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Circadian rhythms of the living organisms are 24hr oscillations found in behavior, biochemistry and physiology. Under constant conditions, the rhythms continue with their intrinsic period length, which are rarely exact 24hr. In this paper, we examine the effects of light on the phase of the gene expression rhythms derived from the interacting feedback network of a few clock genes, taking advantage of a computer simulation with Cell Illustrator. The simulation results suggested that the interacting circadian feedback network at the molecular level is essential for phase dependence of the light effects, observed in mammalian behavior. Furthermore, the simulation reproduced the biological observations that the range of entrainment to shorter or longer than 24hr light-dark cycles is limited, centering around 24hr. Application of our model to inter-time zone flight successfully demonstrated that 6 to 7 days are required to recover from jet lag when traveling from Tokyo to New York.

The mammalian hypothalamic suprachiasmatic nucleus (SCN) is the master oscillator that regulates the circadian rhythms of the peripheral oscillators. Previous studies have demonstrated that the transplantation of embryonic SCN tissues into SCN-lesioned arrhythmic mice restores the behavioral circadian rhythms of these animals. In our present study, we examined the clock gene expression profiles in a transplanted SCN and peripheral tissues, and also analysed the circadian rhythm of the locomotor activity in SCN-grafted mice. These experiments were undertaken to elucidate whether the transplanted SCN generates a dynamic circadian oscillation and maintains the phase relationships that can be detected in intact mice. The grafted SCN indeed showed dynamic circadian expression rhythms of clock genes such as mPeriod1 (mPer1) and mPeriod2 (mPer2). Furthermore, the phase differences between the expression rhythms of these genes in the grafted SCN and the locomotor activity rhythms of the transplanted animals were found to be very similar to those in intact animals. Moreover, in the liver, kidney and skeletal muscles of the transplanted animals, the phase angles between the circadian rhythm of the grafted SCN and that of the peripheral tissues were maintained as in intact animals. However, in the SCN-grafted animals, the amplitudes of the mPer1 and mPer2 rhythms were attenuated in the peripheral tissues. Our current findings therefore indicate that a transplanted SCN has the capacity to generate a dynamic intrinsic circadian oscillation, and can also lock the normal phase angles among the SCN, locomotor activity and peripheral oscillators in a similar manner as in intact control animals.

Most biological phenomena, including behavior and metabolic pathways, are governed by an internal clock system that is circadian (i.e., with a period of approximately 24 h) and is reset by light exposure from outside. In order to understand the molecular basis of the resetting mechanism of the clock, we attempted to isolate light-inducible transcripts in the suprachiasmatic nucleus, where the master clock resides, using a new gene expression profiling procedure. We identified 87 such transcripts, successfully cloned 60 of them and confirmed their light inducibility. Six of the 60 were already known to be light inducible and 17 are protein-coding transcripts registered in the public database that were not known to be light inducible. Induction is subjective night specific in most of the transcripts. Interestingly, 6 of the transcripts exhibit rhythmic expression in a circadian manner in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady light : dark (L : D = 12 : 12 h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA-positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double-labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA-containing neurons co-expressed either vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA-containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2-expressing neurons in the SCN.

Three mammalian Period (Per) genes, termed Per1, Per2, and Per3, have been identified as structural homologues of the Drosophila circadian clock gene, period (per). The three Per genes are rhythmically expressed in the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. The phases of peak mRNA levels for the three Per genes in the SCN are slightly different. Light sequentially induces the transcripts of Per1 and Per2 but not of Per3 in mice. These data and others suggest that each Per gene has a different but partially redundant function in mammals. To elucidate the function of Per1 in the circadian system in vivo, we generated two transgenic rat lines in which the mouse Per1 (mPer1) transcript was constitutively expressed under the control of either the human elongation factor-1alpha (EF-1alpha) or the rat neuron-specific enolase (NSE) promoter. The transgenic rats exhibited an approximately 0.6-1.0-h longer circadian period than their wild-type siblings in both activity and body temperature rhythms. Entrainment in response to light cycles was dramatically impaired in the transgenic rats. Molecular analysis revealed that the amplitudes of oscillation in the rat Per1 (rPer1) and rat Per2 (rPer2) mRNAs were significantly attenuated in the SCN and eyes of the transgenic rats. These results indicate that either the level of Per1, which is raised by overexpression, or its rhythmic expression, which is damped or abolished in over expressing animals, is critical for normal entrainment of behavior and molecular oscillation of other clock genes.

Knowledge of molecular biological systems is increasing at an amazing pace. It is becoming harder to intuitively evaluate the significance of each interaction between the molecules of the complex biological systems. Hence, we need to develop an efficient computational method to explore the biological mechanisms. In this study, we employed a hybrid functional Petri net in order to analyze mammalian circadian genetic control mechanisms, which consists of feedback loops of clock genes and generates endogenous near 24 h rhythms in mammals. We constructed a computational model based on the available biological data, and by using Genomic Object Net, we performed computer simulations of the time courses of clock gene transcription and translation. Although the original model successfully reproduced most of the circadian genetic control mechanisms, two discrepancies remained despite a wide selection of the parameters. We found that addition of a hypothetical path into the original model result in successful simulation of time courses and phase relationships among clock genes. This also demonstrates the usefulness of the hybrid functional Petri net approach to biological systems.

The regulatory factor X (RFX) family of transcription factors is characterized by a unique and highly conserved 76-amino acid residue DNA-binding domain. Mammals have five RFX genes, but the physiological functions of their products are unknown, with the exception of RFX5. Here a mouse RFX4 transcript was identified that encodes a peptide of 735 amino acids, including the DNA-binding domain. Its expression was localized in the suprachiasmatic nucleus, the central pacemaker site of the circadian clock. Also, light exposure was found to induce its gene expression in a subjective night-specific manner. Polyclonal antibodies were prepared, and an 80-kDa band was detected in the suprachiasmatic nucleus by Western hybridization. A histochemical study showed a localization of the products in the nucleus. This is the first report on mouse RFX4, which contains the RFX DNA-binding motif. Our investigation may provide clues to the physiological function of RFX4.

The induction of Per1 gene in the suprachiasmatic nucleus, the center of the circadian clock, is assumed to play significant roles in the adjustment of the internal clock. cAMP is one of the intracellular mediators which activates Per1 transcription. Here, we showed that the amount of the rat Per1 (rPer1) transcript induced by forskolin (FK) was significantly upregulated by the inhibition of phosphodiesterase type 4 (PDE4), a specific phosphodiesterase for cAMP, in rat-1 fibroblasts. Administration of rolipram, a specific inhibitor of PDE4, increased intracellular cAMP concentration, phosphorylation of cAMP response element binding protein (CREB) and enhanced rPer1 induction at their peaks. However, in the falling phase of rPer1 induction, the inhibition of PDE4 hardly affected the profile of rPer1 expression. These findings suggest the involvement of PDE4 for the regulation of rPer1 expression via cAMP metabolism at peak of the induction but little or no participation of PDE4 in the decreasing phase of the gene expression.

The mammalian master clock driving circadian rhythmicity in physiology and behavior resides within the suprachiasmatic nuclei (SCN) of the hypothalamus. SCN neurons contain a molecular oscillator composed of a set of clock genes that acts in intertwined negative and positive feedback loops [1]. In addition, all peripheral tissues analyzed thus far have been shown to contain circadian oscillators [2]. This raises the question of whether the central circadian pacemaker in the SCN is sufficient to evoke behavioral rhythms or whether peripheral circadian clockworks are also required. Mice with a mutated CLOCK protein (a transcriptional activator of E box-containing clock and clock output genes) or lacking both CRYPTOCHROMES, mCRY1 and mCRY2 proteins (inhibitors of E box-mediated transcription), lack circadian rhythmicity in behavior [3,4]. Here, we show that transplantation of mouse fetal SCN tissue into the hypothalamus restores free-running circadian behavioral rhythmicity in Clock mutant or mCry1/mCry2 double knockout mice. The periodicity of the emerged rhythms is determined by the genetic constitution (i.e., wild-type or mCry2 knockout) of the grafted SCN. Since transplanted mCry1/mCry2-deficient mice do not have functional circadian oscillators [5] other than those present in the grafted hypothalamus region, these findings suggest that the SCN can generate circadian behavioral rhythms in the absence of distant peripheral oscillators in the brain or elsewhere.

We identified the Dexamethasone-induced RAS protein 1 (Dexras1) gene as a cycling gene in the suprachiasmatic nucleus (SCN). Investigation of the whole brain using in situ hybridization demonstrated the localization of the expression of the gene in the SCN, thalamus, piriform cortex and hippocampus. However, rhythmic expression of the gene was observed only in the SCN. The rhythmic change in gene expression during 1 day was approximately five-fold, and the maximum expression was observed during subjective night. Real-time PCR using the SCN, paraventricular nucleus and cortex confirmed these results. Next, we analyzed the expression of the Dexras1 gene in the SCN of cryptochrome (Cry) 1 and 2 double knockout mice. We found that the rhythmic expression disappeared. The results indicate that Dexras1 rhythmicity and levels are dependent upon CRYs. This is the first time that the G protein, which may be involved in the input pathway, has been isolated as a cycling gene in the SCN.

It is well known that light induces both mPer1 and mPer2 mRNA in the suprachiasmatic nucleus. We have reported that mPer1 antisense oligonucleotides (ODNs) inhibited the light-induced phase delays of mouse locomotor rhythm. In this study, we asked whether both or either mPer1 or mPer2 expression is necessary to induce the phase shift. We examined the effects of inhibition of mRNA expression on light-induced phase delays of mouse circadian behavior rhythm. Light-induced phase delays were moderately attenuated by microinjection of mPer1 or mPer2 antisense ODN, but not by mPer3 antisense or mPer1, mPer2 scrambled ODNs, whereas following simultaneous injection of both mPer1 and mPer2 antisense ODNs they disappeared. The present results suggest that acute induction of mPer1 and mPer2 gene play an additive effect on photic entrainment.

Responsiveness of the vasoactive intestinal polypeptide (VIP) content to light and darkness in the rat suprachiasmatic nucleus (SCN) was examined by enzyme immunoassay of micropunched tissues. VIP content in the SCN has been shown to decrease monotonically in animals maintained in illumination. Decreases in VIP content in the SCN in response to both 6-h light and dark pulses depended on the phase of the circadian cycle when the pulses were applied. Light imposed at circadian time (CT) 18 or CT 22 was more effective in suppressing VIP levels than light exposure of the same intensity imposed at CT 0 or CT 6. Darkness interrupting continuous light was more effective at around CT 0 and less effective at around CT 12. These results suggest that VIP responsiveness to light and darkness in the SCN is regulated by the circadian clock in different ways and are correlated with phase-dependent phase shifts in the activity rhythm after light and dark pulses.

Light responsiveness of the vasoactive intestinal polypeptide (VIP) content in the suprachiasmatic nucleus (SCN) of the rat with pupils dilated by atropine was examined by enzyme immunoassay. After exposure to 6 h light at 3-1000 lux VIP levels in the SCN decreased as a monotonic function with a working range from 3 to 300 lux. At 12 h, 30 lux light decreased the VIP content to the minimum level that was attained by 300 lux light exposure in 6 h, suggesting that brighter illumination decreases VIP levels more rapidly, but light at a luminance of 0.05 lux for 3 days did not suppress VIP levels. These results suggest that VIP in the SCN codes visual information on luminance with a small working range and a relative high threshold.

Daily profiles of somatostatin mRNA expression were investigated in the rat suprachiasmatic nucleus (SCN) by semiquantitative in situ hybridization histochemistry. Under 12 h light/12 h dark conditions, somatostatin mRNA signals were higher during the day time (Zeitgeber time (ZT) 1) than during the night time (ZT 16). This day-night difference was still maintained in constant darkness where the somatostatin mRNA was higher in the subjective day (circadian time (CT) 1) than in the subjective night (CT 16). Together with previous Northern blot hybridization studies, the present observation suggests that the level of somatostatin mRNA in SCN neurons is controlled by the circadian clock, independent of photic environment.

We investigated photic response of the concentration of vasoactive intestinal polypeptide (VIP) and neuropeptide Y in the rat suprachiasmatic nucleus (SCN) by enzyme immunoassay (EIA). The content of VIP in the SCN did not show circadian rhythms in constant darkness (DD). Under light-dark (LD) condition, VIP contents decreased over the course of the light period and then recovered during the dark period. When the light was continuously delivered to rats, VIP levels were monotonically decreased and did not return to the basal level. Accordingly, VIP in the SCN may code photic information on duration. On the other hand, there is a daily bimodal pattern in NPY content in the SCN under light-dark conditions. When rats were exposed to continuous light, the NPY level steadily increased and reached a peak in 2 h before returning to a basal level. The amount of increase did not depend on duration of light exposure. Thus, NPY in the SCN may code visual information on transitions, which is different from that conveyed by VIP. These results indicate that the two peptides act on a different stage of photic processing and may mediate distinct photic information to the circadian pacemaker.

Dissociated cultures of rat hypothalamic cells showed spontaneous oscillations in intracellular Ca2+ with periods of 5-8 s (ultradian rhythm). Replacement of H2O by deuterium oxide in the culture medium lengthened the period of this oscillation, while tetrodotoxin did not inhibit the deuterium oxide sensitive Ca2+ oscillation. Since these characteristics resemble those of the mammalian circadian pacemaker, the ultradian Ca2+ rhythms may be involved in the circadian rhythm or have a similar rhythm generation mechanism. The gap junction inhibitor, halothane, stopped this oscillation. Gap junctions may be necessary in order to generate rhythmic oscillations.

The expression of vgf gene, first isolated as a gene induced by nerve growth factor in PC12 cells, was investigated in neurons of the suprachiasmatic nucleus (SCN) by in situ hybridization. In the rat forebrain, the vgf mRNA was found most densely in the SCN. Neurons which express vgf mRNA were found both in the dorsomedial and ventrolateral subdivisions. Soluble-labeling of vgf in situ hybridization and peptide immunocytochemistry demonstrated that vgf mRNA was expressed in most vasopressin- and neurophysin-immunoreactive neurons in the dorsomedial part and in vasoactive intestinal peptide (VIP)- and peptide histidine isoleucine amide (PHI)-immunoreactive neurons in the ventrolateral part. These findings suggest that vgf is a highly expressed gene in both vasopressin/neurophysin neurons and VIP/PHI neurons which were speculated to be involved in the generation and entrainment of circadian rhythm.

Neuronal activity of the suprachiasmatic nucleus (SCN) is known to be regulated by two major extrinsic factors conveyed by three anatomically distinct pathways to the SCN: photic stimulus by the direct retinohypothalamic tract (RHT) and the indirect geniculohypothalamic tract (GHT), and information from the brainstem by ascending forebrain serotonergic (5-hydroxytryptamine: 5-HT) tract. It has been shown that VIP mRNA level in neurons of the SCN is altered by external light, but remains stable in constant darkness. In the present study, by using the in situ hybridization technique combined with computer-assisted image analysis, we examined VIP mRNA expression in the SCN of rats in which the two major factors were eliminated, i.e. photic stimulus by exposing animals in total darkness and 5-HT transmission by three-day successive administration of p-chlorophenyl-alanine methylester (an inhibitor of tryptophan hydroxylase, 200 mg/kg, daily). In saline-treated controls, VIP mRNA levels remained almost constant throughout the day. In contrast, in PCPA-treated rats, a significant rhythm of VIP mRNA was observed with a peak at CT 4 and a trough at CT 20. These observations suggest that the removal of photic and 5-HT influence induces VIP mRNA rhythm in the SCN, indicating that VIP mRNA is controlled not only by photic information but also by the circadian clock.

To establish an in vitro system which would enable us to observe SCN functions over a long period, we developed a new method to culture SCN tissues by means of a roller tube. Coronal slices containing the SCN were prepared from 5- to 7-day-old rat brains. Slices were maintained for several weeks in plasma clots on coverslips that were immersed in a culture medium within a rotating test tube. In this preparation, vasopressin- and vasoactive intestinal polypeptide-immunoreactive or their mRNA expressing neurons were found in the subdivisions, similar to those found in the SCN in vivo. Moreover, amounts of vasopressin released from the SCN culture into the medium revealed circadian oscillation during the 48-h sampling period. Therefore, SCN in these slice culture conditions in vitro is able to maintain for several weeks not only topographical organization but also functional integrity as a circadian pacemaker.

The suprachiasmatic nucleus (SCN) of the hypothalamus contains a circadian pacemaker responsible for several circadian rhythms. Retinal cell projections to the SCN carry light information that phase shifts the pacemaker through the release of excitatory amino acids. To study this pathway, the Ca(2+)-sensitive dyes Fluo-3 and Fura-2 were used in organotypic slice cultures of rat SCN to visualize changes in intracellular Ca2+ of individual cells. After at least two weeks of culture, Ca2+ responses were measured in response to agonists of glutamate receptors in the presence of tetrodotoxin (TTX). Cells that showed a Ca2+ increase in response to N-methyl-D-aspartate (NMDA) and non-NMDA agonists also showed immunoreactivity towards vasoactive intestinal peptide (VIP), providing further evidence that VIP-containing neurons receive direct retinal input. The cells differed in their responses to the NMDA and non-NMDA agonists, suggesting that the cells contain differing densities of glutamate receptor subtypes.

For the analysis of neural activity over a long period of time, the organotypic culture of mammalian brain slices provides excellent specimens. To effectively utilize the slice culture, we developed a device for automatic sampling of the culture medium. This device is a computer-controlled combination of a multichannel peristaltic pump to remove the media from the glass culture containers, a fraction collector designed to allow quick freezing of the samples, and a multichannel syringe pump to deliver new media to the containers. Using this device, substances released as results of neural activities can be collected at regular intervals over several days. We monitored the circadian release of arginine-vasopressin from cultured suprachiasmatic nuclei. We also monitored tonic releases of lactate dehydrogenase from cultured hippocampi phasically treated with an excitotoxin and from those transiently deprived of oxygen/glucose.

The circadian rhythm in mammals is under control of the pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. This tiny nucleus contains a number of neurochemicals, including peptides, amines and amino acids. Heterogeneous distribution of these neurochemicals defines the substructures of the SCN. In the present review, functional significance of such neurochemical heterogeneity in the SCN is discussed in the light of circadian patterns of the concentrations of these neurochemicals in the SCN and their effects on SCN neurons in in vitro slice preparation. In particular, the hypothesis that the dorsomedial SCN is involved in maintaining the circadian rhythm, while the ventrolateral SCN is involved in adjusting the phase of the rhythm, is critically discussed. These considerations suggest that distinct sub-components of the SCN as marked by neurochemicals, interact with each other and this organizational architecture could be the basis of the proper operation of the circadian time keeping system in this nucleus.

Signal transduction by second messengers has long been hypothesized to be involved in the circadian system in the suprachiasmatic nucleus (SCN) of the mammalian brain. The present study reports that the concentration of adenosine-3',5'-monophosphate (cyclic AMP or cAMP) in the SCN region exhibited a circadian fluctuation with two peaks during a day under constant conditions. While a sharp peak at around the end of the subjective night was observed only in the SCN region, the other peak in the late subjective day was also found in the anterior hypothalamic area. The distinct cAMP peak at the late subjective night contrasts with the daytime peaks in electrical and metabolic activity rhythms of the SCN.

Arginine-vasopressin (AVP) gene expression in the rat suprachiasmatic nucleus (SCN) is subject to daily rhythmic changes. To determine whether this variation is endogenously generated, temporal changes in the SCN AVP mRNA level in constant dark (DD) condition was compared with changes occurring under the light-dark (LD) condition. In both lighting conditions, the presence of a rhythm in AVP mRNA level was observed in the SCN. In LD condition, peak level of AVP mRNA was found during the latter part of the day (zeitgeber time or ZT 8) and trough value during the night at ZT 20. Correspondingly, peak level of AVP mRNA under DD condition was observed during the latter part of the subjective day (circadian time or CT 8) and a trough during the subjective night (CT 20). Under both lighting conditions, a rapid increase and decrease of mRNA around the peak time was also observed. On the other hand, no significant daily variation in AVP mRNA was found in the supraoptic nucleus in both LD and DD conditions. These results provide evidence that a rhythmic change in AVP mRNA level is regulated by a circadian clock intrinsic to the SCN. The phase relationship of AVP mRNA rhythm to peptide rhythm in the SCN is discussed.

Neuropeptide Y, which is present in the sympathetic nerves innervating pineal glands, has been shown to affect N-acetyltransferase activity and melatonin production. Using enzyme immunoassay, we measured daily patterns of neuropeptide Y (NPY) in rat pineal glands in light-dark cycles (LD) and in constant darkness (DD). NPY within rat pineal glands displayed daily rhythms in LD with a peak at zeitgeber time 16. Similar rhythms in the NPY content were also observed in DD. These rhythms have a similar phase-relationship to that of the rhythm of NAT activity, suggesting that NPY in rat pineal glands plays a role in circadian rhythms of NAT activity.

Administration of a somatostatin (SS) depletor, cysteamine, markedly reduced SS levels in rat suprachiasmatic nucleus (SCN). At the same time, cysteamine administration induced a circadian rhythm of vasoactive intestinal polypeptide (VIP) content in the SCN, which otherwise remains constant under constant environmental conditions. These results suggest that the stable level of VIP in the SCN under constant conditions is not an intrinsic property of VIP neurons but a consequence of interactions with other components in the SCN.

Daily variations of serotonin (5-HT) in the suprachiasmatic nuclei (SCN) were measured in rats kept under various lighting conditions to elucidate the serotonergic contribution to the mechanism underlying SCN function on circadian rhythmicity. Animals kept in 12-h light-12-h dark (LD) cycles showed a peak 5-HT level during the light period and a trough during the dark period. In constant darkness (DD), rhythmic 5-HT variation was out of phase to changes observed in LD. Rats that have been kept in DD and then exposed to constant light (LL) showed transitory increases in 5-HT just after lights on. Taken together, these results show that 5-HT variation in the SCN is generated by an endogenous pacemaker and is also influenced by photic cues.

Neuropeptide Y (NPY)-like immunoreactivity (LI) in the iris-ciliary body of rats kept under constant darkness (DD) and in 12 h light-12 h dark cycle (LD) was determined by enzyme immunoassay. NPY-LI contents in the iris-ciliary body were found to oscillate in circadian fashion under DD and LD conditions, with a peak at about circadian time 12 (CT 12) and a trough at around CT 0. Unilateral superior cervical ganglionectomy caused a significant decrease in NPY-LI levels in the sympathectomized eye compare to the contralateral intact eye, independent of lighting phase. These results suggest the presence of an endogenous circadian rhythm in NPY-LI content in the rat iris-ciliary body, and the possible involvement of a sympathetic input from the superior cervical ganglion.

The content of substance P (SP)-like immunoreactivity (LI) within the suprachiasmatic nucleus (SCN) of rats was determined by enzyme immunoassay to evaluate the effect of light on SP-LI in the rat SCN. Male rats were kept under various lighting conditions: light-dark cycles, constant darkness, continuous light exposure for 24 h or light pulse interrupting constant darkness. Animals were also subjected to ocular enucleation. The present study showed that SP-LI in the SCN was unaffected by environmental lighting conditions or by bilateral ocular enucleation. Immunohistochemical studies also confirmed that SP immunoreactivity, which was found in the ventrolateral (VL) subdivision of the SCN, was not reduced significantly even after ocular enucleation. These results suggest that, in contrast to other neurotransmitters in the VL portion of the SCN such as vasoactive intestinal polypeptide (VIP), gastrin releasing peptide (GRP) and neuropeptide Y (NPY), SP level in the SCN is quite stable to light and arises from an area other than the retina.

The suprachiasmatic nucleus (SCN) of the hypothalamus has been established as the locus of the circadian pacemaker in mammals. The SCN is histochemically divided into two subdivisions: dorsomedial and ventrolateral subfields. The dorsomedial SCN is characterized, in part, by dense somatostatin-like immunoreactivity (SS-LI), but its functional significance in circadian pacemaking remains unclear. Our previous study revealed that 24 h SS-LI contents of the SCN in rats kept under light-dark (LD) conditions or blinded by orbital enucleation showed a distinct circadian rhythm. In the present study, 24 h SS-LI contents of the SCN in sighted rats kept under constant darkness (DD) conditions for prolonged periods were measured by enzyme immunoassay. Cellular contents of SS-LI exhibited a clear circadian rhythm on the third day of constant darkness (DD) with a peak at circadian time (CT) 5, corresponding to the time of peak levels found in LD conditions and in enucleated animals. This endogenous free-running rhythm continued to oscillate without attenuation of the amplitude even at 14 days in constant darkness. Moreover, SS-LI rhythm was found to be similar to the vasopressin rhythm in the SCN. In summary, these findings further strengthen the idea that the cellular content of SS-LI in the SCN is under the control of the endogenous circadian pacemaker.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a circadian pacemaker in mammals. We determined the circadian profiles of arginine-vasopressin (AVP), a major peptide in the dorsomedial SCN, in rats under light-dark (LD), constant dark (DD) and constant light (LL) conditions. Under LD conditions, AVP levels in the SCN showed circadian rhythmicity with a peak at early light phase and a broad trough during the dark phase. This rhythm in the AVP contents was maintained even after 14 days of free-running under DD conditions and 3 days under LL conditions. These circadian patterns of AVP are similar to those of somatostatin, another peptide in the dorsomedial SCN. This indicates a common mode of regulation for peptides in this subfield of the SCN.

Circadian profiles of somatostatin and vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI) messenger RNA levels in the suprachiasmatic nucleus (SCN) of blinded rats were measured by Northern analysis using synthesized oligonucleotide probes; the SCN is the site of a circadian oscillator. Somatostatin mRNA level in the SCN had a clear circadian rhythm with the peak around the onset of the subjective day and the trough around the onset of the subjective night. This rhythmicity was not observed in the adjacent anterior hypothalamus or the cortex and was thus specific to the SCN. No significant circadian fluctuation was observed in VIP/PHI mRNA levels in the SCN in this experiment. These results indicate the presence of an intrinsic circadian rhythm in the mRNA content of somatostatin, one of the peptides in dorsomedial SCN, and suggest a potential role of the peptide in circadian oscillation and the possible involvement of the transcriptional process in circadian rhythm generation in the SCN.

Twenty-four hour patterns in somatostatin (SS)-like immunoreactivity (LI) within the suprachiasmatic nucleus (SCN) were determined by enzyme immunoassay in rats blinded by bilateral orbital enucleation or kept sighted under light-dark conditions. A remarkable circadian rhythm was found in the concentration of SS-LI in the SCN under blinded conditions. The peak time appeared at about circadian time (CT) 4 in the early subjective day and the trough at around CT 16 or 20 in the subjective night. Light-dark cycles did not alter the circadian patterns of SS-LI observed in the blinded rat SCN. These results indicate the presence of an endogenous circadian rhythm in SS-LI in the rat SCN, independent of environmental lightning cycles.

To investigate daily variation in responsiveness of the suprachiasmatic nucleus (SCN) to exposure to light, the evoked change in multiple unit activity of the SCN within the retino-hypothalamic island with the retino-hypothalamic tract (RHT) spared, was measured in the rat at 6 h intervals across a 24 h day. It is found that SCN multiple unit discharge rates increase during a 1 h exposure to light which interrupts constant darkness irrespective of the time of day. A non-parametric statistical analysis did not indicate a significant daily variation in light-responsiveness of the SCN. The present results suggest that the phase-dependent shift of the SCN rhythm after a light exposure is induced not by a rhythm in light-responsiveness of the SCN mediated by the RHT, but by a more complicated machinery within the SCN.

The suprachiasmatic nucleus (SCN) of the hypothalamus contains a circadian pacemaker that regulates many circadian rhythms in mammals. Experimental work in microorganisms and invertebrates suggests that protein synthesis is required for the function of the circadian oscillator, and recent experiments in golden hamsters suggest an acute inhibition of protein synthesis can induce phase shifts in a mammalian circadian pacemaker. To determine whether protein synthesis in the SCN region is involved in the generation of circadian rhythms in mammals, a protein synthesis inhibitor, anisomycin, was microinjected into the SCN region, and the effect on the circadian rhythm of locomotor activity of hamsters was measured. A single injection of anisomycin into the SCN region induced phase shifts in the circadian activity rhythm that varied systematically as a function of the phase of injection within the circadian cycle. These results suggest that protein synthesis may be involved in the generation of circadian rhythms in mammals and that the anatomic site of action of anisomycin is within the hypothalamic suprachiasmatic region.

1. The effect of the extracellular calcium and magnesium concentrations and calcium ionophore, X-537A, on the frequency of miniature excitatory post-synaptic potentials (MEPSPs) was studied in cockroach leg muscle fibres. 2. The frequency of MEPSPs increased as the calcium concentration was increased from 0.1 to 10 mM. In the presence of 10 mM magnesium, however, raising the calcium concentration from 0.1 to 1 mM slightly depressed the frequency. In saline containing elevated potassium (20.8 mM), increasing the calcium concentration produced a much higher frequency than that in the normal potassium saline (10.8 mM) in the absence of magnesium. Raising the extracellular potassium concentration was without effect unless the bathing solution contained calcium. 3. The frequency of the miniature potentials was reduced as the magnesium concentration was raised from 0 to 10 mM, depending on the presence of calcium ions. On the contrary, a slightly increased frequency was observed in the low calcium saline as the magnesium concentration was raised from 1 to 10 mM. The reciprocal relationship between calcium and magnesium and the time course of the effect suggest that both ions act at the same surface sites in the presynaptic membrane. 4. X-537A elicited a transient increase in frequency followed by a fall of the frequency to a very low rate. Further application of the ionophore was without effect. The effect of X-537A on the spontaneous release of transmitter at the insect neuromuscular junction was comparable with that on the spontaneous acetylcholine release in vertebrate neuromuscular junctions.

1. Miniature excitatory post-synaptic potentials (MEPSPs) were recorded extra- and intracellularly from the coxal depressor muscle of the cockroach, Periplaneta americana. 2. Statistical analysis of the time intervals between MEPSPs showed that the variance-to-mean curve for the extracellular data sets lay significantly above the line predicted for a Poisson process. A shuffling procedure, however, made the series almost random. 3. The serial correlation coefficients for the extracellular data sets exceeded the confidence limits for various lags and most of them were positive. In comparison with the intensity function, the departure from the random process appeared to be mainly due to large positive correlation of intervals. 4. It is concluded that in insect neuromuscular junctions, the spontaneous release of transmitter at individual sites is more clustered than that predicted by a Poisson process.

The time intervals between miniature excitatory postsynaptic potentials and the counts of them in the cockroach, Periplaneta americana, were analyzed, using a computer program to test for properties of a Poisson process. The miniature potentials occurred basically in random manner at this neuromuscular junction. Although the distribution of the potentials did not fit the criteria for a Poisson process when the muscle fiber exhibited the short burst of high-frequency discharges, it was suggested that the primary process of such a distribution is Poisson, which is occasionally contaminated by the burst phase of the release rates.

Multiple unit activities (MUAs) of the ventromedial hypothalamic nucleus (VMH) were recorded from intact free-moving male rats. Rhythmic activities observed in the VMH were not distinguishable from those recorded in other places of the brain. When exposed to restricted daily feeding schedules, animals with suprachiasmatic nucleus (SCN) lesions developed a rhythm in motor activity and a rhythm of the VMH with a peak of activity immediately after and a trough right before the feeding. While neural activities of the SCN revealed circadian rhythms even after a lesion of the VMH, circadian rhythms in MUA of the VMH were completely abolished by a lesion of the SCN. Although the VMH is involved in the synchronization of rhythms to periodic feeding, the present results indicate that, unlike the SCN which contains an oscillator associated with light, the VMH does not contain another self-sustained oscillator associated with food.

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.

Anticipatory activities which precede food delivery under restricted daily feeding schedules were compared in rats with septal, suprachiasmatic and ventromedial hypothalamic lesions. Whereas rats with lesions of the septal nucleus and of the suprachiasmatic nucleus showed a normal pattern of increase in activity several hours prior to feeding, the rats with the ventromedial hypothalamic lesions did not show any activity bouts prior to feeding time. The semi-circular knife cut anterior to the ventromedial hypothalamic nucleus also eliminated the anticipatory activities. These findings suggest that the ventromedial hypothalamic nucleus is required to produce the anticipatory activities of restricted daily feeding schedules.

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.

The possible involvement of efferent pathways from the suprachiasmatic nucleus (SCN) in the photoperiodic regulation of reproduction was studied by measuring the testis size of hamsters bearing a horizontal knife cut either ventral or dorsal to the hypothalamic paraventricular nucleus (PVN) that were transferred from photostimulatory long days (light:dark (L:D) 14:10 h) to non-stimulatory short days (L:D 6:18 h). Knife cuts placed either ventral or dorsal to the PVN blocked testicular regression induced by exposure to short days. These results indicate that efferent fibers running dorsally from the SCN to the PVN are involved in relaying photoperiodic information from the SCN to the PVN. Furthermore, recently-defined efferents that leave the PVN dorsally and terminate in the spinal cord appear to be responsible for relaying seasonal information about day-length to the pineal-reproductive axis of hamsters.