REVIEWS Sleep is a global state, the control mechanisms of which are manifested at every level of biological organization, from genes and intracellular mechanisms to networks of cell populations, and to all central neuronal systems at the organismic level, including those that control movement, arousal, autonomic functions, behaviour and cognition. Recent genetic findings indicate that the molecular mechanisms that control CIRCADIAN RHYTHMS, which set the stage for sleep and are inseparable from sleep in a deep biological sense, are highly conserved phylogeneti- cally1,2. Molecular and behavioural conservation indi- cates that sleep conferred a selective advantage on ancestral mammals that might persist in modern popu- lations. Prolonged sleep loss impairs temperature con- trol, dietary metabolism and immune function, and leads ultimately to death3. In the mammalian nervous system, genetic instruc- tions are expressed at the progressively higher levels of gene transcription, protein synthesis and intracellular events, individual neuronal and neuronal-network dynamics, and ultimately behaviour, of which cognition is a specific covert form. In this review, we discuss the genetic mechanisms, cellular neurophysiology and subcortical neuronal-population networks that are involved in sleep. In a forthcoming review4, we will dis- cuss the cognitive neuroscience of sleep at the levels of subcortico-cortical neuronal-population networks, behaviour and cognition. Many brain loci have specific functions in sleep at each of these organizational levels (FIG. 1a). The bottom tier of subcortical structures is addressed here. FIGURE 1b shows a schematic presentation of the molecular, cellu- lar and subcortical networks. For a discussion of the peripheral physiology of sleep, sleep medicine and sleep in psychiatry, which are beyond the scope of this review, see REF.5. The circadian pacemaker Starting with the first demonstration of a circadian gene in the fruitfly6, genetic approaches have begun to illu- minate the intranuclear and cytoplasmic events that are associated with circadian rhythms and sleep. Most notable has been the elucidation of the genetic control of the mammalian circadian pacemaker2,7, which can now explain the near-perfect 24-h rhythmicity of the THE NEUROBIOLOGY OF SLEEP: GENETICS, CELLULAR PHYSIOLOGY AND SUBCORTICAL NETWORKS Edward F. Pace-Schott and J. Allan Hobson To appreciate the neural underpinnings of sleep, it is important to view this universal mammalian behaviour at multiple levels of its biological organization. Molecularly, the circadian rhythm of sleep involves interlocking positive- and negative-feedback mechanisms of circadian genes and their protein products in cells of the suprachiasmatic nucleus that are entrained to ambient conditions by light. Circadian information is integrated with information on homeostatic sleep need in nuclei of the anterior hypothalamus. These nuclei interact with arousal systems in the posterior hypothalamus, basal forebrain and brainstem to control sleep onset. During sleep, an ultradian oscillator in the mesopontine junction controls the regular alternation of rapid eye movement (REM) and non-REM sleep. Sleep cycles are accompanied by neuromodulatory influences on forebrain structures that influence behaviour, consciousness and cognition. CIRCADIAN RHYTHMS Biological rhythms of physiology and behaviour that have a 24-h periodicity, which have evolved in response to the 24-h astronomical cycle to which all organisms are exposed. NATURE REVIEWS | NEUROSCIENCE VOLUME 3 | AUGUST 2002 | 591 Laboratory of Neurophysiology, Department of Psychiatry, Harvard Medical School, Massachusetts Mental Health Center, 74 Fenwood Road, Boston, Massachusetts 02115, USA. Correspondence to E.F.P.-S. e-mail: edward_schott@ hms.harvard.edu doi:10.1038/nrn895 �� 2002 Nature Publishing Group

592 | AUGUST 2002 | VOLUME 3 www.nature.com/reviews/neuro R E V I E W S human circadian clock8. More details on the molecular circadian clock than can be provided here are available in REFS 1,2,7,9. The genetically controlled molecular circadian clock is synchronously expressed collectively and individu- ally10 by each of the ~20,000 cells of the mammalian SUPRACHIASMATIC NUCLEUS (SCN)2, which is situated bilater- ally in the hypothalamus, just above the optic chiasm. The SCN contains the ���master clock��� mechanism, the entrainment of which by the daily light���dark cycle sets the 24-h rhythm for all other physiological rhythms in the organism. The SCN does this, in part, by control- ling molecularly related peripheral cellular oscillators that exert local control over physiological rhythms at sites closer to the expression of these rhythms2. Transplantation of the SCN of hamsters that have nor- mal circadian rhythms into hamsters that have mutant rhythms restores normal periodicity in the implanted hamsters mutant-to-normal transplantation has the opposite effect11. Mammalian circadian rhythms are maintained intra- cellularly by interlocking positive- and negative-feedback control of the transcription (and subsequent translation to protein) of three period genes (Per1���3), two crypto- chrome genes (Cry1,2), and the Clock and Bmal1 (brain and muscle ARNT-like 1) genes2,7 (FIG. 2a). The products of Clock and Bmal1 exist as a heterodimer that is a key component of a transcription factor (abbreviated Clock:Bmal1) that promotes the transcription of Per and Cry genes by binding to their regulatory DNA sequences (E-box elements2,7). The Per and Cry messenger RNAs are translocated to the cytoplasm for translation to pro- teins that form complexes that then re-enter the nucleus to exert feedback control on the Clock:Bmal1 transcrip- tion factor.Products of several other genes modulate this intracellular mechanism2,7. For example, the product of the tau (Csnk1e) gene,casein kinase 1�� (REF. 12),phospho- rylates Per proteins, which affects their translocation between the cytoplasm and the nucleus13. Briefly, molecular feedback control of the circadian clock in SCN neurons operates as follows (see REFS 2,7 for details).At the beginning of the organism���s SUBJECTIVE DAY, CIRCADIAN TIME (CT) 0 (FIG.2b),the transcription and trans- lation of Per and Cry are accelerated by Clock:Bmal1 heterodimers that have accumulated over the previous subjective night (CT 12���24). The levels of the Per and Cry complexes peak at the beginning of the organism���s subjective night (CT 12).Protein complexes that contain the products of the Cry gene exert negative feedback on the Clock:Bmal1 promoter, thereby slowing the tran- scription of Per and Cry (FIG.2a).At the same time,a pro- tein complex that contains Per2 exerts positive feedback by promoting the transcription of Bmal1 (FIG. 2a). Bmal1 begins to accumulate as raw material for new Clock:Bmal1 heterodimers during the subjective day, and its level peaks during the subjective night (FIG. 2b). Negative feedback on the Clock:Bmal1 promoter causes the levels of Per and Cry to reach a minimum during the subjective night (FIG. 2b). Simultaneously, positive feedback on Bmal1 transcription raises the levels of Clock:Bmal1 heterodimers, which, when released from b Sleep���wake control systems a Hypothalamus and basal forebrain Hypothalamic transmitters of circadian signal Mesopontine brainstem Ultradian REM���NREM oscillator Ascending brainstem arousal systems Sleep homeostat Integration of homeostatic and circadian input Wake���sleep switch Diencephalic ascending arousal systems Circadian oscillator Environmental light and photoperiod Central and peripheral biological rhythms Cortex Subcortex (limbic, striatal) Thalamus 12 3 6 9 Retina Origin and expression of circadian rhythms Hypothalamic nuclei: ��� Suprachiasmatic ��� Subparaventricular ��� Dorsomedial Thalamocortical control of NREM sleep rhythms EEG activation and deactivation Hippocampal���cortical control of memory consolidation Diencephalic control of sleep onset Hypothalamic nuclei: ��� Ventrolateral preoptic ��� Lateral ��� Tuberomammillary Basal forebrain Pontine control of the REM���NREM cycle Mesopontine nuclei: ��� Laterodorsal tegmental ��� Pedunculopontine ��� Dorsal raphe ��� Locus coeruleus Forebrain areas key to the neuropsychology of dreaming Prefrontal cortex: ��� Ventromedial ��� Dorsolateral Anterior limbic structures: ��� Amygdala, anterior cingulate, ventral striatum Posterior cortices: ��� Inferior parietal ��� Visual association Figure 1 | Brain regions and regulatory circuits involved in sleep. a | Brain regions of current interest to the neurobiology of sleep. This review considers the bottom tier of subcortical regions (blue boxes), which control sleep���wake transitions and, within sleep, REM���NREM sleep alternation. The top tier includes areas that are key to the generation of the EEG rhythms of sleep, the subjective experience of sleep mentation or dreaming, and sleep���s effects on cognition these are considered in REF. 4. b | Schematic representation of the regulatory circuits that control sleep���wake and REM���NREM transitions, as well as their key inputs and outputs. Parts of this network are considered in more detail in the main text. EEG, electroencephalogram NREM, non-REM REM, rapid eye movement. �� 2002 Nature Publishing Group

NATURE REVIEWS | NEUROSCIENCE VOLUME 3 | AUGUST 2002 | 593 R E V I E W S with autosomal-dominant transmission16. This finding might constitute the first step in identifying molecular components of the human circadian system that are analogous to those described in animals. The precise timing of the appearance of these endogenously reliable signals relative to the astronom- ical day can be entrained to ambient light���dark cycles by light impinging on the retina. The reliable circa- dian output of SCN cells results not only from the endogenous cycling of transcriptional/translational signals described above, but also from temporally ordered sensitivity of these clock cells to input from the retina and other brain structures9. Such neurochemical feedback allows control of the SCN by neuronal responses it has itself previously elicited9. Light-mediated entrainment of SCN cells is believed to result from glutamatergic stimulation of NMDA (N-methyl-D-aspartate) receptors through the retino- hypothalamic tract (RHT)9.Administration of glutamate to SCN slices in vitro effects phase shifts of cell firing that presumably reflect the molecular mechanisms of light- induced entrainment9,17,18. Photic and glutamatergic feedback inhibition, can again begin to promote the transcription of Per and Cry as the organism begins its new subjective day. The combined action of positive- and negative- feedback loops creates a suite of molecular signals that reliably recur at precise times over 24-h cycles. These molecular signals can be read by cytoplasmic mecha- nisms in SCN cells and translated into reliably recur- ring cellular events, such as changes in membrane potential2. Such signals, in turn, can be transmitted to connecting neurons and, ultimately, to those neural structures that control physiological processes with a circadian rhythmicity. Many of the details of the genetic mechanisms of mammalian circadian rhythms have been elucidated by studies of mutations in these genes2. For example, a mouse mutant of the Clock gene shows lengthening of the circadian period14. Cloning of these mammalian genes, beginning with Clock 15, has allowed the precise molecular analysis of normal and mutant circadian genes2.A recent study in humans has reported a herita- ble, familial trait for advanced sleep-phase syndrome b 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 Relative level (% of max.) Relative level (% of max.) Relative level (% of max.) Clock Bmal1 C B Cry Per1 Per2 Per3 + + + + Cry Per1 Per2 Per3 Per2 Cry Cry Per1 C B Per2 Cry + Per3 Cry Per1 + ��� Nucleus Cytoplasm Cry Cry Per1 Per3 Per1 a 0 6 12 Circadian time (h) 18 24 0 6 12 18 24 0 6 12 18 24 Per1 Per2 Per3 Cry1 Bmal1 Bmal1 (expected) Per1 Per2 Cry1 Cry2 RNA levels Protein levels Figure 2 | The molecular control of circadian rhythms. a | The molecular basis of the circadian clock expressed in a single cell of the suprachiasmatic nucleus of the anterior hypothalamus. Clock:Bmal1 (labelled C and B) is a protein heterodimer that is a key component of a transcription factor that promotes the transcription of period (Per) and cryptochrome (Cry) genes. The RNA products of Per, Cry, Bmal1 and Clock are translocated to the cytoplasm, where they are translated to proteins. Per and Cry proteins form complexes that are translocated back into the nucleus to exert feedback effects on Clock:Bmal1. Protein complexes that contain Cry exert negative feedback control on the Clock:Bmal1 heterodimer, and slow the transcription of Per and Cry, whereas complexes that contain Per2 proteins enhance the transcription of Bmal1. The promotion of Per and Cry gene expression by Clock:Bmal1 causes the levels of Per and Cry to peak at the end of the subjective day, at which point the feedback inhibition of Clock:Bmal1 (by complexes composed of Per and Cry) reverses this trend, causing their levels to fall to a minimum during the following subjective night (b). During the subjective day, however, protein complexes that contain Per2 also enhance Bmal1 transcription, causing the levels of Bmal1 to peak during the subjective night (b). This favours the formation of new Clock:Bmal1 heterodimers these promote the transcription of Per and Cry when released from inhibition by minimal levels of Per and Cry as the next subjective day begins, thus restarting the cycle. Note that this is a highly simplified schematic, in which important feedback- related cofactors in protein complexes, as well as factors that favour the translocation of gene products within the cell2, are omitted. Circled + or ��� indicates promotion and inhibition of gene transcription (or heterodimer formation), respectively. Light arrows represent gene transcription. Medium arrows represent gene promotion/inhibition and protein combination. Heavy arrows represent translocation of RNA transcripts and proteins between the nucleus and the cytoplasm. b | Variation over the circadian day in the levels of messenger RNA and protein products of the core circadian clock genes ��� Per1, Per2, Cry1 and Cry2, and Bmal1 mRNA and protein (dashed line). Bars indicate subjective night (purple) and subjective day (yellow). Modified, with permission, from REF. 2 �� 2001 Annual Reviews. SUPRACHIASMATIC NUCLEUS The mammalian circadian pacemaker, or ���master clock���, which consists of two tiny, bilaterally symmetrical nuclei in the anterior hypothalamus, located just above the optic chiasm (where the main fibre tracts, or optic nerves, from the two eyes meet). It is therefore ideally situated to receive photic input from the retina through the retinohypothalamic tract, which follows these nerves. SUBJECTIVE DAY AND SUBJECTIVE NIGHT The time during which an organism is normally active is referred to as the subjective day. The subjective night describes the period during which an organism is normally inactive and in which its sleep normally occurs. Therefore, a nocturnal animal���s subjective day occurs during the astronomical night. �� 2002 Nature Publishing Group