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The effects of gastrin-releasing peptide (GRP) on the circadian clock in the suprachiasmatic nucleus (SCN) are dependent on the activation of N-methyl-d-aspartate (NMDA) receptors in the SCN. In this study, the interaction between GRP, glutamate and serotonin in the regulation of circadian phase in Syrian hamsters was evaluated. Microinjection of GRP into the third ventricle induced c-fos and p-ERK expression throughout the SCN. Coadministration of an NMDA antagonist or 8-hydroxy-2-di-n-propylamino-tetralin [a serotonin (5-HT)1A,7 agonist, DPAT] with GRP limited c-fos expression in the SCN to a region dorsal to GRP cell bodies. Similar to the effects of NMDA antagonists, DPAT attenuated GRP-induced phase shifts in the early night, suggesting that the actions of serotonin on the photic phase shifting mechanism occur downstream from retinorecipient cells. c-fos and p-ERK immunoreactivity in the supraoptic (SON) and paraventricular hypothalamic nuclei also increased following ventricular microinjection of GRP. Because of this finding, a second set of experiments was designed to test a potential role for the SON in the regulation of clock function. Syrian hamsters were given microinjections of GRP into the peri-SON during the early night. GRP-induced c-fos activity in the SCN was similar to that following ventricular administration of GRP. GRP or bicuculline (a γ-aminobutyric acidA antagonist) administered near the SON during the early night elicited phase delays of circadian activity rhythms. These data suggest that GRP-induced phase-resetting is dependent on levels of glutamatergic and serotonergic neurotransmission in the SCN and implicate activity in the SON as a potential regulator of photic signaling in the SCN., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The antidiuretic hormone vasopressin (VP) promotes water reabsorption from the kidney and levels of circulating VP are normally related linearly to plasma osmolality, aiming to maintain the latter close to a predetermined set point. Interestingly, VP levels rise also in the absence of an increase in osmolality during late sleep in various mammals, including rats and humans. This circadian rhythm is functionally important because the absence of a late night VP surge results in polyuria and disrupts sleep in humans. Previous work has indicated that the VP surge may be caused by facilitation of the central processes mediating the osmotic control of VP release, and the mechanism by which this occurs was recently studied in angled slices of rat hypothalamus that preserve intact network interactions between the suprachiasmatic nucleus (SCN; the biological clock), the organum vasculosum lamina terminalis (OVLT; the central osmosensory nucleus) and the supraoptic nucleus (SON; which contains VP-releasing neurohypophysial neurones). These studies confirmed that the electrical activity of SCN clock neurones is higher during the middle sleep period (MSP) than during the late sleep period (LSP). Moreover, they revealed that the excitation of SON neurones caused by hyperosmotic stimulation of the OVLT was greater during the LSP than during the MSP. Activation of clock neurones by repetitive electrical stimulation, or by injection of glutamate into the SCN, caused a presynaptic inhibition of glutamatergic synapses made between the axon terminals of OVLT neurones and SON neurones. Consistent with this effect, activation of clock neurones with glutamate also reduced the excitation of SON neurones caused by hyperosmotic stimulation of the OVLT. These results suggest that clock neurones in the SCN can mediate an increase in VP release through a disinhibition of excitatory synapses between the OVLT and the SON during the LSP., (© 2012 The Authors. Journal of Neuroendocrinology © 2012 Blackwell Publishing Ltd.)

The core molecular clockwork in the suprachiasmatic nucleus (SCN) is based on autoregulatory feedback loops of transcriptional activators (CLOCK/NPAS2 and BMAL1) and inhibitors (mPER1-2 and mCRY1-2). To synchronize the phase of the molecular clockwork to the environmental day and night condition, light at dusk and dawn increases mPer expression. However, the signal transduction pathways differ remarkably between the day/night and the night/day transition. Light during early night leads to intracellular Ca(2+) release by neuronal ryanodine receptors (RyRs), resulting in phase delays. Light during late night triggers an increase in guanylyl cyclase activity, resulting in phase advances. To date, it is still unknown how the core molecular clockwork regulates the availability of the respective input pathway components. Therefore, we examined light resetting mechanisms in mice with an impaired molecular clockwork (BMAL1(-/-)) and the corresponding wild type (BMAL1(+/+)) using in situ hybridization, real-time PCR, immunohistochemistry, and a luciferase reporter system. In addition, intracellular calcium concentrations (Ca(2+)(i)) were measured in SCN slices using two-photon microscopy. In the SCN of BMAL1(-/-) mice Ryr mRNA and RyR protein levels were reduced, and light-induced mPer expression was selectively impaired during early night. Transcription assays with NIH3T3 fibroblasts showed that Ryr expression was activated by CLOCK::BMAL1 and inhibited by mCRY1. The Ca(2+)(i) response of SCN cells to the RyR agonist caffeine was reduced in BMAL1(-/-) compared with BMAL1(+/+) mice. Our findings provide the first evidence that the mammalian molecular clockwork influences Ryr expression and thus controls its own photic input pathway components.

Unlabelled: The development of adult circadian function, particularly sexual dimorphism of function, has been well studied only in rapidly developed rodents. In such species development is complete by weaning. Data from adolescent humans suggest that significant development occurs during the pubertal period. We hypothesized that a more slowly developing rodent might better mimic the changes in circadian function around puberty in humans and allow us to determine the underlying neural changes. Entrained and free-running circadian rhythms were analyzed and correlated with pubertal development in male and female Octodon degus (degu) that remained gonadally intact or were gonadectomized at weaning. Brains were collected during development to measure androgen and estrogen receptors in the suprachiasmatic nuclei (SCN) Adult circadian period does not develop until 10-12 months of age in degus, long after the onset of gonadal maturation (3-5 months). The timing of circadian period maturation correlates with the appearance of steroid receptors in the SCN. Changes in free-running rhythms only occurred in gonadally intact degus. Adult phase angles of activity onset develop between 2 and 3 months of age (comparing results of two experiments), soon after the onset of pubertal changes., Conclusion: The development of sexually dimorphic adult circadian period occurs after gonadal puberty is complete and requires the presence of gonadal steroids. The delay in development until after gonadal puberty is likely due to the delayed appearance of steroid receptors in the SCN. Phase is not sexually dimorphic and changes in the absence of steroid hormones.

To determine whether the daily rhythms of spike activity in the supraoptic nucleus (SON) were accompanied by changes in the behaviour of its inputs, we used conventional extracellular single cell recordings from cells in the SON of anaesthetized rats while stimulating the contralateral optic nerve and the ipsilateral suprachiasmatic nucleus (SCN). Neurones in the SON region were identified by antidromic activation and classified as oxytocin or vasopressin cells, on the basis of their spontaneous firing patterns. Approximately 27% of both oxytocin (29/108) and vasopressin (39/147) neurones were excited by stimulation of the optic nerve, and the majority of responses had a long latency (>20 ms). Very few oxytocin (3/108) and vasopressin cells (2/147) were inhibited by stimulation of the optic nerve. The pattern of the responses (excitatory, inhibitory or nonresponsive) of oxytocin and vasopressin cells to stimulation of the optic nerve was significantly related to the time of day (chi-square test; P = 0.012, oxytocin cells; P = 0.006, vasopressin cells). The proportion of oxytocin cells excited by stimulation of the optic nerve was highest at ZT 4-8 and lowest at ZT 20-24. For vasopressin cells, it was highest at ZT 12-16 and lowest at ZT 20-24. The proportion of excitatory, inhibitory and complex responses seen in oxytocin and vasopressin cells following stimulation of the SCN also changed and was significantly different at different times of day (oxytocin cells: highest proportion of excitatory responses at ZT 12-16, P = 0.029; chi-square test; vasopressin cells: highest proportion of excitatory responses at ZT 0-4, P = 0.005; chi-square test). Thus, inputs to oxytocin and vasopressin neurones from the optic nerve and some outputs from the SCN changed during the light/dark cycle. Such changes may contribute to the generation of 24-h rhythms in activity of oxytocin and vasopressin neurones and release of the peptides.

In the present study, diurnal rhythm and regulation of body temperature were monitored during and several weeks following pyrithiamine-induced thiamine deficiency (PTD group, n=8) or pairfeeding (control group, n=9). A significant decline of core body temperature and a disruption of its diurnal rhythm were observed at varying stages of PTD treatment. Following thiamine administration and return to thiamine-fortified chow, body temperature continued to fall and several days transpired before body temperature and its diurnal rhythm were returned to normal. When exposed to warm and cold environments, no significant group differences were observed in either the maximum temperature change or the time elapsed to reach maximal temperature change. Histological examination revealed necrotic lesions in thalamus and mammillary body in the PTD group characteristic of Wernicke's encephalopathy. No significant damage was observed in the medial preoptic and suprachiasmatic nuclei, brain regions involved in the regulation of body temperature and circadian rhythm. These findings suggest that hypothermia and disruption of the diurnal rhythm of body temperature can be reversed by restoration of adequate thiamine levels and are related to biochemical and physiological disturbances rather than gross structural changes.

Secretion of vasopressin (VP) and oxytocin (OT) displays a daily rhythm. Using electrophysiological methods, we investigated the projections from the optic nerve to the supraoptic nucleus (SON) and its perinuclear zone (PNZ) which might underlie the rhythm. Extracellular recordings were made from magnocellular cells in the SON and its PNZ in 22 urethane-anaesthetized female Wistar rats while stimulating the optic nerve. The responses of magnocellular and PNZ cells were classified as orthodromic excitatory (OD+) or inhibitory (OD-) after creating peri-stimulus time histograms (PSTHs). Twenty-six of 73 (35.6%) VP and OT cells and 16 of 42 (38.1%) PNZ cells were excited by optic nerve stimulation. PNZ cells displayed both short (for 7 cells 30 ms or less) and long (> 60 ms) latency responses. Most (6/7) short latency responses had a short duration but longer latency responses were longer. No magnocellular cells showed responses with both short latency and short duration. Short latency responses with a short duration probably reflect direct monosynaptic inputs whereas longer latency responses with longer duration may reflect complex inputs. Thus the retina projects to the PNZ and to the SON but the PNZ receives a stronger direct input. Such projections might provide a light-related input to SON cells and suggest a role for the PNZ in this input.

Vasopressin (VP) gene expression in the rat suprachiasmatic nucleus (SCN) is subject to a cyclical mode of regulation which is indicative of a close association with the circadian clock intrinsic to this area of the hypothalamus. Previous studies show that both the amount and size (due to differential polyadenylation) of VP mRNA are reduced during the dark phase of the daily cycle. We have now identified the cellular site wherein these changes are mediated. By transcriptional run-on analysis of nuclei isolated at different time points from the SCN we have shown that an attenuation of transcriptional activity can account for the dark-phase reduction in VP mRNA levels; by comparison with other genes expressed in this tissue, a significant, VP gene-specific reduction was observed which resulted in dark-phase transcriptional activity at 30% of light-phase activity (P less than 0.005). A similar diurnal variation was not found in the supraoptic nucleus. In addition, by Northern analysis of sub-cellular RNA pools, we have demonstrated that the smaller, dark-phase-specific VP RNA species is located, in abundance, within the nuclear fraction. These results provide clear evidence that the cyclical changes in SCN VP mRNA expression are primarily regulated within the nucleus, indicating that any potential regulation in the cytoplasm is of secondary importance. Further analysis of the molecular components which mediate the cyclical changes in transcriptional activity of the VP gene may identify fundamental aspects of neuronal timing mechanisms.

The circadian rhythms of plasma ACTH and corticosterone and of locomotor activity were explored in chronically cannulated female rats, after elimination of serotoninergic (5HT) innervation of the SCN (suprachiasmatic nuclei) either by stereotaxic lesion of the median and dorsal midbrain raphe nuclei (RX) or by local injection of SCN with the neurotoxin 5,7-dihydroxytryptamine (5,7DHT). Completeness of 5HT denervation was checked on serial sections of the hypothalamus either by the Falk-Hillarp technique or by radioautography. Neither lesion eliminated the intrahypothalamic 5HT system, which, however does not take part in the 5HT innervation of the SCN. In both experimental series, the circadian rhythms of the three parameters investigated were maintained in unchanged phase relationships compared to the sham-lesioned controls, and with respect to the photoperiod (12 light-12 dark). However, the estimated amplitudes of the ACTH rhythms dropped by 43% (RX) to 47% (5,7DHT) and their mean levels by 44% (RX) to 60% (5,7DHT), whereas the corticosterone rhythm displayed normal amplitude and its mean level rose by 24% (RX) or 38% (5,7DHT). In regard to locomotor activity rhythm, the most noticeable alteration was a 25-55% increase in the light-phase activity of both experimental groups with a correlative increase in the L/D activity ratio. The essential role of 5HT innervation of the SCN therefore seems to be to facilitate circadian control of the ACTH rhythm.

Neural activity of the suprachiasmatic nucleus (SCN) in male albino rats was recorded under restricted daily feeding schedule. Circadian rhythm in multiple unit activity from the SCN showed essentially no interference with daily food cyclicity, while significant re-distribution of motor activities occurred during such a schedule. Since the central pacemaker is presumed to be unaffected by food availability, these results provide more support to the widespread notion that the SCN is an anatomical locus of the circadian pacemaker in rodents.

The role of retino-hypothalamic pathways in the re-entrainment of drinking rhythms after a 12 hr phase shift in the light-dark cycle was investigated by comparing the rate of re-entrainment of unilaterally blinded (UB) rats, with split optic chiasms (OCS) and controls. As reported previously, UB rats required more days to invert drinking rhythms than controls. The number of days required to re-entrain OCS rats fell between controls and UB rats but was not statistically significant from either group. Since OCS rats had the fewest retino-hypothalamic connections, the amount of retinal input to the surachiasmatic nuclei does not appear to play an important role in the rate of re-entrainment. The results are consistent with the hypothesis that the unequal distribution of retiono-hypothalamic fibers in UB rats interferes with the entrainment process. Compared to controls, both UB and OCS rats showed a small but significant post-operative reduction in the nocturnality of drinking.

The identification of a direct retinohypothalamic tract (RHT) terminating in the supra-chiasmatic nuclei (SCN) has focused attention on the role of these structures in the entrainment and generation of circadian rhythms in mammals. Light effects on circadian rhythms are mediated by both the RHT and portions of the classical visual system. The complex interactions of these systems are reflected both in their direct anatomical connections and in the functional changes in entrainment produced by interruption of either set of projections. Destruction of the RHT/SCN eliminated both normal entrainment and normal free-running circadian rhythms. No circadian rhythms has survived SCN ablation in rodents, but a variety of non-circadian cycles can be generated by lesioned animals. The complex behavioral patterns produced by SCN-lesioned hamsters suggest that circadian oscillators continue to function in these animals, but that their activity is no longer integrated into a single circadian framework. The available evidence indicates that the mammalian pacemaking system comprises a set of independent oscillators normally regulated by the SCN and by light information that is transmitted via several retinofugal pathways.

The circadian rhythm of activity in vertebrates often splits into two components after continuous exposure to constant light. This observation suggests that at least two circadian pacemakers underlie the activity rhythm. After unilateral ablation of the hypothalamic suprachiasmatic nuclei in hamsters, the splitting phenomenon was eliminated and a single rhythm of activity was established. The period of the new circadian activity rhythm different from the periods of the split rhythm and that preceding the split. These results suggest an interaction between the bilaterally paired suprachiasmatic nuclei in the generation of the circadian rhythm of activity.

Daily rhythms in many behavioral, physiological, and biochemical functions are generated by endogenous oscillators that function as internal 24-hour clocks. Under natural conditions, these oscillators are synchronized to the daily environmental cycle of light and darkness. Recent advances in locating circadian pacemakers in the brain and in establishing model systems promise to shed light on the cellular and biochemical mechanisms involved in the generation and regulation of circadian rhythms.

The diurnal variations in content of arginine-vasopressin in the supraoptic nucleus, the paraventricular nucleus and the suprachiasmatic nucleus of rats were determined using radioimmunoassay. In the supraoptic nucleus and the paraventricular nucleus the arginine-vasopressin level was relatively constant during the light phase (the inactive phase). When it became dark, the level of arginine-vasopressin lowered during the early and middle dark phase and then increased to the highest level during the late dark phase. In the suprachiasmatic nucleus the level was stable during the light phase, while in the early and the late dark phase it was significantly higher than that in the middle dark phase.

Bilateral destruction of the suprachiasmatic nuclei (SCN) eliminated light-dark (L/D) variations in water intake and urine output in albino rats. The lesions abolished also the circadian rhythm of food intake, without changing significantly the 24 hour number of meals, total meal duration and 24 hour food intake. Only the L/D distribution of the number of meals was changed from 5.6/16.9 in control period to 12.7/12.9 after lesions. In contrast, the L/D distribution of sodium, potassium and chlorides excretions demonstrated attenuated but persistent nocturnal type. These data imply that SCN play a role of driving oscillator for the circadian rhythm of food intake, but probably are not the main synchronizer for the rhythms of electrolyte excretions.

Lateral or medial fornix suction ablations were performed on adult male rats in order to selectively ablate or leave intact, respectively, fibers which terminate in the region of the suprachiasmatic nucleus and hypophysiotropic area of the hypothalamus. Plasma adrenal corticosteroid secretion, locomotor activity, body temperature, and food and water intake were recorded at 4 h intervals over a period of 48 h in individual animals 7-10 days postoperatively. Lateral fornix ablation specifically disrupted adrenal corticosteroid periodicity. A least-squares spectrum analysis of the data indicated that corticosteroid may be under ultradian control after this lesion. All animals, regardless of treatment, exhibited normal circadian locomotor activity patterns. Aberrations in feeding, drinking and body temperature rhythms were occasionally observed. This represents a temporary dissociation between the rhythmic expression of corticosteroid secretion and activity, temperature, feeding and drinking. The evidence presented lends support to the multi-oscillator theory of circadian organization, and suggests that the anteroventral subiculum, via the medial corticohypothalamic tract, is important in the regulation of some, but not all, circadian parameters. In addition to the observations on the rhythmicity of locomotor activity, the extent to which the animals are active is also significantly different between groups; ie., the hyperactivity of fornix-transected animals previously reported by others was found to be associated with lateral and not medial fornix ablation.

Twenty-four-hour (circadian) rhythms in urinary 3-methoxy-4-hydroxyphenylglycol (MHPG) excretion, motor activity, and oral temperature were studied in 14 normal subjects and ten manic-depressive patients. In both groups, a daily rhythm in MHPG excretion was present, with daytime peaks and nighttime lows. This pattern of urinary MHPG excretion may reflect a rhythm in central noradrenergic function. The physiological changes in levels of MHPG excretion associated with the circadian rhythm were at least as great as pathological changes associated with manic-depressive illness. Compared with controls, the timing or phase of circadian rhythms in each variable was one to three hours earlier in the patients, whether depressed or manic. Although the presence of circadian rhythms complicates the task of designing clinical research procedures, their early timing in manic-depressives suggests that disturbances in central biological clocks may be an integral part of the pathophysiology of affective illness and may be related to disturbances of sleep and neuroendocrine function associated with depression.

In situ hybridization of an oligonucleotide probe complementary to vasopressin messenger RNA (mRNA) in sections from normal or Brattleboro rat hypothalami revealed hybridization densities in each of three vasopressin-rich nuclei: the supraoptic, paraventricular, and suprachiasmatic. When entrained to a daily light-dark cycle, each rat strain displayed diurnal variation in hybridizable mRNA in the suprachiasmatic, but not in the supraoptic or paraventricular nuclei. The higher values for suprachiasmatic mRNA in the morning correlate well with previously elucidated morning increases in vasopressin immunoreactivity in the cerebrospinal fluid. These results support the utility of in situ hybridization techniques for elucidating physiological influences on regional peptidergic function, are consistent with a prominent role for vasopressinergic suprachiasmatic neurons in generating the cerebrospinal fluid vasopressin rhythm, and suggest that regulation of this mRNA rhythm is not dependent on release of intact peptide.

(1) Action potentials were recorded from single neurons in suprachiasmatic nuclei of hypothalamus in urethane anesthetized male rat. The rate of firing ranged from less than 1 to over 10/sec but was generally 4-8/sec, and it varied from one cell to another in the same animal. (2) Repetitive stimulation of optic nerve or light acting on the eye augmented the activity of approximately half of the the suprachiasmatic neurons examined (67 out of 159 cells) while 37 neurons (23%) showed clear inhibition by the same stimuli. (3) Stimulation of the suprachiasmatic nuclei strongly inhibited the electrical activity of cervical sympathetic nerves. Light or optic nerve stimulation also inhibited activity of cervical sympathetic nerves. (4) The rate of discharge of suprachiasmatic nuclei showed, at times, some short duration oscillations occurring every 3-5 min, but at other times the same neuron showed a steady low frequency of firing. (5) Projection of optic nerves to the suprachiasmatic nuclei was demonstrated by anterograde migration of horseradish peroxidase placed in the vitreous body. (6) It was suggested that light excites certain groups of neurons in the suprachiasmatic nuclei which exert inhibitory action on cervical sympathetic nerve. This, in turn, caused a reduction in norepinephrine release by nerve fibers innervating the pineal and a reduction in pineal enzyme production. Thus, neurons in these nuclei contribute to the suppression of pineal enzyme production produced by light.

The experiment was performed in order to investigate whether the suprachiasmatic nucleus is a potent circadian pacemaker also in mice. Cortical EEG activity and neck muscle EMG were continuously recorded in 19 albino mice under 12 h light-12 h dark cycles. Computer analysis revealed that after bilateral suprachiasmatic nucleus (SCN) lesions, the diurnal rhythm in slow wave sleep (SWS) and paradoxical sleep (PS) stages was completely eliminated although ultradian rhythms with 2-4 h periodicity persisted. However, the daily amount of either slow wave sleep or paradoxical sleep did not show any significant change after small hypothalamic lesions with a complete destruction of SCN.

Stimulation of the uterine cervix (CS) induced a nocturnal surge of prolactin at 04.00 h and a diurnal surge at 17.00h in normal ovariectomized rats. However, the CS-induced prolactin propionate during the neonatal period. Chronic bilateral lesions of the suprachiasmatic nucleus (SCN) completely abolished the CS-induced nocturnal and diurnal surges of prolactin release which were observed in sham-lesioned, ovariectomized rats. Furthermore, bilateral lesions of the medial basal part of the suprachiasmatic area (MBSC), lying rostral to the SCN, were also effective in blocking the CS-induced nocturnal and diurnal surges. Lesions which destroyed mainly the optic chiasma and extended partially into the MBSC and SCN did not block the CS-induced prolactin surges. These results suggest that one reason for the failure of ovary-grafted male rats and neonatally androgenized female rats to maintain pseudopregnancy is the extinction of the circadian rhythm of the two daily prolactin surges, and that the MBSC, in addition to the SCN which is known to be a generator of other circadian rhythms, is involved in generation of the rhythm of prolactin surges.

The circadian organization of squirrel monkey drinking behavior was examined in constant illumination following lesions which totally destroyed the suprachiasmatic nuclei. Although these lesions eventually led to a severe disruption in the temporal organization of drinking behavior, a statistically significant circadian pattern of drinking was observed in each monkey for at least 4-6 weeks after lesioning.

The pineal gland plays a cental role in the circadian organization of birds, although it is clearly only one component in a system with other components that have not yet been positively identified. The relative importance of the pineal and other components may vary from one group of birds to another. In the most thoroughly studied species, the house sparrow, pineal removal abolishes circadian rhythmicity; rhythmicity is restored by transplantation of a donor bird's pineal and the restored rhythm has the phase of the donor. This, and other evidence, argues convincingly that the pineal is a pacemaker in the sparrow circadian system. The pineal of the chicken has circadian rhythms in several biochemical parameters that result in the rhythmic synthesis of melatonin. The activity of one enzyme in this pathway is rhythmic for at least two cycles in organ culture. In view of this result it is interesting that pineal removal does not abolish circadian rhythmicity in chickens. The fact that lesions of the suprachiasmatic nuclei abolish circadian rhythms in sparrows, several mammalian species, and perhaps Japanese quail and reptiles, suggests that vertebrate circadian organization may be based on differentially weighted interactions between the pineal, the suprachiasmatic nuclei, and perhaps other brain regions.

The suprachiasmatic nuclei (SCN) of the hypothalamus in mammals, including nonhuman primates, contain a key pacemaker of the circadian timing system. Examination of the histology of the anterior hypothalamus in human fetal, child, and adult brains indicates that there is a cluster of neurons which may be homologous to SCN. These neurons are more diffusely organized and laterally placed in human brains than is the SCN of nonhuman primates.

Squirrel monkeys (Saimiri sciureus) demonstrate prominent circadian (approx 24 h) rhythms in many behavioral and physiological variables including drinking and body temperature. Both of these rhythms can be entrained by a 24-h light-dark cycle (LD 12:12) but will free-run with an endogenous period in a constantly illuminated (LL:600 lx) environment free of time cues. After radio-frequency lesions were placed stereotaxically in the suprachiasmatic nuclei (SCN) of five monkeys, the circadian rhythm of drinking behavior was disrupted when the monkeys were maintained in LL. However, the circadian rhythm in core body temperature in these animals persisted in LL with a significant circadian spectral component following destruction of the SCN. The SCN thus appear to be of fundamental importance for regulating the circadian organization of drinking; however, an oscillator located elsewhere in the squirrel monkey is capable of generating the core body temperature rhythm.

The experimental work described tested the prosposition that the suprachiasmatic nucleus of the hypothalamus is an autonomous circadian pacemaker. Simultaneous recording from two extracellular electrodes indicated neural (multiple unit) activity at two sites in the brain, one of which is in or near the suprachiasmatic nucleus and the other in one of many other brain locations. Both sites in intact rats displayed clear circadian rhythmicity of spontaneous neural activity. In experimental animals, a Halasz knife was used to create an island of hypothalamic tissue that contained the suprachiasmatic nuclei. In such animals that were also blinded by bilateral ocular enucleation, circadian rhythmicity was lost at all brain locations recorded outside the island, but it persisted within the island that contained the suprachiasmatic nuclei. The rhythmicity of the island is thus not dependent on afferent inputs from elsewhere in the brain.

Neonatal ablation of the suprachiasmatic nucleus in the rat has two important consequences. First, the direct projection from the retina to the suprachiasmatic nucleus fails to develop and no other retinal projection to any hypothalamic nucleus is formed. Second, circadian rhythms in drinking and spontaneous locomotor activity to not appear in these rats when they are tested as adults, and the females exhibit constant vaginal estrus. These observations indicate that the central neural mechanisms responsible for the generation and entrainment of circadian rhythmicity in the rat are not capable of either the functional or morphological plasticity characteristic of other developing neural systems.

Photoentrainment of circadian rhythms in mammals is mediated by the retinohypothalamic projection to the suprachiasmatic nucleus of the hypothalamus. It should therefore be possible to mimic or block the effects of light on the circadian pacemaker with appropriate pharmacological agents. Such agents and their effects should be useful in identifying the neurotransmitters involved in photoentrainment and their mechanisms of action on the circadian pacemaker. The effects of light on the circadian rhythm in rat pineal serotonin N-acetyltransferase activity are described. Carbachol, a cholinergic agonist, was found to mimic the effects of light on this rhythm, including the acute reduction of nocturnal activity and phase-shifting of the free-running rhythm. These results raise the possibility that acetylcholine is involved in the photoentrainment of mammalian circadian rhythms.

The influence of the light-dark schedule (12 h-12 h) on the sleep-waking cycle has been studied in anophthalmic mice: the 'eyeless' ZRDCT/An strain. The complete anophthalmic mice or the heterozygotous mice of the same strain with unilateral or bilateral eyeballs present a circadian organization of the sleep-waking cycle which is not dependent on the light-dark cycle. These results are different from sleep rhythms of C57Br mice recorded under the same experimental conditions. They indicate that the structures responsible for the circadian rhythmicity of sleep exist in all the 'eyeless' ZRDCT/An mice, but are not functionally linked with the visual system even in the mice with unilateral and bilateral eyeballs.

Daytime restriction of food and water availability in nocturnal animals phase shifts the circadian periodicity of plasma corticosteroid concentrations and body temperature. These shifted rhythms persist in animals with lesions of the suprachiasmatic nuclei who are arrhythmic under normal conditions. These findings suggest the existence of an additional "clock" that may be involved in the generation of the rhythm.

Multiunit activity (MUA) in the suprachiasmatic nucleus (SCN) and adjacent hypothalamic areas were compared in albino rats anesthetized with thiopental, immobilized with succinylcholine chloride and artificially respired. During adaptation to light lasting up to 2 h, MUA in the SCN increased gradually, whereas MUA in the other locations outside the SCN decreased. During adaptation to the dark, MUA in the SCN decreased whereas MUA outside the SCN increased. This reversal was apparent not only when recording in the SCN and adjacent hypothalamus simultaneously, but also while advancing an electrode from the optic chiasm through the SCN to the anterior hypothalamus.

Male rats were treated daily with oil or 100 micrograms of the antioestrogen, ethamoxytriphetol (MER-25), for the first 10 days of life and, when adult, lesions were made in the suprachiasmatic nuclei (SCN) of the hypothalamus or control lesions were made above the SCN and the rats were tested for sexual behaviour. Treatment with MER-25 enhanced the daily rhythmicity in both mounting and lordosis behaviour and SCN lesions disrupted these behavioural rhythms and the rhythm in the mounting behaviour of oil-treated rats. Rats treated with MER-25 and with SCN lesions showed high levels of mounting and lordosis behaviour throughout the light : darkness cycle. These results support the hypothesis that sexual differentiation by perinatal androgen stimulation uncouples the central rhythm generator from the neural substrates of sexual behaviour in rats.