New Insights Into the Mammalian Circadian Clock

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1 Sleep, 19(8): American Sleep Disorders Association and Sleep Research Society State of the Art Review New nsights nto the Mammalian Circadian Clock *Joseph D. Miller, tlawrence P. Morin, :f:william J. Schwartz and Robert Y. Moore *Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S,A.; tdepartment of Psychiatry, SUNY, Health Sciences Center, Stony Brook, New York, U,S.A.; f.department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, U.S.A.; and Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, U.S.A. Summary: The focus of this review is recent studies of the mammalian circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. The anatomy of the SCN and its major afferents from the retina. raphe, and intergeniculate leaflet (GL) of the thalamus are considered, with a special emphasis on the effects of afferent interaction on the circadian timekeeping system. What is known of the endogenous clock mechanism is reviewed in comparison with known molecular circadian mechanisms in other species. Efferents of the SCN are also discussed with a view toward understanding how circadian information is transmitted to the rest of the central nervous system. Where possible, anatomical, electrophysiological, neuropharmacological, molecular, and behavioral data are integrated in an attempt to illuminate the mechanisms of circadian timekeeping. Key Words: Suprachiasmatic nucleus Hypothalamus-Raphe-ntergeniculate leaflet-retinohypothalamic tract-circadian rhythm-neuropeptide Y Enkephalin-Vasoactive intestinal polypeptide-substance P-5HT. The circadian system can be divided into three relatively discrete areas of research interest. The first is the suprachiasmatic nucleus (SeN). This structure is the focus of a large research effort because it is a circumscribed brain region with a very specific function and intrinsically interesting anatomical characteristics (1,2). The function is generation of circadian rhythmicity, for which the SeN is the site of the master oscillator or clock. The SeN is anatomically well delineated by the organization of neuromodulator-containing neurons and fibers that are intrinsic or afferent to the nucleus, ssues pertaining to entrainment of circadian rhythms are the focus of the second division of rhythm research interest, and the majority of these fall into the domain of the "circadian visual system" (Fig. 1). The daily photoperiod is the primary environmental stimulus to the circadian clock. t is through the circadian visual system that the clock is synchronized (entrained) to the photoperiod. The nuclei and pathways contributing to entrainment and circadian rhythm re- Accepted for publication May Address correspondence and reprint requests to Joseph D. Miller, Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A. 641 sponse to light constitute a specialized division of the vertebrate visual plan (3). The third circadian system division consists of the SeN-efferent pathways and their role in regulating measurable rhythms in physiology and behavior, Although SeN-efferent neural projections have been described (4-7), relatively little is known about their function. The study of efferent pathways is rendered more difficult by the increasing evidence that a humoral connection may link the SeN with rhythmic expression of physiology and behavior (8,9). This review is an attempt to highlight "the cutting edge" of research on the components of the mammalian circadian timekeeping system. t is based on a workshop entitled "The Suprachiasmatic Nucleus (Basic Mechanisms)", which was presented at the Eighth Annual APSS meeting, June ORGANZATON, CONNECTONS, AND FUNCTON OF THE SCN While subtle species-specific differences abound, it is constructive to consider the organization of the SeN in a representative mammal; specifically, the hamster, a favorite target of circadian investigations because of its high cycle-to-cycle circadian timing accuracy. The

2 D. MLLER ET AL. DRer----_ MR PLi ~ \ RGT /1 _ /'" OT.,,' ( \- - - ~ RETNA FG. 1. Wiring diagram of the "circadian visual system". Broken lines indicate retinal projections; solid lines indicate connections between brain nuclei. Abbreviations: DR, dorsal raphe nucleus; DTT, dorsal thalamic tract; GHT, geniculohypothalamic tract; GL, intergeniculate leafiet; GLc, contralateral GL; lt, inter-intergeniculate tract; MR, median raphe nucleus; OT, optic tract; PLi, posterior limitans nucleus; RGT, retinointergeniculate tract; SeN, suprachiasmatic nucleus. hamster SCN is not easily subdivided, but can be characterized by a loosely interpreted "core and shell" arrangement of neuromodulator-specific cell groups or fibers. The core of the SCN consists of substance P (SP)-R neurons (Fig. 2C). This small, discrete population of cells (10) is partially enveloped by a dense serotonin-r fiber plexus (10,11), encompassing approximately of arc (Fig. 2A). Only the dorsolateral SCN contains relatively few serotonergic fibers. Populations of several neuromodulator-specific neurons also contribute overlapping sections of the SCN shell (6,11). For example, vasopressin-r cells are spread through the dorsomedial area (Fig. 2H) and vasoactive intestinal peptide neurons occupy the ventral region (Fig. 2F). Cholecystokinin neurons tend to have the same shell sector distribution as serotonin fibers, with a relative paucity only in the dorsolateral area (12). The shell characteristic is also emphasized by distributions of fibers in addition to those that are serotonin-r. SP fibers are relatively sparse within the SCN, but form a curved area of dense fibers around the nucleus from the chiasm to the base of the third 1 ventricle. n a complementary fashion, a curving zone just inside this arc, extending from the chiasm to the midline, is relatively devoid of NPY fibers (Fig. 2D). Although the core/shell analogy is adequate for certain descriptive purposes, it is too simplistic for others. Retinal innervation of the SCN occupies the entire nucleus plus adjacent hypothalamic regions (13). Likewise, NPY -R fibers from the intergeniculate leaflet (GL) occupy (except for the zone mentioned above) the entire SCN (Fig. 2D) (10). Conversely, fibers and terminals immunoreactive to galanin are specifically excluded from the SCN, thereby identifying the nuclear location as a void against a fairly homogenous background (Fig. 2G). Clearly, form and function are related, but the nature of this relationship in the hamster SCN is not yet apparent. Many of the anatomical features just discussed are generally represented in the rodent SCN and, more generally, in the mammalian SeN (see 14,15 for review). t appears that the great majority of SCN neurons are GABAergic in the species so far studied (16-18). Vasopressin neurons tend to be situated dorsomedially in the SCN, while VP and GRP neurons occupy the ventral region. Small populations of somatostatin, CCK, and Substance P neurons have been observed in the rat SCN, but they are neither as dense nor as localized as in the hamster. Calcium-binding proteins are present in the SCN of at least two species (19,20) and may have a critical role to play in rhythm generation (21). n the rat, dorsolateral SCN neurons are characterized by substantial intracellular levels of calretinin (19), whereas another calciumbinding protein, calbindin, is localized to the central region of the hamster SCN (20). The retinohypothalamic tract (RHT), the GL projection, and the 5HT projection from the raphe nuclei terminate more exclusively in the ventrolateral region of the rat SCN than in the hamster SCN, often on vasoactive intestinal polypeptide (VP) or gastrin-releasing peptide (GRP) neurons (22-24). A cholinergic projection primarily from the basal forebrain magnocellular cholinergic neurons (25) and a histaminergic projection from the mammillary hypothalamus (26) have also been described in the rat. There is substantial documentation that the SCN is the site of circadian rhythm generation in mammalian species. For instance, bilateral lesions eliminate circadian periodicity in the hamster (1) and transplantation of perinatal SCN to an SCN-esioned arrhythmic host restores rhythmicity with the period of the donor SCN (27,28). Numerous neuromodulators or agonists have been infused onto the SCN in vivo and the resultant phase shifts have been described (see 29 for a review). Similarly, electrical stimulation of the SCN phase-shifts the behavioral rhythm (30). Circadian

3 THE MAMMALAN CRCADAN CLOCK 643 E F '\, \. ~. '- \ '\, H~./ 1 ":.f'.. :t~" :',1... \ ",' FG. 2. Chemical neuroanatomy of the hamster SCN. A. Serotonin. B. Enkephalin. C. Substance P. D. Neuropeptide Y. E. Gastrinreleasing peptide. F. Vasoactive intestinal polypeptide (VP). G. Galanin. H. Vasopressin. V3, third ventricle. Bar = 200 ).Lm.

4 D. MLLER ET AL. XT3 - A, o, Hr FG. 3. Loss of entrainment after transsection of the RHT (at the arrow). Animal XT3 remained entrained to the photoperiod after an incomplete cut, whereas animal XTl2 showed a free-running rhythm despite being exposed to a daily light-dark cycle. (Photoperiod bar is at the top of each running record.) After Johnson et al. (38). rhythmicity is maintained in vitro in the SCN slice or even in SCN cell culture, in terms of rhythms in neuronal firing and transmitter release. There is increasing interest in the role of VP and GRP neurons (Fig. 2E and F) as endogenous SCN regulators of hamster and rat rhythmicity. nfusion studies implicate one (31) or more (32) of these peptides in the phase control of hamster circadian rhythmicity. While no compelling evidence thus far implicates any particular neurotransmitter-identified cellular type in the SCN as a pacemaker cell, a recent report (33) of circadian rhythms in firing rate in single neurons in culture suggests that endogenous circadian rhythms are a product of intraneuronal mechanisms. ORGANZATON, CONNECTONS, AND FUNCTON OF THE RHT Circadian pacemakers are self-sustaining oscillators that free-run with a stable period under constant conditions. Under normal conditions, however, the period and phase of circadian pacemakers are established by environmental stimuli, particularly the solar light-dark cycle. This process of environmental control of the pacemaker is termed "entrainment" and the primacy of light in the process is recognized by its designation as the zeitgeber. The intent here is to summarize our current understanding of the neurobiology of photic entrainment of circadian rhythms. The importance of light for entrainment indicates that photic entrainment mechanisms must consist of photoreceptors and visual pathways that couple the photoreceptors to the circadian pacemakers. Early studies indicated that the eyes are necessary for entrainment but that ablating all central retinal projections beyond the optic chiasm had no effect on entrainment even though the animals were behaviorally blind (34,35). No other central retinal projections were known at that time and the preliminary work leading to the studies noted above clearly suggested that another, unknown, visual pathway must be mediating the entrainment effects. The most likely candidate was a direct retinal projection to the hypothalamus, an RHT, and this was first shown in 1972 (36,37). The RHT projection to the SCN of the hypothalamus is sufficient to maintain entrainment (35). Selective transection of the RHT at its entry into the SCN abolishes entrainment (Fig. 3), indicating that the pathway is also necessary to preserve the function (38). Retina The retinal portion of the entrainment pathway has three components: photoreceptors, intrinsic retinal pathways, and retinal ganglion cells (RGCs) whose axons comprise the RHT. There are data to indicate that the retinal photoreceptors that transduce photic entrainment effects may be a separate population. n mutant mice in which photoreceptors undergo a genetically determined degeneration, the rd and rds mice, Sleep. Vol. 19. No

5 A, ",,:' THE MAMMALAN CRCADAN CLOCK '. :." ".. " '.r." ",." ' ~.,'. ~..', ~' 1;-"1. 1: '... " - '. --.~". :-..' ',.. ~., ')...,...~.,,1 " '..,..,, :1 '.... ',' ~-$:.':.,1. f:.,!,,~.. ':-:-~. \.: "". '..,.... ';. if...., " ".,., 0' ~,, ',"" f... ~ ". " ".., _ J, FG. 4. Photomicrographs showing retinal ganglion cells projecting in the retinohypothalamic tract. The cells are labeled with pseudorabies virus shown by an antiserum to virus. Scale bar = 50 flm. there is preservation of entrainment despite apparent loss of all photoreceptors (39). The preserved function can be attributed either to a novel photoreceptor or to a combination of known photoreceptors (40). The nature of the retinal pathways beyond the photoreceptors is not known, but recent data (41) indicate that there is a distinct set of RGCs that gives rise to the RHT. These neurons belong to the class ganglion cell population of Perry (42). They are small, averaging m in diameter and m2 in area, with sparse, thin radiating dendrites that have few branches (Fig. 4), and are distributed over the entire retina with a slightly greater density in the temporal than in the nasal retina and represent less than 1 % of the total RGC population in the rat (41). A population of very similar ganglion cells projecting to the SCN has been described in the cat retina (43). A subset of such specialized rat retinal ganglion cells apparently projects to the SCN and the thalamic GL, the two major nuclei of the circadian visual system, but not to other classical visual areas (44). n the hamster, individual ganglion cells contributing to the circadian visual system can project to both the GL and the SCN (45). This and other data (46) suggest that retinal contributions to the rat circadian system are specialized for rhythm regulation, although similar information has not yet been obtained in the hamster. RHT Projections The axons that arise from this population of ganglion cells project through the optic nerve into the optic chiasm. Unlike most of the retinal projections in the rat, which are almost exclusively crossed in the chiasm, RHT axons are approximately two thirds crossed and one third uncrossed (13,47). n the rat, the major site of projection of the RHT is to the ventrolateral SCN (13,48; Fig. 5) but there is a sparse projection to the dorsomedial SCN. n addition, there are projections to a number of areas in the medial and lateral hypothalamus outside the SCN. The densest of these is to the retrochiasmatic area and there are projections to the medial preoptic area, the anterior hypothalamic area/subparaventricular zone, and the tuberal hypothalamus immediately caudal to the retrochiasmatic area (13,48). The projections outside the SCN appear to develop from projections into the SCN (49). The function of RHT axons that innervate hypothalamic areas outside the SCN is unknown. Collaterals of RHT axons projecting to the SCN continue in the optic tract to produce a dense, bilateral innervation of the intergeniculate leaflet of the lateral geniculate complex (50). Cholera toxin 13 (CT3) fragment immunohistochemistry nicely demonstrates the RHT innervation of both the SCN and adjacent hypothalamus and the GL Sleep, Vol, /9, No, 8, 1996

6 D. MLLER ET AL. A.. 1.r.' --'.... OC~: B';'~,,..., f... i \. \. ~;,:r..,. FG. 5. Photomicrographs showing retinohypothalamic projections in the rat as demonstrated by anterograde transport of cholera toxin injected into the eye. A. RHT projection into the SeN (arrows). B. RHT projection to the retrochiasmatic area (arrows). c. RHT projection to the intergeniculate leaflet (arrows). Abbreviation: oe-optic chiasm. Scale bar = 150 J.1m (A), 100 J.1m (B and e). (Figs. 6 and 7). CT3 is transported anterogradely from retinal ganglion cells and is a very sensitive label of axons and terminals of the visual system. The RHT forms complex synaptic interactions in the SCN. RHT terminals in the rat range from 0.5 to 2 f..lm in diameter and contain a lucent cytoplasmic matrix with small, lucent spherical vesicles and occasional dense core vesicles. These boutons can be distin-

7 THE MAMMALAN CRCADAN CLOCK 647 asymmetric synapses but a substantial number, approximately 25%, forming symmetric synapses (53). Physiology of the RHT The initial mechanism of entrainment is the transduction of photic information into nerve impulses in the RHT. t is not possible to record directly from the RHT because it is embedded in the optic nerve and chiasm among axons traveling to the geniculate and tectum. Early studies recording from the SeN in anesthetized animals showed that visual SeN neurons function as detectors of diffuse temporal luminance gradients (54). Subsequent studies have confirmed these data and shown that SeN neurons that respond to visual stimuli alter their discharge rate as a monotonic function of luminance (55,56). The intensity-response curve is described by a function with a small working range between threshold and saturation and a relatively high threshold, which indicates that the input to the SeN neurons, and their responses, are specialized for luminance coding in the range of light intensities occurring around dusk and dawn (55). Furthermore, the magnitude of the response of SeN neurons does not change as a function of time of day (57), indicating that induction of a phase change requires more than SeN neuron activation. The magnitude of phase shifts appears to be monotonically related to light-induced changes in SeN neuron discharge rate during sensitive portions of the light phase response curve (PRe) (56). n terms of spectral response of the circadian visual system, roost of what we know is derived from studies in the hamster. Hamster rhythm phase response is maximally sensitive to wavelengths near 500 nm, although it is not clear whether phase shifts are rod- or cone-mediated (58). RHT Transmitters FG. 6. Retinal innervation of the hamster SCN illustrated by cholera toxin 13 fragment immunoreactivity following a unilateral intraocular injection. A. Rostral. B. Middle. C. Caudal. Abbreviations: OC, optic chiasm; V3, third ventricle. Bar = 100 f.lm. guished from others containing lucent spherical vesicles by their mitochondria, which contain a network of tubules rather than standard cristae (51,52). n the ventral SeN, RHT boutons frequently form glomeruli with SeN neuron dendrites. The boutons typically synapse on distal dendrites, with the majority forming The synaptic vesicle morphology of RHT boutons in the SeN, small, lucent spherical vesicles with some dense core vesicles, suggests that the RHT contains a typical small molecule neurotransmitter colocalized with one or more peptides. All of the available evidence indicates that glutamate (GLU) is the small molecule neurotransmitter, although the source of GLU may be at least in part the dipeptide N-acetyl aspartyl glutamate, which is very rapidly metabolized to its constituent amino acids, glutamate and aspartate (59). GLU is present in RHT terminals in the SeN (60-62). GLU is released by hypothalamic slices containing SeN after electrical stimulation of the optic nerve (63), and GLU produces excitatory responses from SeN neurons in a dose-dependent manner in slices in

8 648 j, D, MLLER ET AL. CT~ B. ~: NPY A "j, '",\. ;:, FG. 7. Chenical neuroanatomy (NPY, ENK) and retinal innervation (CT-3) of the hamster GL. A. Rostral. B. Middle. C. Caudal. Arrows point to peptide-immunoreactive neurons in the GL. Bar = 100 /-Lm.

9 THE MAMMALAN CRCADAN CLOCK PHASE SHFT o CTa CT12 CT24 CRCADAN TME FG. 8. Phase response curve for light (solid line) and for application of glutamate to SCN in vitro (solid dots). SCN data from Shirakawa and Moore (64). vitro (64). Antagonists of NMDA or non-nmda receptors block light-induced phase shifts of locomotor activity (65-67). Most importantly, GLU applied to the SeN in vitro produces phase shifts of the circadian curve that are similar to those produced by light (Fig. 8) (68,69), an effect that appears to depend on nitric oxide release (69). There are also data indicating that some RHT axons terminating in the rat SeN contain SP (70,71). SP applied to the SeN in vitro produces increases of firing rate in SeN neurons in a dose-dependent manner and SP potentiates GLU responses (68). SP also produces phase shifts in the rhythm of SeN neuron firing rate with a PRe similar to light (72). Thus, SP produces functional effects consistent with what would be expected from a peptide colocalized with GLU in RHT terminals. Transduction, transcription, and translation n pioneering work on marine molluscs (73), the cascade of events that comprise the circadian pacemaker's entrainment pathway has in part been identified and traced. Ultimately this pathway must terminate on components of the oscillatory machinery in order to cause phase shifts of overt rhythmicity. Photic entrainment is viewed as a signal transduction process that couples an extracellular stimulus (light) to a lasting cellular response (a permanent phase shift of the pacemaker's oscillation). One possible mechanism for such a long-term change is the regulation of gene transcription, the control of which is mainly accomplished by the interaction of nuclear DNA-binding proteins and specific cis-acting regulatory DNA sequences. Recently, a number of laboratories initiated the study of the photic and temporal regulation of such proteins in the rodent SeN (74-78), especially the transcription factor activity known as Activator Protein-l (AP-), which is primarily composed of structurally related DNA-binding proteins belonging to the fos and jun protooncogene families. Environmental light physiologically regulates AP-l DNA-binding activity in the SeN, in part by altering the composition of its constituent proteins (79). Fos Band lun D proteins may be the predominant components forming AP-l-binding complexes in the SeN during darkness, whereas c-fos and lun B appear to compete for binding after photic stimulation. These kinds of changes in AP-l protein composition are known to lead to alterations in the DNA-binding affinity and stability of the AP-l complex and may even specify conformational differences in the DNA helix itself. These effects profoundly influence the transcriptional regulation of target genes that have AP--binding sites among the response elements on their promoters. Thus, the combinatorial power of a limited set of transcriptional regulatory proteins might be responsible for orchestrating a lightinduced program of gene expression in the SeN. The functional roles and target genes for AP-l proteins are unknown, and they appear to be expressed in a heterogeneous population of SeN cells (80). There is correlative evidence to suggest that the proteins are

10 D. MLLER ET AL. somehow involved in the mechanism for the photic entrainment of circadian rhythms to the light-dark cycle (for reviews, see 81,82). First, their expression in the SCN is anatomically specific. The pattern of labeling in the ventrolateral SCN is similar to that in the site of termination of visual inputs, and the only other retinorecipient area besides the SCN to express c-fos to ambient light is the GL. Second, c-fos expression in the SCN is correlated with photic phase shifts of overt behavioral rhythms. n hamsters, the illumination threshold for induction of c-fos mrna in the SCN is similar to the threshold for light-induced phase shifts of the locomotor rhythm, and pharmacological agents that block these behavioral phase shifts also block the photic stimulation of c-fos in specific regions of the SCN. On the other hand, c-fos is not activated when non photic stimuli are used to generate such phase shifts. Of note, the photic stimulation of fos and jun gene expression in the SeN is a function of circadian phase, with a phase dependence similar to that already well described for light-induced phase shifts of overt locomotor rhythmicity. Thus, these genes are clockcontrolled; the phase-dependent mechanism that gates their photic activation is unknown, but it appears to be specific to the SCN, persists in vitro in tissue explants (83), and is regulated (at least in part for c-fos) by the phosphorylation of the cyclic-amp response elementbinding protein (CREB) (84). n turn, CREB phosphorylation in SCN neurons may result from the large increase in intracellular calcium and concomitant activation of calcium/calmodulin-dependent kinase (CaM kinase), resulting from the occupation of both NMDA and non-nmda receptors by glutamate presumably released from RHT terminals (63). Blockade of either glutamate receptor subtype prevents phase shifts to light or glutamate (67,85). However, it appears that at least one other pathway is activated by a glutamate-induced elevation in intracellular calcium. Nitric oxide synthase (NOS) activity is also elevated by glutamatergic stimulation in the SCN and NOS, in turn, is thought to activate guanylyl cyclase, cyclic GMp, and presumably Protein Kinase G (PKG), at least at certain circadian times (86). Light- or glutamate-induced phase shifts are blocked by NOS inhibition and light-induced phase advances (but not delays) are blocked by cyclic PKG inhibition (70,86). t is possible that one target of the AP-l transcription complex is the NOS gene itself. Alternatively, the NOS and CREB cascades may be parallel and independent transduction pathways activated by an increase in intracellular calcium. A recent study (87), compatible with either of these hypotheses, demonstrates that NOS inhibition does not prevent light-induced c-fos expression in the SCN. Current studies are now focusing on whether c-fos expression in the SCN is similarly required for photic phase shifts of behavioral rhythmicity. A critical experiment-demonstrating that light's phase-shifting effects can be blocked by the experimental reduction of intracellular c-fos levels-has been presented by Wollnik et al. (88). These authors have reported that a light-induced phase delay of the rat locomotor (wheelrunning) rhythm was prevented by the intracerebroventricular injection (6 hours earlier) of antisense phosphorothioate oligodeoxynucleotides to both c-fos and june. The treatment lowered immunoreactive c-fos and Jun B protein levels in the SCN by about half. njection of nonsense sequences had no effect. Although the concentrations used in this study were very high (1 mm of each oligo), the authors claim that immunoreactive Fos Band c-jun protein expression was not affected and argue for the sequence-specificity of their oligos. Another approach has been taken by Honrado et al. (89), who have reported that mice homozygous for a c-fos null mutation entrained to a light-dark cycle and generated phase-dependent phase shifts of their wheel-running rhythms to very bright light pulses. While these findings in knockout mice do not rule out a necessary physiological role for c-fos in normal mice, they do emphasize the potential redundancy of the components of the photic entrainment system. To summarize, light is the dominant zeitgeber for circadian function. Photic information is transduced in the retina by photoreceptors and intrinsic retinal pathways that are not yet elucidated. This information is transmitted to the circadian pacemaker, the SCN, by the RHT, a pathway which contains the axons of a distinct subset of retinal ganglion cells. The major transmitter of the RHT appears to be GLU, with SP as a colocalized peptide modulator. SCN neurons are responsive to changes in luminance mediated through the RHT but the induction of phase changes in the SCN requires clock-dependent processes within the neurons themselves. Such processes probably include an elevation in intracellular calcium and activation of calcium-dependent pathways, including both the transcription and translation of AP-l proteins and activation of NOS. ORGANZATON, CONNECTONS, AND FUNCTON OF THE GL Photic information is known to have access to the circadian clock both through a direct RHT projection (55-57,90) and via an indirect path through the GL (91-93) which, in turn, projects through the geniculohypothalamic tract (GHT) to the SCN (Fig. 1). When GL-esioned animals are phase-shifted, rate of reentrainment after a photoperiod phase advance or delay

11 ;;:; lie!.... ~ x... '" '" 4( X L c o 0 00 THE MAMMALAN CRCADAN CLOCK : 12 CRCADAN TME (HRS) ;;:; /e!.... ~ X."...." 4( X L o,, ', 1#, ' \.,. " ~~ ",,... ".",, "" ". 12 CRCADAN TME (HRS) FG. 9. Phase response curve (left) to excitotoxic stimulation by NMDA injected into the hamster GL or (right) to NPY infusion into the SeN. The NPY phase response data are taken from Albers and Ferris (101); the figure as a whole is after Johnson et al. (94). is retarded (94). However, the effects of GL lesions are most obvious under constant light (LL) conditions. Normally, LL causes the circadian period to lengthen. n contrast, GL lesions block the effect of LL on period (95,96). The GL lesions apparently reduce circadian system response to a tonic photic stimulus. However, studies in which most of the visual projections, including the retinointergeniculate projection, are destroyed yield somewhat different results. n the one instance in which the direct retinal projection to the GL was destroyed without concomitant loss of the RHT, animals tended to have very long circadian periods (97,98). On the other hand, destruction of both the retinal projection to the GL and the GL neurons or destruction of the GL neurons alone initially has no obvious effect on entrained rhythm phase (94-96). These somewhat variant findings have yet to be completely resolved. The primary candidate for mediating the above effects is the GHT connecting the GL to the SeN (94-96). n the hamster, at least four different types of neurons are identifiable in this path (99). NPY- and ENK R neurons project to the SeN (Fig. 7). A subset of each also is immunoreactive to the other peptide. ENK-R neurons in the rat do not project to the SeN (100). A specific neuromodulator has not yet been associated with the fourth cell type, but these cells are probably GAB Aergic, as in the rat (18). The possibility that the GL regulates rhythm phase control is supported by experiments manipulating NPY in the SeN. Application of the peptide directly to the SeN will modify circadian rhythm phase according to a PRe distinctly different from that achieved with photic stimulation, but quite similar to that of dark pulses superimposed on a background of dim illumination (Fig. 9) (101,102). A similar result is achieved by excitatory chemical (Fig. 9) (94) or electrical (103) stimulation of the GL. n each case, it is thought that activation of the neurons contributing to the GHT produce release of NPY, thereby modulating peptide availability in the SeN and phase of the circadian clock. The GL and GHT may also play a nonphotic role in circadian rhythm regulation. When the short-acting benzodiazepine, triazolam, is administered to hamsters in LL or DD, the circadian rhythm shifts in accordance with an NPY-type PRe (104). Such a phase response can be eliminated by GL lesions (Fig. 10) whether or not the benzodiazepine utilized induces locomotion ( ). Likewise, GL lesions eliminate the NPYtype phase response ordinarily induced by novel wheel-induced locomotion (109,110). Furthermore, direct intra-sen administration of an NPY antibody eliminates the locomotor-activity-induced phase shift (111). t is unlikely that benzodiazepines act on the GL to modify phase (112) but, to date, little is known of their locus of action or the anatomical routes afferent to the GL through which locomotor activity or benzodiazepines exert their effects on the circadian clock. Figures 1 and 7 not only diagram the GHT, but illustrate the fact that GL neurons project to the contralateral GL as well. n the hamster, these neurons are neither ENK- nor NPY-R (99), whereas analogous neurons in the rat are ENK-R. The number of contralaterally projecting neurons is quite substantial, suggesting that the commissural pathway has an equally substantial function. The structural position of the GL within the visual system seems to suggest a function wider than a simple secondary mechanism moderating rhythm response to light (or light offset, see above). The GL sends a rostroventral NPY- and ENK-R projection to the SeN (99). The SeN also receives direct retinal innervation (13,50,113). Similarly, the GL sends a caudodorsal NPY- and ENK-R projection to the posterior limitans

12 D. MLLER ET AL. NMA A SHAM ~ o, o 24 o 24 Hrs 2.0 B -<t ~ :: w c{ 0.0 :: ** C CT 6 CT 21 FG. 10. A. (left) Absence of phase response to triazolam (TZ) injection (dot) in a hamster with bilateral GL lesions or (right) normal phase advance in response to TZ by a sham-operated animal. B. Phase response histogram showing advances and delays of GL-esioned (open bars) and sham-operated (hatched bars) animals after TZ injection at CT6 or CT21. After Johnson et a!. (l05). nucleus (PLi; 99) which also receives direct retinal innervation (Morin and Blanchard, unpublished data). This does not imply a similar function for the PLi and SeN, but suggests that either the PLi has a significant role in rhythm regulation or the GL is the only nucleus that communicates with both the circadian visual system and the pretectal visual system (and possibly with the tectum as well; 114). n addition, it is becoming increasingly apparent that the midbrain serotonergic system can have a major impact on visual regulation of the circadian system (see below). t is also becoming clear that the SeN and GL have many more connections than simply between themselves and that few have been tested for Sleep. Vol. 19. No rhythm regulatory function (see below). To the extent that these additional efferent and afferent paths are, in the future, proven to contribute to circadian rhythm regulation, they will comprise the "extended" circadian visual system. THE RAPHE 5HT SYSTEM AND CRCADAN RHYTHMS The 5HT projection to the SeN: anatomical and behavioral correlates The RHT and GHT constitute two of the three major pathways afferent to the SeN. The third is the sero-

13 ,._ " '_ THE MAMMALAN CRCADAN CLOCK 6S3 DHT/TZ DHT/V <-LESON 8hr PHASE ADVANCE <1111-8hr PHASE ADVANCE - -' '.. e,. e,- -_.. Be e Q_ eee_' e.,,... t_! F '_',,'_ le._ t. "_ ", "- - P" - "_., --- ",,:g're"'\'~de.r,_ ' <l1li-00,..,!!,!!!!! J o CLOCK TME! t',, t, '!!, "! '! o CLOCK TME FG. 11. Effects of serotonin depletion by intraventricular 5,7-DHT (dot) on onset, offset, and duration of the activity phase. The onset and offset of the activity phase prior to DHT are demarcated by the parallel vertical lines. The initial photoperiod is shown as the lightdark bar at the top of each running record; the shifted photoperiod is the second light-dark bar. At CT6 (triangle) on the day of the 8-hour advance in the photoperiod, one animal (DHTrrZ) was injected with TZ and the other received vehicle (DHTN). TZ was associated with a large phase jump and more rapid reentrainment than seen in vehicle-treated animals. DD-animal placed in constant darkness. After Smale et al. (116). tonergic (SHT) input (Fig. 2A) from the midbrain (11S). Despite the robust presence of the SHT terminal plexus in the SeN, there has been relatively little study of its function. Smale et al. (116) utilized the SHTselective neurotoxin S,7-DHT to destroy serotonergic input to the SeN. Almost immediately on treatment, activity onset advanced, offset delayed, and the active phase was lengthened (Fig. 11). This robust change is uniformly characteristic of animals with successful lesion of SHT input to the SeN following either intraventricular (1S-117) or direct midbrain raphe nucleus (118) placement of the neurotoxin. Successfully lesioned animals continued to show large phase shifts in response to triazolam (116). However, contradictory data exist (119,120), suggesting that the extent of SHT depletion may be critical. Subsequent evaluation of rhythmic and anatomical sequelae to the serotonin neurotoxin treatment has been revealing (117). The lengthened activity phase persists in constant darkness without an effect of serotonin loss on the circadian period. n constant light, however, period rapidly lengthens and normal circadian rhythmicity is quickly lost (Fig. 12). None of the observed effects is unexpected in control animals ex-

14 654 J. D. MLLER ET AL. 8 LL- 0',,,,,,,,, '12',,,,,,,,, '24',,,,,,,,, '36',,,,,,,,, 48 0""""" '12',,,,,,,,, 24',,,,,,,,, '36',,,,,,,,, 48 H R FG. 12. Patterns of wheel running after depletion of brain serotonin following intraventricular 5,7-DHT (dot). n all cases, activity onset was later and the activity phase was longer. Most animals showed "splitting" (A), many showed a more complex form of splitting (B), and several became almost arrhythmic (C). All three patterns are found in normal animals, but with a much reduced incidence. After Morin and Blanchard (117). posed to constant light, but the magnitude of the effects following serotonin depletion is much greater than normal. The role of endogenous 5HT appears to be a normal tonic inhibitor of sensitivity to light. n the absence of serotonin, the rhythmic changes seen under constant light in the normal animal are hyperexpressed. t is also useful to note that the changes in rhythmicity during LL are consistent with the increased phase shifts to light observed in one particular portion of the PRC of serotonin-depleted hamsters (117). Preliminary anatomical studies, performed as a necessary prelude to the experiments evaluating the effects of the neurotoxin on circadian rhythmicity, showed total loss of 5HT-R fibers in the SCN following intraventricular administration of 5,7-DHT. This was associated with an approximately 91% loss of 5HT-R neurons in the dorsal raphe (116,117). However, in the first experiment (116), some residual fibers were evident in brains from animals killed 60 days after neurotoxic lesion. n a second experiment (117), many more fibers were present after 180 days. Therefore, the possibility of fiber regeneration in the SCN and GL was studied in animals allowed to recover for 2, 8, 14, or 20 weeks after intraventricular treatment with the neurotoxin (115). The results showed clearly that, in the short term, intraventricular 5,7-DHT eliminates 5HT innervation of both the SCN and GL with concomitant induction of the expected activity phase changes (Fig. 13). The behavioral effects persisted through the entire 20-week test period despite the fact that there was gradual recovery of innervation by serotonergic fibers in both SCN and GL (without restoration of lost raphe neurons). Clearly, the substantial reinnervation evident 20 weeks postsurgery does not signify restoration of function. Despite the implication of the above studies that dorsal raphe neurons are involved in the 5,7-DHT-induced rhythm changes, the specific loss of neurons in the median raphe was not fully evaluated. The anatomical literature based on rat studies does not permit an unambiguous determination that only neurons of the dorsal raphe innervate the SCN. The only relevant hamster paper (121) simply shows that serotonergic innervation of the SCN and GL occurs at distinctly different developmental ages, the former being postnatal (P3) and the latter being prenatal (E13). Periventricular regions are generally innervated later than lateral areas in the diencephalon. The large difference between developmental dates of SCN and GL inner-

15 THE MAMMALAN CRCADAN CLOCK 655 vation may be related to differences in midbrain sites at which the serotonergic projections to the two nuclei originate (Table ). t is now apparent from both retrograde (Fluoro-Gold, Fast Blue, Cholera toxin ~ fragment) and anterograde (Phaseolus vulgaris leucoagglutinin) tracing studies that the median raphe projects to the SCN (but not to the GL) and that the dorsal raphe projects to the GL (but not to the SCN; 118). The tracing studies are supported by the effects of site-specific 5,7-DHT lesions on both rhythmic behavior and neuroanatomy (118). Selective destruction of median raphe 5HT neurons eliminates the 5HT innervation of the SCN (but not the GL). Destruction of the 5HT neurons in the dorsal raphe eliminates 5HT innervation of the GL (but not the SCN). Behaviorally, the median raphe lesions mimic the effects of intraventricular 5,7-DHT; administration of the neurotoxin to the dorsal raphe has no effect on the circadian system (Table ). The SHT projection to the SeN: neurochemical and behavioral correlates Circadian rhythms in 5HT reuptake in the SCN (122); the 5HT metabolite, 5-hydroxyindoleacetic acid (5HAA) (123,124); and responsiveness of the postsynaptic neurons in the SCN to iontophoresed 5HT (125) are all in phase, with maximum levels (acrophases) occurring during the dark (active) phase of the rat circadian locomotor rhythm. As in other monoaminergic systems, cellular contents of the transmitter (5HT) (126,127) are in antiphase with the metabolite (5HAA), with acrophase during the light (sleep) phase, whereas releasable 5HT is in phase with its metabolite with acrophase during the dark phase (Fig. 14). Such observations are in agreement with data showing that raphe unit activity is greatest during waking and least during the deepest stages of sleep (128). The persistence of rhythms in 5HT content in the SCN (127) and 5HT release in the SCN slice (Miller, unpublished data) in constant conditions suggests that the release of this major afferent transmitter in the SCN is under circadian control by SCN efferents, the essence of a feedback loop. n contrast, 5HT content in the entire anterior hypothalamus seems to be driven primarily by the ighudark cycle (129). Elevation of synaptic levels of 5HT by systemic administration of the specific monoamine oxidase type A inhibitor clorgyline or by the 5HT reuptake blocker FG. 13. Recovery of serotoncrgic innervation in the SCN following depletion by 5,7-0HT. Normal control (A) and 2 (B), 8 (C), 14 (0). and 20 (E) weeks postlesion. After Morin (115). Sleep. Vo/. 19. No, R, 1996

16 6S6 TABLE 1. J. D. MLLER ET AL. Summary of anterograde and retrograde dorsal or median raphe tracing results and the effects of localized serotonin neurotoxin lesions on innervation of the SeN and GL and wheel-running rhythmicity Tracer/toxin locus Anterograde in MR Anterograde in DR Retrograde in SCN Retrograde in GL Toxin in MR Toxin in DR Primary anatomical result Fibers to SCN No fibers to SCN Cells in MR Cells in DR No fibers in SCN No fibers in GL Secondary anatomical result No fibers to GL Fibers to GL No cells in DR No cells in MR Fibers in GL Fibers in SCN Behavioral effect Rhythm changes No rhythm changes Abbreviations used: SCN, suprachiasmatic nucleus; GL, intergeniculate leaflet; MR, median raphe nucleus; DR, dorsal raphe nucleus. imipramine produces phase delays in locomotor rhythms (130), whereas depletion of SHT by parachlorophenylalanine (PCPA) produces phase advances in rat locomotor and corticosterone rhythms (131,l32). While the time course and pharmacodynamics of such agents are difficult to judge with respect to the SCN, maximum effectiveness should occur when there is appreciable 5HT tonus in the synaptic cleft of the 5HT terminal in the SCN, i.e. in the dark phase. A more direct test of the function of 5HT in the SCN involved the direct application of the nonspecific 5HT agonist quipazine to the SCN in vitro (133). These studies showed that quipazine administered in projected day in the SCN slice produces phase advances, whereas administration in projected night produces phase delays (Fig. S). The phase shifts were not sensitive to the interruption of synaptic transmission, most likely suggesting a direct effect of quipazine on clock cells bearing the appropriate SHT receptor (l34). The phase delays were consistent with the theoretical mode of action of indirect SHT agonists (see above). These studies were replicated in vivo (l35). After a protracted series of studies (l36) it was concluded that the daytime phase advance was solely mediated by the 5HTA receptor, whereas the nighttime phase delay was mediated by a thus far uncharacterized 5HT receptor. This conclusion was predicated on the efficacy of 8-0H-DPAT, the reference 5HTlA ag- 250~ ~ 200 Q) C) s::::: 150 a:s.r:. 0 ~ o Circadian Time FG. 14. Circadian rhythm in the release of 5HT in the SCN slice. Eight SCN slices from four male Wistar rats previously entrained to a 12: 12 light/dark cycle were maintained for 24 hours in a slice chamber. Perfusates were collected every hour via a refrigerated fraction collector. The slices were superfused with normal medium (15 f,llminute) supplemented with ascorbic acid to inhibit oxidation (100 f,lm), pargyline to inhibit MAO (50 f,lm), L-tryptophan as a substrate for 5HT synthesis (25 f,lm), and the antibiotic gentamycin (0.025%). Samples were analyzed for 5HT via HPLC with electrochemical detection. Basal 5HT under these conditions is approximately 6 nm.

17 -0::: :J: , ~ CT (HR) FG. 15. Phase response curve for quipazine in the SCN slice. Quipazine (10 J.LM) phase-shifts the single-unit rhythm. Circadian time (CT) in hours is plotted on the x-axis; the magnitude of the phase shift in hours (t.</» is plotted on the y-axis. After Prosser et al. (134). THE MAMMALAN CRCADAN CLOCK +' C OJ '- 5 o OJ c ro '-.D UK) in SeN primary culture 5001 E 200 OJ E t f f i command potential (mv) 6S7.. l. ~ ritanserin &... _/ 8-0H-DPAT."... 8-OH-DPAT FG H-DPAT (10 J.LM), a 5HT7 agonist, elevates a voltage-sensitive potassium current in whole cell-clamped SCN neurons in primary culture. onist, in producing daytime phase advances, a result replicated by other groups in vitro and in vivo ( ). However, the phase response to 8-0H-DPAT was essentially identical to that of nonhydrolyzable analogues of camp (140). n a later study SHTergic phase advances were completely blocked by antagonists of camp or protein kinase A (141). These results suggested that SHTA receptor activation in the SeN led to an elevation of camp, in contradiction to most reports indicating that this receptor depresses camp through its coupling to the G protein, Gi. This conundrum was resolved by the discovery of a novel SHT receptor in the SeN, SHT7, at which 8-0H-DPAT is also a full agonist (142). This receptor is now known to elevate camp through its coupling to the G protein, Gs. nitial results by the polymerase chain reaction (per) demonstrated its presence in the SeN and more generally in the hypothalamus (142). More recent results (Michael Rea, personal communication, 1995) by in situ hybridization and immunohistochemistry have demonstrated the presence of both the mrna and the receptor protein for SHT7 in the ventral SeN. Perhaps the most convincing evidence that the receptor mediating SHTergic phase advances in the SeN is SHT7 derives from the observation that 8-0H-DPAT-induced phase advances are potently antagonized by ritanserin, but unaffected by pindolol (142). Such results are consistent with the pharmacology of the SHT7 receptor and completely inconsistent with the SHTA receptor. A recent report indicates that both elevation of camp and the opening of a calcium-dependent potassium channel are necessary for the phase advance presumably mediated by the SHT7 receptor in the SeN (141). Whole cell data from cultured SeN neurons indicate that 8-0H-DPAT produces a large increase in an outward current, probably potassium, which can be antagonized by the SHT7 antagonist, ritanserin (Fig. 16). f this is indeed a calcium-activated current, the SHT7 receptor may also elevate intracellular calcium in the SeN (Fig. 17) distally to the activation of the camp pathway or perhaps in parallel via coupling to a G protein in addition to Gs. t is important to consider the functional correlates of activation of the SHT input to the SeN. A large body of work suggests that the most effective stimulus for elevating SHTergic neurotransmission is rhythmic locomotor activity (143). A strong correlation has been observed between wheel locomotion and SHT level in the SeN of blinded rats in both subjective day and subjective night (144). Furthermore, spontaneous wheel locomotion is itself a zeitgeber, with a PRe very similar to that of quipazine (14S). Recent data suggest that this zeitgeber effect of locomotion can be eliminated by destruction of SHT afferents in the SeN by the SHT neurotoxin, S,7 dihydroxytryptamine (D. Edgar, unpublished data). Similarly, the phase-shifting effects of triazolam, which seem to be mediated by an increase in locomotor activity, may be antagonized by destruction of these afferents to the SeN (119,120, but see 116). t is tempting to conclude that the feedback effects of locomotor activity on the SeN circadian clock are mediated by the SHT input. This would also be consistent with the similarity between the PRe for dark pulses superimposed on a dim light background (146) and the PRe for either quipazine or spontaneous wheel locomotion, since light offset in nocturnal ro-

18 D. MLLER ET AL umdpat Ca2+ Free + 1 mm EGTA Ca2+ Free + 1 mm EGTA 20 umdpat 20 um Quipazine 250 ~ 200 c N CO (.) ~ o O MN FG H-DPAT (20 LM) mobilizes intracellular calcium in SCN cells (previously loaded with Fura-2) in primary culture. The elevation occurs in a calcium-free medium, suggesting mobilization of an intracellular pool. This particular cell is an astrocyte, but similar responses are also seen in SCN neurons. dents stimulates locomotor activity. However, the phase response curve for NPY, a major transmitter of the GL input to the SeN (see above), is also very similar to that of spontaneous wheel locomotion (101). An antibody against NPY administered directly into the SeN also blocks the zeitgeber effect of locomotor activity (111). t appears that both the NPY and the 5HT projections are necessary for the feedback effect of locomotor activity on the biological clock. This may in tum reflect an intimate anatomical (22,23) and developmental interaction (147) between these nonretinal SeN afferents. AFFERENT NTERACTON N THE SCN Electrical stimulation of the optic nerve or direct application of glutamate agonists to the SeN in vitro in rats produces a PRe equivalent to that for photic stimulation (64,70,148,149). n contrast, GL stimulation (94,107) or neuropeptide Y applied directly to the SeN (101,102) produces a PRe that is very similar to that of dark pulses or locomotor activity (see above). Similarly, 5HTergic stimulation of the SeN produces a "dark-pulse-like" PRe (see above). The anatomical juxtaposition of the glutamatergic projection from the retina, the NPY-containing projection from the GL, and the 5HTergic projection from the raphe in the SeN (22-24) suggests that in addition to interactive effects at the postsynaptic neuron, there may be a large degree of presynaptic interaction among them. For instance, one function of serotonin may be to modulate photic input to the clock by gating glutamate release from the RHT. A microdialysis study has shown that local perfusion of the SeN with a 5HTlAl5HT7 agonist (8- OH-DPAT) reduced extracellular glutamate, whereas systemic administration of the 5HT2/5HT7 antagonist ritanserin elevated extracellular glutamate in the SeN (150). As the authors note, one caveat here is that the mechanism of glutamate modulation is not known. t may depend on presynaptic 5HT receptors on RHT terminals or other glutamatergic afferents, or possibly 5HT receptor-mediated glutamate uptake by astrocytes (150) which are prominent in the SeN (151) and may play an important role (152) in the organization of circadian rhythmicity. But local perfusion of the SeN in vitro with 8-0H-DPAT does block optic-nerve-stim-

19 THE MAMMALAN CRCADAN CLOCK 659 ulation-induced field potentials (probably a direct effect on RHT terminals), whereas systemic administration of the agonist causes a large attenuation in lightinduced phase advances or delays (153). t is important to note that all of these effects of 8-0H-DPAT were seen at circadian times (dark cycle) when 8-0H-DPAT does not itself generate phase shifts, apparently a postsynaptic effect (134). A likely explanation of these results is that the 5HT7 receptor may be present on RHT terminals, as well as on postsynaptic neurons in the SeN. and may act as a terminal heteroceptor in modulating transmitter release. A probable role of the 5HTB receptor in modulating glutamate release has also been demonstrated (150). A substrate for postsynaptic interaction of the RHT and 5HTergic projection is suggested by the observation that photic ally activated SeN neurons are generally inhibited by the nonspecific 5HT agonist quip a zine (154). However, this same study showed that general 5HT receptor blockade did not influence photic responses in the SeN. But since the study was conducted in late morning and early afternoon when 5HT tonus is thought to be very low in rats, there may simply have been very little endogenous 5HT to antagonize. n fact, an iontophoretic study in the in vivo hamster SeN, which largely avoided the projected light period, found that 5HT or agonists effective at 5HT7 (8-0H-DPAT, 5eT) tended to reduce the photic response of SeN neurons, whereas the general 5HT antagonist metergoline tended to facilitate it (155). Similarly, photic ally induced or basal Fos protein in the SeN is greatly attenuated by 5HT receptor agonists ( ). On the other hand, a recent study in the rat SeN in vitro has shown that 5HT can greatly facilitate glutamatergic neuronal responses in the SeN during the projected light period (159). But most observations do suggest an inhibitory action of 5HT on the functional effects of RHT activation (see above), although it is not clear in many of these studies whether the photic/5ht interaction is presynaptic or postsynaptic. While the majority of studies have concentrated on the photicl5ht interaction, some work has examined the photiclnpy interaction. An enhanced phase shift to light has been observed following administration of an antiserum against NPY directly into the SeN (160), in analogy to the effects of 5HT denervation (117). nterestingly, light can reciprocally block the NPY-induced phase advance, even when the light pulse is given at a circadian time when light does not phase-shift the circadian clock (161). This suggests that photic stimulationrht glutamate may interact with the NPY signal transduction pathway in the SeN neuron proximal to the point at which glutamate induces a phase shift (analogous to the way in which 5HT7 receptor stimulation appears to alter RHT function at circadian times when 5HT7 postsynaptic stimulation is ineffective). Finally, almost nothing is known of any physiological interaction between the raphe/5ht and GLNPY afferents in the SeN. The extended circadian visual system consists of the neural systems modifying circadian function of the SeN. Only the retinal and GL contributions to this extended system have been explored in any detail. Our understanding is beginning to encompass the circadian functions of the raphe nuclei. The median raphe appears to modulate rhythmicity, probably via its direct projection to the SeN. Serotonin-specific neurotoxins have been used to lesion raphe input to the circadian system, but the serotonin system must now be stimulated. t is also time to more fully examine GL-diencephalic connections. This issue has not been adequately addressed and must be studied in order to achieve a reasonable understanding of the relationship between the GL, SeN, light, and nonphotic zeitgebers. EFFERENT OUTPUT OF THE SeN There are four major SeN-efferent projections in the hamster. with many of the fibers containing VP- or VP-R (11). These include (i) an anterior projection to the anterior paraventricular thalamus, ventral lateral septum, and bed nucleus of the stria terminalis; (ii) a generalized peri ventricular fiber system innervating much of the medial hypothalamus from the preoptic region to the premammillary area; (iii) a lateral thalamic projection to the GL; and (iv) a posterior projection to the posterior paraventricular thalamus, precommissural nucleus, and olivary pretectal nucleus (7). Many of these projections exhibit vasopressin or VP immunoreactivity (6,11). This same general projection system is seen in rats and is probably characteristic of mammals (4,5). n both rat and hamster there is a substantial projection to a region ventral to the paraventricular nucleus of the hypothalamus, called the subparaventricular zone (spvz) (4,5). This region in tum projects to most of the same sites as the direct efferents of the SeN. Knife cuts dorsal and posterior to the SeN in the rat abolish drinking and locomotor rhythms (162). Efferents traveling to the spvz, paraventricular nucleus of the hypothalamus (PVN), paraventricular nucleus of the thalamus. and locally in the medial hypothalamus would all have been interrupted by such cuts (as well as efferents from the retrochiasmatic area). This in tum suggests a special role for at least some subset of these efferents in transmitting the output signal from the SeN circadian clock. The projection to the PVN is part of a multi synaptic pathway projecting from there to the superior cervical ganglion and finally to the pineal Sleep. Vol. 19. No

20 660 J. D. MLLER ET AL. gland (34, but see 163,164 for an alternative interpretation). This pathway is the only one thus far identified with a specific circadian output, the circadian rhythm in melatonin synthesis. However, a recent report suggests that a monosynaptic projection from the SCN to a region in the ventral lateral preoptic nucleus may be the means by which the SCN regulates sleep in a circadian fashion (165). Hypothalamic knife cuts (SCN island preparation; 166) that isolate the SCN from synaptic contact with the rest of the CNS have been reported to result in the loss of neuronal rhythms both in other nuclei and in behavioral rhythms. This result has recently been challenged (9). Apparently, if the SCN island is large enough, allowing preservation of the ventricular and vascular systems as well as SCN proper, behavioral circadian rhythms may be maintained. The mechanism may involve an endocrine or paracrine discharge of some circadian output molecule into the third ventricle. This result gains further support from the recent observation that SCN transplants can restore rhythmicity when placed in the lateral ventricle, in the apparent absence of synaptic contact with any other brain structures (27,167,168). Furthermore, SCN transplants, which neither reinnervate nearby tissue nor are themselves reinnervated, can restore rhythmicity in SCNlesioned rodents, possibly through the release of a diffusible factor such as mentioned above (R. Silver, personal communication, 1996). All such results must be considered in light of the observation that the blockade of fast sodium-channel-dependent action potential generation in the SCN by tetrodotoxin (TTX) does abolish overt circadian rhythmicity (169) for the duration of TTX administration. This in turn suggests that the release of such an endocrine or paracrine factor into the CSF or vascular system is probably dependent on action potential generation, otherwise rhythmicity should not have been eliminated by TTX. The cellular elements that release such a factor could be neurons themselves or nonneuronal elements strongly influenced by neuronal activity. Thus, there appear to be two output mechanisms for the circadian clock, both mediated by neuronal activity: i) a primarily intrahypothalamic projection system utilizing classical synaptic transmission and ii) a potentially global system allowing secretion of diffusible output transmitters into the cerebrospinal fluid (CSF) or cerebral vasculature. Transmitters of the output pathways n the SCN, the most intensively studied molecular rhythm is a circadian rhythm of the levels of the neuropeptide vasopressin. Rhythmic vasopressin levels have been measured in the CSF of rats, guinea pigs, rabbits. sheep, goats, cats, and rhesus monkeys, with high levels in all during the light phase of the lightdark cycle (or during the projected light phase in constant darkness) (170). The circadian regulation of hormone release into CSF is insulated from the osmotic regulation of peptide secretion into blood, and vasopressin in CSF appears to originate in the parvocellular peptide-containing neurons of the SCN. The circadian rhythm of vasopressin release from rat SCN neurons persists in vitro in tissue explants (171), slices (172), organotypic slice cultures (173,174), and dissociated cell cultures (175,176). Levels of both the vasopressin peptide (177) and mrna ( ) exhibit circadian rhythmicity in the rat SCN (but not in supraoptic or paraventricular nuclei), and nuclear run-on analysis has demonstrated that mrna abundance is regulated by transcription (182). Such circadian clock control of transcription is already well known in other systems ( ). nterestingly, the peak levels of vasopressin mrna in the rat SCN occur during the latter half of the light phase (i.e., in the afternoon) while the peak levels of the peptide in the nucleus and CSF occur during the morning. This observation hints that other, perhaps posttranscriptional, mechanisms may contribute to the temporal pattern of SCN vasopressin expression. n fact, there is a circadian rhythm of vasopressin mrna polyadenylate tail length, with long tails (~200 nucleotides) during the light phase and short tails (~30 nucleotides) during the dark phase (188,189). Since the appearance of the long-tailed vasopressin mrna species in the morning coincides with the rise of peptide content in the SCN and CSF, it is possible that polyadenylation state accounts for rhythmic peptide levels by altering mrna stability and translational efficiency. There are previous examples of circadian clocks exerting some of their effects by a posttranscriptional control of peptide synthesis. Such a mechanism appears to be responsible for the rhythm of the amount of luciferin-binding protein in Gonyaulax (190) and contributes to the rhythm of the per protein in the fruit fly Drosophila (191,192). The role of vasopressin rhythmicity in circadian clock function is unknown. The peptide does not appear to be an essential component of the circadian oscillatory mechanism per se because Brattleboro rats, with a mutation in the vasopressin gene that prevents peptide synthesis, display normal mrna rhythms of vasopressin and vasopressin Va receptor levels in the SCN ( ), as well as normal behavioral rhythms. However, it is quite possible that Brattleboro rats are compensated mutants; that is, over the course of maturation and development some other transmitter may co-opt the function of vasopressin. For this reason an acute inducible knockout of the vasopressin gene would be a stronger test of the role of vasopressin in

21 THE MAMMALAN CRCADAN CLOCK 661 circadian function. n any event, it is certainly possible that the vasopressin rhythm works as an output timing signal ("hands of the clock") and may modulate the amplitude of the circadian rhythm of sleep and wakefulness ( ). There is evidence that other SeN neuropeptides also show rhythmic properties (196). Levels of both the somatostatin peptide and mrna exhibit circadian rhythmicity in the rat SeN, with the mrna peak preceding the peptide peak by about 4 hours (197). The peptide and mrna levels of GRP and VP-which are colocalized in some SeN neurons-exhibit oppositely-phased rhythms during the light-dark cycle, with high levels of GRP during the light and of VP during the dark (198). These rhythms are not found in constant darkness, suggesting that GRP and VP rhythmicity is driven by the external lighting cycle and not an endogenous clock. Recently, however, VP peptide rhythmicity has been observed in vitro in organotypic slice cultures (173), and perhaps in vivo under special experimental conditions (199,200). Finally, an endogenous rhythm in GABA content in the SeN has been reported (201). THE ENDOGENOUS MECHANSM OF THE CLOCK Although much is now known about the anatomy, physiology, and pharmacology of the SeN, the molecular processes that make up the actual oscillatory machinery in the nucleus are not understood. Still, a number of properties are exhibited by this intrinsic "clockworks" in the SeN. First, synaptic transmission and calcium-dependent neurotransmitter release are not essential to the timekeeping mechanism. Acute or chronic interference with action potential generation in vivo (169) or more generally with synaptic transmission in vitro (134,202) will disrupt the behavioral or neuronal expression of rhythmicity for the duration of the interfering treatment, but once that treatment terminates, rhythmicity recovers at a phase consistent with persistence of occult circadian timekeeping throughout the interfering treatment. Similarly, circadian rhythmicity in 2-deoxyglucose uptake occurs prenatally in the SeN prior to synaptogenesis (203). Furthermore, circadian rhythms in body temperature in hibernating ground squirrels are observed well below temperatures at which it is no longer possible to record action potentials in the SeN (204,205). n all likelihood, synaptic activity is an output function of the clock which usually communicates phase information to the rest of the nervous system, but is not necessary for rhythm generation per se. Secondly, circadian function is homogeneous throughout the SeN. Rhythmicity may persist after a lesion of as much as 90% of SeN volume (e.g. 206), suggesting a large degree of cellular redundancy for the maintenance of circadian timekeeping. mportantly, lesion locus within the SeN is relatively unimportant. Similarly, composite circadian rhythms in neuronal activity in the SeN slice are evident, even though such rhythms represent the mean firing of neurons throughout the SeN (e.g. 207). This could not occur unless virtually all SeN neurons are circadian oscillators or neuronal activity in the SeN is driven and synchronized by a subset of cellular oscillators in the SeN. Thirdly, a recent report (33) suggests that virtually all SeN neurons may be circadian oscillators (or that only the oscillator population survives the culture procedure). n this work, simultaneously recorded neurons in dissociated SeN cell cultures continued to exhibit circadian rhythms in neuronal firing. nterestingly, the neuronal rhythms were typically out of phase, in contrast to neuronal rhythms in the SeN slice. While these data do not exclude the possibility of nonneuronal circadian oscillators in the SeN (e.g. astrocytes), it appears unlikely that the individual neuronal oscillations are the product of a coupled network of ultradian (as in 208) or circadian cellular oscillators. f such a coupled network were necessary for circadian expression, the individual neuronal oscillations would have been in phase. Such considerations suggest two important endogenous mechanisms in the SeN: i) autonomous cellular oscillators and ii) some mechanism for coupling or synchronizing the activity of such oscillators, present in the SeN slice but sometimes absent in cell culture (but note that peptide rhythms are sometimes present in culture, suggesting that synchronization can be at least occasionally maintained in culture; 173,175). Furthermore, synchrony of neuronal discharge in the SeN may persist even during the blockade of calciumdependent synaptic transmission (209). Possible synchronizing factors include extracellular chloride and/or potassium, which may contribute to circadian regulation of resting membrane potential (14); small diffusible molecules such as nitric oxide (210) or arachidonic acid (211); or neural cell adhesion molecules (212). Furthermore, it is possible that such factors could be modulated by astrocytes intercommunicating via gap junctions (14). nterference with gap junctions or glial metabolism can, in turn, disrupt circadian rhythmicity (152). Such mechanisms of synchronization have been extensively reviewed (14,15). n comparison, the intrinsic mechanism of the cellular oscillator in the SeN is almost completely opaque. This ignorance may soon yield to the application of the powerful techniques of genetics and mo- ~,:

22 662 J. D. MLLER ET AL. lecular biology (213-2l7), and here we outline where some of these advances are being made. Generating mutant circadian clocks The techniques of induced mutagenesis and modem molecular genetics have yielded remarkable results identifying single genetic loci, period (per) and timeless (tim) in Drosophila and frequency (jrq) in the fungus Neurospora, that affect pacemaker behavior ( ,2l7). Of note, cell bodies in the SeN are labeled by an antibody directed against a small domain of the per protein that is tightly conserved among Drosophila species (218), a mouse DNA probe homologous to the Thr-Gly repetitive sequence of the per gene hybridizes to sections of rat SeN (219), and putative per homologs have been reported from mammalian cdna libraries (220). A number of circadian clock mutations in mice are known (references in 221): there are congenitally anophthalmic mice that do not entrain to light-dark cycles, mice with hypogenesis of the SeN that show disorganized circadian locomotor rhythmicity, mice that do not synthesize pineal melatonin, and mice with retinal hypopigmentation that show reduced light sensitivity (222) (and retinally degenerate mice that do not; 39). The development of the murine clock is genetically programmed independently of the environment (223), and studies of inbred mouse strains indicate that one or more genetic loci influence the value of the endogenous period (1") (221). A vertebrate single-gene mutation affecting 1" was discovered spontaneously as an autosomal, semidominant allele in golden hamsters (tau) that reduced 1" of temperature and locomotor rhythmicity to about 20 hours in homo zygotes (224). These animals cannot entrain to the 24-hour light-dark cycle, and photoperiodic responsiveness is also dramatically altered (225). nterpulse intervals in the secretion of luteinizing hormone and cortisol are lengthened (226), while other rhythmic phenomena (estrous cyclicity, heart rate) remain unaffected (227,228). Recently, two laboratories have initiated chemical mutagenesis programs with N-ethyl-N-nitrosourea and have isolated single-locus semidominant autosomal mutations in mice that alter 1". Clock lengthens 1" and abolishes circadian locomotor rhythmicity in constant darkness in homozygotes (229). Wheels also lengthens 1", but, unlike clock, exhibits a complex phenotype including bidirectional circling, hyperactivity, and inner ear anomalies (230). These mutations have been mapped to the midportion of chromosome 5 and the subcentromeric portion of chromosome 4, respectively, and identification of the genes is now underway. 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