RESUMO
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.
Assuntos
Neuropeptídeo Y/fisiologia , Peptídeo Intestinal Vasoativo/fisiologia , Animais , Ritmo Circadiano/fisiologia , Cricetinae , Estimulação Luminosa , Ratos , Núcleo Supraquiasmático/fisiologiaRESUMO
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.
Assuntos
Núcleo Supraquiasmático/metabolismo , Animais , Sequência de Bases , Imuno-Histoquímica , Hibridização In Situ , Técnicas In Vitro , Dados de Sequência Molecular , Neurônios/metabolismo , RNA Mensageiro/biossíntese , Ratos , Núcleo Supraquiasmático/citologia , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
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.
Assuntos
Arginina Vasopressina/biossíntese , Ritmo Circadiano , Expressão Gênica , RNA Mensageiro/metabolismo , Núcleo Supraquiasmático/metabolismo , Análise de Variância , Animais , Northern Blotting , Masculino , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
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.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Escuridão , Fenclonina/farmacologia , RNA Mensageiro/biossíntese , Núcleo Supraquiasmático/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/genética , Análise de Variância , Animais , Sequência de Bases , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Estimulação Luminosa , Ratos , Ratos Wistar , Serotonina/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMO
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.
Assuntos
Ritmo Circadiano/fisiologia , Neuropeptídeo Y/metabolismo , Glândula Pineal/metabolismo , Animais , Escuridão , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Neuropeptídeo Y/imunologia , Glândula Pineal/imunologia , Ratos , Ratos WistarRESUMO
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.
Assuntos
Cálcio/metabolismo , Hipotálamo/efeitos dos fármacos , Tetrodotoxina/farmacologia , Animais , Células Cultivadas , Ritmo Circadiano , Óxido de Deutério/farmacologia , Relação Dose-Resposta a Droga , Junções Comunicantes/efeitos dos fármacos , Halotano/farmacologia , Ratos , Ratos WistarRESUMO
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.
Assuntos
Arginina Vasopressina/metabolismo , Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/metabolismo , Animais , Técnicas Imunoenzimáticas , Masculino , Fotoperíodo , Ratos , Ratos WistarRESUMO
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.
Assuntos
Cálcio/metabolismo , Núcleo Supraquiasmático/metabolismo , Potenciais de Ação/efeitos dos fármacos , Compostos de Anilina , Animais , Diagnóstico por Imagem , Corantes Fluorescentes , Fura-2 , Imuno-Histoquímica , Neuroglia/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Receptores de Glutamato/metabolismo , Núcleo Supraquiasmático/anatomia & histologia , Núcleo Supraquiasmático/citologia , Tetrodotoxina/farmacologia , Peptídeo Intestinal Vasoativo/metabolismo , XantenosRESUMO
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.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Proteínas Nucleares/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Núcleo Supraquiasmático/efeitos dos fármacos , Animais , Proteínas de Ciclo Celular , Ritmo Circadiano/fisiologia , Luz , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Atividade Motora/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Núcleo Supraquiasmático/metabolismo , Fatores de TranscriçãoRESUMO
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.
Assuntos
Ritmo Circadiano , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiologia , Aminoácidos/metabolismo , Animais , Humanos , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Núcleo Supraquiasmático/anatomia & histologiaRESUMO
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.
Assuntos
Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Análise de Variância , Animais , Escuridão , Análise dos Mínimos Quadrados , Luz , Masculino , Ratos , Ratos Wistar , Núcleo Supraquiasmático/efeitos da radiaçãoRESUMO
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.
Assuntos
Encéfalo/metabolismo , Meios de Cultivo Condicionados , Manejo de Espécimes/métodos , Animais , Arginina Vasopressina/metabolismo , Automação , Ritmo Circadiano , Estudos de Avaliação como Assunto , Ácido Glutâmico/farmacologia , Hipocampo/metabolismo , Hipóxia/metabolismo , L-Lactato Desidrogenase/metabolismo , Técnicas de Cultura de Órgãos , RatosRESUMO
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.
Assuntos
Condicionamento Psicológico , Comportamento Alimentar , Núcleo Hipotalâmico Ventromedial/fisiologia , Animais , Ritmo Circadiano , Masculino , Atividade Motora , Ratos , Ratos EndogâmicosRESUMO
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.
Assuntos
Hipotálamo/fisiologia , Retina/fisiologia , Núcleo Supraquiasmático/fisiologia , Vias Visuais/fisiologia , Animais , Ritmo Circadiano , Potenciais Evocados Visuais , Luz , Masculino , Estimulação Luminosa , Ratos , Ratos EndogâmicosRESUMO
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.
Assuntos
Ritmo Circadiano , Comportamento Alimentar/fisiologia , Núcleo Hipotalâmico Ventromedial/fisiologia , Ciclos de Atividade , Animais , Potenciais Evocados , Masculino , Ratos , Ratos Endogâmicos , Núcleo Supraquiasmático/fisiologiaRESUMO
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.
Assuntos
Núcleo Hipotalâmico Paraventricular/fisiologia , Núcleo Supraquiasmático/fisiologia , Testículo/fisiologia , Animais , Mapeamento Encefálico , Cricetinae , Luz , Masculino , Mesocricetus , Vias Neurais/fisiologia , Periodicidade , Glândula Pineal/fisiologia , Estações do AnoRESUMO
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.
Assuntos
Ritmo Circadiano , Luz , Serotonina/metabolismo , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiologia , Análise de Variância , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Fotoperíodo , Ratos , Ratos WistarRESUMO
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.
Assuntos
Ritmo Circadiano , Peptídeos/metabolismo , Núcleo Supraquiasmático/metabolismo , Animais , Córtex Cerebral/metabolismo , Hipotálamo Anterior/metabolismo , Técnicas Imunoenzimáticas , Masculino , Ratos , Ratos Wistar , Somatostatina/metabolismo , Distribuição TecidualRESUMO
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.
Assuntos
Ritmo Circadiano , AMP Cíclico/fisiologia , Hipotálamo Anterior/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Masculino , Ratos , Ratos WistarRESUMO
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.