Neuropeptide-mediated calcium signaling in the suprachiasmatic nucleus network

Neuroactive peptides and the intracellular calcium concentration ([Ca(2+) ](i) ) play important roles in light-induced modulation of gene expression in the suprachiasmatic nucleus (SCN) neurons that ultimately control behavioral rhythms. Vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) are expressed rhythmically within populations of SCN neurons. Pituitary adenylate cyclase-activating peptide (PACAP) is released from retinohypothalamic tract (RHT) terminals synapsing on SCN neurons. Nociceptin/orphanin FQ (OFQ) receptors are functionally expressed in the SCN. We examined the role of several neuropeptides on Ca(2+) signaling, simultaneously imaging multiple neurons within the SCN neural network. VIP reduced the [Ca(2+) ](i) in populations of SCN neurons during the day, but had little effect at night. Stimulation of the RHT at frequencies that simulate light input signaling evoked transient [Ca(2+) ](i) elevations that were not altered by VIP. AVP elevated the [Ca(2+) ](i) during both the day and night, PACAP produced variable responses, and OFQ induced a reduction in the [Ca(2+) ](i) similar to VIP. During the day, VIP lowered the [Ca(2+) ](i) to near nighttime levels, while AVP elevated [Ca(2+) ](i) during both the day and night, suggesting that the VIP effects on [Ca(2+) ](i) were dependent, and the AVP effects independent of the action potential firing activity state of the neuron. We hypothesize that VIP and AVP regulate, at least in part, Ca(2+) homeostasis in SCN neurons and may be a major point of regulation for SCN neuronal synchronization.     

Regional pacemakers composed of multiple oscillator neurons in the rat suprachiasmatic nucleus

Regional specificities of the dorsal and ventral regions of the suprachiasmatic nucleus (SCN) were examined to elucidate the structure of multioscillator circadian organization. The circadian rhythms of arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) release, and of electrical activity of individual neurons were measured in an organotypic, static slice culture of the SCN obtained from neonatal rats. Five days after the start of culture, robust circadian rhythms were detected in AVP release with a peak located consistently at the middle of the original light phase, while the 24 h profiles of VIP release were either arrhythmic or rhythmic. In the latter case, a phase delay of 5-7 h was observed in the circadian peak from the AVP rhythm. Multi-channel, extracellular recording revealed that 51 (76.1%) out of 67 firing neurons, examined in the SCN, showed circadian rhythms in their firing rate. The percentage of rhythmic neurons was significantly larger in the dorsal (86.8%) than in the ventral (62.1%) region of the SCN, where the AVP and VIP containing neurons predominate, respectively. Twenty-seven percent of the firing rhythms were almost antiphasic from the majority of rhythms. There was no regional specificity in the distribution of the antiphasic rhythm. These findings, that the dorsal and ventral regions of the SCN both contain circadian pacemakers with different properties that regulate the AVP and VIP release separately, is probably due to differences in the number and, hence, the coupling strength of oscillating neurons.     

Vasoactive intestinal peptide and gastrin-releasing peptide play distinct roles in the suprachiasmatic nucleus

Vasoactive intestinal peptide and gastrin-releasing peptide levels were measured in the punched-out suprachiasmatic nucleus tissue from rats kept under a prolonged dim light (in vivo). Vasoactive intestinal peptide content increased from 4 to 8 h, returned to the baseline level at 12 to 16 h, and then increased again until 36 h after the light was switched off (dim light). Gastrin-releasing peptide level, in contrast, showed no significant changes, but a slight decrease from 1 to 4 h was detected under the dim light. In suprachiasmatic nucleus explant-slice culture, in vitro, Arg-vasopressin release increased transiently or showed a decrease at 30 min after exposure to vasoactive intestinal peptide or gastrin-releasing peptide, respectively. Treatment with anti-vasoactive intestinal peptide and anti-gastrin-releasing peptide antibodies reversed these effects. These findings suggest reciprocal roles of vasoactive intestinal peptide and gastrin-releasing peptide in Arg-vasopressin release.     

Role of vasoactive intestinal peptide in seasonal encoding by the suprachiasmatic nucleus clock

The neuropeptide vasoactive intestinal peptide (VIP) is critical for the proper functioning of the neural circuit that generates circadian rhythms. Mice lacking VIP show profound deficits in the ability to generate many behavioral and physiological rhythms. To explore how the loss of VIP impacts on the intact circadian system, we carried out in vivo multiunit neural activity (MUA) recordings from the suprachiasmatic nucleus of freely moving VIP knockout (KO) mice. The MUA rhythms were largely unaltered in the VIP KO mice, with no significant differences being seen in the amplitude or phase of the rhythms in light-dark conditions. Robust differences between the genotypes were revealed when the mice were transferred from light-dark to constant darkness conditions. In addition, the ability of the VIP KO mice to encode changes in photoperiod was examined. Strikingly, the behavioral and physiological rhythms of VIP KO mice showed no adaptation to short or long photoperiods. The data indicate that the intact circadian system can compensate for some of the consequences of the loss of VIP, whereas this peptide is indispensable for endogenous encoding of seasonal information.     

Transgenic approach reveals expression of the VPAC2 receptor in phenotypically defined neurons in the mouse suprachiasmatic nucleus and in its efferent target sites

Circadian rhythms in mammals depend on the properties of cells in the suprachiasmatic nucleus (SCN). The retino-recipient core of the mouse SCN is characterized by vasoactive intestinal peptide (VIP) neurons. Expression within the SCN of VPAC2, a VIP receptor, is required for circadian rhythmicity. Using transgenic mice with beta-galactosidase as a marker for VPAC2, we have phenotyped VPAC2-expressing cells within the SCN and investigated expression of the VPAC2 marker at sites previously shown to receive VIP-containing SCN efferents. In situ hybridization and immunohistochemistry demonstrated identical distributions for VPAC2 mRNA and beta-galactosidase and coexpression of the two signals in the SCN. Double-label confocal immunofluorescence identified beta-galactosidase in 32% of the VIP and 31% of the calretinin neurons in the SCN core. Of the arginine-vasopressin neurons that characterize the SCN shell, 45% expressed beta-galactosidase. In contrast, this marker was not apparent in astrocytes within the SCN core or shell. Cell bodies containing beta-galactosidase were detected at sites reportedly receiving VIP-containing SCN efferents, including the subparaventricular zone and lateral septum and the anteroventral periventricular, preoptic suprachiasmatic, medial preoptic and paraventricular hypothalamic nuclei. The detection of a marker for VPAC2 expression in the SCN in almost one-third of the VIP and calretinin core neurons and nearly half of the arginine-vasopressin shell neurons and also in cell bodies at sites receiving VIP-immunoreactive projections from the SCN indicates that VPAC2 may contribute to autoregulation and/or coupling within the SCN core and to the control of the SCN shell and sites distal to this nucleus.     

In vitro entrainment of the circadian rhythm of vasopressin-releasing cells in suprachiasmatic nucleus by vasoactive intestinal polypeptide

Mammalian circadian pacemaker is located in suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is entrained by light-dark cycle; the photic information is transmitted primarily via the retino-hypothalamic tract (RHT). The main neurotransmitter of the tract is glutamate. RHT fibers end on the ventrolateral part of the nucleus, where vasoactive intestinal peptide (VIP)-immunopositive neurons are localized. They send their axons into dorsomedial SCN, where most of the vasopressinergic (AVP) neurones are located. The AVP neurons retain the clock-like properties in vitro. Vasopressin release from the cultured neurons shows circadian rhythm peaking in the middle of subjective day. VIP induces phase-shifts of the rhythm, magnitude and direction of the shift depending on timing of the application. VIP applied 6-12 h before the peak of vasopressin rhythm induces advances, application 4-8 h after the peak induces delays. The lowest concentration required to induce the phase-shift is 30 nM, further increase of the concentration does not affect the magnitude of the shift. In contrast, glutamate has no effect on the phase of vasopressin rhythm, although in high concentrations it transiently stimulates vasopressin release. The data indicate that the vasopressinergic cells in the SCN contain circadian oscillators, whose rhythms run mutually synchronized in our cultures. VIP acts directly on the vasopressinergic cells to shift the phase of their pacemakers; glutamate has no such effect presumably because in vivo it acts through the VIP-ergic cells but the neuronal network is altered after the dissociation of the cells.     

Human brain contains vasopressin and vasoactive intestinal polypeptide neuronal subpopulations in the suprachiasmatic region

The suprachiasmatic nuclei (SCN) and retinohypothalamic tract ( RHT ) in the anterior hypothalamus have been postulated to play an important role in the timing of daily biological rhythms in mammals. Although physiological studies have described circadian rhythms in man, the presence of an RHT or SCN has not been conclusively demonstrated in the human brain. Immunocytochemical identification of distinct ventral vasoactive intestinal polypeptide (VIP) containing and dorsal vasopressin containing neuronal subpopulations in the human suprachiasmatic region provides correlative evidence of neuronal clusters which are homologous to discrete cell groups in the SCN of other mammalian species. Manipulation of the circadian system has been used to treat some affective illnesses and other physiological timing disorders. Characterization of the neural substrates underlying human circadian rhythms could be useful in the development of future treatment modalities and is essential for understanding normal human circadian organization.     

Differential contributions of intra‐cellular and inter‐cellular mechanisms to the spatial and temporal architecture of the suprachiasmatic nucleus circadian circuitry in wild‐type,cryptochrome‐null and vasoactive intestinal peptide receptor 2‐null mutant mice

To serve as a robust internal circadian clock, the cell‐autonomous molecular and electrophysiological activities of the individual neurons of the mammalian suprachiasmatic nucleus (SCN) are coordinated in time and neuroanatomical space. Although the contributions of the chemical and electrical interconnections between neurons are essential to this circuit‐level orchestration, the features upon which they operate to confer robustness to the ensemble signal are not known. To address this, we applied several methods to deconstruct the interactions between the spatial and temporal organisation of circadian oscillations in organotypic slices from mice with circadian abnormalities. We studied the SCN of mice lacking Cryptochrome genes (Cry1 and Cry2), which are essential for cell‐autonomous oscillation, and the SCN of mice lacking the vasoactive intestinal peptide receptor 2 (VPAC2‐null), which is necessary for circuit‐level integration, in order to map biological mechanisms to the revealed oscillatory features. The SCN of wild‐type mice showed a strong link between the temporal rhythm of the bioluminescence profiles of PER2::LUC and regularly repeated spatially organised oscillation. The Cry‐null SCN had stable spatial organisation but lacked temporal organisation, whereas in VPAC2‐null SCN some specimens exhibited temporal organisation in the absence of spatial organisation. The results indicated that spatial and temporal organisation were separable, that they may have different mechanistic origins (cell‐autonomous vs. interneuronal signaling) and that both were necessary to maintain robust and organised circadian rhythms throughout the SCN. This study therefore provided evidence that the coherent emergent properties of the neuronal circuitry, revealed in the spatially organised clusters, were essential to the pacemaking function of the SCN.     

Light-induced c-Fos expression in suprachiasmatic nuclei neurons targeting the paraventricular nucleus of the hamster hypothalamus: phase dependence and immunochemical identification.

The suprachiasmatic nuclei (SCN) contain a master clock driving the majority of circadian rhythms in mammals. It is believed that the SCN confers circadian rhythmicity as well as light responsiveness to pineal melatonin secretion via a direct projection to the paraventricular nucleus of the hypothalamus (PVN). Neurons in the SCN respond to light during subjective night with an expression of the immediate early gene c-fos. The number and distribution of c-Fos protein-containing neurons depend on the zeitgeber time (ZT) at which the light stimulus is presented. To investigate whether this phase-dependent activity is present in the SCN output neurons targeting the PVN, we combined retrograde cholera toxin subunit B (ChB) tracing from the PVN with c-Fos immunohistochemistry. Male golden hamsters were injected iontophoretically with ChB into the PVN area and 7 days later given a 1.5-hr light stimulus at either ZT 14 or ZT 19 followed by vascular fixation. Light stimulation at ZT 19 gave rise to more c-Fos containing neurons in the SCN than light presented at ZT 14. Double immunostaining for ChB and c-Fos revealed that light stimulation at ZT 14 induced c-Fos expression in 26.6% +/- 2.8% of the retrogradely filled perikarya, whereas light-stimulation at ZT 19 increased this fraction to 40.7% +/- 1.9%. This demonstrates the presence of a phase-dependent c-Fos induction in the suprachiasmatic-paraventricular projection system. Triple immunohistochemistry showed that light-activated output neurons contained both gastrin-releasing peptide and vasoactive intestinal polypeptide and to a lesser extent vasopressin. The present findings provide functional evidence of light activation of central pathways involved in the regulation of circadian output rhythms.     

Altered vasoactive intestinal polypeptide gene expression in the fetal rat suprachiasmatic nucleus following prenatal serotonin deficiency

This study has evaluated the possible role of serotonin, a potential morphogen, in the regulation of vasoactive intestinal polypeptide (VIP) gene expression in the target neurons of the suprachiasmatic nucleus (SCN) before and after the onset of the serotonin neurotransmitter function. VIP gene expression was quantified by in situ hybridization of the corresponding mRNA on cryostat sections with subsequent film autoradiography and densitometry. The content of VIP mRNA was measured in the SCN in fetuses at the 21st embryonic day (E21) and in postnatal rats at day 11 (P11) following chronic depletion of serotonin by p-chlorophenylalanine, an inhibitor of serotonin synthesis. This inhibitor was daily injected to pregnant rats for E13–20 or to postnatal animals for P2–10. Results of this study indicate that prenatal serotonin depletion caused a significant increase in VIP mRNA content in the SCN compared to control fetuses. On the contrary, the same treatment performed postnatally did not change VIP mRNA levels in the SCN. These data suggest that the VIP gene expression in differentiating target neurons of the SCN might be under serotonin inhibitory control during prenatal neurogenesis, prior to the onset of the serotoninergic neurotransmission.     

Behavioural rhythm splitting in the CS mouse is related to clock gene expression outside the suprachiasmatic nucleus

CS mice exhibit a spontaneous splitting in the circadian rhythm of locomotor activity under constant darkness, suggesting that they contain two weakly coupled oscillators in the circadian clock system regulating locomotor activity rhythm. In order to clarify whether the two oscillators are located in the suprachiasmatic nucleus (SCN), a site of the master circadian pacemaker in mammals, circadian rhythms in mRNA of mouse Period genes (mPer1, mPer2 and mPer3) in the SCN and cerebral cortex were examined during rhythm splitting by in situ hybridization. In the SCN, mPer1 and mPer2 showed a circadian rhythm with a single peak in both split and unsplit mice. The rhythms of mPer1 and mPer2 were slightly phase delayed during rhythm splitting in reference to the activity onset, but the phase relationship between the two rhythms was not changed. In the cerebral cortex, the expression of mPer1 and mPer2 underwent the bimodal fluctuation with peaks temporally corresponding to split activity components. The unsplit mice showed the circadian rhythms with a single peak. There was no difference in the mPer3 rhythms in either the SCN or the cerebral cortex between the split and unsplit mice. These results indicate that the circadian oscillations of mPer1, mPer2 and mPer3 in the SCN are not related to the rhythm splitting of CS mice. The split rhythms of the CS mice are suggested to be caused by uncoupling of oscillators located outside the SCN from the SCN circadian pacemaker.     

Daily rhythms and sex differences in vasoactive intestinal polypeptide, VIPR2 receptor and arginine vasopressin mRNA in the suprachiasmatic nucleus of a diurnal rodent, Arvicanthis niloticus

Diurnal and nocturnal animals differ with respect to the time of day at which the ovulatory surge in luteinizing hormone occurs. In some species this is regulated by the suprachiasmatic nucleus (SCN), the primary circadian clock, via cells that contain vasoactive intestinal polypeptide (VIP) and vasopressin (AVP). Here, we evaluated the hypothesis that chronotype differences in the timing of the luteinizing hormone surge are associated with rhythms in expression of the genes that encode these neuropeptides. Diurnal grass rats ( Arvicanthis niloticus ) were housed in a 12/12-h light–dark cycle and killed at one of six times of day (Zeitgeber time 1, 5, 9, 13, 17, 21; ZT 0 = lights-on). In-situ hybridization was used to compare levels of vip , avp and VIP receptor mRNA ( vipr2 ) in the SCN of intact females, ovariectomized females, ovariectomized females given estradiol and intact males. We found a sex difference in vip rhythms with a peak occurring at ZT 13 in males and ZT 5 in intact females. In all groups avp mRNA rhythms peaked during the day, from ZT 5 to ZT 9, and had a trough in the dark at ZT 21. There was a modest rhythm and sex difference in the pattern of vipr2 . Most importantly, the patterns of each of these SCN rhythms relative to the light–dark cycle resembled those seen in nocturnal rodents. Chronotype differences in timing of neuroendocrine events associated with ovulation are thus likely to be generated downstream of the SCN.     

Changes in vasoactive intestinal Peptide binding site densities in the female rat central nervous system and pituitary during lactation

Using quantitative autoradiography, vasoactive intestinal peptide (VIP) binding site densities were investigated in the female rat during lactation. Plasma prolactin levels were significantly (P<0.01) elevated on days 3 and 10 of lactation. In the brain, there was a significant (P<0.05) increase in VIP binding sites during lactation in the striatum, thalamus, anterior portion of the paraventricular nucleus of the hypothalamus, and several cortical areas compared to virgin female diestrous rats. VIP binding density in the anterior pituitary was significantly higher (P<0.01) when measured on day 3 of lactation, and decreased to diestrous levels by day 10. Pup removal resulted in a significant reversal of the lactation-induced increase in VIP binding density in the thalamus, parietal and insular cortices, and the anterior portion of the paraventricular nucleus. Taken together, modifications in VIP binding site density in the pituitary and in several central areas are associated with changes in the level of prolactin secretion. Moreover, the pattern of VIP receptor induction during lactation suggests a role for VIP in the brain that extends beyond the regulation of prolactin secretion.     

Monoaminergic control of vasopressin and VIP expression in the mouse suprachiasmatic nucleus

We studied the effects of serotonin and noradrenaline on the expression of arginine-vasopressin (AVP) and vasoactive intestinal peptide (VIP) in the suprachiasmatic nucleus (SCN). We used transgenic Tg8 mice knockout for the MAO-A (monoamine oxidase A) gene, which are characterized by increased amounts of serotonin and noradrenaline in brain compared to wild-type mice (C3H). The MAO-A deficiency caused an increase in AVP and VIP expression (determined by immunohistochemistry, enzyme immunoassay, and in situ hybridization) compared to C3H mice. The number of peptidergic neurons was also increased. Inhibiting serotonin or noradrenaline synthesis in Tg8 mice by the administration of parachlorophenylalanine or alpha-methylparatyrosine, respectively, the amounts of AVP, VIP and their mRNAs were decreased, but not the number of peptidergic neurons. This study indicates that serotonin and noradrenaline stimulate AVP and VIP expression, and could participate in the differentiation of the neurochemical phenotype in the mouse SCN.     

Segmental distribution of corticotropin-releasing factor-like and vasoactive intestinal peptide-like immunoreactivities in presumptive sympathetic preganglionic neurons of the cat

The distribution of corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP), and luteinizing hormone-releasing hormone (LH-RH) in cell bodies of sympathetic autonomic nuclei of the thoracolumbar spinal cord was studied immunohistochemically in cats after intrathecal administration of colchicine. Neurons containing CRF-like immunoreactivity (CRFir) and VIP-like immunoreactivity (VIPir), but not LH-RH-like immunoreactivity, were found in the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf). On the basis of identification in previous studies and the size, shape, and location of the immunoreactive cells, it is suggested that the neurons are sympathetic preganglionic neurons. Most of the neurons with CRFir (85.5%) were found in the IMLp in segments T2-T7 and L2-L3 and the remaining 14.5% were found in the IMLf in segments T2-T5. The largest proportion of neurons with VIPir (93.7%) was found in the IMLp in segments T2, T4-T7, and T9-T13. Only 6.3% of the neurons containing VIPir were found in the IMLf in segments T2, T4, T5, and T10. These findings suggest that CRF and VIP may participate in peptide-specific pathways to peripheral organs.     

Intrinsic vasoactive intestinal polypeptide (VIP)-containing neurons in the baroreceptor nucleus of the solitary tract in rat

Vasoactive intestinal polypeptide (VIP)-immunoreactive cell bodies, dendrites and nerve terminals in the nucleus of the solitary tract have been identified with electron microscopic immunocytochemistry in rats. The relatively high concentration of VIP in this nucleus, which corresponds to the primary baroreceptor center, was depleted only insignificantly by uni- or bilateral transection of the solitary tract, which eliminates peripheral neuronal (via glossopharyngeal and vagal nerves) input to the nucleus. Both immunocytochemical and biochemical data suggest that the majority of VIP in the nucleus of the solitary tract is present in intrinsic neurons.     

Circadian PER2::LUC rhythms in the olfactory bulb of freely moving mice depend on the suprachiasmatic nucleus but not on behaviour rhythms

The temporal order of physiology and behaviour in mammals is regulated by the coordination of the master circadian clock in the suprachiasmatic nucleus (SCN) and peripheral clocks in various tissues outside the SCN. Because the circadian oscillator(s) in the olfactory bulb (OB) is regarded as SCN independent, we examined the relationship between the SCN master clock and the circadian clock in the OB. We also examined the role of vasoactive intestinal peptide receptor 2 in the circadian organization of the OB. We continuously monitored the circadian rhythms of a clock gene product PER2 in the SCN and OB of freely moving mice by means of a bioluminescence reporter and an optical fibre implanted in the brain. Robust circadian rhythms were detected in the OB and SCN for up to 19 days. Bilateral SCN lesions abolished the circadian behaviour rhythms and disorganized the PER2 rhythms in the OB. The PER2 rhythms in the OB showed more than one oscillatory component of a similar circadian period, suggesting internal desynchronization of constituent oscillators. By contrast, significant circadian PER2 rhythms were detected in the vasoactive intestinal peptide receptor 2‐deficient mice, despite the substantial deterioration or abolition of circadian behavioural rhythms. These findings indicate that the circadian clock in the OB of freely moving mice depends on the SCN master clock but not on the circadian behavioural rhythms. The circadian PER2::LUC rhythm in the cultured OB was as robust as that in the cultured SCN but reset by slice preparation, suggesting that culturing of the slice reinforces the circadian rhythm.     

The role of the neuropeptides PACAP and VIP in the photic regulation of gene expression in the suprachiasmatic nucleus

Previously, we have shown that mice deficient in either vasoactive intestinal peptide (VIP) or pituitary adenylate cyclase‐activating polypeptide (PACAP) exhibit specific deficits in the behavioral response of their circadian system to light. In this study, we investigated how the photic regulation of the molecular clock within the suprachiasmatic nucleus (SCN) is altered by the loss of these closely‐related peptides. During the subjective night, the magnitude of the light‐induction of FOS and phosphorylated mitogen‐activated protein kinase (p‐MAPK) immunoreactive cells within the SCN was significantly reduced in both VIP‐ and PACAP‐deficient mice when compared with wild‐type mice. The photic induction of the clock gene Period1 (Per1) in the SCN was reduced in the VIP‐ but not in the PACAP‐deficient mice. Baselines levels of FOS, p‐MAPK or Per1 in the night were not altered by the loss of these peptides. In contrast, during the subjective day, light exposure increased the levels of FOS, p‐MAPK and Per1 in the SCN of VIP‐deficient mice, but not in the other genotypes. During this phase, baseline levels of these markers were reduced in the VIP‐deficient mice compared with untreated controls. Finally, the loss of either neuropeptide reduced the magnitude of the light‐evoked increase in Per1 levels in the adrenals in the subjective night without any change in baseline levels. In summary, our results indicate that both VIP and PACAP regulate the responsiveness of cells within the SCN to the effects of light. Furthermore, VIP, but not PACAP, is required for the appropriate temporal gating of light‐induced gene expression within the SCN.