesion where they generated astrocytes, few oligodendrocytes but no neurons. Recruitment and differentiation at the lesion site occurred in due time with respect to the chronology of repair of lysolecithin induced lesions, suggesting their contribution to myelin repair of the lesion. Since lysolecithin-induced demyelination requires injection of the gliotoxin in the brain, therefore creating a small trauma, which is likely to participate to the SVZ reactivation we explored this phenomenon in models in which CNS demyelination is induced by feeding animals with a Cuprizone diet, therefore, allowing to rely the SVZ reactivation essentially to demyelination. Cuprizone is a mitochondrial cytochrome oxydase inhibitor that leads to a selective loss of the oligodendrocytes, when administrated at a concentration of 0.2% (Matsushima and Morell, 2001). In this model, large lesions of myelin prevail in the corpus callosum. Demyelination is fully accomplished after 6 weeks of Cuprizone feeding and spontaneous remyelination, is achieved within 4 weeks following the end of the treatment (Jurevics et al., 2001; Arnett et al., 2002). We found that the SVZ of Cuprizone-treated animals were expanded at 4 and 6 weeks of the Cuprizone diet and this expansion lasted throughout the remyelination period. Immunostaining for PSA-NCAM showed the presence of numerous neural progenitors in the corps callosum with small chains of PSA-NCAM, leaving the SVZ and RMS to reach the demyelinated white matter. Immunolabelling with various cell type specific markers indicated that PSA-NCAM positive cells detected in the lesioned corpus callosum were mainly astrocytes and few cells of this population expressed markers of the oligodendrocyte precursors, such as Olig1 (Fig. 9.2), and NG2 proteoglycan. The location of these cells in the corpus callosum suggested that they were issued from the SVZ. To unambiguously confirm the origin of these cells and their fate, we are currently developing a selective sable labeling of the SVZ progenitor cells that will allow us to follow their fate in demyelinating conditions. Interestingly experiments performed with retroviral labeling of the adult SVZ cells in mouse indicate that emigrated SVZ cells also differentiated into oligodendrocytes in the corpus callosum and cortex in response to cortical injury (Goings et al., 2004). The lysolecithin and Cuprizone lesions were mainly localized in the vicinity of the SVZ and RMS pathway. Therefore, it seemed interesting to explore the potentialities of the SVZ in models reflecting the multifocal pathology of multiple sclerosis (MS). In experimental allergic encephalitis (EAE)-induced mice, the closest model of multi-focal auto-immune demye-

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Figure 9.2. Re-activation of the adult SVZ after Cuprizone induced demyelination (A) Amplification of the SVZ as evidenced by PSA-NCAM immunostaining, at 6 weeks after Cuprizone diet; (B) Control SVZ stained for PSA-NCAM; (C) Gliosis is detected around the lateral ventricle and (D) in Cuprizone-induced demyelinating lesions of the corpus callosum. (E) Immuno-detection of PSA-NCAMþ cells (red) and Olig1þ oligodendrocyte precursors (green) in the corpus callosum adjacent to the lateral ventricle and (F) in the lesions of the corpus callosum of Cuprizone feeded mice.

lination to MS, demyelinated lesions spread in the entire CNS white matter. Tracing SVZ cells with BrdU, we found that SVZ cells were mobilized not only in the corpus callosum but also in the fimbria and the striatum (Picard-Riera et al., 2002). However, cells were not recruited by white matter areas more remote from the SVZ such as the cerebellum suggesting that outside of the normal pathway of migration SVZ cells have a more limited capacity of migration in the adult CNS. Moreover, inflammatory demyelination potentiated the migration of the neuroblasts migrating

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through the RMS into the olfactory bulb. We also found that the differentiation potential was dependent of the structure and the disease type. In mice injected only with the inflammatory inducer, mobilized cells differentiated into astrocytes in gliogenic areas such as the corpus callosum and in neurons, in the neurogenic olfactory bulb. However, in EAE mice, SVZ cells differentiated in astrocytes and oligodendrocytes in gliogenic areas and in astrocytes and oligodendrocytes in addition to neurons in the olfactory bulb. While the newly born oligodendrocytes could remain the target of other demyelinating insults, we found that the newly generated oligodendrocytes survived at least 3 weeks after EAE induction and we were unable to detect apoptotic cells assessed by tunnel reaction within this time frame (Picard-Riera, pers. comm.). It appears from these multiple studies that oligodendrogenesis clearly occurs in rodents in response to demyelination. However, the nature of the reactivated cells is unknown. While it is clear that PSA-NCAMþ cells (Type A cells) are found within the lesions, the possibility that other cell types such type C cells or type B are also activated remains to be elucidated. The tracing studies also indicate that cells from the SVZ have the capacity to undergo neurogenesis and gliogenesis according to lesion-specific signals. These signals vary with the lesion type and location. The nature of the signals that trigger reactivation and those involved in cell specification remains to be unraveled. The contribution of the SVZ in myelin repair seems geographically restricted to areas surrounding the germinative zone. Moreover, migration and cell replacement remain limited, implying that strategies aiming at promoting SVZ-derived migration and differentiation will have to be developed to achieve successful auto-repair of the demyelinated CNS.

Migration Pattern of the Adult SVZ in Response to Demyelination The SVZ is certainly the most striking germinative zone of the adult CNS due to the migrative properties of its resident cells (Lois and Alvarez-Buylla, 1994; Lois et al., 1996). During development, new neurons migrate from their site of genesis to their final destination following radial or tangential pathways and migrate according to the gliophilic or axonophilic modes. In the gliophilic mode, cells slide along extensions of radial glia while in the axonophilic mode, they migrate along axons. In the adult SVZ, cells retain the capacity of tangential migration over long distances (3-5 mm) in the rostral migratory stream (RMS) to reach the olfactory bulb (Lois and Alvarez-Buylla, 1994). Rather than migrating according to the axonophilic or gliophilic modes, cells use homophilic interactions leading to migrationin-chain of cells enchased in an astroglial furrow (Jankovski and Sotelo, 1996; Lois et al., 1996; (Peretto et al., 1997). According to the classification proposed by Alvarez-Buylla and colleagues, the migrating cells are type A

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neuroblasts which express in addition to nestin and beta3-tubulin, a repertoire of cell-adhesion molecules such as PSA-NCAM, CD24, CD100, doublecortin, Ephrins, integrins (Murase and Horwitz, 2002), Slit1 and Slit2 (Nguyen-Ba-Charvet et al., 2004), all of which play a role in their ability to migrate towards the olfactory bulb. While PSA-NCAM facilitates neuroblast migration in the astroglial tube (Tomasiewicz et al., 1993; Chazal et al., 2000), the interaction of extracellular matrix molecules with integrins such as avb3 and avb6 (Murase and Horwitz, 2002) seems to act on cell translocation and the complex a6b1 on directed migration (Emsley and Hagg, 2003). While the astroglial furrow does not seem to be necessary for chain migration, it seems to contribute to the directed migration of neuroblasts to the olfactory bulb by secreting tenascin C, which would prevent cell dispersion from the chains, and Mia, a factor which induces the migration activity of the neuroblasts (Mason et al., 2001). Chemorepulsive factors such as Slit 1/2, which are secreted by the septum, may also help targeting the neuroblast to the olfactory bulb (Hu, 2001; Wu et al., 1999). However, the recent finding that Slit 1/2 are also expressed in the SVZ and the RMS, suggests that these molecules may participate in the chain orientation since their disruption leads to caudal migration of SVZ cells and cell dispersion in the RMS (Nguyen-Ba-Charvet et al., 2004). Other molecules such as tenascin R and reelin control the dispersion of neuroblasts arriving in the olfactory bulb (Rochefort et al., 2002). Thus, the olfactory bulb-directed migration seems to result from the combination of multiple repulsive-attractive/inhibitorystimulatory factors (Mason et al., 2001), some of which remain to be unraveled (Pennartz et al., 2004). While the fore-mentioned studies unraveled the cellular and molecular mechanisms involved in the SVZ cell migration in the intact brain, studying their modulation in pathological situations may help to gain further insights on the precise role of these molecules in neuroblast migration as well as to identify ways to promote migration of SVZ-derived precursors to lesions of the diseased CNS. From the above studies in animal models of demyelination, it is obvious that demyelination triggers mobilization of SVZ or RMSderived cells in the adjacent white matter tissue thus changing the orientation of cell migration as well as modifying the pattern of migration of the neuroblasts from a chain to a radial mode. The mechanisms allowing the migrating cells to deviate from the RMS and to be recruited by the lesion are not yet understood. These modifications must undoubtably induce cellular and molecular changes. Disruption of the astrocyte furrow seems to occur in response to demyelination providing leakiness in the astroglial channels and helping chain dispersion (Nait-Oumesmar et al., 1999). However, this is unlikely to be the sole mechanism involved. Since PSA-NCAM is highly expressed by the migrating neuroblasts (Bonfanti and Theodosis, 1994) and plays a crucial role in the chain migration in vitro (Decker et al., 2000; Hu, 2000) and in vivo (Cremer et al., 2000; Hu, 2000; Ono et al., 1994; Tomasiewicz et al., 1993), it appeared that studying its modulation in the SVZ in

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response to CNS demyelination seemed to be a reasonable goal to achieve. In fact, deletion of PSA-NCAM generates irregular homophilic interactions thereby favoring the emergence of specialized heterophilic junctions between the migrating neuroblasts and the astrocytes of the glial tube (Chazal et al., 2000). Thus, PSA residues may favor chain formation by limiting heterophilic (astrocyte/neuroblast) interactions and enhancing homophilic (neuroblast/neuroblast) interactions, allowing cells to slide on each other in the astroglial tubes. We induced demyelination in the NCAM-/-mice and found that such as in wild-type mice, the migration of SVZ cells was delayed in the olfactory bulb. However, unexpectedly, their recruitment was enhanced in the lesion either due to a traffic jam in the RMS at the level of the lesion, and/or possibly to chain disruption helping their exit from the RMS and their recruitment by the lesion. Recent in vitro and in vivo experiments showed that the absence of PSA-NCAM enhanced neural progenitors to mature into NG2þ, CNPþ or GalCþ oligodendrocytes, thus indicating that PSA on NCAM may also promote migration by preventing premature differentiation of the migrating cells in a phenotype which capacities of migration are reduced (Chazal et al., 2000; Decker et al., 2002a). In fact, in demyelinated animals, the deletion of PSA enhanced the differentiation of the recruited cells in oligodendrocytes. This unexpected finding suggest a novel role for PSA in oligodendrocyte differentiation which could be mediated through cell signaling via integrins and/or growth factor receptors and their ligands in the lesions, all of which are known to modulate the biology of oligodendrocytes (Colognato et al., 2002). Finally, comparing demyelination in the NCAM-/- mice or after removal of PSA by endoneuraminidase injections in wild-type mice, provided insights for a differential effect of NCAM and PSA on neural precursor behavior in response to demyelination. While NCAM plays a role in neural progenitor proliferation, PSA facilitates their migration and prevents their premature differentiation (Decker et al., 2002a).

Effect of Growth Factors on SVZ Activation Several growth factors regulate the proliferation, mobilization and fate of the SVZ cells. Receptors to Ciliary Neurotrophic Factor (CNTF), Insulinlike Growth factor (IGF I), Epithelial Growth factor (EGF) and Fibroblastgrowth Factor (FGF) are abundantly expressed in the SVZ (Doetsch, 2003; Emsley and Hagg, 2003). All of these growth factors promote SVZ proliferation when delivered in the rodent brain (Craig et al., 1996; Kuhn et al., 1997; Wagner et al., 1999). In addition to promoting the SVZ proliferation, treatment with EGF and FGF-2 triggers the mobilization of the adult SVZ cells in the adjacent parenchyma (Craig et al., 1996) and BDNF and FGF-2 treatment induce the differentiation of the SVZ precursors in neurons

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(Zigova et al., 1998) as well as glia including oligodendrocytes (Craig et al., 1996; Lachapelle et al., 2002). CNS injury induces changes in the SVZ growth factor gene expression (Arvidsson et al., 2001) and administration of growth factors enhances CNS injury-induced proliferation in the SVZ, mobilization of SVZ cells in the injured parenchyma and their differentiation in neurons (Dempsey et al., 2003; Fallon et al., 2000; Matsuoka et al., 2003; Wada et al., 2003). Since demyelination in the forebrain triggers proliferation in the SVZ, we investigated the effect of growth factor delivery on SVZ proliferation and recruitment by lysolecithin-induced demyelination in the corpus callosum. One single intraperitoneal injection of FGF-2 not only enhanced the proliferation of SVZ and RMS cells but also favored their recruitment at the lesion site (Decker et al., 2002b). Although long-term intrathecal administration of FGF-2 promotes SVZ-derived mobilization and oligodendrogenesis in the intact brain (Craig et al., 1996; Lachapelle et al., 2002), its role in SVZderived oligodendrogenesis in response to demyelination has not been studied so far. A recent study showed that injection of NGF into a EAE-injured brain is translocated from the SVZ to the parenchyma and is associated with differentiation of oligodendrocytes (Aloe and Micera, 1998). Whether FGF2 also remains associated to its receptor and transported by a similar mechanism to the demyelination site should be further investigated. Several studies have highlighted the differences that exist between young and aged neural precursors in terms of proliferation and migration capacities in normal conditions. While proliferation persists in the SVZ during adulthood (Levison et al., 1999), there is a dramatic age-dependent decrease in proliferation in the SVZ (reviewed in Chen et al., 2003; Maslov et al., 2004) with few cells proliferating in the SVZ and rare ones in the RMS (Decker et al., 2002b; Mirich et al., 2002; Tropepe et al., 1997). Senescence-induced decrease in proliferation of the SVZ cells occurs but cell density remains unchanged, suggesting the lengthening of the cell cycle of the aging neural progenitors (Tropepe et al., 1997). As telomerase is active in adult neural precursors and as its activity may reflect the number of SVZ cells (Caporaso et al., 2003) one cannot exclude a variation in its regulation during aging. Despite a lower proliferation activity, SVZ cells from aged brains seem to retain the same capacity of migration than in young adults in vitro. Moreover, aged brains retain a similar ability to respond to exogenous growth factors than young adult ones. IGF-1 infusion in senescent rat brain restores the size of the RMS and neurogenesis (Lichtenwalner al Neuroscience 01). Infusions (Tropepe et al., 1997), intraperitoneal injections of FGF-2, TGF-a, and EGF (Decker et al., 2002b) and intracerebroventricular administration of FGF-2 and HB-EGF (Jin et al., 2003) restores the proliferation rate of SVZ cells in aged animals to that of young adults. Moreover, these exogenous growth factor treatments not only play a role on SVZ cell proliferation, but also modify the functional behavior as meas-

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ured by the increase of ChAT and BDNF synthesis after the combined intracerebroventricular injection of NGF and EGF (Tirassa et al., 2003). We investigated the role of age on the migration and proliferation capacities of the adult SVZ cells in response to demyelination and showed that aged neural precursors from the SVZ maintain a lower rate of proliferation compared to young ones in response to demyelination, and the recruitment by the lesion were less efficient in aged mice than in young ones (Decker et al., 2002b). While the capacity of aged neural precursors to respond to growth factor treatment opened new therapeutical perspectives in age-associated pathologies, FGF-2 treatment stimulates young neural precursor recruitment to the lesion but not aged ones. Since the migration capacity of the aged neural progenitors was not affected in vitro, we speculated a loss of the capacities of the neural precursors to respond to demyelinationinduced signals or modifications of the aged extracellular matrix were responsible for the observed defect (Decker et al., 2002b). Indeed, changes in ECM with age (Pagani et al., 1991) can directly affect cell-cell contacts and transmembrane complexes that interact with the cytoskeleton and can therefore alter cell response to growth factors and consequently signaling involved in cell migration.

What is the Relevance of the SVZ in Human Myelin Diseases? The SVZ is also present in the adult human brain. SVZ cells express PSANCAM, the EGFR and are multipotent in vitro (Bernier et al., 2000; Kirschenbaum et al., 1994; Pincus et al., 1998; Alvarez-Buylla, 2000). However, it seems to differ from the rodent structure in shape as well as organization. Indeed, Bernier and colleagues (2000) highlighted the heterogeneity in the thickness of the adult human SVZ, with single rows of cells present in the dorsal part and multiple rows in the ventral part. Moreover, AlvarezBuylla and colleagues reported that the adult human SVZ is essentially constituted of a ribbon of GFAP-positive astrocytes lining the lateral ventricles that proliferate in vivo and behave as multipotent progenitor cells in vitro (Sanai et al., 2004). Surprisingly, PSA-NCAMþ cells do not seem to form chains of migrating cells or RMS-like structure in the non-injured human brain. One could, therefore, speculate that the modes of migration of these cell ribbons may differ from the rodent species. Although the presence of stem cells in the adult human brain has opened new therapeutic avenues for the treatment of neurological diseases, these observations may have some barring on the capacity of SVZ cells to be mobilized in the injured parenchyma. The involvement of these pools of neural stem in auto-repair of the diseased human brain was only recently addressed. A recent post-mortem analysis of the SVZ from Huntington disease-afflicted patients showed increased PCNA staining in this region which correlated with the severity

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of the disease (Curtis et al., 2003). However, proliferation and neurogenesis was decreased in the SVZ of patients affected with Parkinson’s disease (Hoglinger et al., 2004). Our preliminary data indicate that cells of immature phenotype expressing PSA-NCAM are present in MS lesions and that cellular activation of the SVZ seems also to occur. Further investigation should determine whether PSA-NCAMþ cells in the lesions originate from the SVZ and if these cells are more likely to proliferate and differentiate in myelin-competent cells than the oligodendrocyte progenitors recently identified in MS lesions (Chang et al., 2002; Wolswijk, 2000). Although these pioneer studies clearly show that the adult human brain contains cells with great plasticity, future studies should aim at gaining insights in their migration and differentiation potential in the demyelinated brain, as well as in the signals involved in their recruitment and remyelination process (AlvarezBuylla and Lim, 2004; Fuchs et al., 2004; Wagers and Weissman, 2004). Acknowledgment. We are grateful to Franc¸ois Lachapelle, Virginia Avellana-Adalid, Danielle Pham-Dinh, Roland Liblau and current numbers of Anne Baron-Van Evercooren’s lab for helpful discussions and critical reading of this manuscript. Studies cited in this chapter have been supported by grants from INSERM, MNRT, ARSEP, ELA, AFM, FRM and Myelin Project (USA).

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Chapter 10 Auxiliary Proliferative Zones in the Developing and Adult Central Nervous System: Lessons from Studies on the Effects of Ethanol Michael W. Miller1,2 and Marla B. Bruns1

Introduction Early exposure to alcohol is a major health problem; estimates are that alcohol affects the well-being of as many as 3% of all infants (e.g., Abel and Sokol, 1992; Abel and Hannigan, 1995; Sampson et al., 1997; West et al., 1998). The array of defects and deficits is broad, including anatomical, physiological, and behavioral abnormalities. The consequences of prenatal alcohol exposure include microencephaly, a constellation of characteristic craniofacial malformations (including short palpebral fissures, the absence of a philtrum, a thin upper lip, and a narrow nasal bridge), deficits in learning and memory, and mental retardation (MR) (Lemoine et al., 1968; Smith and Jones, 1973; Astley and Clarren, 1995, 2001). Indeed, fetal alcohol is the major cause of MR in the Western world (Abel and Hannigan, 1995; Sampson et al., 1997). Children with fetal alcohol-induced problems also have high incidences of attention deficit hyperactivity disorder (e.g., Weinberg, 1997; Coles, 2001; O’Malley and Nanson, 2002) and autism (Nanson, 1992; Miles et al., 2003). Clinical abnormalities and dysfunction caused by ethanol result from disturbances in basic developmental processes. A prime target of ethanol is a proliferating cell (Miller, 1992a; Miller and Luo, 1998). Ethanol-induced changes in cell proliferation are site- and time-dependent. This chapter will explore the effects of the ethanol on the structure and function of the cortical proliferative zones. Studies conducted on ethanol provide unique insight into the activity of the various proliferative zones. 1 Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse NY13210 2 Research Service, Veterans Affairs Medical Center, Syracuse NY13210 Mailing Address: Michael W. Miller, Department of Neuroscience and Physiology SUNY-Upstate Medical University, 750 East Adams Street, Syracuse NY 13210, Tel.: 315-464-7729, Fax: 315-464-7725; E-mail: [email protected]

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Sites of Cell Proliferation in the Forebrain Neurons are produced in the developing and adult brain. This production is not ubiquitous and haphazard. Rather, it is highly organized and occurs within discrete sites in the central nervous system (CNS). Four segments of the CNS have been identified as neuronal generators: the ventricular zone (VZ), the subventricular zone (SZ), the intrahilar zone (IHZ), the ganglionic eminence (GE), and the external granule cell layer (EGCL). Embryologically, the VZ is the first proliferative zone to appear. The VZ is a pseudostratified epithelium that forms the lining of the neural tube and its derivatives, the ventricles (Fig. 10.1) (e.g., Sauer, 1935, 1936; Seymour and Berry, 1975; Miller, 1989; Bayer and Altman, 1991). Thus, all cells contact the basement membrane at the ventricular surface, though not all cells reach the apical limit of the VZ. Proliferating VZ cells pass through the cell cycle in an organized manner (e.g., Sauer, 1936; Watterson et al., 1956; Sauer and Chittenden, 1959; Jacobson, 1991). Mitotic cells are located along the basement membrane and cells in the S phase are distributed in the external (apical) third of the zone. Cells passing through the G1 and G2 phases are found in intermediate positions. This organization results from the rhythmic movement of the nucleus as VZ cells course through the cell cycle, so called interkinetic nuclear migration. The VZ is evident through development and adulthood. [n.b. In the adult, the VZ is also referred to as the ependymal zone.] In contrast to the VZ, the SZ, IHZ, GE, and EGCL are derived proliferative zones. That is, these are not evident in the early fetus and the cells in these zones are seeded by VZ cells. Each of the derived zones is a true, stratified epithelium (e.g., Ramon y Cajal, 1909–1911; Shoukimas and Hinds, 1978; Palay and Chan-Palay, 1974; Rakic et al., 1974; Doetsch et al., 1997. This means: (a) that all constituent cells do not contact a basement membrane and; (b) that there is no apparent cell cycle-associated organization, i.e. cells in the four stages of the cell cycle are intermingled and distributed throughout its depth (Fig. 10.1). Derived proliferative zones are not distributed throughout the CNS; each zone is site-specific. The SZ, IHZ, GE, and EGCL are associated with the neocortex, dentate gyrus, striatum, and cerebellum, respectively. There are dramatic changes in these derived zones during development. In fact, the EGCL is only evident during development. In summary, there are distinct differences between the VZ and the derived zones, e.g., the organization, distribution, and history of the zones. Presumably, these differences are indicative of specific roles played by the various zones. A question that has intrigued developmental neurobiologists is what cells the VZ and the derived zones generate.

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Figure 10.1. Neocortical proliferative zones. These micrographs show the cortical proliferative zones on gestational day (G) 13 (Top), G17 (Middle), and G20 (Bottom). The cerebral wall in normal rats contains two proliferative zones that are located near the ventricular surface, the ventricular zone (VZ) and the subventricular zone (SZ). The VZ contains the radially oriented elongate somata of cycling cells and the SZ comprises a stratified epithelium with pleomorphic cells. Mitotic cells (arrows) are located at the ventricular surface of the VZ and throughout the SZ. Prenatal

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Neural Proliferation During Normal Development Ventricular Zone Our contemporary understanding of the roles of the various proliferative zones began with studies at the end of the nineteenth century. Early neurobiologists described the changing structure of the VZ (e.g., His, 1887; Schaper, 1897; Ramon y Cajal, 1894) and identified mitotic cells in the VZ and the large representation of the VZ in the fetal cerebral wall. Events defining the proliferation of VZ cells were only identified after the advent of tritiated thymidine autoradiography (Leblond and Walker, 1956; Sidman et al., 1959; Sauer and Walker, 1959; Angevine and Sidman, 1961; Martin and Langman, 1965. Autoradiography was used to trace the movement of the nuclei of proliferating cells and assess cell cycle kinetics by measuring the temporal change in the frequency of radiolabeled mitotic cells (e.g., Quastler, 1959; Atlas and Bond, 1965; Waechter and Jaensch, 1972). The dynamics of cell proliferation is best appreciated in neocortex. The VZ is the dominant zone during the first half of neocortical neuronogenesis (e.g., Miller, 1989; Bayer and Altman, 1991)(Fig. 10.2). In the rat cerebral vesicle, the VZ is deepest between gestational day (G) 16 and G18. After G18, the VZ thins, and by G21 (the end of neocortical neuronogenesis in normal rats), it is half its maximal depth. In developing rodents, at the beginning of the neuronogenetic period, virtually all VZ cells are actively cycling (Takahashi et al., 1993). Over time, however, this, number falls so that by the end of neuronogenesis, only 80% of the VZ cells are cycling (Miller and Nowakowski, 1991). The length of the cell cycle increases as neuronogenesis proceeds. At the beginning of neuronogenesis, a VZ cell in the mouse takes 8 hours to transit through the four phases of the cell cycle (Takahashi et al., 1993). At the end, a VZ cell takes 19 hours (Miller and Nowakowski, 1991).

Subventricular Zone In the rat, the SZ appears in the cerebral wall at the end of the second week of gestation as a thin layer external to the VZ (Miller, 1989). By G17, it doubles in depth and it becomes the predominant neocortical proliferative zone. A cumulative labeling method with bromodeoxyuridine (Nowakowski et al., 1989) shows: (a) that the total length of the cell cycle (TC ) in rat neocortical SZ on G21 is 16 hours; and (b) that the frequency of actively cycling cells (the growth fraction—GF) is 0.12 (Miller and Nowakowski, exposure to ethanol affects both zones. Though the general appearance and organization of the two zones appears normal in control (Ct) and ethanol-treated (Et) fetuses, the VZ is thinner and the SZ is wider in Et-treated fetuses. Scale bars are 50 mm. With permission from Miller, M.W. (1989). J. Comp. Neurol. 287: 326–338.

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Figure 10.2. Effect of ethanol on the ventricular zone Top. The radial depth of the ventricular zone was measured in fetuses from dams fed an ethanol-containing diet (Et), a control diet (Ct), or chow and water (Ch). This depth varies during the period of cortical neuronogenesis, i.e., between gestational day (G) 13 and G23, and it is

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1991). This number is substantially lower than it is for the VZ because the SZ is more heterogeneous. For example, in addition to proliferating cells, it contains VZ derivatives that are radially migrating through the SZ to their ultimate residence in the cortical plate.

Ganglionic Eminence The GE, a modified segment of the telencephalic SZ, gives rise to cells that will become neocortical local circuit neurons (LCNs) (e.g., Tamamaki et al., 1997; Zhu et al., 1999; Anderson et al., 2001; Wichterle et al., 2001). The GE has at least two compartments, the medial GE and the lateral GE. The former is the source of the cortical LCNs and the latter generates striatal neurons (e.g., Marin et al., 2000; Letinic et al., 2002; Stenman et al., 2003; Xu et al., 2004). Neurons from the medial GE migrate tangentially into cortex along one of several pathways, most of which are through the VZ, SZ, and IZ (Zhu et al., 1999; Jimenez et al., 2002; Powell et al., 2001; Ang et al., 2003). Subsequently, these neurons associate with radial glia and migrate into the cortex via an inside-to-outside sequence (Miller, 1985, 1986a, 1992b; Faire´n et al., 1986; Cobas and Faire´n, 1988; Jackson et al., 1989). This radial migration proceeds along with the co-generated projection neuron cohorts derived from the VZ and SZ (Miller, 1985, 1987a, 1988a, 1997).

Effects of Ethanol upon the Proliferation of Cortical Cells Overall Effects Chronic prenatal exposure to moderate amounts of ethanol (that result in blood ethanol concentrations of 80–150 mg/dl) does not grossly affect the neocortical proliferative zones in fetal rats. Both zones are discriminable and the total thickness of the proliferative zones is unaffected (Kennedy et al., 1985; Miller, 1989). On the other hand, some specific effects are evident. The cycling activity of cells garnered from longitudinal studies of the cerebral

reduced by prenatal exposure to ethanol. With permission from Miller, M.W. (1989). J. Comp. Neurol. 287: 326–338. Bottom. A cumulative labeling method with bromodeoxyuridine (BrdU) was used to determine cell cycle kinetics for fetuses on G21. Pregnant dams were injected with BrdU every two hours and fetuses were collected half-hour post-injection. The curves depict the change in the frequency of BrdU-labeled cells in the VZ and the table below describes the derived data. With permission from Miller, M.W., Nowakowski, R.S. (1991). Alcohol Clin. Exp. Res. 15: 229–232. Each datum represents the mean ( the standard errors of the means) of four independent measures. Asterisks denote statistically significant (p < 0.05) differences.

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wall is altered (Miller and Kuhn, 1995). Whereas in normal rats the TC increases from 11 to 17 hr over the period of neocortical neuronal generation, in ethanol-treated rats the TC remains steadily at 17–19 hr throughout neuronogenesis. A second factor that defines cycling activity is the GF. GFs of 1.0 and 0 mean that all or none of the cells, respectively, are cycling. In normal rats, the GF falls 15-fold from 0.9 on G13 to <0.1 on G21 (Miller and Kuhn, 1995). A decline is also evident for ethanol-treated rats, so that on G21 the GF is 0.2. Interestingly, from G17 on, the GF for the ethanol-treated rats is significantly greater than the GF for the controls. These in vivo changes in cycling activity are remarkably similar to those described in cell culture studies of the cerebral wall (Jacobs and Miller, 2001). Post-proliferative activity is affected by ethanol. For example, the time cells remain within the proliferative zones before commencing their migrations (the release time) is significantly increased by ethanol (Miller, 1993). This effect, however, is only evident for late-generated neurons. The combination of the TC , GF, and release time contribute to a proliferation index. This index falls in control rats during the period of neocortical neuronal generation (Miller, 1988b). Likewise, the proliferation index decreases in the ethanol-treated rats with two notable differences: the index is generally lower in the ethanol-treated rats and there is a late surge in the index during the waning days of neuronogenesis. The above data represent the sum of activity in the VZ and SZ. Various studies have explored specific effects of ethanol on these two neocortical proliferative zones.

Ventricular Zone The basic organization of the VZ is unaffected by ethanol, implying that interkinetic nuclear migration continues in ethanol-treated rats (Miller, 1989). That said, ethanol does affect the transit time through the cell cycle. In rats, VZ cells take 25% longer to pass through the four phases of the cell cycle (Miller and Nowakowski, 1991). Ethanol also reduces the number of cells in the VZ (Miller, 1989), though it does not affect the proportion of cycling VZ cells (Miller and Nowakowski, 1991). The net result of a smaller and more slowly cycling population is that ethanol compromises the product of the VZ. The proliferation of VZ cells at other levels of the neuraxis is depressed by ethanol. This is most easily examined in parts of the brain where the VZ is the only source of neurons (e.g., pons). The generation of neurons in the principal sensory nucleus of the trigeminal nerve (PSN) is reduced by prenatal exposure to ethanol (Miller and Muller, 1989; Miller, 1995a, 1999). As in many structures of the developing CNS, an excess of neurons is produced in the PSN (Miller and Al-Ghoul, 1993). Ethanol reduces the peak number of neurons in the PSN by 20% (Miller, 1999). The generation

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of Purkinje cells in the cerebellum also appears to be reduced by prenatal exposure to ethanol (Bauer-Moffett and Altman, 1977). Purkinje cells are derived from the rhombencephalic VZ (Uzman, 1960; Miale and Sidman, 1961; Inouye and Murakami, 1980). Likewise, prenatal exposure to ethanol depresses proliferative activity in the hippocampal VZ (Miller, 1995b).

Derived Proliferative Zones The SZ in ethanol-treated rats is deeper and contains more cells than does the SZ of controls (Fig. 10.3)(Miller, 1989). Moreover, the proportion of cycling cells in the SZ and the frequency of mitotic cells in the SZ are greater in ethanol-treated rats (Kennedy et al., 1984; Miller, 1989; Miller and Nowakowski, 1991). The length of the cell cycle for SZ cells, however, is not affected by ethanol. Taken together, cell production in the SZ is increased by ethanol exposure. This increase in SZ cell proliferation links with the increases in the number of neurons born late in the period of neuronogenesis (Miller, 1986b, 1988b). Non-SZ derived proliferative zones are also affected by ethanol. Early postnatal exposure to ethanol (between postnatal day (P) 4 and (P) 10) affects IHZ cell proliferation in a dose-dependent manner (Miller, 1995b). Exposure to a low dose (125 mg/dl) stimulates cell proliferation and leads to an increase in the number of the granule cells in the dentate gyrus. These are the same doses that stimulate SZ cell proliferation (Miller and Nowakowski, 1991). In contrast, high doses (300 mg/dl) lead to a reduction in cell proliferation and a reduction in granule cell number. We do not know whether these changes in cell proliferation reflect changes in the TC or GF, but if it is like the neocortical SZ, we can predict that the ethanol-induced change in granule cell production results from an increase in the GF at low doses and an increase in the TC at high doses. The effects of ethanol on cell proliferation in the EGCL of the cerebellum has been examined (Bauer-Moffett and Altman, 1977; Borges and Lewis, 1983). Ethanol hampers cell proliferation in this germinal zone. Using percent-labeled mitoses method, it was shown that the cell cycle is 21% longer in ethanol-treated rats than in controls. Most of this change reflects an increase in the length of the G1 phase. No direct studies have examined the effects of ethanol on the GE or its derivatives. It appears, however, that cell production in the GE is compromised by ethanol. MRI studies of children with fetal alcohol syndrome (Mattson et al., 1994, 1996; Clark et al., 2000; Archibald et al., 2001) and biochemical and anatomical studies of rats prenatally exposed to ethanol (Frumkina, 1985; Clarren et al., 1990; Mattson et al., 1994; Nagahara and Handa, 1995) reveal a smaller, dysfunctional basal ganglia. It is unknown whether this effect is caused by a reduction in cell proliferation, an increase in cell death, or both. This demands careful morphological analyses of the

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Figure 10.3. Effect of ethanol on the subventicular zone. See notations for Fig. 10.2.

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developing striatum. Recent data show that prenatal exposure to ethanol does not alter the expression of the local circuit neurons in monkey somatosensory cortex (Miller, submitted). This may be indicative of an equivalent effect of ethanol on the GE relative to the VZ and SZ. No studies have examined the effects of early exposure to ethanol on the population of neocortical local circuit neurons.

Contributions of VZ Cells, SZ Cells, and Other Proliferative Cells to Populations of Cortical Cells Opposing Concepts An early understanding of neocortical neurogenesis was that the primary roles of the VZ and SZ was to generate neurons and glia, respectively (e.g., Berry, 1974; Smart and McSherry, 1982; Bayer and Altman, 1991). This conclusion was based upon the timing of the prominence of the VZ early in neocortical neuronogenesis and later prominence of the SZ at a time when primarily, or solely, glia are generated. Various studies have called this understanding into question, including an analysis of the proliferation among cultures of presumptive VZ cells and neurospheres of SZ cells (Martens et al., 2000). Accordingly, cells from the VZ are solely a self-replacing population in which cell generation is matched by cell death. SZ cells, in contrast, are responsible for generating the neurons and glia in neocortex. These studies concur with ideas proposed by Alvarez-Buylla and colleagues who studied the anterior SZ in the adult (Doetsch et al., 1997; 1999; AlvarezBuylla et al., 2002). Furthermore, these investigators conclude that neurons are derived from GFAP-positive glia distributed in the internal segment of the SZ. It is unclear whether these data for the adult, anterior SZ can be generalized to the developing CNS.

Insights from Ethanol Studies An alternative conceptualization for the generation of neocortical cells is based on studies of ethanol-treated rats. These studies argue that neocortical neurons are generated by both the VZ and the SZ. In normal rats, neocortical neuronogenesis occurs over a protracted period; it begins on G11 and ends on G21 (e.g., Al-Ghoul and Miller, 1989; Bru¨ckner et al., 1976; Lund and Mustari, 1977; Miller, 1985). Ethanol delays this period by one day and extends it two days to G23 (Miller, 1986b, 1988b; Miller and Robertson, 1993a) (Fig. 10.4). The effects of ethanol are heterogeneous. Ethanol depresses neuronal generation between G12 and G19 and stimulates neuronogenesis between G20 and G22 (Miller, 1986b, 1988b). This parallels changes in the proliferative activity in the VZ and the SZ (Miller, 1989, 1996). That

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Figure 10.4. Effect of prenatal exposure to ethanol on neuronal generation. This plot depicts the numbers of cortical cells in mature rats that were heavily radiolabeled with an injection of [3 H]thymidine on a single day during the period from G12 to G23. For ethanol-treated rats, separate counts were made for cells that were appropriately (layer II/III) and inappropriately distributed (other laminae). Ethanol delayed and extended the period of neuronal generation and it caused substantial numbers of late-generated neurons to end up in ectopic sites. The points and T-bars signify the means and standard errors of the means for four samples. With permission from Miller, M.W. (1986). Science 233: 1308–1311.

is, exposure to ethanol throughout the period of neocortical neuronogenesis or selectively during the period of VZ prominence: (a) depresses VZ proliferation; and (b) reduces the number of neurons in the adult. In contrast, exposure to ethanol during the period of SZ prominence stimulates SZ proliferation and underlies a surge in neuronal production during the waning days of neuronogenesis (Miller, 1986b, 1988b). The effects of ethanol on the laminar distribution of neurons with particular times of origin provides insight into the site of origin of neuronal subpopulations. Early-generated neurons, which become deep neocortical neurons, are VZ derivatives, whereas late-generated neurons, which normally populate superficial laminae, are SZ derivatives (Miller, 1986b, 1989, 1996). Interestingly, the VZ derivatives in ethanol-treated rats most often find their way to a proper site in cortex. SZ derivatives are not as successful. For example, in control rats, neurons generated on G20 become layer II/III neurons. In ethanol-treated rats, some neurons born on G20 migrate to layer II/III, but most distribute to deep cortex. Regardless of when a neuron is born and to which layer it migrates, the neuron retains its connectional phenotype (Miller, 1987a, 1997). This

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includes corticospinal and callosal projections neurons. In normal rats, corticospinal neurons have cell bodies distributed in layer Vb (e.g., Hicks and D’Amato, 1977; Wise et al., 1979; Donoghue and Wise, 1982; Miller, 1987b) and most are born on G16 (Miller, 1987a). In ethanol-treated rats, the distribution of the somata of corticospinal neurons is broader, including superficial layer II/III, and most corticospinal neurons (regardless of their laminar residence) are generated one day late (on G17)(Miller, 1987a; Miller et al., 1990). This suggests that neuronal lineage is defined before migration begins. These findings from ethanol studies are strikingly analogous to heterochronic transplants, i.e. transplanting post-mitotic neurons into proliferative zones at a time other than their time of origin (McConnell, 1988). In vitro studies of cortical cells show that ethanol neither affects the time of neural determination nor the proportion of cycling and determined cells (Jacobs and Miller, 2001).

Cell Death A factor that may define the effect of ethanol on the outcome of VZ and SZ cell proliferation is a potential differential effect of ethanol on neuronal death in the germinal zones. In normal fetuses, cell death among proliferating populations does occur, but it affects only a small number (5%) of the cells (Blaschke et al., 1996; Thomaidou et al., 1997). Analyses of organotypic neocortical cultures do not reveal any differences in the incidence of death among VZ or SZ cells (Mooney and Miller, unpublished results). Recent data suggest that ethanol may affect the survival of cycling cells. The amount of death among primary cultures of fetal neocortical cells is significantly increased by ethanol (Jacobs and Miller, 2001). In such cultures, 90% of the cells express neuronal markers, 28% of all cells are actively cycling (of VZ and SZ origin). Ethanol does not affect these parameters. On the other hand, whereas 35% of the cells are dying daily in the untreated cultures, 40% die daily in the ethanol-treated cultures (Jacobs and Miller, 2001; Miller, 2003a). Unresolved issues (for both untreated and ethanol-treated cultures) are whether the dying cells are among the cycling or post-cycling population, and whether the dying cells are of VZ or SZ origin. No studies have directly explored the survival of the post-migratory progeny of VZ and/or SZ cells, much less the effects of ethanol on those progeny. That said, we can glean some insight into the neuronal death of presumed VZ and SZ derivatives in normal rat cortex. The amount of death among neurons in the various laminae has been described (Miller, 1995c). The rate of death among neurons generated on G15 (neurons in layers V and VIa) is 49.5% and the rate for neurons generated on G20 (i.e. those in layer II/III) is 58.9%. Based on the premises: (a) that early-generated neurons migrate to infragranular layers and are VZ derivatives; and (b) that late-

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generated, layer II/III neurons are mostly SZ derivatives, it appears that the incidence of death among VZ and SZ derivatives is equivalent.

Non-SZ Derived Proliferative Zones Cell proliferation in the IHZ and EGCL are affected by ethanol. As in the SZ, proliferation of cells in the IHZ can also be stimulated by ethanol. These effects are concentration-dependent (Miller, 1995b). Lower doses are stimulatory, whereas higher doses are inhibitory. The frequency of mitotic cells in the SZ increases after chronic exposure to ethanol during gestation (Kennedy et al., 1984). In contrast, the proliferation in the EGCL is reduced by exposures to high concentrations of ethanol (Bauer-Moffett and Altman, 1977; Anderson and Sides, 1979). At least in part, this reduction results from a significant increase in the TC (Borges and Lewis, 1983). Unfortunately, no studies have examined the effects of low ethanol exposures on cerebellar neurogenesis. Therefore, it is impossible to determine if all derived proliferative zones are affected by ethanol in parallel fashions. Three compelling conclusions about the SZ can be drawn from the data described above: (1) The extended neuronogenetic activity in the SZ may be an attempt to compensate for the depressed production from the VZ. (2) Both the VZ and the SZ gives rise to neocortical neurons. (3) The VZ largely generates neurons in infragranular laminae, whereas the SZ produces cells in layer II/III. On the other hand, global conclusions about the effects of ethanol on cell proliferation in the derived zones cannot be made. Based on the available data, ethanol affects these zones in dissimilar ways. The effects are site, concentration, and temporally specific. It is likely that these differences reflect intra-site variations in the expression of ligand-receptor systems or the menu of intracellular signaling systems (e.g., Luo and Miller, 1998, 1999a, 1999b).

Radial Glia Up until the early part of the twentieth century, controversy swirled about whether VZ cells extended processes from the VZ and to the pial limit of the developing brain. Such cells were referred to as spongioblasts and were considered to be the intermediary for VZ cells to become neocortical neurons. This issue was largely resolved by the elegant electron microscopic studies of Rakic (e.g., 1971; Rakic et al., 1974) showing that young migrating neurons may have contact with neither the basement membrane nor the subpial limit. The migrating neurons are closely associated with long processes that stretch from the ventricular to the pial limits of the cerebral wall. These are the processes of radial glial cells. Radial glia are derived from and have cell bodies in both the VZ and SZ.

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Many studies have shown that radial glia transform into astrocytes after they have served their role in guiding migration (e.g., Schmechel and Rakic, 1979; Lu et al., 1980; Voigt, 1989; deLima et al., 1997). Radial glia express nestin (Hockfield and McKay, 1985; Tohyama et al., 1992; Miller and Robertson, 1993b), a marker of neural stem cells (e.g., Lendahl et al., 1990). Radial glia are the precursors of neurons (Gray and Sanes, 1992; Malatesta et al., 2000; Hartfuss et al., 2001; Levers et al., 2001; Noctor et al., 2001; Alvarez-Buylla and Garcia-Verdugo, 2002). That is, radial glia are neural stem cells that generate daughters that differentiate into neurons or glia. The expression of GFAP by radial glia (or, for that matter, some SZ cells; Doetsch et al., 1997; 1999; Alvarez-Buylla et al., 2002) does not mean that these cells are astrocytes. In other words, radial glia are misnamed and they should be reconsidered as spongioblasts. Prenatal exposure to ethanol affects the appearance and disappearance of neocortical radial glia (Gressens et al., 1992; Miller and Robertson, 1993b; Liesi, 1997; Miller, 2003b). These cells prematurely lose their attachment to the subpial glial limitans. Concurrently, GFAP-positive astrocytes appear precociously in deep cortex. Moreover, early exposure to ethanol can increase GFAP expression in deep cortex (Fletcher and Shain; 1993; Goodlett et al., 1993). These events are in concert with the concept that ethanol causes the premature transformation of radial glia into astrocytes. Evidence that radial glia can differentiate into neurons compels a reconsideration of this conclusion. This possibility is supported by findings that prenatal exposure leads to the generation of ectopic neurons in deep cortex (Miller, 1986b, 1988b, 1997).

Neurogenesis in the Adult The occurrence of neuronal generation in the adult cortex has been a topic of discussion for many years. It is widely acknowledged that cortical neurons are generated by the anterior SZ and the IHZ (e.g., Jacobson, 1991; Conover and Allen, 2002). These two structures generate cells that differentiate into granule neurons in the olfactory bulb and the dentate gyrus. Though debated (Rakic, 2002), data have also been forwarded that neuronal generation can occur in the adult at intracortical sources (Altman, 1962; Kaplan and Hinds, 1977, 1980; Kaplan, 1981) and/or in developmental proliferative zones (e.g., the VZ, SZ, and IHZ) (e.g., Hinds, 1968; Jacobson, 1991; Gage, 1994; Gage et al., 1995; Kuhn et al., 1996; Gould et al., 1999, 2001). Two studies have examined the effects of ethanol on the incorporation of bromodeoxyuridine in the adult rat (Nixon and Crews, 2002; Pawlak et al., 2002). Nixon and Crews show that ethanol (peak blood ethanol concentrations (BECs) of >300 mg/dl) reduces cell proliferation in the IHZ. These findings are interesting in the light of studies linking loss of IHZ cell generation and memory loss. Neurogenesis in the IHZ is reduced by

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treatment with the anti-mitotic agent, methylazoxymethanol and this reduction is associated with deficits in forming trace memories (Shors et al., 2001). Ethanol causes losses in short term memory (e.g., Walker and Freund, 1971; Walker and Hunter, 1978) and hippocampal damage (Walker et al., 1980). Presumably, the ethanol-induced memory loss results from reductions in IHZ neurogenesis. Pawlak et al., (2002), using a pair-feeding paradigm that generates animals with BECs of 150 mg/dl (Miller, 1992c), show that ethanol causes a 2-fold increase in the proliferation of IHZ cells. This compares with a 20% loss among CA1 and CA2 pyramidal neurons. Thus, there appears to be a concentration-dependent effect of ethanol on IHZ cell proliferation. The changes in the IHZ of the adult are identical with those described in younger rats (Miller, 1995b). Accordingly, exposure causing: (a) a low BEC increases cell proliferation in the IHZ; and (b) a high BEC reduces IHZ cell proliferation. Hence, the effect of ethanol is not age-dependent.

Conclusions The differential effects of ethanol on the proliferative zones support the thesis that both neocortical proliferative zones generate neurons. Proliferative activity in the VZ is depressed by ethanol regardless of the location along the neuraxis and regardless of the ethanol concentration. In contrast, proliferation within derived zones (at least the SZ and IHZ) is bimodally affected by ethanol in a concentration-dependent manner. Indeed, the unique responses of the VZ and SZ may reflect an interaction between the two zones. For example, the increased proliferative activity in the SZ or IHZ at low concentrations results from a recruitment of new cells into the cycling population (increased GF). This may be an compensatory response to offset the depressed proliferation of VZ cells. After all, the derived zones are seeded by the VZ. Acknowledgment. Research support was provided by grants from the National Institute of Health (AA06916, AA07568, and AA15245) and by the Department of Veterans Affairs.

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Abbreviations CNS EGCL G GE GF IHZ LCN MR P SZ Tc VZ

central nervous system external granular cell layer gestational day ganglionic eminence growth fraction intrahilar zone local circuit neuron mental retardation postnatal day subventricular zone total length of the cell cycle ventricular zone

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Michael W. Miller and Marla B. Bruns

Many names and abbreviations are used to describe the proliferative zones. For example, some investigators use SVZ and SGZ for subventricular zone and supragranular zone, respectively. We use VZ, SZ, and IHZ to minimize redundancy and confusion such as between VZ and SVZ and SVZ and SGZ. Similarly, various names are used to refer to prenatal (e.g., E or ED for embryonic day or F or FD for fetal day) and postnatal (e.g., PND for postnatal day) ages. Embryonic properly refers to the period before implantation and fetal to the period post-implantation. In contrast, reference to a prenatal age as a gestational day eliminates the scientific inaccuracy. Therefore, we use G and P to improve both clarity and accuracy.

Index Aging, 162 Alcohol, 281, 288; see also ethanol Astrocyte Differentiation 17-18 Progenitors 1, 11, 12, 16, 17, 18, 20, 21, 22, 40, 89, 91, 92, 136, 139, 140, 141, 145, 186, 188, 230, 232, 262, 263, 265, 266, 267, 269, 294 Reactive, 226, 228, 229, 230, 232, 236, 252 Ataxia Leangiectasia, 160, 167 Brain Regions Anterior Commissure, 226 Corpus Callosum, 10-14, 57, 86, 98, 126, 130, 135, 138, 162–163, 170– 172, 215, 224, 226 –227, 230, 247, 251, 263, 266–272 Cerebral Cortex, 4, 57, 117–118, 122– 128, 133–142, 164, 169, 210 –216, 221–227, 230, 232–233, 254, 262 Forebrain, 5, 7, 9, 12–15, 19–22, 32, 40, 50, 87–89, 117–145, 162, 210, 222, 224, 230, 243, 260, 262, 272–274 Frontal Cortex, 10, 137, 219, 224, 230 Neocortex, 2, 12–17, 21, 86, 136 –138, 169–174, 282, 284, 290, Olfactory Bulb, 5–9, 12, 21, 36, 44 – 48, 58, 84 –86 –91, 93, 104, 124, 161, 171, 185, 210–211, 219–222, 230, 243, 249, 261–263, 267–271, 274, 294 Olfactory Peduncle, 219, 230 Cell Cycle Kinetics, 50–51

Phases, 46, 51–53, 55–56, 130, 193, 216–222, 227, 282, 284, 287–288 Regulators p16Ink4A, 53–55 p19Arf, 53–55 P21cip, 48, 55–57 p27Kip, 53–57 P53, 51, 54, 56–57 b–Catenin, 43, 46, 128, 212 Retinoblastoma, 52 Cell Death Apoptosis, 37–38, 41, 43– 44, 48, 56–58, 105, 129–131, 160, 166, 169–174, 190, 192–193, 223, 244 Necrosis, 187–189, 194, 244 Cell Proliferation Labeling Index, 193–195, 216, 220, 222 Markers Of Proliferation Proliferating Cell Nuclear Antigen (PCNA), 124, 128, 158, 221, 249, 273 Ki67, 221 Bromodeoxyuridine, 7, 9, 19, 36, 44, 88, 128, 130, 161, 167, 170, 172–173, 192–194, 215–223, 228, 230 –232 Tritiated Thymidine, 188, 221, 267, 284 Modes of proliferation, 30 –31, 46, 50, 123, 196–197, 252 Regulatory Proteins p16Ink4A, 53–55 p19Arf, 53–55 P21cip, 48, 55–57 p27Kip1, 53–57

305

306

Index

Cell Proliferation (Continued ) P5351, 51, 54, 56–57 Retinoblastoma, 52 Cerebrospinal Fluid (CSF), 173, 211, 216 Chemokines, 142–144 Chemotherapy, 201, 203 Chromatin, 32, 40, 51, 54, 131–132, 170, 244 Edema, 211, 234, 244 Ependyma, 2, 19, 23, 36, 38, 55, 85, 88, 92, 128, 166, 212, 248, 261, 264, 282 Experimental Allergic Encephalomyelitis, 168, 265, 267, 269, 272, 274 Extracellular Matrix Proteins Chondroitin Sulfate Proteoglycan (CSPG), 226, 233 Polysialylated Neural Cell Adhesion Molecule (PSA–NCAM), 4, 7–8, 35–36, 96 –100, 124, 136, 158, 161, 163, 168, 223, 226 –233, 246 – 247, 252, 261, 266 –274 Tenascin, 45, 223, 225, 233, 270 Ethanol, 281, 284 –295 Fetal Alcohol Syndrome, 281, 288 Germinal Zones External Granule Cell Layer, 282 Ganglionic Eminence, 3, 4, 6, 46, 117–119, 123, 124, 126, 128, 133–135, 261, 267, 282 Subgranular Zone, 200 Ventricular Zone, 1–2, 6, 30, 33–34, 52, 57, 106, 120 –121, 126, 127, 143, 232, 283, 285 Growth Factors Brain Derived Neurotrophic Factor (BDNF), 36, 46– 47, 59, 83, 104, 160–162, 172, 175, 232, 254, 271, 273, Epidermal Growth Factor (EGF), 19, 20, 33–35, 40– 42, 45, 48, 57, 85, 93–94, 129, 160, 162, 172– 173, 231–232, 254, 264 –265, 271– 273, Fibroblast Growth Factor –2 (FGF–2), 6, 32–34, 40 – 45 Nerve Growth Factor (NGF) 36, 143, 162, 272–273

Sonic Hedgehog (Shh), 41–43, 45, 93, 136–137, 259, 26 Transforming Growth Factor–a (TGF–a), 33–38, 93–94, 143, 160, 163–164, 233, 272 Wnts, 42–43, 45 Hippocampus Dentate Gyrus, 104, 166, 200, 282, 288, 294, Intrahilar Zone, 282, 303 Subgranular Zone, 200 Human, 6–7, 48, 91, 95, 117–145, 165–168, 175, 211–216, 222, 228, 230, 234, 243, 248, 256, 260, 265, 273–274 Hypoxia, 43, 44, 120, 248, 254 Injury Aspiration Cortical Lesion, 216–218, 222–223, 228, 228–229, 232–233 Traumatic Brain Injury, 210, 221 Chemical Injury, 19, 162, 202, 203, 264, 250, 266, 267, 272 Stroke; see also, Ischemia Irradiation, 56, 168, 186–204 Ischemia, 45, 68, 120, 168, 242–255 Macrophage, 123, 129, 133, 142, 144, 222 Microglia, 123, 131, 133, 135, 140 –145, 222, 230 Mouse, 2–3, 6, 9, 11, 19, 34, 44 –45, 53, 87–88, 99, 120, 124, 128, 131, 141, 171, 173, 188, 191, 193, 215–217, 219, 221–222, 228–229, 233–234, 262, 266 –267, 284 Migration, 7, 11, 37, 58, 96 –99, 185, 189, 223, 233, 252, 254 –255, 270, 273 Neural Stem Cells, 2, 18–19, 23, 32–59, 84–94, 133, 140–141, 166, 167, 254, 261–267, 294 Neuroblasts, 5, 8, 9, 12, 14, 21, 23, 31–38, 50, 56, 86, 90–91, 95–100, 103, 105, 185, 190, 198, 200, 202–203, 222–223, 226 –229, 251–252, 261, 268, 270–271

Index Neurons, Differentiation, 20, 35, 38, 40– 43, 92, 161, 170, 172, 175, 230, 253 Interneurons, 5, 8–11, 21–22, 36, 84, 86, 100–105, 117, 121, 126, 128, 133–134, 136, 138, 140, 144 –145, 169, 210, 228, 262–263 Neurogenesis, 1, 5, 22, 32–38, 41–50, 57, 84–85, 89, 91–94, 102–106, 159, 161, 165–175, 186, 198, 203, 223, 229, 231–232, 243, 253–255, 264, 269, 272, 274, 290, 293, 295 Migration, 37, 84, 85, 95, 98, 139, 167 Progenitors, 10, 33–34, 41, 44, 48, 84 –85, 93, 104, 166, 246, 250, 252, 261, 264 –266, 271–273 Projection, 13, 164, 170–174

307

Alzheimer’s Disease, 48 160, 162, 165–166 Multiple Sclerosis, 142, 160, 167–168, 267 Parkinson’s Disease, 35, 160, 163, 274 Radial Glia, 1, 2, 8, 11–14, 89–92, 99, 124, 126, 134, 137, 139–145, 172, 221–224, 227, 254, 261, 269, 286, 293–294 Repair, 30, 32, 59, 145, 159–160, 169, 175, 186, 189, 192, 210, 212, 231–232, 265–267, 269, 273 Rostral Migratory Stream, 7, 53, 55, 57, 58, 95, 123–124, 158, 161, 171–173, 185, 212, 261–263, 269

Oligodendrocytes Differentiation, 17, 142, 144, 271–272 Migration, 136 Progenitors, 6, 50, 123–126, 133–136, 140–144, 229, 243–244, 247–248, 253, 263, 274

Transcription Factors Dlx, 3–6, 10, 20, 93, 124 –125, 133, 136, 138, 261–262 Emx, 6, 57–58, 250 Nkx, 123–125, 134 –136, 138 Olig1, 2, 20, 261, 264 Vax, 58 Tuberous Sclerosis, 160, 167

Plasticity, 103–106, 120, 159, 198, 202–203, 212, 256, 265, 274 Progenitors; see also Neuroblasts, Astrocyte Progenitors, Oligodendrocyte Progenitors Psychiatric Disorders, 121, 160, 164, 167–168 Neurological Disorders

Ventricular Zone, 1–2, 6, 30, 33–34, 52, 57, 106, 120 –121, 126, 127, 143, 232, 283, 285 Viruses Adenoviruses, 21 Retroviruses, 6–17, 20 –24, 27, 87, 89, 92, 126, 134, 136, 140–141, 218, 224, 228, 230, 252, 262, 267