Additive effect of mPer1 and mPer2 antisense oligonucleotides on light-induced phase shift.

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.

Phase dependent response of vasoactive intestinal polypeptide to light and darkness in the suprachiasmatic nucleus.

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.

Luminance-dependent decrease in vasoactive intestinal polypeptide in the rat suprachiasmatic nucleus.

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.

Differences of somatostatin mRNA in the rat suprachiasmatic nucleus under light-dark and constant dark conditions: an analysis by in situ hybridization.

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.

TTX-resistant Ca2+ oscillation in cultured hypothalamus: similarity to the mammalian 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.

In situ hybridization histochemistry of vgf mRNA in the rat suprachiasmatic nucleus: co-localization with vasopressin/neurophysin and VIP/PHI.

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.

Circadian change of VIP mRNA in the rat suprachiasmatic nucleus following p-chlorophenylalanine (PCPA) treatment in constant darkness.

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.

Photic information coded by vasoactive intestinal polypeptide and neuropeptide Y.

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.

Calcium imaging in organotypic cultures of the rat suprachiasmatic nucleus.

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.

Automated collection of conditioned medium from organotypically cultured brain slice.

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.

Neurochemical organization of circadian rhythm in the suprachiasmatic nucleus.

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.

Circadian fluctuations of cAMP content in the suprachiasmatic nucleus and the anterior hypothalamus of the rat.

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.

Circadian variation of arginine-vasopressin messenger RNA in the rat suprachiasmatic nucleus.

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.

Circadian variations of neuropeptide Y-like immunoreactivity in the rat pineal gland.

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.

Emergence of VIP rhythmicity following somatostatin depletion in the rat suprachiasmatic nucleus.

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.

Diurnal and circadian changes of serotonin in the suprachiasmatic nuclei: regulation by light and an endogenous pacemaker.

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.

Organotypic slice culture of the suprachiasmatic nucleus.

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.

Circadian rhythm of neuropeptide Y-like immunoreactivity in the iris-ciliary body of the rat.

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.

Substance P-like immunoreactivity in the suprachiasmatic nucleus of the rat.

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.