Biological Signals Relevant to Sleep

Biological Signals Relevant to Sleep

Guest Editor

Ricardo A. Velluti, Montevideo

26 figures, 3 tables, 2000

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Vol. 9, No. 6, 2000

Contents

Biological Signals Relevant to Sleep Guest Editor: Ricardo A. Velluti, Montevideo 279 Influence of the Temperature Signal on Sleep in Mammals Parmeggiani, P.L. (Bologna) 283 The Amygdala: A Critical Modulator of Sensory Influence on

Sleep Morrison, A.R.; Sanford, L.D.; Ross, R.J. (Philadelphia, Pa.) 297 Reciprocal Actions between Sensory Signals and Sleep Velluti, R.A.; Peña, J.L.; Pedemonte, M. (Montevideo) 309 Bright Light during Nighttime: Effects on the Circadian

Regulation of Alertness and Performance Daurat, A.; Foret, J. (Toulouse); Benoit, O. (Créteil); Mauco, G. (Toulouse) 319 Adenosine as a Biological Signal Mediating Sleepiness

following Prolonged Wakefulness Basheer, R.; Porkka-Heiskanen, T.; Strecker, R.E.; Thakkar, M.M.; McCarley, R.W. (Brockton, Mass.) 328 A Critical Assessment of the Melatonin Effect on Sleep in

Humans Monti, J.M.; Cardinali, D.P. (Montevideo/Buenos Aires)

340 340 341 342 after 342

Author Index Vol. 9, No. 6, 2000 Subject Index Vol. 9, No. 6, 2000 Author Index Vol. 9, 2000 Subject Index Vol. 9, 2000 Contents Vol. 9, 2000

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Influence of the Temperature Signal on Sleep in Mammals Pier Luigi Parmeggiani Department of Human and General Physiology, University of Bologna, Bologna, Italy

Key Words Temperature W Sleep W Thermoregulation

Abstract The influence of the temperature signal on sleep may be considered physiologically specific if it entails thermoreceptor activation. Experimental evidence shows that sleep time peaks at neutral ambient temperature. Copyright © 2000 S. Karger AG, Basel

Introduction

Temperature is a physical variable which not only directly and unspecifically affects cellular activities (thermophysical and thermochemical effects), but also indirectly and specifically influences the entire somatic and autonomic activity of the organism by excitation

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of specific thermoreceptors. On this basis, temperature must be considered a signal of both physicochemical and biological relevance. However, this distinction is only conceptual, since a functional loop is operative from the regulatory viewpoint. Particularly in mammals, thermoregulatory mechanisms respond to the temperature signal to maintain a substantial homeothermy of the body core. This control optimizes the unspecific effects of temperature according to the metabolic needs of body tissues within a wide range of ambient temperatures. The thermoregulatory responses depend on both somatic (thermally adequate postural and motor repertoires) and autonomic (vasomotion, piloerection, shivering, nonshivering thermogenesis, thermal tachypnea, sweating) activity of the organism. From a biological viewpoint, however, thermoregulatory responses to the same temperature signal show a clear-cut behavioral

Prof. Pier Luigi Parmeggiani Dipartimento di Fisiologia Umana e Generale Piazza Porta San Donato 2 I–40127 Bologna (Italy), Tel. +39 051 244499 Fax +39 051 251731, E-Mail [email protected]

Table 1. Thermoregulatory responses during the wake-sleep cycle

Responses

Wakefulness NREM sleep REM sleep

Specific

somatic autonomic

somatic autonomic

– –

Unspecific

vigilance

arousal

arousal

state dependency during the wake-sleep cycle (table 1). The cycle consists of a single sequence of at least three behavioral states which may be called quiet wakefulness, nonrapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Changes in threshold and gain are commonly observed in the transition from wakefulness to NREM sleep [1, 2]. The responsiveness to peripheral and central thermal loads in NREM sleep is consistent with homeothermic regulation at a decreased level of core temperature. In contrast, the disappearance or depression of somatic and autonomic thermoregulatory responses during REM sleep is particularly dramatic [1, 2]. This stresses the operative difference between a physical signal and a biological signal. Biologically, temperature is a signal that ought to be regarded as both an endogenous and an exogenous stimulus of the organism. The endogenous stimulus is compartmentalized by physiological mechanisms into the body core and the body periphery. Core temperature is further compartmentalized in the central nervous system by behavioral statedependent selective brain cooling in several species [3]. The fact that selective brain cooling includes the hypothalamus (the term refers also to the preoptic region in this context) but not the brain stem is of thermoregulatory relevance [4]. In the ambient thermal zone for vasomotor regulation of core temperature, se-

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lective hypothalamic cooling underlies a thermal feedback which is maintained by a temperature error signal of the extrahypothalamic core [5]. This feedback differentiates the relative functional weights of hypothalamic and extrahypothalamic thermoreceptors inputs in the behavioral states of wakefulness and NREM sleep. Evidently, these considerations do not apply to depressed selective hypothalamic cooling in REM sleep [6], since hypothalamic thermosensitivity is altered in this behavioral state [7–11]. In conclusion, the physical temperature signal is so differently compartmentalized as an endogenous stimulus to assume a very complex role as a biological signal affecting not only temperature regulation but also the whole behavior of the wake-sleep cycle.

A Brief Overview of the Influence of the Temperature Stimulus on Sleep Behavior

The structure of the wake-sleep cycle at ambient temperature within thermoneutrality is conventionally considered as the normal reference [12]. Thermoneutrality, which varies in the different species [13], is defined as the range of ambient temperatures within which the metabolic rate decreases to the minimum at rest and temperature regulation is implemented by physical mechanisms alone. Long-term adaptation to ambient temperatures without the limits of thermoneutrality induces shifts and/or modifications of the ambient temperature range well tolerated by the different species in terms of normal structure of the wake-sleep cycle. Moreover, the amount of thermal load does not depend only on ambient temperature since other factors, like body size, thermal insulation, age, sexual cycle, feeding, season and humidity, as well as regional differences in body sensitivity to

Parmeggiani

temperature, consistently influence individual thermoregulatory responses. The set of interacting variables, therefore, ought to be considered very complex from the behavioral and physiological viewpoint. Sleep time peaks around the upper limit of the ambient thermoneutrality range [14–19]. Sleep time declines above and below such range but the rate of decline is larger above it than below. Deviations of ambient temperature from thermoneutrality not only increase the waking time but also modify the structure of sleep [14, 15, 17, 19–22]. In particular, NREM sleep stages and/or REM sleep episodes may be selectively affected. Experimentally induced changes in the temperature of the hypothalamus affect the wake-sleep cycle. Cooling increases waking time [19], and warming promotes both NREM and REM sleep [12, 19, 22–26]. At this point, it is sufficient to note that the thermoregulatory activity elicited by cooling is opposite of the thermolytic adjustments induced by NREM sleep processes. In contrast, warming elicits a change in thermoregulatory activity, which is synergic of NREM sleep changes in somatic and autonomic activity. NREM sleep processes are, therefore, specifically affected by the influence of moderate central thermal loads at ambient thermoneutrality, since cooling promotes wakefulness and warming promotes sleep, and as a consequence REM sleep occurrence is also affected.

Conclusion

The relation of warming to sleep and cooling to wakefulness is generally relevant in respect to the influence of temperature as both an endogenous and an exogenous stimulus. However, unspecific effects of exogenous thermal stimuli, related to the amount of ther-

Temperature Signal and Sleep

mal comfort or stress, may more readily affect the wake-sleep cycle independently of thermoregulatory mechanisms. During wakefulness and sleep, thermal loads may induce or oppose the occurrence and persistence of sleep, respectively, depending on whether sensory influences (EEG synchronizing or desynchronizing) [27] and thermoregulatory somatic and autonomic activities are consistent or inconsistent with sleep processes. NREM sleep is characterized by postural and motor quiescence, functional prevalence of the parasympathetic over the sympathetic activity, lowering of metabolic heat production (decrease in muscle tone and heart and breathing rates) and brain and body temperatures (vasodilation of heat exchangers, sweating) [28, 29]. Thus, a moderately warm ambient temperature or a slight increase in core temperature promotes sleep: in this case, the onset of sleep is a synergic concomitant of thermoregulation in adaptation to the positive thermal load [12]. Since sleep may also occur under adverse ambient conditions, the sleep-promoting role of thermoregulatory mechanisms is only facultative in wakefulness, and its functional importance is inversely related to the degree of sleep pressure. During NREM sleep, thermoregulatory mechanisms are involved in the control of REM sleep occurrence according to the actual thermal load [12]. On the other hand, in the poikilothermic condition of REM sleep, the temperature signal as a biological stimulus affecting both thermoregulation and sleep becomes temporarily subliminal. Heavy thermal loads elicit arousal in every sleep state (table 1). This stereotyped effect of temperature is adequate as a survival response of the organism since only the waking state underlies the full expression of somatic and autonomic thermoregulatory responses.

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References 1 Parmeggiani PL: Temperature regulation during sleep: A study in homeostasis; in Orem J, Barnes CD (eds): Physiology in Sleep. Research Topics in Physiology. New York, Academic Press, 1980, vol 3, pp 97– 143. 2 Heller HC, Glotzbach SF: Thermoregulation and sleep; in Eberhardt RC, Shitzer A (eds): Heat Transfer in Biological Systems: Analysis and Application. New York, Plenum, 1985, pp 107–134. 3 Hayward JN, Baker MA: A comparative study of the role of the cerebral arterial blood in the regulation of brain temperature in five mammals. Brain Res 1969;16:417–440. 4 Satinoff E: Neural organization and evolution of thermal regulation in mammals. Science 1978;201:16–22. 5 Azzaroni A, Parmeggiani PL: Changes in selective brain cooling across the behavioral states of the ultradian wake-sleep cycle. Brain Res 1999;844:206–209. 6 Azzaroni A, Parmeggiani PL: Mechanisms underlying hypothalamic temperature changes during sleep in mammals. Brain Res 1993;632: 136–142. 7 Parmeggiani PL, Azzaroni A, Cevolani D, Ferrari G: Responses of anterior hypothalamic-preoptic neurons to direct thermal stimulation during wakefulness and sleep. Brain Res 1983;269:382–385. 8 Glotzbach SF, Heller HC: Changes in the thermal characteristics of hypothalamic neurons during sleep and wakefulness. Brain Res 1984; 309:17–26. 9 Parmeggiani PL, Azzaroni A, Cevolani D, Ferrari G: Polygraphic study of anterior hypothalamic-preoptic neuron thermosensitivity during sleep. Electroencephalogr Clin Neurophysiol 1986;63:289–295.

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10 Parmeggiani PL, Cevolani D, Azzaroni A, Ferrari G: Responses of anterior hypothalamic-preoptic neurons during the waking-sleeping cycle: A study in brain functional states. Brain Res 1987;415:79–89. 11 Alam MN, McGinty D, Szymusiak R: Preoptic/anterior hypothalamic neurons: Thermosensitivity in rapid eye movement sleep. Am J Physiol 1995;269:R1250–R1257. 12 Parmeggiani PL: Interaction between sleep and thermoregulation: An aspect of the control of behavioral states. Sleep 1987;10:426–435. 13 Altman PL, Dittmer DS: Environmental Biology, Bethesda, FASEB, 1966. 14 Parmeggiani PL, Rabini C: Sleep and environmental temperature. Arch Ital Biol 1970;108:369–387. 15 Schmidek WR, Hoshino K, Schmidek M, Timo-Iaria C: Influence of environmental temperature on the sleep-wakefulness cycle in the rat. Physiol Behav 1972;8:363–371. 16 Valatx JL, Roussel B, Cure M: Sommeil et température cérébrale du rat au cours de l’exposition chronique en ambiance chaude. Brain Res 1973;55:107–122. 17 Sichieri R, Schmidek WR: Influence of ambient temperature on the sleep-wakefulness cycle in the golden hamster. Physiol Behav 1984;33: 871–877. 18 Obal F Jr, Tobler I, Borbély A: Influence of ambient temperature on the 24-hour sleep wake-cycle in normal and capsaicin-treated rats. Physiol Behav 1983;30:425–430. 19 Sakaguchi S, Glotzbach SF, Heller HC: Influence of hypothalamic and ambient temperatures on sleep in kangaroo rats. Am J Physiol 1979; 237:R80–R88. 20 Haskell EH, Palca JW, Walker JM, Berger RJ, Heller HC: The effects of high and low ambient temperatures on human sleep stages. Electroencephalogr Clin Neurophysiol 1981;51: 494–501.

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21 Sewitch DE, Kittrell EMW, Kupfer DJ, Reynolds CF III: Body temperature and sleep architecture in response to a mild cold stress in women. Physiol Behav 1986;36:951– 957. 22 Parmeggiani PL, Cianci T, Calasso M, Zamboni G, Perez E: Quantitative analysis of short term deprivation and recovery of desynchronized sleep in cats. Electroencephalogr Clin Neurophysiol 1980;50:293– 302. 23 von Euler C, Soederberg U: The influence of hypothalamic thermoceptive structures on the electroencephalogram and gamma motor activity. Electroencephalogr Clin Neurophysiol 1957;9:391–408. 24 Roberts WW, Robinson TCL: Relaxation and sleep induced by warming of preoptic region and anterior hypothalamus in cats. Exp Neurol 1969;25:282–294. 25 Roberts WW, Bergquist EH, Robinson TCL: Thermoregulatory grooming and sleep-like relaxation induced by local warming of preoptic area and anterior hypothalamus in opossum. J Comp Physiol Psychol 1969;67:182–188. 26 Parmeggiani PL, Zamboni G, Cianci T, Agnati LF, Ricci C: Influence of anterior hypothalamic heating on the duration of fast-wave sleep episodes. Electroencephalogr Clin Neurophysiol 1974;36:465–470. 27 Moruzzi G: The sleep-waking cycle. Ergeb Physiol 1972;64:1–165. 28 Parmeggiani PL: The autonomic nervous system in sleep; in Kryger MH, Roth T, Dement WC (eds): Principles and Practice of Sleep Medicine. Philadelphia, Saunders, 1994, pp 194–203. 29 Sagot JC, Amoros C, Candas V, Libert JP: Sweating responses and body temperatures during nocturnal sleep in humans. Am J Physiol 1987;252:R462–R470.

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The Amygdala: A Critical Modulator of Sensory Influence on Sleep Adrian R. Morrison a, b Larry D. Sanford a Richard J. Ross b, c a Laboratory

for Study of the Brain in Sleep, Department of Animal Biology, The School of Veterinary Medicine and b Department of Psychiatry, The School of Medicine, The University of Pennsylvania and c Veterans Affairs Medical Center, Philadelphia, Pa., USA

Key Words Sleep W Amygdala W Rapid-eye-movement sleep W Ponto-geniculo-occipital waves W Fear conditioning W Serotonin

(PGO) into NREM. Stimuli conditioned by pairing with aversive stimuli in a fear-conditioning paradigm significantly increased sound-elicited PGO and reduced REM. Copyright © 2000 S. Karger AG, Basel

Abstract The influence of external stimuli and the memories of both unpleasant and pleasant conditions clearly can have a considerable impact on the quality of sleep. The amygdala, a structure that plays an important role in coding the emotional significance of stimuli and is heavily interconnected with brainstem nuclei known to be involved in sleep control, has received little attention from sleep researchers. We report on a series of studies, focusing on its central nucleus (Ace). Presence of serotonin (5-HT) in Ace caused a rapid change of state when injected in rapideye-movement sleep (REM) compared with non-REM (NREM) injections. A 5-HT antagonist released ponto-geniculo-occipital waves

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Introduction

Homeostatic, circadian and ultradian processes explain the internal ‘drive’ for sleep and for the influence of periodical zeitgebers such as light on the normal distribution of sleep and wakefulness (W) throughout the day. A complete understanding of the control of the sleep-wake cycle must account for these processes. Examining their roles in the regulation of sleep generally requires the exclusion of extraneous stimuli and influences that could interfere with their expression. However, both inside and outside the laboratory, an organism must contend with life conditions that are not static. Fluctuations in its

Adrian R. Morrison, DVM, PhD Laboratory for Study of the Brain in Sleep Department of Animal Biology, School of Veterinary Medicine University of Pennsylvania, Philadelphia, PA 19104-6045 (USA) Tel. +1 215 898 8891, Fax +1 215 573 2004

internal milieu and periodically occurring and random environmental events can have varying degrees of significance for its survival. The impact of external stimuli may be most apparent in behavior emitted during W. However, myriad stimuli and events an organism experiences can determine when and for how long sleep occurs as well as mentation during sleep in the form of dreams and nightmares and other parasomnias in humans. While this influence may be obvious, especially for situations that produce mental discomfort in the form of perceived real or potential danger, sleep may also be disrupted without immediately observable cause. Examples of the latter include emotions driven by memories of past experiences and motivational states such as hunger. The mechanisms by which such factors affect the occurrence of sleep are poorly understood, and the topic has received little attention from researchers studying the neurophysiology of sleep. This may be because a possible neural substrate has not been identified. We are beginning to examine limbic influences on sleep and alerting, focusing on the amygdala, a structure relatively neglected by the field of sleep research. In the context of the present discussion, we note that the amygdala ‘invests’ sensory input with emotional significance. The amygdala evaluates the emotional significance of stimuli and initiates appropriate neurobehavioral responses [1]. Clearly, the amygdala must be considered a critical structure in determining whether a bout of sleep will occur and whether it will be a comfortable success. The issue, then, is how the amygdala interacts with the more traditionally considered structures, that is the hypothalamus and caudal brainstem, armed with immediate information from the environment and memories of past events. We can begin by considering the connectivity of the amygdala, which natu-

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rally forms the framework for its functional role(s). Each of the pontine cell groups that have been the focus of interest of sleep researchers for more than thirty years, the serotonergic dorsal raphe nucleus (DRN), the noradrenergic locus coeruleus and the pedunculopontine/laterodorsal tegmental (PPT/LDT) nuclei connect reciprocally with amygdalar nuclei [2–10]. LDT and PPT are home to a group of cholinergic (ACh) neurons thought to be instrumental in initiating rapid-eye-movement sleep (REM) [11] but the ACh contribution to the central nucleus (Ace) is sparse at best [12]. The Ace of the amygdala is the source of the projections to the pons by way of the ventral amygdalotegmental pathway and to the hypothalamus [13], and the amygdala interconnects with the basal forebrain [13]. Cholinoceptive neurons there promote W [14]. The amygdala’s interactions with the caudal brainstem will be the focus of this paper.

Serotonin and the Amygdala

The amygdala originally captured our attention because of the massive projection from Ace to the pons and more caudally [2] and then the report that electrical stimulation of Ace increased ponto-geniculo-occipital wave (PGO) frequency by 30% during REM [15]. Briefly, PGO are macropotential waves traditionally recorded in the pons, lateral geniculate body and the occipital cortex (hence the acronym). They appear spontaneously just prior to and during REM (fig. 1). PGO arise from activity in the dorsal pontine ACh neurons [11], and a number of studies using a variety of approaches have shown them to be suppressed by the presence of serotonin (5HT) [16]. An extensive series of investigations in our laboratory [17], beginning with the demonstrations that auditory stimuli elicit

Morrison/Sanford/Ross

Fig. 1. Polygraph record from a cat illustrating the change in the EEG and EMG as PGO

appear in the lateral geniculate body at the transition from NREM to REM. In REM, the EMG recorded from nuchal muscles demonstrates atonia, and the electro-oculogram reveals rapid eye movements. Time = 1 s.

Fig. 2. Polygraph record showing NREM after (a) saline and after (b) methysergide microin-

jected into the amygdala. Reprinted from [26] with permission.

PGO waves (PGOE) in REM and other states in the cat’s lateral geniculate body [18, 19] and the rat’s pons [20], generated the hypothesis that PGO are phasic expressions of the ‘peculiar’ resemblance to alertness that characterizes the brain during REM. The characteristics of the EEG support this idea as do other observations reported below. Given that PGO are inhibited by a 5-HT mechanism thought to be dependent on activity of DRN neurons, which project to PPT [21], we postulated that local injection of 5HT or its agonists and/or antagonists would affect both PGO and PGOE occurrence. In

fact, we were unable to exert any influence either by injections in the PPT of cats or rats [22–24] or the LDT of rats [25], independent of behavioral state. In contrast to these negative results, those obtained by manipulation of Ace were dramatically positive [26]. In 9 rats, the broadspectrum antagonist methysergide injected in non-REM (NREM) after two complete sleep cycles significantly increased PGO frequency in W and NREM (p ! 0.045 and 0.043) but not in REM (fig. 2). Because 5-HT neurons in DRN, which projects to Ace [9], are normally silent during REM [27, 28], one would expect

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Fig. 3. Cumulative frequency distributions of changes of state from (a) REM and (b) NREM

after saline and 5-HT. For example, 62% of microinjections of 5-HT during REM resulted in a change of state within 60 s compared to 15% after saline. In contrast, 29% of microinjections of 5-HT during NREM resulted in a change of state within 60 s compared to 25% after saline. Reprinted from [26] with permission.

that methysergide would have no effect on PGO in REM. Methysergide significantly (p ! 0.011) increased sleep efficiency (total sleep/ total recording time) but no other sleep parameters. 5-HT had a prominent effect on behavioral state. When injected in REM (n = 10), the time course of changes in behavioral state differed significantly from injections of 5-HT during NREM (p ! 0.028) as well as saline in either state (fig. 3). This suggests that the presence of 5-HT in Ace is most incompatible with REM and promotes arousal. Because not all animals aroused (awakened) when injected during either phase of sleep, we could not perform statistical analysis of this phenomenon although the latency to arousal was generally shorter from REM. This result finds support in those obtained by Rueter and Jacobs [20]. They observed

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increased 5-HT release in the amygdala using microdialysis during behavioral manipulations that increased alert W. A similar increase occurred at the onset of darkness, a time when rats are most active. Also, preliminary measures of 5-HT via microdialysis in humans revealed the lowest concentration to be in REM with the amount recovered in NREM intermediate between REM and W [30]. Much more remains to be learned about the actions of 5-HT in the amygdala in relation to its effects on sleep though. Iontophoresis of 5-HT into the lateral nucleus (LA) of the amygdala inhibited glutamate-activated action potentials via GABAergic interneurons [31]. This nucleus is the major receiving area of the amygdala [13]. Thus, one might predict that the absence of 5-HT in REM would make the LA more reactive at that time. Indeed, in

Morrison/Sanford/Ross

natural sleep 50% of the neurons in LA increased their activity in sleep, and some cells had greater firing rates in REM than in NREM [32]. Clearly, the complexity of interactions in the amygdala during changes in behavioral state will have to be unraveled before we understand what mechanisms reduce the ability of sensory input to arouse an animal behaviorally.

Differentiating Alerting from Arousal

any sleeping cat, the ears and vibrissae are pricked forward. And the cats will pounce as if attacking prey (see fig. 6 in Hendricks et al. [35]). One does not observe the piloerection that occurs in a cat during affective defense when awake [37], which is to be expected given the suppression of sympathetic activity during REM [38]. Such cats are inordinately aggressive toward other cats when awake [39]. We examined whether damage to the pathway extending from Ace might contribute to these expressions of aggressive behavior during W and REM-A and found that they do [40]. Unilateral lesions were placed successfully in Ace of 6 of 12 cats with pontine lesions located in the region that does not lead to attack behavior in REM-A. All 6 were extremely aggressive toward conspecifics, but only the rare cat exhibited such behavior toward the experimenters. These cats exhibited no changes in their predatory behavior toward mice. Four of these 6 had adequate behavior in REM-A to permit study of expressions of aggressive-like behavior. In all of them, the same predatory-like attack occurred. Thus, damage to fibers stemming from Ace appears to contribute all or in part to the syndrome observed in a subset of cats with pontine lesions alone exhibiting REM-A [35, 40].

The studies described thus far point to two conclusions: (1) a brain that seems alert (in terms of EEG characteristics and PGO occurrence) does not necessarily equate with a behaviorally aroused animal, and (2) the amygdala may be a key structure in deciding whether alerting stimuli are important enough to arouse an animal into full W. No more dramatic demonstration that the brain can be alert in sleep exists than in the case of animals exhibiting REM without atonia (REM-A). Bilateral pontine lesions result in cats that exhibit complex behavior during every episode of REM, ranging from simple lifting of the head to quadrupedal locomotion [33, 34]. The degree of complexity is consistent within cats and varies according to lesion site [35]. Yet, auditory stimuli can elicit orienting reactions that resemble those that accompany orienting when cats are awake. These include turning of the pinnae or head toward a sound source. Even a move toward the sound was observed in one animal capable of locomotion during REM-A, but it did not awaken [36]. Manipulation of the amygdala can alter the behavior one observes. Cats with lesions placed rostroventrally in the pons exhibit a behavior during REM-A closely resembling that during predatory attack. Although the nictitating membranes obscure the eyes as in

PGO are a sign of alerting. In REM they occur without arousing an animal, even those capable of behavior in REM-A, indicating that the brain is insulated against arousing effects of alerting signals to a degree at least. PGOE can be elicited in REM, and their amplitudes do not differ significantly from the highest-amplitude spontaneous PGO. This is all the more remarkable considering that the

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Effects of Amygdalar Stimulation on PGO and Sleep

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Fig. 4. OR and PGOE across trials for each block in W and in NREM and REM considered as a percentage of the initial trial. Note that the W data came from day 1 of testing and the data for NREM or REM could have been collected on either day 1 or 2 of testing (n = 13). Relative change across trials is a reflection of proportion as well as amplitude and difference score. Reprinted from [41] with permission.

highest-amplitude PGOE occur during W when an animal orients [41]. Tones (90–100 dB SPL; 1,000 or 4,000 Hz sine wave; 90 ms duration; 5 ms rise time; 2 ms interstimulus interval) were presented in a minimum of 5 blocks of at least 40 stimuli spaced 20 min apart and then throughout NREM or REM. A final block of 40 was presented 20 min after sleep. Comparing PGOE with a quantitative measure of behavioral orienting in W [42] revealed that the highest PGOE amplitudes occurred at the beginning of a block of 40 stimuli when the orienting score was highest (fig. 4). Although PGOE continued to be elicited throughout a stimulation block, their amplitudes were lower than the initial wave at

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the onset of the block of stimulation. This suggests that the amplitude of the PGOE may represent the degree of significance of the stimulus. Because the amygdala invests sensory stimuli with emotional tone based on the current emotional state of an organism, one would expect that it would influence a measure such as PGO, which represents detection of environmental events. To test this idea, we have undertaken a series of experiments to explore the effects of manipulating the amygdala. We first examined the effects of stimulating the amygdala on spontaneous PGO [43]. Although such stimulation in cats increased

Morrison/Sanford/Ross

Fig. 5. Polygraph excerpts from one rat illustrating the effect of electrical stimulation of Ace on PGO wave activity in waking, NREM and REM. Each panel contains an excerpt from an episode (a) without stimulation that occurred at approximately the same time during baseline recordings and an excerpt (b) with electrical stimulation indicated by an asterisk. No significant effects were found in waking. In NREM, PGO

wave frequency was reduced by electrical stimulation of Ace. For comparison, examples of PGO during NREM (a) without stimulation are indicated by arrowheads. The two excerpts during REM illustrate the increase in PGO wave amplitude (b) during stimulation of Ace compared to (a) without stimulation. Reprinted from [43] with permission.

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289

Fig. 6. Representative responses

from one rat demonstrating a PGOE in response to presentation of the auditory stimulus alone (a). No response appeared when Ace was electrically stimulated without being paired with an auditory stimulus (b), and a relatively higher amplitude PGOE resulted when the auditory stimulus was preceded by 1.0 ms by electrical stimulation of Ace (c). Asterisks = Onset of the auditory stimulus (a, c) or of electrical stimulation of Ace (b). Reprinted from [44] with permission.

the frequency of PGO in REM [15], this did not happen in rats. Six rats were stimulated while in the recording chamber for 6 h during the light phase (12:12) via bilateral electrodes placed in Ace (single cathodal pulses, 0.1 ms at 150 ÌA). Ten stimuli were administered during each vigilance state at 10-second intervals with at least 1 h between each series of stimuli. One animal’s stimulating electrodes were misplaced, and so it was eliminated from the data

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pool. Amygdalar stimulation had no effect on the amount of time spent in each state. During REM, PGO amplitudes were significantly increased (p ! 0.05) (fig. 5), but frequency did not change. Surprisingly, given the results from cats, the frequency of PGO occurrence was significantly reduced in NREM (p ! 0.03). Thus, the amygdala can further excite the PGO generator neurons in PPT without arousing animals or affecting their sleep.

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Fig. 7. a Magnitude (summed amplitude of responses/ No. of stimulus stimulations) of PGOE during habituation to an auditory stimulus over 50 trials, expressed relative to the mean PGOE magnitude over the 50 habituation trials (= 100%). Asterisks = Trials that differ significantly from the last 30 habituation trials (p ! 0.05, post-hoc Bonferroni after significant ANOVA). b Magnitude of PGOE elicited by an auditory stimulus preceded by (or at the same time as) electrical stimula-

tion of Ace (time between electrical and auditory stimulus ranged from 0 to 100 ms; values expressed as in a), and PGOE magnitude after auditory (A) and electrical (E) stimulation alone. Asterisks = Differences from auditory stimuli alone (p ! 0.05, post-hoc Duncan after significant ANOVA). CNA = Central nucleus of the amygdala. Reprinted from [44] with permission.

During W, electrical stimulation of the amygdala shortly before a tone will also increase the amplitude of PGOE [44], which have all the characteristics of the spontaneous PGO of REM other than their mode of origin [45]. In 10 rats habituated to a recording chamber, a sequence of 50 auditory stimuli (white noise, 100 dB, 50 ms, 5 ms rise time, 5 s interstimulus interval) was delivered during the light period. After habituation, bilateral stimulation of Ace (single cathodal pulse, 0.1 ms at 150 ÌA) was presented simultaneously with and at 1, 3, 5, 10, 25, 50 and

100 ms before the onset of the white noise. White noise and electrical stimulation were also presented alone. Ten trials (15 s interstimulus intervals) of each of the stimulus conditions were presented in a randomized Latinsquare design. PGOE were recorded in the pons [20] and accepted as such if they occurred with latencies between 20 and 100 ms. Figure 6b demonstrates that Ace stimulation itself did not elicit a PGOE, but that electrical stimulation 1 ms prior to white noise (fig. 6c) led to a significant (p ! 0.05) enhancement of the magnitude of the wave (fig. 7).

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Influence of Fear Conditioning on Elicited PGO Waves and Sleep/Wake Parameters

Fear conditioning involves creating an association between a neutral stimulus (generally a light or auditory stimulus) or situational context and an aversive stimulus (usually a foot shock) [46]. The physiological consequences of fear conditioning closely mimic those seen in human anxiety disorders [46]. Indeed, the concepts of anxiety and fear are closely related although fear is considered to be stimulus-specific whereas anxiety is more generalized. Both anxiety and fear appear to involve the amygdala. Fear-potentiated startle, the output measure usually studied after fear conditioning, has been used as a model of anticipatory anxiety [46]. Fear-potentiated startle appears to be regulated by the amygdala because lesions of the central nucleus of the amygdala block the effects of conditioned fear on the acoustic startle response [47]. In addition, several indices of fear, such as freezing, changes in heart rate and blood pressure, and increased startle can be induced by electrical stimulation of the central nucleus of the amygdala [48–52]. We sought to determine the effects of fear conditioning on sleep and found that a fear-conditioned stimulus would significantly increase PGOE amplitudes and suppress REM [53]. Six rats were adapted to the sleep recording chamber and cable for 6 h during the light period (11:00 a.m. to 5:00 p.m. EST). On a subsequent day, a 4-hour recording of EEG, EMG and PGO wave activity was obtained to determine baseline sleep amounts and to determine whether each rat exhibited spontaneous PGO waves during REM. Following baseline sleep monitoring, the rats were trained in a fear conditioning procedure. The rats were presented with 15 light-shock pairings (light: 5 s duration; foot shock (0.5 mA)

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via a grid floor, on during the last 0.5 s) on each of 4 consecutive days. Immediately after each training session, the rats were taken to a separate room for polygraphic monitoring of W and sleep. On each fear conditioning day, 4-hour polygraphic studies were conducted between 12 p.m. and 4 p.m. Trained observers determined the presence of W, NREM and REM. Postconditioning sleep was compared to sleep during the same hours on the baseline days. Twenty-four to forty-eight hours after the last conditioning trial, the rats were presented with 200 white noise bursts (100 dB, 20 ms duration, 3 s ISI) to habituate the PGOE response. During the subsequent testing phase, the rats were presented with 25 additional white noise bursts alone and 25 in the presence of light. All test trials were presented in a randomized order with a 5- to 10-second ISI. Both training and test trials occurred in a darkened, sound-dampened chamber. For training, the animals were placed in a cage with a grid floor. Testing was conducted with the rats in their home cage, but in the same chamber as that used for training. The experiments were controlled, and the PGOE data were collected, using DataWave Experimenter’s Workbench software. Data were collected beginning immediately with the onset of the light-conditioned stimulus, and the white noise stimulus was presented 3.5 s after light onset so that it would not coincide with the exact time that shock had been presented. For control trials, data collection was begun immediately prior to the onset of the white noise stimulus. Overall, the amplitudes of PGOE were significantly (p = 0.007) greater in the trials in which the light was present compared to those during posthabituation trials (fig. 8). No other comparisons were significant. The amplitudes of PGOE elicited in response to auditory stimuli during testing without the presence of light

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Fig. 8. Mean PGOE amplitudes across blocks. 25 test trials are grouped in blocks of 5 stimuli. PGOE amplitudes were significantly increased when light was presented with white noise (WN + light) compared with posthabituation values in the first (p = 0.001) and second block (p = 0.005). Prehabituation and posthabituation are values from the first and last 25 of 200 presentations of WN alone. Lack of significant effects in the last three blocks indicates that habituation occurred in all conditions during the test day itself. Reprinted from [53] with permission.

Fig. 9. REM over 4 h after con-

ditioning on each of 4 days. REM was significantly suppressed, whereas NREM was not reduced. Base = Preconditioning recording; CF1–CF4 = postconditioning days 1–4. * p = 0.001 vs. baseline. Reprinted from [53] with permission.

had intermediate values between those of prehabituation and those with the light, and those after habituation. A second prominent finding of this study was the relatively selective (p = 0.001) suppression of REM produced by fear conditioning. The finding of minimal effects on W and

NREM supports this conclusion. Total minutes in NREM and W were fairly consistent across conditions, suggesting that the significant increase in NREM percentage was due to actual decreases in REM and not to an increase in the amount of NREM.

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The average duration of REM episodes was not significantly altered. The suppression of REM was most pronounced during the first 2 h of recording and had returned to baseline levels by hour 4 (fig. 9). Comparisons among means also revealed that fear conditioning significantly increased REM latency on day 1 of conditioning. REM latency was also increased on day 2.

Conclusions

The internal emotional and drive status of the organism, its environment and the interactions between these internal and external factors can clearly influence sleep and W. In general, these influences have received little attention in basic sleep research, though their impact is recognized in many human sleep disorders. It may be that the role of the amygdala in evaluating and storing the emotional significance of events is a key component of its involvement in sleep-wake regulation. The use of fear conditioning, a validated behavioral model for studies in W, may be useful in gaining insight into the processes by which the amygdala affects the regulation of brainstem alerting systems and behavioral arousal and contributes to the problems of posttraumatic stress disorder.

Emotional situations incite the cataplectic attacks of narcolepsy [54]. Clearly, the amygdala is a prime candidate for exploration in understanding this disease. Indeed, the amygdala is one area that shows neuronal degeneration, at least in the case of growing narcoleptic dogs at the time cataplexy first makes its appearance [55]. Furthermore, the amygdala would seem to be an important structure to study in the case of insomnia. Understanding the role the amygdala plays in mediating emotional and environmental influences on sleep may give insight into some causes of this very common problem. For tackling this and the other sleep problems mentioned, it will be important to examine the roles of intra-amygdalar and other forebrain connections in addition to those with the brainstem in naturally sleeping animals.

Acknowledgments The work reported here was supported by NIH grants MH42903 and NS35281 and the Department of Veterans Affairs. We thank Graziella L. Mann and Antoinette M. Mann for their excellent technical assistance and Eileen P. Conner for administrative help.

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26 Sanford LD, Tejani-Butt SM, Ross RJ, Morrison AR: Amygdaloid control of alerting and behavioral arousal in rats: Involvement of serotonergic mechanisms. Arch Ital Biol 1995;134:81–99. 27 McGinty DJ, Harper RM: Dorsal raphe neurons: Depression of firing during sleep in cats. Brain Res 1976; 101:569–575. 28 Trulson ME, Jacobs BL: Raphe unit activity in freely moving cats: Correlation with level of behavioral arousal. Brain Res 1979;163:135–150. 29 Rueter LE, Jacobs BL: Changes in forebrain serotonin at the light-dark transition: Correlation with behaviour. Neuroreport 1996;7:1107– 1111. 30 Wilson CL, James WL, Yang S, Behnke EJ, Fried I, Bragin A, et al: Direct measures of extracellular serotonin change in the human forebrain during waking, non-REM sleep and REM-sleep (abstract). Soc Neurosci Abstr 1997;23:313. 31 Stutzmann GE, LeDoux JE: GABAergic antagonists block the inhibitory effects of serotonin in the lateral amygdala: A mechanism for modulation of sensory inputs related to fear conditioning. Neurosci 1999; 19:(RC8)1–4. 32 Bordi F, LeDoux JE, Clugnet MC, Pavlides C: Single-unit activity in the lateral nucleus of the amygdala and overlying areas of the striatum in freely behaving rats: Rates, discharge patterns, and responses to acoustic stimuli. Behav Neurosci 1993;107:757–769. 33 Jouvet M, Delorme F: Locus coeruleus et sommeil paradoxal. C R Séances Soc Biol Fil 1965;159:895– 899. 34 Henley K, Morrison AR: A re-evaluation of the effects of lesions of the pontine tegmentum and locus coeruleus on phenomena of paradoxical sleep in the cat. Acta Neurobiol Exp 1974;34:215–232. 35 Hendricks JC, Morrison AR, Mann GL: Different behaviors during paradoxical sleep without atonia depend on pontine lesion site. Brain Res 1982;239:81–105. 36 Morrison AR, Sanford LD, Ball WA, Mann GL, Ross RJ: Stimuluselicited behavior in rapid eye movement sleep without atonia. Behav Neurosci 1995;109:972–979.

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37 Leyhausen P: Cat Behavior. New York, Garland Press, 1979. 38 Parmeggiani PL, Morrison AR: Alterations in autonomic functions during sleep; in Loewy AD, Spyer KM (eds): Central Regulation of Autonomic Functions. New York, Oxford University Press, 1990, pp 367–386. 39 Morrison AR: Behavioral capabilities of cats during different behavioral states; in Oomura Y (ed): Neuronal and Endogenous Chemical Control Mechanisms on Emotional Behavior. New York, Springer, 1986, pp 241–254. 40 Zagrodzka J, Hedberg CE, Mann GL, Morrison AR: Contrasting expressions of aggressive behavior released by lesions of the central nucleus of the amygdala during wakefulness and rapid eye movement sleep without atonia. Behav Neurosci 1998;112:589–602. 41 Sanford LD, Morrison AR, Ball WA, Ross RJ, Mann GL: The amplitude of elicited PGO waves: A correlate of orienting. Electroencephalogr Clin Neurophysiol 1993;86:438– 445. 42 Ball WA, Sanford LD, Morrison AR, Ross RJ, Hunt WK, Mann GL: The effects of changing state on elicited ponto-geniculo-occipital (PGO) waves. Electroencephalogr Clin Neurophysiol 1991;79:420–429.

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43 DeBoer T, Sanford LD, Ross RJ, Morrison AR: Effects of electrical stimulation in the amygdala on ponto-geniculo-occipital waves in rats. Brain Res 1998;793:305–310. 44 DeBoer T, Sanford LD, Ross RJ, Morrison AR: Electrical stimulation in the amygdala increases the amplitude of elicited PGO waves. Physiol Behav 1998;66:119–124. 45 Ball WA, Morrison AR, Ross RJ: The effects of tones on PGO waves in slow wave sleep and paradoxical sleep. Exp Neurol 1989;104:251– 256. 46 Davis M: The role of the amygdala in fear and anxiety. Annu Rev Neurosci 1992;15:353–375. 47 Hitchcock J, Davis M: Lesions of the amygdala, but not of the cerebellum or red nucleus, block conditioned fear as measured with the potentiated startle paradigm. Behav Neurosci 1986;100:11–22. 48 Applegate CD, Kapp BS, Underwood MD, McNall CL: Autonomic and somatomotor effects of amygdala central n. stimulation in awake rabbits. Physiol Behav 1985;31: 353–360. 49 Iwata J, Chida K, LeDoux JE: Cardiovascular responses elicited by stimulation of neurons in the central amygdaloid nucleus in awake but not anesthetized rats resemble conditioned emotional responses. Brain Res 1987;418:183–188.

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50 Kapp BS, Gallagher M, Underwood MD, McNall CL, Whitehorn D: Cardiovascular responses elicited by electrical stimulation of the amygdala central nucleus in the rabbit. Brain Res 1985;234:251–262. 51 Rosen JB, Davis M: Enhancement of acoustic startle by electrical stimulation of the amygdala. Behav Neurosci 1988;102:195–202. 52 Rosen JB, Davis M: Enhancement of electrically elicited startle by amygdaloid stimulation. Physiol Behav 1990;48:343–349. 53 Sanford LD, Silvestri AJ, Ross RJ, Morrison AR: Influence of fear conditioning on elicited ponto-geniculooccipital waves and rapid eye movement sleep. Arch Ital Biol, in press. 54 Guilleminault C: Cataplexy; in Dement WC, Passouant P, Guilleminault C (eds): Advances in Sleep Research. 3. Narcolepsy. New York, Spectrum Publications, 1976, pp 125–143. 55 Siegel JM, Nienhuis R, Gulyani S, Ouyang S, Wu MF, Mignot E, Switzer RC, McMurry G, Cornford M: Neuronal degeneration in canine narcolepsy. J Neurosci 1999;19: 248–257.

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Reciprocal Actions between Sensory Signals and Sleep Ricardo A. Velluti José L. Peña Marisa Pedemonte Neurofisiologı´a, Departamento de Fisiologı´a, Facultad de Medicina, Universidad de la Repu´blica, Montevideo, Uruguay

Key Words Sleep W Wakefulness W Sensory signals W Processing, auditory W Processing, visual W Hippocampus W Theta rhythm

Abstract To the best of our knowledge, there is no simple way to induce neural networks to shift from waking mode into sleeping mode. Our best guess is that a whole group of neurons would be involved and that the process would develop in a period of time and a sequence which are mostly unknown. The quasi-total sensory deprivation elicits a new behavioral state called somnolence. Auditory stimulation as well as total auditory deprivation alter sleep architecture. Auditory units exhibiting firing shifts on passing to sleep (augmenting or diminishing) are postulated to be locked to sleep-related networks. Those (F50%) that did not change during sleep are

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postulated to continue informing the brain as in wakefulness. A rhythmic functional plasticity of involved networks is postulated. A number of auditory and visual cells have demonstrated a firing phase locking to the hippocampal theta rhythm. This phase locking occurs both during wakefulness and sleep phases. The theta rhythm may act as an organizer of sensory information in visual and auditory systems, in all behavioral states adding a temporal dimension to the sensory processing. Sensory information from the environment and body continuously modulates the central nervous system activity, over which sleep phenomenology must develop. It also produces a basal tonus during wakefulness and sleep, determining changes in the networks that contribute to sleep development and maintenance and, eventually, it also leads to sleep interruption. Copyright © 2000 S. Karger AG, Basel

Ricardo A. Velluti, MD, DSc Departamento de Fisiologı´a, Facultad de Medicina Av. Gral. Flores 2125, ROU–11800 Montevideo (Uruguay) Tel. +5982 924 3414 ext. 3409, Fax +5982 924 8784 E-Mail [email protected]

Introduction

There is not a single known physiological signal that may evoke sleep. Wakefulness (W), on the other hand, can be obtained by several signals coming from the internal world – the body – and from the environment. To the best of our knowledge, there is no simple way to induce neural networks to shift from the W mode to the sleeping mode. Our best guess is that a whole group of neurons would be involved and that the process would develop in a period of time in a sequence which is mostly unknown. Since 1916, it has been supposed that sensory input participates in waking and influences sleep [1]. An interaction between sleep and sensory input in general has been recently reported [2]. A landmark paper by Bremer [3] positioned the sensory systems as the main factors to support W while their lack or decreasing actions would lead to sleep. This is known as the ‘passive’ theory of sleep generation. However, sensory information is continuously received and processed throughout sleep and thus, some time later, it was conceived and demonstrated that active processes supporting sleep generation in general should exist [4–8].

Quasi-Total Deafferentation

The surgical section of the olfactory, optic, statoacoustic, and trigeminal nerves, a vagus nerve and the spinal cord posterior paths in cats (quasi-total deafferentation), carried out by Vital-Durand and Michel [9], confirmed a decrease in motor activity previously reported [10, 11]. Then, the erroneous idea of ‘continuous sleep’ (99% of the time) was put forward [11]. The diminished contact with the external world may induce a particular behavior and may also result in the lack of behavioral

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initiative. However, when studied under polygraphic control [9], the animals under quasitotal deafferentation revealed the existence of a sleep-waking cycle with the following characteristics: (1) The W time was reduced from 44.9 to 18.5%, and when asleep, the cats could be easily awakened. The EEG characteristics were those of normal cats with the exception of slow-wave bursts in the visual cortex [12]. (2) The time spent in slow-wave sleep (SWS) was reduced from 41.7 to 29.6%. A quasi-constant ‘somnolence’ was described, characterized by the sphinx position and a sequential fast and slow EEG activity. In contrast, the subcortical, hippocampus and amygdala activity was that of a quiet W indicative of a distinct state, both from a behavioral as well as from a bioelectrical viewpoint. (3) The total amount of paradoxical sleep (PS) (or rapid eye movement sleep) was slightly diminished (from 13.4 to 11.2%) with normal episode length and frequency. PS exhibited the characteristic rapid eye movements, and muscular twitches were present previous to the cervical spinal cord lesion. Muscle activity inhibition as well as cortical EEG activation, hippocampal theta rhythm and pontogeniculo-occipital waves on the visual cortex were also present. Five days after a cat was blinded, the visual cortex showed the most profound differences. While the sensorimotor cortex had an active, desynchronized EEG and the hippocampus exhibited a normal theta rhythm, the visual cortex showed short periods of flat EEG between bursts of highamplitude fast activity. Thus, it appears that after sensory deprivation, a behavioral state called ‘somnolence’ develops, characterizing what we are postulating now as a new behavioral state.

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The Auditory System during Sleep and Waking

As the only sensory system remaining active while asleep (at least in a micro-osmatic animal), a special relation may exist between the auditory system and sleep. The auditory system seems to act as a constant guardian to signal, e.g., danger, a predator or perhaps also prey. A second characteristic that makes the auditory system unique is its conspicuous efferent or descending component, featuring a complex anatomy located in parallel to the classic ascending pathway [13–15]. It is postulated to function as an input controller particularly through the action of its most peripheral stages, the olivo-cochlear system [16–20]. Effects of Sound Stimulation on Sleep Organization The organization of human sleep is extremely sensitive to acoustic stimuli, [21] and noise generally exerts an arousing influence on it [22]. A noisy nighttime environment leads to a decrease in total sleep time, particularly that of delta wave sleep (stage 4) and PS with the consequent increase in time spent in stage 2 and in W [23, 24]. Moreover, the remarkable sleep improvement after noise abatement [25] suggests that the environment is continuously scanned by the auditory system. This notion is also supported by unitary recording in sleeping animals as it is shown below. Upon intense auditory stimulation specifically during PS, the number of PS episodes was increased without changing its total amount [26]. Recent experimental data on auditory stimulation carried out in rats led to the conclusion that the pattern of PS occurrence (frequency and single episode duration) was affected by auditory stimulation during nonselective sleep phases, while the total amount of PS was always preserved [27].

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Auditory stimulation in animals as well as in humans seems to produce more general actions on the sleep process, still not clearly defined, because stimulation during SWS or PS results in similar changes in the sleep pattern. Effects of Total Auditory Deprivation on Sleep Organization Total auditory deprivation in guinea pigs (by surgical removal of both cochleae) enhances SWS and PS in similar proportion while reducing W, studied up to 45 days after lesion [28]. The SWS and PS increments cited above were determined mainly by an increase in the number of episodes, but there was no change in the duration of a single episode. The authors assert that the relative isolation from the outside world may be part of the change observed in deaf guinea pigs. Thus, the elimination of an input to a complex set of networks, such as those that regulate the sleepwaking cycle, would introduce functional shifts particularly if such input has some significance, as appears to be the case with the behavior under study. Contrasting results have been reported although all of them are indicative of a correlation between both auditory input and the sleep-waking processes, and they are supported by the stimulation experiments as well as by the opposite approach, i.e., sleep recording of deaf guinea pigs. More experimental data are evidently needed in the particular field of auditory sensory processes and their interaction with sleep.

Sleep and Wakefulness Auditory Information Processing

Auditory Evoked Potentials Human auditory responses have been reported by several investigators using similar approaches and obtaining similar results. In

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all subjects, the major changes in the auditory evoked response, on passing from the awake state to the four stages of SWS, were consistent increases in peak-to-peak amplitude while during PS the amplitude was lower and similar to W. The later waves of the response were of longer latency during both sleep phases, SWS and PS [29–32]. The experimental data gathered using the far-field evoked-potential recording technique in humans arrived at some interesting although debatable conclusions [33] and showed no sleep effects on the brainstem auditory evoked potentials [34–37]. The brainstem auditory evoked potentials, a coarse representation of auditory function, do not reflect the effects of sleep on the compound auditory nerve action potential or on the cochlear microphonic described in guinea pig recordings [20]. Moreover, another phenomenon demonstrates actions on the receptor itself; namely the transiently evoked oto-acoustic emissions (sounds emitted by the cochlea reflecting the outer hair cell motility controlled by the efferent system) have been reported in humans as being modified by sleep in general although independently of the sleep phase [38]. Auditory System Unitary Activity in Wakefulness and Sleep The effects of sleep and W on the auditory evoked activity of cats’ single units at the mesencephalic reticular formation was reported by Huttenlocher [39]. The evoked activity of those units was greater during quiet W and diminished during SWS; a decrease in evoked activity was commonly associated with an increase in spontaneous firing. The most constant effect on evoked neuronal firing was observed during PS. In all the cells studied during sleep, the response to clicks was reversibly diminished or abolished.

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In the anteroventral cochlear nucleus (CN) of guinea pigs, extracellularly recorded responses revealed neurons having clear-cut relationships to the sleep-waking cycle [40, 41]. In agreement with previous results yielding increments in the averaged auditory nerve compound action potential amplitudes during SWS [20], 47% of the CN units responding to sound increased their firing in SWS, 27% did not show any change, while no units decreased their firing. During PS, 20% of sound-responding neurons followed the auditory nerve amplitude trend while, on the other hand, a great proportion of units failed to follow the auditory nerve pattern. It was postulated that the auditory efferent system modulates the auditory input at the level of the CN during sleep and W. The probability of firing and the changes in the pattern of discharge are thus dependent on both the auditory input to the cochlear nucleus and the brain’s functional state, e.g., asleep or awake [41]. Most neurons from guinea pig lateral superior olive showed a firing rate modulation on passing from W to SWS; 80% of the recorded cells changed their firing during binaural or ipsilateral sound stimulation. In addition, shifts in the discharge pattern were observed in 15% of the cells recorded on passing from W to SWS while the most striking change, observed in decreasing firing units, was the near-absence of responses in PS during the last 40 ms as judged from the post-stimulus time histogram (stimuli: 50-ms tone bursts). Furthermore, the waking cues for binaural directional detection in these experiments disappeared during SWS; one possible interpretation of these results is that the binaural function of some (11.5%) lateral superior olive cells is impaired during SWS [42]. Similar results were obtained from the guinea pig inferior colliculus [43]. Most neurons (63%) exhibited evoked firing rate in-

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Fig. 1. Grouped changes in the auditory evoked activity throughout the wakefulness-sleep cycle for 78 primary cortex-recorded units.

creases or decreases on passing from W to SWS; during PS, all units recorded shifted their evoked firing while only 11% did not show changes. The inferior colliculus auditory neurons send descending connections to regions such as the dorsal pontine nuclei, known to mediate sleep processes, making this locus suitable for sleep-auditory system interactions. Connections were demonstrated between brain stem auditory loci and known pontine regions that generate paradoxical sleep signs after cholinergic local stimulation [44]. Pedunculo-pontine sleep-related neurons that respond to auditory stimuli have also been reported [45]. Moreover, cerebral cortex auditory areas descending projections to regions such as the dorsal pontine nuclei (known to mediate sleep processes) make the pedunculopontine region also suitable for sleep-auditory system interactions [15]. The auditory areas of the cerebral cortex receive complex ascending inputs, originating from both ears and, in turn, sending projections to thalamic, midbrain targets [15] and ipsilateral-contralateral cortical loci. Thus, the auditory cortex may control the whole auditory system and, at the same time, be dependent on the general state of the brain, asleep or awake. Extracellular recordings of

sound-evoked responses from the primary auditory cortex (A1) have been shown to be highly dependent on the state of the brain, e.g., W, SWS or PS. In guinea pigs, auditory cortical units recorded at their characteristic frequency varied their firing-evoked activity. During SWS and PS, the cortical units exhibited decreased and increased firing while, most important, a proportion (F50%) of units did not show firing rate changes, discharging as in W (fig. 1). Sleep and W behavior modifies auditory cortical processing of simple sound stimuli [46]. The auditory cortex may control the whole auditory system and, at the same time, be dependent on the general state of the brain, asleep or awake. However, it is important to remark that more than half of the neurons sampled fail to show discharge changes and thus would be able to preserve auditory ascending information. Moreover, we propose that the neurons whose discharge is about the same in sleep as in W are continuously informing the brain of auditory events. On the other hand, the units exhibiting firing shifts on passing to sleep – augmenting or diminishing – are postulated to be locked to sleeprelated networks, whatever role they may play in sleep.

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Fig. 2. Cortical evoked unitary discharges (guinea pig) by binaural stimulation recorded during 9 min and 15 s (555 s). After a relatively steady discharge level during SWS, a reduction of the response to sound can be observed on passing to PS. The firing level increases during a short period of W to regain a higher discharge level when a new SWS appears.

Figure 2 shows a cortical neuron responding to simple sound stimuli while changing the behavioral state. A repeated pattern appears throughout sleep, i.e., there are rhythmic firing changes most probably representing the auditory cortical networks involved that vary according to the actual behavioral state. Behavioral-related repeated network shifts are the basis for what we postulate as functional rhythmic cortical plasticity.

Auditory and Visual Neuronal Firing Exhibits Hippocampal theta Rhythm Phase Locking

The hippocampus exhibits a rhythmic electrographic activity in rodents called theta ( ) with a frequency ranging from 4 to 12 per second [47]. This rhythm, prominent during active W and PS, is also present in SWS [48, 49]. Two important sensory systems demonstrated a special relationship with the hippocampal   rhythm (Hipp  ), i.e., phase locking between the unitary firing of auditory and visual neurons and the hippocampal field potential   waves.

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An increasing number of auditory cells, from the CN up to the primary auditory cortex, have demonstrated a firing phase locking to the Hipp  . This phase locking occurs both during W and sleep phases, SWS and PS. The hypothesis proposed was that theta rhythm is a temporal organizer for sensory unit processing in all behavioral states, adding a temporal dimension to lateral superior olive, inferior colliculus and cortical auditory sensory processing [50]. In figure 3, an auditory unit from the lateral superior olive, exhibiting changes in its firing rate, is shown. The firing is modified according to the behavior, being enhanced during SWS and showing the greater increment during PS (autocorrelation). The interaction between the auditory unit and the Hipp  , demonstrated by the crosscorrelation, exhibits a phase locking during W and PS at the same Hipp   phase, while in SWS it fails to appear in spite of the Hipp   presence as shown in the SWS power spectrum (fig. 3). An auditory cortical neuron spontaneous and evoked activity is shown during SWS (fig. 4). The pattern of discharge changed as well as the cross-correlation to Hipp   when

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Fig. 3. Functional relationship between the lateral superior olive neuronal spontaneous discharges and Hipp   during different behavioral states. The auditory unit discharge is phase-locked with Hipp   during W and PS. The correlation is lost during SWS. On the other hand, the firing rate increases during both SWS and

particularly during PS (autocorrelogram). The power spectra exhibit a strong   frequency band power (black bars) in each state. Inset: Post-stimulus time histogram (PSTH) of the unit; binaural sound stimulation (tone burst at characteristic frequency: 2.0 kHz, 48 dB SPL).

the neuron was sound-stimulated. While the spontaneous activity did not show any correlation to   rhythm, after click stimulation (8/ s), i.e., during evoked neuronal activity, the phase locking with the Hipp   appears evident. The only change was the beginning of sensory stimulation while the behavior was the same SWS episode. The function Hipp   may perform during sleep is not known. Nevertheless, it was corre-

lated to movement (muscular twitches) and ponto-geniculo-occipital spikes as phasic PS phenomena; it has also been correlated to auditory unitary firing in response to sound during sleep [50] and autonomic activity (heart rate) in PS [51]. The reported phase locking of a proportion of auditory units that persist throughout behavior suggests that it may subserve a function in W that persists during both sleep

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Fig. 4. Relationship between Hipp   and auditory cortical unit during SWS. Upper traces: Raw data showing (from top to bottom) digitized units, sound stimuli (clicks), hippocampal field electrogram (Hipp) and auditory cortical unitary discharges. The spontaneous activity is shown left to the vertical line, and in the right part the activity during sound stimulation (8/s) is

shown. The crosscorrelogram did not exhibit phase locking with the Hipp   during the auditory unit spontaneous discharge. However, when the sound stimulation started, the neuron began to discharge in close correlation with a particular   rhythm phase. Calibrations: Hipp = 1 mV; unit = 50 ÌV.

phases. Perhaps, in the three behavioral situations the same temporal organization of sensory information is present although holding a particular relation to the sensory need of each behavioral state, i.e., the phase locking is

present and perhaps aimed to different functional targets. The Hipp  -auditory unit interactions were also found in lateral geniculate (LG) visual neurons. Figure 5 shows an LG neuron-

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Fig. 5. LG neuronal evoked discharges phase-locked

with Hipp   during two different behavioral states. Upper traces: Raw data showing (from top to bottom) light stimuli (flashes, 6/s), electromyogram (EMG), hippocampal field electrogram (Hipp) and unitary LG discharges. The crosscorrelogram shows that the phase

locking is enhanced during PS in comparison with the crosscorrelogram obtained during the previous W. A: Hippocampal field electrogram wave correlation; B: hippocampal power spectrum (Hipp   band: black bars). Calibrations: EMG = 100 ÌV; Hipp = 1 mV; unit = 50 ÌV.

evoked activity during W and PS. This particular case demonstrates that the unit discharge and Hipp   phase locking is not only the consequence of Hipp   power. A lower Hipp   power during PS, as compared to the W one,

exhibited a higher cross-correlation with the LG neuron. Thus, perhaps the PS is the important condition to determine the stronger degree of correlation of sensory neurons to the Hipp   rhythm.

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Conclusions

Although we have an incomplete understanding of the underlying processes at a synaptic level, the phenomenon of activity-dependent development has been demonstrated in the sensory systems most studied, the visual, auditory and somatosensory [52–54]. The activity-dependent development is surely a mechanism that directs neural connectivity in the brain. Thus, ‘... a function of waking in early life is directing central nervous system maturation’ [55], while we are suggesting now that early-life long-sleeping time may participate in CNS maturation to a great extent because the sensory information continues to enter the system at a high degree. Thus, during early ontogenetic development, the sensory information that reaches the CNS (occurring mainly during sleep) may be a function directing its connectivity; the sensory input that ‘sculptures’ the brain is active during sleep. Several points of interaction between sensory input and behaviors of W, SWS and PS are cited here: (1) the quasi-total lack of sensory input determines drastic changes in the sleep-waking cycle as well as in CNS bioelectrical activity; (2) a group of neurons continues to inform the sleeping brain in a similar fashion as during W; (3) a second group of neurons, however, exhibits repeated firing shifts, increasing or decreasing, on passing to sleep, suggesting they belong to sleep-related neural networks; (4) the interdependence of firing rate and sleep phases observed at the auditory nuclei and cortex supports the notion of a functional ultradian rhythmicity of plastic phenomena in the brain, and (5) sensory information from the environment and body is continuously modulating CNS activity, over which sleep phenomenology must develop. Sensory input exerts an influence maintaining a basal tonus during W and sleep,

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determining changes in the networks that contribute to sleep development and maintenance and, eventually, to sleep interruption. Among several functions postulated for Hipp   rhythm, the fluctuation of cell excitability is the best-known, providing a probable substrate for distant interactions among neurons discharging in a close temporal relation. The temporal relationship between the sensory neuronal firing and the Hipp   field activity is a changing phenomenon whose variation is dependent on the interaction of three signals or sets of signals: (1) the Hipp   rhythm, (2) the actual behavioral state of the brain, and (3) the specific sensory incoming information. A neuronal network may shift its discharge as a function of the three cited types of input. The interactions among them following a novel stimulus may produce a new phase locking and, besides, can evoke a Hipp   with higher power that may also shift brain condition and behavior. Reciprocally, the shift of the sensory neural behaviorally related network could induce hippocampal bioelectrical changes and may condition waking or sleep behavior. The parallel recording of hippocampal field activity ( ) and cortical auditory multiunit firing revealed a precise temporal structure of population events during W and both sleep phases, SWS and PS. Our results can support the hypothesis of a discontinuous exchange of information between the hippocampus and the neocortex [56], because the phase locking of cortical auditory units and Hipp   mainly occurs when a novel stimulus or repetition of the same one is applied during W and SWS. Discharge level and pattern changes in response to a constant stimulus represent a way to show that the CNS is able to modulate the incoming information. On the other hand, we have demonstrated that sensory information

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is able to break into the neural system and alter ultradian behavioral cycles in a complex manner. Adaptation implies a fine trade between the organism and the environment necessary for survival. The sensory systems represent the way information enters the CNS. Although this input is always present, biological constraints determine the necessity to proceed in alternate states of sleep and W that

imply shifts in the processing of sensory information. Thus, when the brain switches to different levels of consciousness, it must continue to keep track of the environment and the body. These facts acting together probably represent an evolutionary pressure on the development of complex reciprocal actions between sensory information processing and the neural circuits involved both in sleep and wakefulness.

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Bright Light during Nighttime: Effects on the Circadian Regulation of Alertness and Performance A. Daurat a J. Foret a O. Benoit b G. Mauco c a Laboratoire

Travail et Cognition, Université Toulouse-Le Mirail, Toulouse, d’Etudes du Sommeil, Service Exploration Fonctionnelle, Hôpital Henri-Mondor, Créteil, c Laboratoire Biochimie III, Hôpital La Grave, Toulouse, France

b Laboratoire

Key Words Bright light pulses W Alertness W Performance W EEG bands W Core temperature W Melatonin W Night work

the best time for using the alerting effect of BL. The immediate alerting effect of BL seems to be mediated by a global activation of the central nervous system. Copyright © 2000 S. Karger AG, Basel

Abstract The present studies evaluated to what extent duration (all-night or 4-hour exposures) and timing of nocturnal bright light (BL) (beginning or end of the night) modulate effects on vigilance. The results showed that all-night BL exposure is able to alleviate the nocturnal decrements in alertness and performance. However, under certain circumstances, this continuous BL exposure may induce adverse effects on mood and finally reveal to be counterproductive. Shorter BL exposure (4 h) during nighttime helps improve mood and performance, although the effects of short BL pulses were less efficacious than all-night BL exposure. The latter part of the night appears

ABC

© 2000 S. Karger AG, Basel 1422–4933/00/0096–0309$17.50/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/journals/bsi

Introduction

Bright light (BL) has a major synchronizing influence on the human circadian timing system, as demonstrated in various cases of changes in natural light timing. Seasonal variations in light/dark ratio affect important physiological functions; rapid transfer to another time zone requires a readaptation time to local time. In addition, artificial light permits activity to take place during natural darkness and to ‘trick’ the circadian clock [1, 2]. However, since the paper by Lewy et al. [3] in 1980, we know that light has to be brighter than regular electrical light to have a signifi-

Agnès Daurat Laboratoire Travail et Cognition, Maison de la Recherche Université Toulouse Le Mirail F–31058 Toulouse Cedex 1 (France) E-Mail [email protected]

cant impact on human circadian control via the retino-pineal link. This influence depends on the time and the location on body temperature time course at which BL is presented. A phase-response curve (PCR) for BL exposure has been unquestionably established. Circadian phase is delayed by light exposure if light exposure takes place at the beginning of the night, advanced if it takes place at the end of the night and unaffected by any light exposure during daytime. Phase shift is estimated mainly by the changes observed in the time course of core temperature [4, 5] or hormonal markers [6]. The dephasing effects is an increasing function of light intensity [7] and number of successive exposures. It is likely due to the effect of light on pineal melatonin secretion [8, 9]. In fact, BL at nighttime can inhibit melatonin secretion and suppress its hypothermic effect. In addition to the strictly chronobiological effect on phase, nocturnal light exposure also has an immediate positive influence on arousal level and performances [10, 11]. In contrast to the therapeutic applications of light in order to eventually change the phase of the circadian oscillator in several types of patient, practical applications of light at work require only short-term action. In fact, night work now consists in one or two, rarely three night shifts in a row and shifting the phase of the circadian timing system of workers is no longer desirable. This study was designed in order to utilize some results of fundamental research on biological rhythms for practical purposes. Therefore the experimental procedure mimicked the situation experienced by many industry workers as closely as possible: long night shifts, repetitive work, constant environmental conditions, limited physical activity, confined small groups and, of course, artificial lighting. We chose a ‘quasi-constant routine’, as advocated by Minors and Waterhouse [12],

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i.e. during a period of 24 h or more without sleep, subjects are kept in constant environmental conditions and sitting most of the time. In our experiments, to investigate sleep behavior following nocturnal light exposure, subjects slept either in the morning (24-hour wake) or the following night (36-hour wake). This difference, because they were aware of the expected wake time, likely affected the way they reported mood and subjective alertness. The objectives of this study were first to evaluate to what extent the duration (all-night or 4-hour episodes) and the timing of nocturnal BL (beginning or end of the night) modulate the effect on nighttime vigilance; second to establish whether a phase-shifting effect is superposed on or follows a stimulating effect.

Experimental Procedure In a first project (experiment A), the effects of allnight exposure to BL on arousal, mood and performance were studied during a night without sleep. Experiment A included two experimental protocols: in A1, subjects remained awake for 24 h (9.00 to 9.00 h on the following day) and in A2 they were kept awake from 9.00 h to the following night, that is a 36-hour wake. In both versions, subjects were exposed either to BL or to dim light (DL) throughout the night. A second project (experiment B) compared the effects of a short exposure to BL (4 h) at two different times of the night: 20.00–0.00 h or 4.00–8.00 h. Subjects again were kept awake for 24 h (9.00–9.00 h on the following day) and except during BL exposures, remained in DL. In the two experiments, subjects were young good sleepers (age range: 19–25). After one night of laboratory recording (23.00–7.00 h), subjects remained awake under a quasi-constant routine during the day and the following night. Subjects were allotted to groups of 4 and remained seated around a table without sleeping. They were allowed to read, talk together, write and play games. Light meals were served at 3-hour intervals. Environmental conditions: ambient temperature, humidity, noise were maintained constant. BL was provided by a lighting ceiling fixture and its level was strictly controlled. In experiments A, subjects were exposed to BL from 20.00 to 8.00 h (" 2,000 lx) in one

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Table 1. Results of the statistical analysis for the body temperature and melatonin rhythms

(melatonin was not measured in A1) Time-of-night effect

Main effect of the Time by light light condition conditions interaction

Temperature Experiment A1 Experiment A2 Experiment B

p ! 0.0001 p ! 0.0001 p ! 0.0001

n.s. n.s. n.s.

n.s. p ! 0.0001 p ! 0.0001

Melatonin Experiment A2 Experiment B

p ! 0.0001 p ! 0.0001

p ! 0.001 n.s.

p ! 0.0001 p ! 0.0001

condition and to DL (! 50 lx) in the second condition. In experiment B, they were exposed to BL between 20.00 h and 0.00 h in one condition and between 4.00 and 8.00 h in the second condition. In each experiment (8 subjects divided in groups of 4), light conditions were balanced between the two groups of subjects. The following variables were studied: by means of Thayer’s Adjective Check List [13], the subjective activation/deactivation dimension was evaluated by the GA/DS index (GA = general activation, DS = deactivation sleepiness) and tense-arousal by the HA/GD index (HA = high activation, GD = general deactivation), mood (POMS in experiments A2 and B), quantified EEG test and performance (letter cancelation, logical reasoning and in addition in experiment B, SAM 3 and SAM5 5). Measurements were made every 3 h. Rectal temperature was continuously recorded. In experiment B, blood samplings were collected every 2 h during nighttime from 20.00 h to 8.00 h. Urinary melatonin metabolites (experiment A2) and serum melatonin (experiment B) were determined by a radioimmunoassay. All experiments were carried out between October and March, which means that BL exposure took place during the hours of darkness.

Results

Core Temperature and Melatonin In our study, BL exposure affected nocturnal mean rectal temperature only slightly whereas it affected its temporal pattern more clearly (table 1). In A1, A2 and B, no signifi-

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cant difference was found on average over the night between BL and DL conditions. However, parts of the night were more sensitive to the difference between BL and DL. In A2, rectal temperature was significantly higher between 1.00 h and 7.00 h in BL than in DL (significant light by time interaction; see table 1). In experimental B, rectal temperature was higher during the 4.00- to 8.00-hour BL pulse compared to the 20.00- to 0.00-hour BL condition. Mean urinary melatonin level was decreased during the all-night BL exposure in experiment A2 (not measured in experiment A1) and was reduced (plasma melatonin) during both the 20.00- to 0.00-hour BL pulse and the 4.00- to 8.00-hour BL pulse in experiment B (fig. 1). Effects of 12-Hour BL Exposure (Experiment A) Subjective Evaluation of Alertness The statistical analysis of the comparison between BL and DL conditions for some variables in experiments A1 and A2 is presented in table 2. In experiment A1, BL exposure improved the activation/energy level (GA score) at 3.30 h and 6.30 h (p ! 0.05). In contrast to GA, the level of sleepiness assessed by the DS

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1

3

Fig. 1. Time course of aMT6S (a: experiment A2) and plasma melatonin (b; experiment B) in both light con-

ditions (n = 8; significant differences between the light conditions: * p ! 0.05, ** p ! 0.01, *** p ! 0.001). Fig. 2. Time course of the GA index in both light conditions of experiment A2 (n = 8; significant differences between the light conditions; * p ! 0.05). Fig. 3. Time course of the tension (HA/DG scores; a) and irritability (b) levels in both light conditions of experiment A2 (n = 8; significant differences between the light conditions: * p ! 0.05).

2

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Table 2. Results of the statistical analysis

Time-of-night effect

Main effect of the Time by light light condition conditions interaction

Experiment A1 GA scores DS scores Letter cancelation Logical reasoning

p ! 0.001 p ! 0.0001 p ! 0.0001 p ! 0.05

n.s. n.s. n.s. n.s.

p ! 0.05 n.s. p ! 0.05 p ! 0.05

Experiment A2 GA scores DS scores HA/DG scores Irritability (POMS) Theta/alpha (A1+A2) Letter cancelation Logical reasoning

p ! 0.05 p ! 0.0001 p ! 0.06 n.s. p ! 0.0001 p ! 0.01 p ! 0.01

n.s. n.s. p ! 0.05 p ! 0.05 n.s. n.s. n.s.

p ! 0.05 p ! 0.05 n.s. n.s. p ! 0.05 p ! 0.0001 n.s.

Experiment B GA scores DS scores Theta power density Letter cancelation Logical reasoning SAM 3 SAM 5

p ! 0.0001 p ! 0.0001 p ! 0.0001 p ! 0.0001 p ! 0.0001 p ! 0.05 p ! 0.1

n.s. n.s. n.s. n.s. n.s. n.s. n.s.

p ! 0.01 p ! 0.1 p ! 0.06 n.s. n.s. p ! 0.1 p ! 0.07

score was not affected by BL exposure and continuously increased throughout the night in both the BL and DL conditions. In experiment A2, GA scores were also improved by the 12-hour BL exposure but only in the first half of the night. In the early morning, GA scores sharply dropped, becoming lower than in the DL condition (fig. 2). The ratio of the tense/arousal indices (HA/GD scores) and the irritability index (POMS) showed some interesting features. In fact, BL exposure resulted in an increase (significant at p ! 0.05) in tense-arousal and irritability levels compared with the DL condition (fig. 3). In the middle of the night, subjects felt less calm and more tense-excited than under DL. Together with the increased activation/energy level, this suggests a state of hyperarousal. In contrast, at

the end of the night, subjects felt more tired, ill at ease, silent, with a loss of interest, than in DL condition. This state could be qualified ‘tense-tiredness’ as proposed by Thayer [13]. In conclusion, experiment A2 suggested that all-night BL exposure may, in certain conditions, induce first a state of hyperarousal and then a state of apathy/tense-tiredness. This state of hyperarousal could be related to the significant decrease in the DS score observed in the middle of the night. In contrast, at the end of the night, DS scores were significantly higher in the BL condition than in the DL condition, which confirmed that subjects experienced greater fatigue/tiredness associated to BL exposure while in A1 the HA/DG and DS scores did not vary between the two light conditions.

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Fig. 4. Time course of the theta/alpha ratio in both

Fig. 5. Time course of the letter cancellation task (time

light conditions of the experiments A (n = 8; significant differences between the light conditions; * p ! 0.05).

of execution) in both light conditions of experiment A2 (n = 8; significant differences between the light conditions: * p ! 0.05; ** p ! 0.01).

Quantified EEG Test A Cz-Pz lead was recorded for 4.5 min while subjects were fixing a spot and then 4.5 min with eyes closed. The theta (4–8 Hz)/alpha (8– 12 Hz) power density ratio was used to quantify physiological sleepiness. Signs of sleepiness are associated with progressively decreased alpha power density, increased theta power density and thus with an increased theta/alpha ratio. As the effects of all-night BL exposure on theta/alpha ratio were similar between experiments A1 and A2, the two sets of results were pooled. Figure 4 displays the time course of the theta/alpha ratio in the DL conditions (experiment A1 + A2; n = 16) and in the BL conditions (experiment A1 +A2; n = 15). When subjects were exposed to BL, the mean level of sleepiness was significantly lower than under DL. At 2.30 and 5.30 h, BL exposure resulted in a significant decrease in the theta/alpha ratio. Afterward, this ratio quickly increased, becoming nonsignificantly different from the DL conditions.

of correct responses) was not affected. In experiment A1, subjects slowed down throughout the night in order to maintain their accuracy in both BL and DL conditions. BL exposure shortened the time needed to complete the letter cancelation task in the later part of the night (6.30 h, p ! 0.05). In A2, and by contrast with A1, speed did not significantly vary in the DL condition, whereas accuracy tended to decline at the end of the night. Under BL, speed performance was better than under DL in the early part of the night. By contrast, when mood begun to deteriorate in the middle of the night, speed performance became worse than under DL (fig. 5). For the logical reasoning task, the effect of time was moderate, with the longest latencies observed at the end of the night (3.30 h and 6.30 h). The changes induced by exposure to BL were similar to those observed for letter cancelation, but without reaching significance level. In experiment A1, the BL condition tended to increase the speed in the later part of the night, but intersubject variability was great. In experiment A2, it tended to improve speed compared with the DL condition in the

Performance BL exposure modified speed of performance in A1 and A2 while accuracy (number

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Fig. 6. Time course of the GA index in both light conditions of experiment B (n = 8; significant differences between the light conditions: * p ! 0.05).

early part of the night and was worse in the later part, that is identical to the letter cancelation task. Effects of 4-Hour BL Exposure (Experiment B) All-night BL exposures took place at parts of the PRC which favored both phase delay (before the temperature trough) and phase advance (after the temperature trough). Therefore there might be a mutual neutralization of BL effects. To avoid this source of confusion, experiment B was designed with either a 4-hour BL exposure either at the beginning or at the end of the night. The effects of the two BL conditions (20.00–0.00 h vs. 4.00– 8.00 h) were compared. Two issues will be examined: (1) Can a short BL pulse help alleviate nocturnal decrements of arousal and performance without negative effects on mood and performance as observed in experiment A2 with all-night exposure? and (2) What is the best timing for a short BL treatment to enhance arousal and performance without modifying the circadian phase and affecting subsequent sleep?

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Fig. 7. Time course of performance (time of execu-

tion) in both light conditions of experiment B (n = 8; significant differences between the light conditions: * p ! 0.05). a Letter cancellation. b SAM 3.

Subjective and Objective Evaluation of Alertness As in experiment A1, BL did not affect subjective sleepiness assessed by the DS scores whatever the time of the BL pulse. In contrast, the activation/energy level (GA) was significantly improved by the 4.00- to 8.00-hour BL pulse (fig. 6) and the theta power density significantly decreased while the 20.00- to 0.00hour BL pulse induced a nonsignificant increase in the GA scores (p ! 0.09) and no difference in the theta band.

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Performances Figure 7 displays performance for two tasks which were affected by both BL exposures. For letter cancellation, SAM 3 and SAM 5 speed decreased throughout the night and was significantly better during both early and late BL exposures compared with the DL condition.

Discussion

For the studies reported here, the discussion must take into account that all BL exposures took place during nighttime. Subjects were sleep deprived at the same time they were exposed to BL. Therefore, when explaining the effects of different levels of illumination at nighttime one should take into consideration not only circadian determinants (time-of-day effect) but also the cumulated sleep deficit, particularly as regards performances. Experiments A1 and A2 showed that BL treatment may be a useful tool to alleviate decrements in both alertness and performance which are the main difficulties experienced by night workers. In good agreement with other studies [7, 10], subjects felt more alert in BL than in DL in both the middle of the night and the early morning. In experiment A2, the increased level of tension/irritability associated to the increased level of activation/energy even suggests a state of hyperarousal which was not observed in A1, although experimental conditions were identical for the part of the study reported here. Individual characteristics might explain this discrepancy. However, the recruitment of subjects was made with the same inclusion criteria and it is unlikely that they had a particularly high natural level of alertness. Higher constraints in A2 than in A1 (longer duration of the constant routine, EEG continuous-

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ly recorded, regular urinary collection and so on) may have facilitated a particularly high arousal level and a feeling of tension. This likely potentiated the alerting effect of BL and eventually favored the development of hyperarousal in the first part of the night. Hyperarousal, internal tension and difficulty to cope with, for example by motor activity, seem to result in a decline in subjective energy at the end of the night. Along with the circadian trough of alertness, it may explain the state of apathy/exhaustion observed in BL-exposed subjects in A2. According to the hypothesis of Thayer [14], a mood of exhaustion ‘could be a predictable biological reaction to diminished physical resources’. This state may contribute to save energy by limiting the energy expenditure due to activity or emotions. On the whole, experiments A1 and A2, which were identical in terms of sleep/wake schedules and light exposures, led to the same results, but, probably because subjects felt different amounts of subjective strain, the time course (but not the average) of a few variables (DS index, HA/DG scores, irritability, temperature) was more sensitive to BL exposures in A2 than in A1. The changes induced by BL on performance globally paralleled those observed on alertness and mood. When BL improved alertness, subjects performed better. Inversely, when BL induced a mood-degrading effect, work efficiency deteriorated. An interesting finding in our studies is that BL specifically affected speed but not accuracy. Lafrance et al. [15] explored the strategy used by their subjects in a task of choice reaction time. They found a decrease in reaction times at the cost of more errors. The authors interpreted these results as a change in the strategy adopted by the subjects in BL. Our results do not support this hypothesis. Search tasks (SAM with various memory loads) were more affected by BL than the logi-

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cal reasoning task. There might be a difference because the short logical reasoning task involves a high-level mental activity and thus is insensitive to alertness variations. Moreover, the hypothesis of a ceiling effect cannot be excluded. Nevertheless, although not significant, the changes induced by BL in this task paralleled those observed for the search tasks. In summary, the BL condition resulted in a decrease of reaction times and in improvement of subjective alertness, which is consistent with a global increase in physiological arousal, as suggested in Lafrance et al. [15]. As a consequence, it increased the capacity to resist sleep in the middle of the night/ early morning when sleepiness normally is highest. Experiment B shows that a 4-hour BL pulse of moderate intensity is less efficacious than 8-hour BL exposure to override the circadian trough of alertness. However, this short BL pulse helped subjects resist sleep. It is likely that a pulse of longer duration starting earlier in the night or of higher intensity could reveal as efficacious as all-night BL exposure. The 20.00- to 0.00-hour BL pulse failed to significantly increase the level of arousal, but sleep need is too weak at this time of the day which corresponded to the ‘wake maintenance zone’ coined by Lavie [16]. In fact, reference EEG tests in the DL condition did not exhibit any variation up to 2.30 h. Thus, it is not surprising that there was no detectable effect of BL on the EEG in the early part of the night. This latter result does not mean that BL was inefficacious at this circadian phase, as shown by the improvement in speed and subjective alertness, in agreement with Myers and Badia [7] and Cajochen et al. [17]. It rather suggests that changes induced by BL were due to an alerting effect. Moreover, melatonin secretion onset which probably mediates the circadian effect of light occurred late in the course of the BL pulse.

Our results are in favor of a global activation effect on the central nervous system. When subjects were exposed to BL, they were more alert and performed faster than under DL at the same time of the night. Furthermore, BL superimposed on stressful conditions can induce hyperarousal which may be detrimental to well-being and efficiency. Thus, the main effect of BL may be to increase physiological arousal, as indicated by the changes in activation index, speed performance and EEG indices. The lack of BL effects on sleepiness scores and sleep need supports the hypothesis [18] that BL acts via a direct or indirect action on the wake structure rather than on those underlying sleep. Nocturnal BL exposure, even moderate in intensity and duration, is able to improve well-being and efficiency, particularly at the end of the night/early morning. This is of practical importance for night and early morning shifts. As previously noted, current practice is to work no more than two or three nights in succession. In this condition, pronounced changes in the circadian phase are not desirable. Thus, the immediate alerting properties of BL may be useful in the case of rapidly rotating shift workers. It may be particularly beneficial for the first night shift which is perceived by shiftworkers as the most difficult, the least safe and the least productive [19].

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References 1 Eastman CI, Boulos Z, Terman M, Campbell SS, Dijk DJ, Lewy AJ: Light treatment for sleep disorders: Consensus report. VI. Shift work. J Biol Rhythms 1995;10:157–164. 2 Dijk DJ, Boulos Z, Eastman C, Lewy AJ, Campbell SS, Terman M: Light treatment for sleep disorders: Consensus report. II. Basic properties of circadian physiology and sleep regulation. J Biol Rhythms 1995;10:113–125. 3 Lewy AJ, Wehr TA, Goodwin FK, Newsome DA, Markey SP: Light suppresses melatonin secretion in humans. Science 1980;210:1267– 1269. 4 Minors DS, Waterhouse JM, WirzJustice A: A human phase-response curve to light. Neurosci Lett 1991; 133:36–40. 5 Czeisler CA, Kronauer RE, Allan JS, Duggy JF, Jewett ME, Brown EN, Ronda JM: Bright light induction of strong (type 0) resetting of the human circadian pacemakers. Science 1989;244:1328–1333. 6 Van Cauter E, Sturis J, Byrne MM, Blackman JD, Leproult R, Ofek G, Lhermite-Baleriaux M, Refetoff S, Turkey FW, Van Reeth O: Demonstration of rapid light-induced advances and delays of the human circadian clock using hormonal phase markers. Am J Physiol (Endocrinol Metab 29) 1994;266:E953–E963.

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7 Myers BL, Badia P: Immediate effects of different light intensities on body temperature and alertness. Physiol Behav 1993;54:199–202. 8 Reiter RJ: The melatonin rhythm: Both a clock and a calendar. Experientia 1993;49:654–664. 9 Claustrat B, Geoffriau M, Brun J, Chazot G: Melatonin in humans: A biochemical marker of the circadian clock and an endogenous synchronizer. Neurophysiol Clin 1995;25: 351–359. 10 Campbell SS, Dawson D: Enhancement of nighttime alertness and performance with bright ambient light. Physiol Behav 1990;48:317–320. 11 Daurat A, Aguirre A, Foret J, Gonnet P, Keromes A, Benoit O: Bright light affects alertness and performance during a 24-h constant routine. Physiol Behav 1993;53:929– 936. 12 Minors DS, Waterhouse JM: The use of constant routines in unmasking the endogenous component of human circadian rhythms. Chronobiol Int 1984;1:205–216. 13 Thayer R: Activation-deactivation adjective check list: Current overview and structural analysis. Psychol Rep 1986;58:607–614.

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14 Thayer R: Toward a psychological theory of multidimensional activation. Motivat Emotion 1978;2:1– 34. 15 Lafrance C, Dumont M, Lespérance P, Lambert C: Daytime vigilance after morning bright light exposure in volunteers subjected to sleep restriction. Physiol Behav 1998;63:803– 810. 16 Lavie P: The 24 hour sleep propensity function: Practical and theoretical implications; in Monk TH (ed): Sleep, Sleepiness and Performance. Chichester, Wiley, 1991, pp 65–93. 17 Cajochen C, Kräuchi K, Von Arx MA, Graw P, Wirz-Justice A: Daytime melatonin administration enhances sleepiness and theta/alpha activity in the waking EEG. Neuroscience Lett 1996;207:209–213. 18 Daurat A, Aguirre A, Foret J, Benoit O: Disruption of sleep recovery after 36 hours of exposure to moderately bright light. Sleep 1997;20:352– 358. 19 Budnick LD, Lerman SE, Nicolich MJ: An evaluation of scheduled bright light and darkness on rotating shiftworkers: Trial and limitations. Am J Ind Med 1995;27:771–782.

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Adenosine as a Biological Signal Mediating Sleepiness following Prolonged Wakefulness Radhika Basheer Tarja Porkka-Heiskanen Robert E. Strecker Mahesh M. Thakkar Robert W. McCarley Neuroscience Laboratory, Department of Psychiatry, Harvard Medical School and VA Medical Center, Brockton, Mass., USA

Key Words Sleep deprivation W Basal forebrain W Adenosine W A1 receptor W NF-ÎB

Abstract Recent reports from our laboratory have shown that extracellular adenosine levels selectively increase in basal forebrain during prolonged wakefulness in cats and rats. Furthermore, microdialysis perfusion of adenosine into the basal forebrain (BF) increased sleepiness and decreased wakefulness in both the species, whereas perfusion of the A1-receptor-selective antagonist, cyclopentyl-1,3-dimethylxanthine resulted in increased wakefulness, an observation similar to that found with caffeine or theophylline administration. The selective participation of the A1 subtype of the adenosine receptor in mediating the effects of adenosine in the BF was further examined by the technique of

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single unit recording performed in conjunction with microdialysis perfusion of selective agonists and antagonists. Perfusion of the A1 agonist cyclohexyladenosine, inhibited the activity of wake-active neurons in the basal forebrain. The effect of prolonged wakefulness-induced increases in adenosine levels were further investigated by determining the changes in the BF in the levels of A1 receptor binding and the levels of its mRNA. We observed that A1 receptor mRNA levels increase after 6 h of sleep deprivation. One of the transcription factors that showed increased DNA-binding activity was nuclear factor ÎB (NF-ÎB) and may regulate the expression of A1 mRNA. We observed, using a gel shift assay, that the DNA-binding activity of NF-ÎB increased following 3 h of sleep deprivation. This was further supported by the increased appearance of NF-ÎB protein in the nuclear extracts and the consequent disappearance of cytoplasmic protein inhibitor

Robert W. McCarley Harvard Medical School & VA Medical Center, Psychiatry, 116A 940 Belmont Street, Brockton, MA 02401 (USA) Tel. +1 508 583 4500 (ext.) 3723, Fax +1 508 586 0894 E-Mail [email protected]

ÎB (I-ÎB). Together our results reviewed in this report suggest that the somnogenic effects of adenosine in the BF area may be mediated by the A1 subtype of adenosine receptor, and its expression might be regulated by induction in the NF-ÎB protein as its transcription factor. This positive feedback might mediate some of long-duration effects of sleep deprivation, including ‘sleep debt’.

In this review, we present the most recent evidence obtained from our laboratory using physiological, pharmacological, biochemical and molecular techniques. Taken together, these data provide convergent evidence that adenosine is an endogenous sleep factor, whose effects are mediated via the A1 adenosine receptor subtype in the cholinergic-cellrich areas of the basal forebrain (BF).

Copyright © 2000 S. Karger AG, Basel

Introduction

Adenosine, a purine nucleoside and a natural byproduct of cellular energy metabolism has been found to covary with sleep and wakefulness in the cat [1] and recently in the rat [2], a finding that is congruent with 30% reduction in cerebral metabolic activity with nonREM sleep compared with wakefulness. This is also consistent with earlier data showing adenosine being higher during the rat circadian activity period and lower during its resting period [3, 4]. Early pharmacological studies with intraperitoneal or intracerebral administration of adenosine or adenosine agonists resulted in increased sleep [5–7], supporting the idea of the role of adenosine as a somnogenic factor. In contrast, caffeine and theophylline, known to be antagonists of adenosine receptors, increased waking [5, 8]. These observations drew attention towards the possibility that one of the functions of adenosine in the central nervous system could be to serve as a ‘sleep factor’. To be categorized as a factor controlling the sleepiness following prolonged wakefulness (endogenous sleep factor), adenosine levels should rise with prolonged wakefulness, and this rise, in turn, should promote sleep. Also, adenosine concentration should decrease with sleep occurring after prolonged wakefulness.

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Extracellular Adenosine Levels Selectively Increase in the BF following Sleep Deprivation

Microdialysis and adenosine measurements, performed in conjunction with EEG recording to monitor the vigilance state, provided the initial evidence that extracellular adenosine levels increase progressively with prolonged sleep deprivation in the BF of cats [1]. The levels declined with subsequent recovery sleep (fig. 1a). The cortex and four other subcortical sites were also examined in cats and none of the areas showed the significant increase in adenosine seen in BF [9]. A more recent study from our laboratory reported similar waking-induced increases in the levels of extracellular adenosine in BF in rats (fig. 1b) [2], thus confirming the presence of this phenomenon in two different species. The physiological significance of such a selective increase in adenosine in BF was further studied by monitoring the effects of increased adenosine levels in BF, either due to direct microdialysis perfusion [2, 10] or due to the use of a transmembrane transporter blocker, S-(4-nitrobenzyl)-6-thioinosine, 1 ÌM [1], on the vigilance state in both species. Elevated levels of adenosine in BF resulted in decreased wakefulness and increased slow-wave sleep and delta activity, whereas no such effect was observed by a similar increase in adenosine in the thalamus [1].

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Fig. 1. a Cat brain. Adenosine con-

centration in different brain areas during sleep deprivation and recovery sleep. In the BF and cortex, adenosine levels increase significantly, whereas in four other areas, the levels decline slowly during 6 h of deprivation. b Rat brain. In rats, during the light period, adenosine levels rise with sleep deprivation for 3 h. CTRL = Control, 10 a.m. to 11 a.m.; D1, D2 and D3 = 1, 2 and 3 h of deprivation, respectively; R1 and R2 = recovery sleep, 1st and 2nd hour, respectively.

Together these studies strongly suggest that the somnogenic effects of adenosine are selectively mediated in the BF area. The BF contains both cholinergic and noncholinergic neurons [11] with cortical projections. The discharge activity of the neurons in

this area is mostly highest during waking and was initially identified as cholinergic in nature [12–15]. This led to the studies using whole-cell and extracelluar recordings on the effect of adenosine in in vitro brain slices of cholinergic neurons in BF demonstrating

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Fig. 2. a Microdialysis perfusion of an A1 antagonist (cyclopentyltheophylline; CPT) in the BF of cats produced a concentration-dependent increase in wakefulness (p ! 0.05). b The electrical discharge activity of neurons in the BF with a wake-selective pattern (n = 4)

was inhibited by local microdialysis perfusion of an A1 agonist, cyclohexyladenosine (CHA, 0.1, 1.0 and 10.0 ÌM ) in freely behaving cats. SWS = Slow-wave sleep.

strong inhibitory tone of adenosine on these neurons [16]. Recently, inhibition of wakerelated unit activity has been reported by in vivo infusion of adenosine in the BF [17, 18]. This suggests that adenosine may mediate its effects by inhibiting the wake-active neurons in BF.

types, A1 receptors are widely distributed in the brain [20, 21] whereas A2a receptors are believed to be concentrated in the striatum, nucleus accumbens, olfactory tubercle and lateral globus pallidus [22, 23]. A3 receptors are reported to be located mainly in peripheral tissues [21]. Both A1 and A2a receptor subtypes have been implicated in mediating the sleep-inducing effects of adenosine. The propensity to sleep and the intensity of delta waves upon falling asleep were increased following intraperitoneal or intracerebroventricular administration of the highly selective A1 receptor agonist, N6-cyclopentyladenosine [5, 24]. On the other hand, the A2a agonist CGS 21680 infused into the subarachnoid space below the rostral BF (i.e. not in the parenchyma) in-

The Effects of Adenosine in BF May Be Mediated by the A1 Receptor Subtype

Adenosine mediates its effects via different receptor subtypes. To date, A1, A2A, A2B and A3 have been described, based on both their affinity for a variety of ligands and also their protein sequences [8, 19]. Among these sub-

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creased slow-wave sleep [25]. The somnogenic effects of prostaglandin D2 have been suggested to be mediated through the A2a receptor in the same area [25–27]. We have recently observed that in BF, contrary to the effects of adenosine, perfusion of the A1 selective antagonist, cyclopentyl-1,3dimethylxanthine (CPT) increased wakefulness and decreased sleep (fig. 2a) [9]. Singleunit recording of wake-active neurons in BFperformed in conjunction with in vivo microdialysis have shown that perfusion of the A1-selective agonist, N6-cyclohexyladenosine, decreased the discharge activity of the wakeactive neurons [17] (fig. 2b), whereas the A1selective antagonist CPT increased the activity of wake-selective neurons. Together, these results suggest that in BF the effects of adenosine may be mediated by the A1 subtype adenosine receptor.

receptor subtypes are different in mediating the somnogenic effects of adenosine, we have examined the ligand-binding efficiency of both the receptor subtypes in BF following prolonged sleep deprivation. Contrary to our initial expectation, the preliminary results obtained showed a tendency for increased receptor binding following 6 h of sleep deprivation [31]. More extensive investigation is ongoing to confirm this paradoxical observation. However, this led to the next question: whether the increase in receptor binding is accompanied by an increase in A1 receptor mRNA levels.

A1 Receptor mRNA Levels Are Increased with Sleep Deprivation

The effects of adenosine A1 receptor are mediated by G-proteins coupled to these receptors [19, 28]. A common functional feature of these receptors is its rapid attenuation in response to the agonists, the commonest response being receptor downregulation, i.e. loss of receptors from the cell surface following prolonged exposure to agonist [29]. In order to maintain reduced response to the overabundant agonist, the effector pathways have been shown to further regulate the synthesis and stability of receptor mRNA (as clearly demonstrated in the case of the ßadrenergic receptor [30]. In view of our previous observations that extracellular adenosine levels selectively increase in BF following sleep deprivation, we first postulated that the adenosine receptor would also be downregulated in BF following sleep deprivation. Although the anatomical location of A1 and A2a

The changes in the levels of A1 receptor mRNA were determined by the reverse transcription-polymerase chain reaction of total RNA from the BF tissue samples. We observed a significant increase in the levels of A1 receptor mRNA in BF following 6 h of sleep deprivation whereas there was no effect on cortex A1 mRNA levels [31] (fig. 3). In addition to being intriguing, these results supported our earlier observations that the effects of wake-induced adenosine levels were specific to BF and these changes may be involved in mediating the somnogenic effects of adenosine, as we will discuss later. Achieving a balance between the synthesis and degradation of receptors is essential in order to maintain a constant level of receptor density and function. Many examples of autoregulation of receptor transcription by agonist binding have been described in the central nervous system. Interestingly, some of these regulatory events appear to be mediated by the same second-messenger systems that are activated by the A1 receptor. For example, the substance P receptor is coupled to phospho-

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a

Fig. 3. a Autoradiograph of the PCR products ob-

tained by RT of total RNA from the BF and cortex of sleep-deprived and control rats using A1 receptor and the housekeeping gene cyclophylline-specific primers. b The optical densities of the PCR products were measured using a molecular imager and the relative amount of the mRNA was determined using the ratio of the value for A1 mRNA to the value of cyclophylline mRNA for each sample. In the BF, the A1 mRNA levels (mean B SEM) were significantly increased (+78%; sleep-deprived 0.679 B 0.095; control 0.380 B 0.0629) following 6 h of sleep deprivation, whereas no significant change was observed in the cortex (sleepdeprived 0.903 B 0.079, control 0.734 B 0.085).

inositol turnover which releases intracellular Ca2+, resulting in the activation of protein kinase C. Activated protein kinase C phosphorylates and activates transcription enhancers for the substance P receptor gene [32]. Another example of the autoregulation is the stimulatory G-protein-coupled receptor, such as the ß2-adrenergic receptor, which generates cAMP upon ligand binding which, in turn, activates protein kinase A; this is capable of phosphorylating the cAMP response element binding protein, which serves to enhance the transcription of the gene for the receptor [33]. In order to investigate the events occurring after receptor binding and to identify the mechanisms that are involved in the regulation of A1 receptor gene expression

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b

as a consequence of prolonged wakefulness, we are now investigating changes in transcription factors.

Effect of Adenosine on Transcription Factors

Several reports show the wake-related induction of the transcription factor, c-Fos in rat brain [34–36]. Recently, we have shown that unilateral administration of adenosine by microdialysis selectively increases the DNAbinding activity of c-Fos in the ipsilateral side without any change in the contralateral region in the same animal [2]. Thus, increase in extracellular adenosine might mediate its ef-

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b

Fig. 4. a Gel shift assays done using the crude nuclear extracts

of BF of rats show that NF-ÎB DNA binding is higher after 3 h of sleep deprivation compared to controls. SD = Sleep deprivation, 3 and 6 h; C = undisturbed circadian controls; P = lane with probe alone; Mut = mutant oligonucleotide. b Western blot of the pure nuclear protein pooled from 3 rats after 3 h of sleep deprivation shows the presence of the p65 NF-ÎB subunit in the nucleus, whereas the undisturbed sleeping controls had no signal in the nucleus. Whereas in the cytoplasm (each lane is from an individual rat), the I-ÎB protein levels declined following 3 h of sleep deprivation, indicating the loss due to degradation compared to sleeping controls.

a

fects, at least in part, through the pathways involving c-Fos as a transcription factor. The A1 adenosine receptor is coupled to the inhibitory G-protein, known to inhibit adenylate cyclase. Recently, much attention has been drawn towards the potential additional signaling pathway involving stimulation of phospholipase C (PLC) [37]. Activation of PLC mobilizes internal Ca2+ and activates protein kinase C. Furthermore, protein kinase C has been shown to phosphorylate the inhibitor ÎB (I-ÎB) protein in the cytoplasm, thus causing the release of the nuclear factor ÎB (NF-ÎB). Once released, it is then permitted to translocate to the nucleus, where it serves as a transcription factor [38, 39]. The most recent experiments from our laboratory have examined the possibility of NF-ÎB activation by determining its nuclear translocation using Western blots of purified nuclear proteins and DNA binding activity determined by gel shift assays. Three hours of sleep

deprivation resulted in nuclear translocation of NF-ÎB and also increased DNA binding in nuclear extracts from BF area (fig. 4). Conformation of the release of NF-ÎB following phosphorylation of I-ÎB was obtained by finding a decrease in cytoplasmic I-ÎB levels, known to degrade after binding to degradatory proteins like ubiquitins [40] enhancing their proteolytic degradation. Recently, a sleep deprivation-induced increase in NF-ÎB DNA binding activity has been reported in the parietal cortex [41]. In our studies we did not observe any change in the DNA-binding activity in the cingulate cortex. While it still has not been definitively shown whether the increase in NF-ÎB activity is directly related to A1 receptor activation, this possibility is intriguing, since NF-ÎB production may regulate the A1 receptor expression. In human A1 receptor gene, an NF-ÎB binding site has been identified in the upstream promoter region. Furthermore, in

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smooth muscle cells, induction of NF-ÎB oxidative stress has been shown to increase A1 receptor expression [42]. It is possible that the increase in A1 mRNA is due to the activation of NF-ÎB. More experiments are in progress to address these issues. In conclusion, the studies detailed above summarize our attempts towards a better understanding of the physiological significance of the selective increase in the extracellular levels of adenosine. Our results show that in the BF area the somnogenic effects of adenosine may be mediated by A1 subtype of adenosine receptor. From the preliminary evidence presented in this report, an interesting hypothesis emerges that selective increase in the extracellular adenosine during prolonged sleep deprivation activates the A1 receptor and might be responsible for maintaining the feed-forward and positive feedback regulation of its own expression via NF-ÎB activation. It is tempting to speculate that transcriptional induction of A1 recetpor may increase the number of surface A1 receptors, thereby providing a mechanism for the production of the ‘sleep debt’ that follows prolonged wakeful-

ness/sleep deprivation, and consists of attention and performance decrements [43]. The increased number of A1 receptor would amplify the effects of endogenous adenosine, so that a decrement in vigilance might be produced at adenosine concentrations that had much less effect prior to accrual of a ‘sleep debt’. In addition, NF-ÎB might also regulate the expression of other genes possibly involved in production of ‘sleep debt’. Further studies are in progress towards gaining better insight into the cellular and molecular mechanisms that are involved in mediating the somnogenic effects of adenosine. Also to elucidate the effects of prolonged A1 receptor stimulation and the subsequent effects on the expression of other genes that may play a role in generating sleep debt.

Acknowledgments This work was supported by awards from the Veterans Administration (RES and RWM) and from the National Institute of Mental Health (MMT, MH01798-01; RWM, MH R37 39,683).

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7 Radulovacki M: Role of adenosine in sleep in rats. Rev Clin Basic Pharmac 1985;5:327–339. 8 Fredholm BB, Bättig K, Holmén J, Nehlig A, Zvartau EE: Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol Rev 1999;51:83–133. 9 Strecker RE, Porkka-Heiskanen T, Thakkar MM, Dauphin LJ, Stenberg D, McCarley RW: Recent evidence that the sleep-promoting effects of adenosine are site specific. Sleep 1999;22:S32. 10 Portas CM, Thakkar M, Rainnie DG, Greene RW, McCarley RW: Role of adenosine in behavioral state modulation: A microdialysis

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34 Basheer R, Sherin JE, Saper CB, Morgan JI, McCarley RW, Shiromani PJ: Effects of sleep on wakeinduced c-fos expression. J Neurosci 1997;17:9746–9750. 35 Cirelli C, Pompeiano M, Tononi G: Neuronal gene expression in the waking state: A role for the locus coeruleus. Science 1997;274:1211– 1215. 36 Grassi-Zucconi G, Menegazzi M, Carcereri De Prati A, Bassetti A, Montagnese P, Mandile P, Cosi C, Bentivoglio M: c-fos mRNA is spontaneously induced in the rat brain during the activity period of the circadian cycle. Eur J Neurosci 1993;5: 1071–1078. 37 Freund S, Ungerer M, Lohse M: A1 adenosine receptors expressed in CHO cells couple to andenylyl cyclase and to phospholipase C. Naunyn-Schmiedeberg’s Arch Pharmcol 1994;350:49–56. 38 Zhong H, Su Yang H, ErdjumentBromage H, Tempst P, Ghosh S: The transcriptional activity of NFÎB is regulated by the I-ÎB associated PKAc subunit through cyclic AMP-independent mechanism. Cell 1997;89:413–424. 39 Jacobs MD, Harrison SC: Structure of an IÎB·/NF-ÎB complex. Cell 1998;95:749–758. 40 Roff M, Thomson J, Rodriguez MS, Jacque JM, Baleux F, ArenzanaSeisdedos F, Hay RT: Role of IÎB· ubiquitination in signal-induced activation of NF-ÎB in vivo. J Biol Chem 1996;271:7844–7850. 41 Chen Z, Gardi J, Kushikata T, Fang J, Krueger JM: Nuclear factor-ÎBlike activity increases in murine cerebral cortex after sleep deprivation. Am J Physiol 1999;276:R1812– R1818. 42 Nie Z, Mei Y, Ford M, Rybak L, Marcuzzi A, Ren H, Stiles GL, Ramkumar V: Oxidative stress increases A1 adenosine receptor expression by activating nueclear factor ÎB. Mol Pharmacol 1998;53: 663–669. 43 Dinges DF, Pack F, Williams K, Gillen KA, Powell JW, Ott GE, Aptowicz C, Pack AI: Cumulative sleepiness, mood disturbances and psychomotor vigilance performance decrements during a week of sleep restricted to 4–5 hours per night. Sleep 1997;20:267.

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A Critical Assessment of the Melatonin Effect on Sleep in Humans Jaime M. Monti Daniel P. Cardinali Farmacologı´a y Terapéutica, Hospital de Clı´nicas, Montevideo, Uruguay, and Departamento de Fisiologı´a, Facultad de Medicina, Universidad de Buenos Aires, Argentina

Key Words Melatonin W Sleep disorders W Pineal gland W Circadian physiology W Melatonin receptors W Alzheimer disease

Abstract Melatonin is synthesized and secreted during the dark period of the light-dark cycle. The rhythmic nocturnal melatonin secretion is directly generated by the circadian clock, located in mammals within the suprachiasmatic nucleus (SCN), and is entrained to a 24hour period by the light-dark cycle. The periodic secretion of melatonin may be used as a circadian mediator to any system that can ‘read’ the message. In addition, direct effects of the hormone on the SCN could explain some of the melatonin effects on the circadian system. Duration of the melatonin nocturnal secretion is directly proportional to the length of the night and it has experimentally

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been demonstrated to be the critical parameter for photoperiod integration. The sites and mechanisms of action of melatonin for circadian and photoperiodic responses are far from being elucidated, but action through specific membrane receptor sites starts to emerge. A possible bicompartmental model of distribution for melatonin, the first compartment in plasma acting on peripheral organs and the second in the cerebrospinal fluid affecting neurally mediated functions at a much higher concentration, has recently been proposed. From earlier studies it was concluded that melatonin administration to humans reduces sleep latency and induces sleepiness and fatigue. More recently, the effect of lower pharmacologic or physiologic doses of melatonin was examined in different laboratories. These studies included young normal volunteers and patients with chronic insomnia, as well as dementia patients exhibiting sundowning syndrome. Irre-

Dr. Jaime M. Monti, Clinical Hospital Department of Pharmacology and Therapeutics, Av. Italia s-n J Zudanez 2833-602, ROU–11300 Montevideo (Uruguay) Tel. +59 82 710 58 07, Fax +59 82 487 37 87 E-Mail [email protected]

spective of the method of assessment, melatonin showed effects in insomniac patients in most studies. With some exceptions, melatonin administration reduced sleep latency and/or increased total sleep time and sleep efficiency. Furthermore, melatonin was more effective when given to elderly insomniacs, or Alzheimer disease patients, although sleep improvement was not strictly correlated with prior levels of the hormone. Copyright © 2000 S. Karger AG, Basel

The Circadian System

Melatonin (N-acetyl-5-methoxytryptamine) is normally synthesized and secreted during the dark phase of the day in all species studied to date. The primary physiological function of melatonin is to convey information about the daily cycle of light and darkness to body physiology. By its pattern of secretion during darkness, melatonin indicates the length of the night, thus representing the chemical code of the scotophase. This information is used for the organization of functions which respond to changes in photoperiod such as circadian and seasonal rhythms [1, 2]. Because the earth rotates on its axis, it presents two environments, i.e. light and darkness; because the earth’s axis of rotation is tilted, the durations of daily periods of darkness and light vary systematically during the course of the year. Through evolution, animals responded to these environmental changes by preferentially adapting to them. This is the origin of biological rhythms that repeat approximately every 24 h, called circadian rhythms (from Latin circa, ‘around’, and dies, ‘day’), and of rhythms that oscillate annually, following the recursive appearance of the seasons [3–6].

Melatonin in Sleep-Disturbed Patients

Thus, when animals switch between diurnal, nocturnal or seasonal modes of their behavior, they are not simply responding passively to changes in external lighting conditions. They are responding to signals generated by a circadian pacemaker that is synchronized with the cycles of the earth’s rotation, anticipates the transitions between day and night, and triggers the appropriate changes in behavioral state and physiological substrates. In this way, the circadian pacemaker creates a day and a night within the organism that approximately mirrors the world outside. Research in animals and humans has shown that only a few such environmental cues, such as light-dark cycles, are effective entraining agents for the circadian oscillator (‘Zeitgebers’). In addition, the sleep-wake schedule may also be an important entraining agent in humans. An entraining agent can actually reset, or phase-shift, the internal clock. Depending on when an organism is exposed to such an entraining agent, circadian rhythms may be advanced, delayed, or not shifted at all. Therefore, involved in adjusting the daily activity pattern to the appropriate time of day is a rhythmic variation in the influence of the Zeitgeber as a resetting factor [3–6]. In humans, light exposure during the first part of the night delays the phase of the cycle; a comparable light change near the end of the night advances it. At other times during the day, light exposure has no phase-shifting influence [7]. Melatonin, the endogenous chemical code of the night, showed an opposite phase response curve to light, producing phase advances during the first half of the night and phase delays during the second half [7]. In mammals, considerable experimental evidence indicates that a region of the hypothalamus, the suprachiasmatic nucleus (SCN), is a major circadian pacemaker. The

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Fig. 1. Possible mechanism of action of melatonin in

the transmission of photoperiodic information. Photic information is conveyed to the SCN, principally through the retino-hypothalamic tract (RHT), where it synchronizes the activity of the circadian oscillator to exactly 24 h. Neuronal efferent pathways from the SCN directly distribute circadian information to different brain areas, including the pineal gland which generates the melatonin rhythm. The neural route for

environmental lighting control of melatonin secretion includes the intermediolateral column of the thoracic chord gray (ILC) and the superior cervical ganglion (SCG). The generated melatonin rhythm might be used by the SCN to distribute its rhythmic information. The possibility that there could be two compartments of melatonin affecting physiological function, with melatonin concentration in the brain about 50 times greater than in plasma, is discussed in the text.

SCN, composed of a cluster of thousands of small nerve cells, can generate circadian rhythms when isolated from other areas of the brain. The integrity of the SCN is necessary for the generation of circadian rhythms as well as for synchronization of rhythms with light-dark cycles. Compelling evidence that the SCN functions as the primary circadian pacemaker comes from animal studies of SCN transplantation. In these experiments, the SCN is de-

stroyed, abolishing circadian rhythms. When fetal brain tissue containing SCN nerve cells is transplanted into the brains of these animals, circadian rhythms are restored [3–5]. Light in the environment activates cells in the eye which in turn activate early genes within cells in the SCN. The output is principally transmitted down the axons of SCN neurons to specific targets in the central nervous system and hence to the rest of the body through neuronal and endocrine pathways (fig. 1).

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Melatonin is synthesized from serotonin through two enzymatic steps. The first is the N-acetylation by serotonin N-acetyltransferase to yield N-acetylserotonin. The physiological regulation of serotonin N-acetyltransferase, with its sharp increase in activity at night, has received considerable attention as a major regulatory step in melatonin synthesis. The second step in melatonin synthesis is the transfer of a methyl group from S-adenosylmethionine to the 5-hydroxy group of N-acetylserotonin to yield melatonin. This reaction is catalyzed by the enzyme hydroxyindole-Omethyl transferase (HIOMT). The day/night changes of HIOMT are less prominent; however, a day/night rhythm of HIOMT gene transcription occurs. In addition to N-acetylserotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol and 5-hydroxytryptophan are also O-methylated by HIOMT; to what extent these secretory products are active pineal products by themselves is less known [1]. Environmental lighting, acting through the eye in adult mammals and in part directly on the pineal body in lower vertebrates and birds, has profound effects on the rhythms in melatonin biosynthesis. Exposure of animals to light at night rapidly depresses pineal melatonin synthesis. Based on denervation or nerve stimulation studies, a simple model of pineal regulation was envisioned, comprising two premises: (1) the neural route for environmental lighting control of melatonin secretion is the neuronal circuit retina – retinohypothalamic tract – SCN – periventricular hypothalamus – intermediolateral column of the thoracic chord gray – superior cervical ganglion – internal carotid nerves – pineal gland (fig. 1); (2) norepinephrine released from sympathetic terminals at night activates postsynaptic ß-adrenoceptors coupled to the adenyl-

ate cyclase-cAMP system, therefore increasing melatonin synthesis and release. However, the presence of ·-adrenoceptors as well as the characterization of central peptidergic pinealopetal pathways have made the subject more complicated [8, 9]. Melatonin appears to be secreted by the pineal gland by simple diffusion, and there is consensus in that the concentration of melatonin in the pineal body is a direct reflection of both its synthesis and its concentration in plasma. The lipophilicity of melatonin contributes to its easy passive diffusion across cell membranes as well as through cell layers. Radioactive melatonin administered intravenously rapidly disappears from the blood with a half-life of about 30 min depending on the species examined. About 60–70% of melatonin in plasma is bound to albumin and none is bound in the cerebrospinal fluid (CSF) [10]. Exogenous oral or intravenous melatonin has a short metabolic half-life (from 20 to 60 min in humans) with a large hepatic firstpass effect and a biphasic elimination pattern [11]. Very large individual variations in peak plasma concentrations (25-fold) after a given oral dose are seen and attributed to differences in absorption. Most of the melatonin in the general circulation is converted to 6hydroxymelatonin in the liver which clears 92–97% of circulating melatonin on a single pass [12]. The 6-hydroxymelatonin formed is conjugated and excreted into urine. The sulfate derivative of 6-hydroxymelatonin amounts for 50–80% and the glucuronide for 5–30% of the excreted melatonin. The remaining melatonin is excreted either unchanged (less than 1%), as 5-methoxyindoleacetic acid (0.5%), or as the nonindolic metabolite N-acetyl-5-methoxykynurenamine (15%). A new melatonin metabolite, cyclic 3-hydroxymelatonin, could be an indicator of the potent, endogenous hydroxyl radical scavenging properties of melatonin [13].

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Melatonin as an Endocrine Arm of the Biologic Clock

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The Nocturnal Melatonin Peak Mediates the Photoperiodic Response

Most species show seasonal variations in their physiology and behavior. The reproductive cycle is timed so that environmental conditions are propitious for the growth of the young, and variations in behavior, pelage, appetite, body weight and fat are such that survival in ambient temperature conditions is optimal. Although the exact mechanisms by which animals anticipate seasons are not entirely known, the SCN and the pineal gland via melatonin secretion are the principal structures involved [14]. Removal of the pineal gland in photoperiodic species abolishes the ability to respond to changing day length. It is also possible to administer melatonin by daily infusion or feeding in order to generate circulating profiles with a duration characteristic of particular photoperiods in the intact or pinealectomized animal. In this way, it has become clear that a particular melatonin duration is a necessary and sufficient condition for the induction of a given seasonal response and is equipotent with a particular photoperiod [1, 14]. In most mammalian species, the duration of the nocturnal melatonin peak is positively correlated with the length of the dark period. These observations led to establish the ‘duration hypothesis’ of melatonin action, holding that the duration of the nocturnal peak of melatonin is the critical parameter in the transmission of photoperiod information. This hypothesis has been validated by administering melatonin to pinealectomized animals to induce daily ‘long day’ or ‘short day’ melatonin concentrations. This is the basis to consider melatonin as a ‘chemical code’ of the night. Using daily programmed melatonin infusions, the importance of the duration of the melatonin signal has been demonstrated in

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several species such as sheep, mink, and Siberian and Syrian hamster [14, 15]. The ‘coincidence hypothesis’ is based on the idea that circadian variation of sensitivity to melatonin is involved in the photoperiodic response. This hypothesis, which implies an effect of melatonin on the circadian clock, was supported by data obtained following daily melatonin injections in Syrian hamsters. Moreover, in pinealectomized animals maintained under long photoperiods, gonadal atrophy was observed only when the animals were injected twice per day with one injection at the beginning of the dark phase and the other at the beginning of the light phase [14, 15]. Indeed, melatonin acceptor sites are present in the SCN and the SCN metabolism is known to be directly affected by exogenous melatonin. In SCN-lesioned rats, daily injections of melatonin cannot entrain free-running locomotor activity. In general, entrainment occurs when melatonin is injected at the onset of activity and coincides with induction of a phase advance [8, 16]. It should be noted that melatonin is not only involved in the photoperiodic response but is also a direct component of the circadian apparatus in that it constitutes a signal driven by the clock and exporting information on time of day to (theoretically) every tissue in the body. Since the melatonin rhythm is an important efferent hormonal signal driven by the clock, its rhythmic secretion can therefore be used as an internal synchronizer (or ‘internal Zeitgeber’) [17]. Therefore the melatonin signal can be considered an efferent hormonal signal downstream from the clock which reflects the functioning of the pacemaker. Melatonin can be used as a mediator to impose circadian rhythmicity upon target structures, directly driving these rhythms (fig. 1).

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Site and Signal Transduction of Melatonin Action Are Multiple

The first experiments on brain melatonin receptor sites were carried out in the late seventies by employing 3H-melatonin as a ligand indicating the existence of melatonin acceptor sites in bovine hypothalamic, cerebral cortex and cerebellar membranes [18]. The introduction of the 2-125I-iodomelatonin analog, a major landmark in the melatonin receptor field, allowed detection of melatonin binding in several brain areas, the choroid plexus and in some brain arteries as well as in peripheral organs, like the Harderian glands, primary and secondary lymphoid organs, the adrenals, heart and lungs, the gastrointestinal tract, the mammary glands, the kidney and male reproductive organs. Indeed, to have an organ devoid of melatonin binding sites may constitute an exception rather than the rule [16]. A first classification of putative melatonin receptor sites into ML1 and ML2 was based on kinetic and pharmacological differences of 2-125I-iodomelatonin binding, and a major achievement in the field has been the cloning of the ML1 melatonin receptors [16, 19]. A nomenclature of melatonin receptors has recently been proposed by the International Union of Pharmacology (IUPHAR) [20]. Besides membrane acceptor sites, evidence has accumulated on nuclear binding of melatonin as well [21]. Melatonin appears to be the natural ligand for the orphan nuclear hormone receptor superfamily RZR/ROR [22]. Among presumptive second messengers for melatonin action, the cAMP generating system has received paramount attention. The main signal transduction pathway of high-affinity MT1 receptors in both neuronal and nonneuronal tissues is the inhibition of cAMP formation through a pertussis-toxin-sensitive inhibitory Gi protein. Coupling of the high-affinity melatonin receptors to other signaling path-

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ways has also been reported. Melatonin augmented cGMP levels, decreased Ca2+ influx and inhibited arachidonate acid conversion and prostaglandin synthesis [8]. Direct effects of melatonin on calmodulin and other intracellular proteins, nuclear receptor activity for melatonin, and the free radical scavenging properties of melatonin should also be considered. One important question in melatonin physiology concerns the actual concentration of melatonin that reaches sites of action in the brain. It must be noted that the levels of a lipophilic substance like melatonin reaching neurons under physiological or pharmacological conditions can differ considerably from circulating hormone concentration. In early studies using high-pressure liquid chromatography [23] or RIA [24, 25], hypothalamic melatonin concentrations were found to be about 50 times greater than in plasma. The possibility that there could be two compartments of melatonin affecting physiological function, the first in plasma acting on peripheral organs, and the second in the CSF affecting neurally mediated functions at a much higher concentration, has recently been proposed in a study demonstrating that third-ventricle CSF melatonin levels were 20-fold higher than nocturnal plasma concentrations [26] (fig. 1).

Studies on the Melatonin Effect on Sleep

Initial studies addressing the effect of melatonin on sleep made use of the intravenous or the intranasal route [27–29] or administered very large doses of the methoxyindole by the oral route [27, 30]. From these early studies it was concluded that melatonin reduces sleep latency and induces sleepiness and fatigue.

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More recently, the effects of lower pharmacologic or physiologic doses of melatonin were examined in different laboratories. These studies included young normal volunteers and patients with chronic insomnia, as well as dementia patients exhibiting sundowning syndrome. Although in most instances melatonin significantly improved subjective and/ or objective sleep parameters in normal subjets, negative results have also been reported.

Studies in Normal Volunteers Polysomnographic Assessment of Sleep The effect of melatonin on the polysomnographic sleep of normal volunteers has been characterized in several studies. In two reports [31, 32], melatonin was given in the evening at 21:00 or 22:45 h and recordings were started at 22:00 or 23:00 h, respectively. The effect of placebo or melatonin 0.3, 1 or 5 mg was assessed in the sleep laboratory during one night. Melatonin 0.3 or 1 mg significantly reduced sleep latency and increased sleep efficiency [32]. In contrast, the 5-mg dose did not significantly modify sleep induction or maintenance [31]. Zhdanova et al. [33] administered melatonin 0.3 or 1 mg at three different times (18:00, 20:00 or 21:00 h) to a group of normal subjects. Either dose of the methoxyindole decreased stage 2 sleep latency irrespective of the time of administration. The effect of melatonin (80 mg) was also tested in subjects with situational insomnia induced by exposure to recorded traffic noise. Melatonin was administered at 21:00 h and polysomnographic recordings started at 22:30 h. The hormone reduced sleep latency and the number of awakening episodes and increased stage 2 sleep and sleep efficiency [34]. Melatonin was also administered during morning and afternoon hours to subjects with

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normal sleep. In a study by Badia et al. [35] in which normal volunteers had a 4-hour sleep opportunity from 12:00 to 16:00 h, melatonin (1, 10 or 40 mg) significantly reduced sleep latency and increased total sleep time. Similar results were observed in subjects treated with 3 or 6 mg and allowed a 2-hour nap beteween 18:00 and 20:00 h, or instructed to attempt to sleep for 7 min every 20 min during 7 h, and treated with 3 mg melatonin [36]. In the study by Dijk et al. [37] subjects were partially sleep-deprived the previous night and allowed a 4-hour nap starting at 13:00 h. Administration of melatonin 30 min prior to the daytime nap did not significantly modify sleep induction or maintenance. On the other hand, EEG power density was increased in the sigma band and reduced in the delta band. Sleep Propensity Assessed Using Multiple Sleep Latency Tests Reid et al. [38] administered either a 5-mg oral formulation of melatonin or placebo at 14:00 h to healthy subjects, and recorded sleep onset latency to stage 1 and stage 2 sleep using an hourly multiple sleep latency test (MSLT). Mean sleep latencies were significantly reduced between 15:00 and 18:00 h as was core temperature. Gilbert et al. [39] compared the effect of daytime melatonin (5 mg) or temazepam (20 mg) administered at 14:00 h to young adults. Sleep onset latency was measured using 20 min MSLT. Compared with placebo, both melatonin and temazepam significantly reduced sleep latency and body temperature. Induction and Quality of Sleep Monitored Using Wrist-Worn Actigraphs Terlo et al. [40] characterized the effect of melatonin (0.1, 0.5 and 5 mg) on induction and quality of sleep, as well as on subjective

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fatigue and mood ratings in the late afternoon (17:00–21:00 h) in healthy volunteers. Sleep quality was objectively monitored using actigraphs and subjectively by using sleep logs. Melatonin did not significantly reduce sleep latency but decreased wake time after sleep onset. In addition, the methoxyindole increased feelings of sleepiness and fatigue. Assessment of Subjective Sleepiness and Mood using Performance Tasks Dollins et al. [41] examined the effects of melatonin (0.1–10 mg) administered at 11:45 h on sleep latency and duration, mood, performance and oral temperature in healthy volunteers. All melatonin doses significantly increased sleep duration as well as self-reported sleepiness and fatigue. In addition, sleep onset latency, oral temperature and the number of correct responses on the Wilkinson vigilance task were significantly reduced. In the study by Cajochen et al. [42] subjective sleepiness and waking EEG power density in the range of 0.25–20 Hz were measured after administration of a 5-mg dose of melatonin at either 13:00 or 18:00 h. Subjective sleepiness was significantly increased 40 and 90 min after melatonin administration (at 13:00 and 18:00 h, respectively). There was also an increase in the theta/alpha frequencies of the waking EEG which appeared before the subjective symptoms of sleepiness became apparent. In summary, melatonin has been administered during nighttime and daytime to young subjects with normal sleep. It has been contended that high pharmacological doses of melatonin are needed to improve sleep of normal volunteers during nighttime [43]. An interesting question, not yet solved, concerns as to what ‘physiological’ means in terms of intracerebral melatonin levels (see above). However, Zhdanova et al. [32] reported that physiologic (0.3 mg) and low pharmacologic

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(1 mg) doses of melatonin given during nighttime significantly reduced sleep latency and increased sleep efficiency, as assessed polysomnographically. The effects of melatonin on normal subjects during daytime were assessed using polysomnography, MSLTs, wrist-worn actigraphs, and performance tasks. Melatonin was administered in doses ranging from 0.1 to 40 mg, 60–120 min prior to the beginning of the evaluation procedure. The volunteers received the methoxyindole at 11:45, 12:00, 14:00, 17:00 or 18:00 h during the test day(s). The subjects had a sleep opportunity that amounted from 7 to 240 min. Melatonin administration during daytime reduced sleep latency, increased total sleep time, induced feelings of sleepiness and fatigue and reduced correct responses on vigilance tasks. The soporific properties of melatonin as manifested in spectral EEG changes during waking (augmented power density in the theta/alpha range) appeared in advance of the subjective symptoms of sleepiness [42].

Young, Middle-Aged and Elderly Patients with Insomnia The effects of melatonin on sleep of insomniac patients were characterized in several studies. Polysomnographic Assessment of Sleep The effect of melatonin on the polysomnographic sleep of insomniac patients was determined in three studies. James et al. [44] administered melatonin 1 or 5 mg 15 min before bedtime in one night. Variables related to sleep induction and maintenance were not significantly modified. On the other hand, REM latency was significantly increased after the 1-mg dose. It must be noted that in this study, the subject population under placebo showed a mean total sleep time of 395 min

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and a sleep efficiency of 88%. Hence any possible effect of melatonin could have been limited by a ceiling effect. Moreover, the absence of a decrease of stage 2 sleep latency could have been the result of the patients taking their medication only 15 min before lights off. In the study by Hughes et al. [45], the patients made use of immediate-release (0.5 mg) and controlled-release (0.5 mg) preparations of melatonin that were taken 30 min before bedtime. Polysomnographic recordings (2 nights) and sleep actigraphy (13 nights) of middle-aged and elderly insomniacs showed melatonin to shorten latencies to persistent sleep. However, the methoxyindole was not effective in sustaining sleep or improving subjective reports of nighttime sleep and daytime alertness. There was no correlation between prior melatonin production and responsiveness to melatonin replacement. Monti et al. [46] administered 3 mg of melatonin during 14 nights to a group of elderly patients with chronic primary insomnia. The hormone was given immediately before turning off the lights in the sleep laboratory. Wake time after sleep onset was significantly reduced whereas total sleep time and sleep efficiency were increased in 5 out of the 10 patients treated with melatonin. No strict correlation was found between prior 6-sulphatoxymelatonin levels in urine and subsequent sleep improvement after receiving melatonin. Sleep Monitored by Wrist Actigraphy or Self-Reported Questionnaires Garfinkel et al. [47] monitored sleep quality by wrist actigraphy in elderly insomniacs who were receiving various medications for chronic illnesses. Controlled-release melatonin (2 mg) was taken 2 h before the desired bedtime during 3 weeks. Melatonin reduced sleep latency and wake time after sleep onset

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and increased total sleep time and sleep efficiency. However, it should be taken into consideration that improvement of sleep could have been related to replacement of endogenous melatonin depleted by the various medications that the patients were receiving. Haimov and Lavie [48] compared the effect of immediate-release (2 mg) and controlled-release (2 mg) melatonin in independently living or institutionalized elderly insomniacs. The methoxyindole was administered 2 h before bedtime during 1 week. The sustained-release preparation improved sleep. Further improvement of sleep maintenance was observed following 2 months’ treatment with 1 mg sustained-release melatonin. In the study by MacFarlane et al. [49] insomniac patients received 75 mg of melatonin at 22:00 h for 7 consecutive nights. Melatonin significantly improved subjective sleep time and subjective daytime alertness. However, more than 50% of patients reported that the treatment had no effects on their subjective feeling of well-being. Wurtman and Zhdanova [50] characterized the effect of melatonin (0.3 mg) given during 3 nights on motor activity and core temperature of middle-aged and elderly patients with chronic insomnia. Melatonin reduced sleep latency, the number of nocturnal awakenings and body movements per night. Core temperature remained unchanged. Ellis et al. [51] administered 5 mg of melatonin at 20:00 h for a week to a group of middle-aged insomniac patients. Effects on sleep and wakefulness were monitored by visual analogue scale and structural interview. Sleep onset latency, estimated total sleep and wake time were not altered by melatonin. In the study by Fainstein et al. [52] melatonin (3 mg) was administered 30 min before the expected bedtime for 21 nights to patients with chronic insomnia. Overall sleep quality and daytime alertness were assessed by means

Monti/Cardinali

of structured clinical interviews and sleep logs completed by the patients. Starting from day 2 or 3 of treatment, melatonin significantly improved sleep quality and decreased the number of awakenings. A retrospective study of 14 Alzheimer’s disease (AD) patients receiving 9 mg of melatonin daily for 22–35 months was published [53]. All patients had cognitive and neuroimaging alterations (cortical and bitemporal atrophy) compatible with different evolutionary stages of AD. After varying periods of time of treatment with melatonin, a significant improvement of sleep quality was found in all cases. Sundowning, diagnosed clinically in all 14 patients examined, was no longer detectable in 12 of them. Neuropsychological evaluation by FAST and Mini-Mental State Examination indicated the absence of significant differences between the initial and the present state of evolution of the disease. This should be contrasted with the significant deterioration of clinical conditions of the disease expected from patients after 1–3 years of evolution of AD. In a case report of two monozygotic twins suffering from AD of 8 years’ evolution (one of them treated with melatonin, 6 mg per os for 36 months), evolution of the disease was halted in the melatonin-treated

subject, as indicated by a stable impairment of mnesic function and a substantial improvement of sleep quality and reduction of sundowning [54]. In summary, irrespective of the method of assessment, melatonin shows effect in insomniac patients in most of the studies. With one exception, melatonin was administered in doses ranging from 0.3 to 5 mg immediately before or 15, 30 or 120 min prior to bedtime, for periods extending from 1 to 21 nights. Melatonin administration reduced sleep latency and/or increased total sleep time and sleep efficacy. Furthermore, melatonin was more effective when given to elderly insomniacs, or AD patients, althoug sleep improvement was not strictly correlated with prior levels of the hormone.

Acknowledgments The studies in the authors’ laboratories were supported by Pedeciba, Montevideo, Uruguay, Elisium, Buenos Aires, the University of Buenos Aires (TM 07), the Consejo Nacional de Investigaciones Cientı´ficas y Técnicas, Argentina (PIP 4156) and the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica, Argentina (PICT 2350).

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32 Zhdanova IV, Wurtman RJ, Morabito C, Piotrovska VR, Lynch HJ: Effects of low oral doses of melatonin, given 2–4 hours before habitual bedtime, on sleep in normal young humans. Sleep 1996;19:423–431. 33 Zhdanova IV, Wurtman RJ, Lynch HJ, Ives JR, Dollins AB, Morabito C, Matheson JK, Schomer DL: Sleep-inducing effects of low doses of melatonin ingested in the evening. Clin Pharmacol Ther 1995;57: 552–558. 34 Waldhauser F, Saletu B, TrinchardLugan I: Sleep laboratory investigations on hypnotic properties of melatonin. Psychopharmacology 1990;100:222–226. 35 Badia P, Hughes R, Murphy BD, Myers BL, Wright K: Effects of exogenous melatonin on memory, sleepiness, and performance after a 4-hr nap. J Sleep Res 1996; 5(suppl 1):11. 36 Nave R, Peled R, Lavie P: Melatonin improves evening napping. Eur J Pharmacol 1995;275:213–216. 37 Dijk DJ, Roth C, Landolt HP, Werth E, Aeppli M, Achermann P, Borbely AA: Melatonin effect on daytime sleep in men: Suppression of EEG low frequency activity and enhancement of spindle frequency activity. Neurosci Lett 1995;201: 13–16. 38 Reid K, Van den Heuvel C, Dawson D: Day-time melatonin administration: Effects on core temperature and sleep onset latency. J Sleep Res 1996;5:150–154. 39 Gilbert SS, van den Heuvel CJ, Dawson D: Daytime melatonin and temazepam in young adult humans: Equivalent effects on sleep latency and body temperature. J Physiol (Lond) 1999;514(pt 3):905–914. 40 Terlo L, Laudon M, Tarasch R, Schatz T, Caine YG, Zisapel N: Effects of low doses of melatonin on late afternoon napping and mood. Biol Rhythm Res 1997;28:2–15. 41 Dollins AB, Zhdanova IV, Wurtman RJ, Lynch HJ, Deng MH: Effect of inducing nocturnal serum melatonin concentrations in daytime on sleep, mood, body temperature, and performance. Proc Natl Acad Sci USA 1994;91:1824–1828.

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339

Author Index Vol. 9, No. 6, 2000

Basheer, R. 319 Benoit, O. 309 Cardinali, D.P. 328 Daurat, A. 309 Foret, J. 309 McCarley, R.W. 319 Mauco, G. 309 Monti, J.M. 328 Morrison, A.R. 283

Parmeggiani P.L 279 Pedemonte, M. 297 Peña, J.L. 297 Porkka-Heiskanen, T. 319 Ross, R.J. 283 Sanford, L.D. 283 Strecker, R.E. 319 Thakkar, M.M. 319 Velluti, R.A. 297

Subject Index Vol. 9, No. 6, 2000

A1 receptor 319 Adenosine 319 Alertness 309 Alzheimer disease 328 Amygdala 283 Basal forebrain 319 Bright light pulses 309 Circadian physiology 328 Core temperature 309 EEG bands 309 Fear conditioning 283 Hippocampus 297 Melatonin 309, 328 – receptors 328 NF-ÎB 319 Night work 309

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Performance 309 Pineal gland 328 Ponto-geniculo-occipital waves 283 Processing, auditory 297 – visual 297 Rapid-eye-movement sleep 283 Sensory signals 297 Serotonin 283 Sleep 279, 283, 297 – deprivation 319 – disorders 328 Temperature 279 Thermoregulation 279 Theta rhythm 297 Wakefulness 297

Author Index Vol. 9, 2000 Author Index of the Abstracts of the International Symposium on Receptor and Non-Receptor Mediated Actions of Melatonin, Hong Kong, November 1999 appears in Vol. 9, No. 1, 2000 Author Index of ‘Ovarian Apoptosis’ appears in Vol. 9, No. 2, 2000 Author Index of ‘Receptor and Non-Receptor Mediated Actions of Melatonin’ appears in Vol. 9, No. 3–4, 2000 Author Index of ‘Biological Signals Relevant to Sleep’ appears in Vol. 9, No. 6, 2000

Aerts, T. 45 Akagawa, K. 231 Cardinali, D.P. 215 Chan, J.S.C. 21 Clauwaert, J. 45 Cutrera, R.A. 215 Di, A.K. 1 Esquifino, A.I. 215 Fujino, I. 231 Ho, M.K.C. 21 Hosoda, Y. 231 Iyengar, B. 260 Koga, N. 267 Kunert-Radek, J. 255 Kuwahara, A. 231 Lachowicz-Oche¸dalska, A. 255 Li, W.W.Y. 29 Lu, G.-W. 38 Magata, K. 267 Makarevich, A.V. 248 Meng, Z. 38

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Pang, C.S. 1 Pang, S.F. 1 Pawlikowski, M. 255 Pei, G. 240 Re¸bas, E. 255 Sato, T. 267 Sirotkin, A.V. 248 Slegers, H. 45 Tsim, K.W.K. 240 Ueyama, A. 267 Wang, Q. 45 Winczyk, K. 255 Wong, Y.H. 21, 240 Wu, X.M. 1 Wu, Y. 45 Xu, J.N. 1 Xu, R.K. 1 Yau, K. 29 Yew, D.T. 29 Yoshida, H. 267 Yung, L.Y. 21, 240

341

Subject Index Vol. 9, 2000 Subject Index of ‘Ovarian Apoptosis’ appears in Vol. 9, No. 2, 2000 Subject Index of ‘Receptor and Non-Receptor Mediated Actions of Melatonin’ appears in Vol. 9, No. 3–4, 2000 Subject Index of ‘Biological Signals Relevant to Sleep’ appears in Vol. 9, No. 6, 2000

Adenylyl cyclase 21, 240 Affinity partition 45 Aging 215 Angiotensin 231 – type 1 receptor 231 AT1 231

Lateral cervical nucleus 38 Leucine uptake 29 Lymph nodes 215

C6 glioma 45 cAMP 248 Cancer, colon 255 Cat 38 Cell number 260 CH-275 255 Circadian rhythms 215 Cloning 231 Cognitive function 215 Cytokines 215

Nonradioisotope assay 267

Melatonin 215 – receptor 1

Octreotide 255 Opioid receptor-like receptor 240 pGH 248 Pineal 1 Plasma membrane 45 Protein kinase A 248 – – C 21 – metabolism 29

Dendricity 260 2-Deoxyglucose 6-phosphate 267 Detachment 29 Dopamine 1

Rat skeletal muscle 267 Retina 29

E2-induced prolactinoma rat 1 Epitope tag 240

Solitary tract nucleus 38 Somatostatin 255 Spinal neurons 38 Sympathetic nervous system 215

Freund’s adjuvant arthritis 215 G protein 21 G2 phase arrest 260 Glucose uptake 267 Gonadotropin-releasing hormone 215 Granulosa cells 248 Guinea pig 231 Hypothalamus 1 IGF-I 248 – receptor 45 Intracellular recordings 38

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Tumor, pituitary 255 Two-phase partitioning 45 Tyrosine kinase 248 – kinases 255 UV response 260 Wheat germ agglutinin 45 Whole-skin organ culture 260

Contents Vol. 9, 2000

No. 2

No. 1

Ovarian Apoptosis

Review 1

Pituitary Prolactin-Secreting Tumor Formation: Recent Developments

Guest Editor: Benjamin K. Tsang, Ottawa

Xu, R.K.; Wu, X.M.; Di, A.K.; Xu, J.N. (Beijing); Pang, C.S.; Pang, S.F. (Hong Kong)

081 The Regulation of Apoptosis in Preantral Ovarian

Original Papers

087 Mammalian Follicular Development and Atresia:

Follicles McGee, E.A. (Lexington, Ky.)

21

The Effect of Protein Kinase C Activation on Gz-Mediated Regulation of Type 2 and 6 Adenylyl Cyclases Ho, M.K.C.; Chan, J.S.C.; Yung, L.Y.; Wong, Y.H. (Hong Kong)

29

Changes in Leucine Uptake in the Retina of the Hamster after Traumatic Detachment Yau, K.; Li, W.W.Y.; Yew, D.T. (Hong Kong)

38

45

Role of Apoptosis Asselin, E.; Xiao, C.W.; Wang, Y.F.; Tsang, B.K. (Ottawa)

096 Granulosa Cell Apoptosis: Conservation of Cell

Signaling in an Avian Ovarian Model System Johnson, A.L. (Notre Dame, Ind.)

102 Proteolytic and Cellular Death Mechanisms in

Ovulatory Ovarian Rupture Murdoch, W.J. (Laramie, Wyo.)

Projection Linkage from Spinal Neurons to Both Lateral Cervical Nucleus and Solitary Tract Nucleus in the Cat

115 N-Cadherin-Mediated Cell Contact Regulates

Meng, Z.; Lu, G.-W. (Beijing)

122 Apoptosis and Chemoresistance in Human Ovarian

Purification of Rat C6 Glioma Plasma Membranes by Affinity Partitioning Wang, Q.; Wu, Y.; Aerts, T.; Slegers, H.; Clauwaert, J. (Antwerp)

Ovarian Surface Epithelial Cell Survival Peluso, J.J. (Farmington, Conn.)

Cancer: Is Xiap a Determinant? Li, J.; Sasaki, H.; Sheng, Y.L.; Schneiderman, D.; Xiao, C.W. (Ottawa); Kotsuji, F. (Fukui); Tsang, B.K. (Ottawa)

131 Author Index Vol. 9, No. 2, 2000 131 Subject Index Vol. 9, No. 2, 2000

Abstracts 53

Receptor and Non-Receptor Mediated Actions of Melatonin International Symposium, Hong Kong, China, November 6–8, 1999

76

Author Index for Abstracts only

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© 2000 S. Karger AG, Basel

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248 Presumptive Mediators of Growth Hormone Action

No. 3–4 Receptor and Non-Receptor Mediated Actions of Melatonin Selected Papers from the International Symposium held in Hong Kong, China, November 6–8, 1999

on Insulin-Like Growth Factor I Release by Porcine Ovarian Granulosa Cells Makarevich, A.V.; Sirotkin, A.V. (Nitra)

255 Effects of Somatostatin and Its Analogues on

Tyrosine Kinase Activity in Rodent Tumors Lachowicz-Oche¸dalska, A.; Re¸bas, E.; Kunert-Radek, J.; Winczyk, K.; Pawlikowski, M. (Lodz)

Editors: S.Y.W. Shiu; S.F. Pang (Hong Kong)

260 Melatonin and Melanocyte Functions Iyengar, B. (New Delhi)

Original Papers 137 Significance of Melatonin in Antioxidative Defense

System: Reactions and Products

267 Nonradioisotope Assay of Glucose Uptake Activity

in Rat Skeletal Muscle Using Enzymatic Measurement of 2-Deoxyglucose 6-Phosphate in vitro and in vivo

Tan, D.-X.; Manchester, L.C.; Reiter, R.J.; Qi, W.-B. (San Antonio, Tex.); Karbownik, M. (Lodz); Calvo, J.R. (San Antonio, Tex.)

Ueyama, A.; Sato, T.; Yoshida, H.; Magata, K.; Koga, N. (Tokushima)

160 Pharmacology and Physiology of Melatonin in the

Reduction of Oxidative Stress in vivo Reiter, R.J.; Tan, D.-X.; Qi, W.; Manchester, L.C.; Karbownik, M.; Calvo, J.R. (San Antonio, Tex.)

No. 6

172 Biological Basis and Possible Physiological

Implications of Melatonin Receptor-Mediated Signaling in the Rat Epididymis Shiu, S.Y.W.; Li, L.; Siu, S.W.F.; Xi, S.C.; Fong, S.W.; Pang, S.F. (Hong Kong)

Biological Signals Relevant to Sleep Guest Editor: Ricardo A. Velluti, Montevideo

188 Photic Regulation of mt1 Melatonin Receptors and

2-Iodomelatonin Binding in the Rat and Siberian Hamster

279 Influence of the Temperature Signal on Sleep in

Mammals

Masson-Pévet, M.; Gauer, F.; Schuster, C. (Strasbourg); Guerrero, H.Y. (Strasbourg/Caracas)

197 Nuclear Receptors Are Involved in the Enhanced IL-6

Parmeggiani, P.L. (Bologna)

283 The Amygdala: A Critical Modulator of Sensory

Influence on Sleep

Production by Melatonin in U937 Cells Guerrero, J.M.; Pozo, D.; García-Mauriño, S.; Carrillo, A.; Osuna, C.; Molinero, P.; Calvo, J.R. (Seville)

Morrison, A.R.; Sanford, L.D.; Ross, R.J. (Philadelphia, Pa.)

297 Reciprocal Actions between Sensory Signals and

Sleep

203 Melatonin and Biological Rhythms Pévet, P. (Strasbourg)

Velluti, R.A.; Peña, J.L.; Pedemonte, M. (Montevideo)

309 Bright Light during Nighttime: Effects on the

Circadian Regulation of Alertness and Performance

213 Author Index Vol. 9, No. 3–4, 2000 214 Subject Index Vol. 9, No. 3–4, 2000

Daurat, A.; Foret, J. (Toulouse); Benoit, O. (Créteil); Mauco, G. (Toulouse)

319 Adenosine as a Biological Signal Mediating

Sleepiness following Prolonged Wakefulness Basheer, R.; Porkka-Heiskanen, T.; Strecker, R.E.; Thakkar, M.M.; McCarley, R.W. (Brockton, Mass.)

No. 5

328 A Critical Assessment of the Melatonin Effect on

Sleep in Humans

Review

Monti, J.M.; Cardinali, D.P. (Montevideo/Buenos Aires)

215 Psychoimmune Neuroendocrine Integrative

Mechanisms Revisited Cardinali, D.P.; Cutrera, R.A.; Esquifino, A.I. (Buenos Aires/Madrid)

Original Papers

340 340 341 342

Author Index Vol. 9, No. 6, 2000 Subject Index Vol. 9, No. 6, 2000 Author Index Vol. 9, 2000 Subject Index Vol. 9, 2000

231 Molecular Cloning of Guinea Pig Angiotensin Type 1

Receptor Hosoda, Y. (Shizuoka); Fujino, I.; Akagawa, K. (Tokyo); Kuwahara, A. (Shizuoka)

240 Immunoglobulin G1 Fc Fragment-Tagged Human

Opioid Receptor-Like Receptor Retains the Ability to Inhibit cAMP Accumulation Yung, L.Y.; Tsim, K.W.K. (Hong Kong); Pei, G. (Shanghai); Wong, Y.H. (Hong Kong)

IV

Biol Signals Recept Vol. 9, 2000

Contents