Desflurane anesthesia shifts the circadian rhythm phase depending on the time of day of anesthesia.

Desflurane is one of the most frequently used inhalational anesthetics in clinical practice. A circadian rhythm phase-shift after general anesthesia with sevoflurane or isoflurane has been reported in mice, but few studies have reported this effect with desflurane. In the present study, we examined the rest/activity rhythm of mice by counting the number of running wheel rotations, and we found that desflurane anesthesia caused a phase shift in the circadian rhythm that was dependent on the time of day of anesthesia. We also found that desflurane anesthesia altered the relative mRNA expression of four major clock genes (Per2, Bmal, Clock, and Cry1) in the suprachiasmatic nucleus (SCN). These results are important for elucidating the effects of desflurane on the SCN, which is the master clock for the mammalian circadian rhythm. Further studies on the relationship between anesthesia and circadian rhythm may lead to the prevention and treatment of postoperative complications related to circadian rhythms.

Maternal Obesity during Pregnancy Alters Daily Activity and Feeding Cycles, and Hypothalamic Clock Gene Expression in Adult Male Mouse Offspring.

An obesogenic diet adversely affects the endogenous mammalian circadian clock, altering daily activity and metabolism, and resulting in obesity. We investigated whether an obese pregnancy can alter the molecular clock in the offspring hypothalamus, resulting in changes to their activity and feeding rhythms. Female mice were fed a control (C, 7% kcal fat) or high fat diet (HF, 45% kcal fat) before mating and throughout pregnancy. Male offspring were fed the C or HF diet postweaning, resulting in four offspring groups: C/C, C/HF, HF/C, and HF/HF. Daily activity and food intake were monitored, and at 15 weeks of age were killed at six time-points over 24 h. The clock genes Clock, Bmal1, Per2, and Cry2 in the suprachiasmatic nucleus (SCN) and appetite genes Npy and Pomc in the arcuate nucleus (ARC) were measured. Daily activity and feeding cycles in the HF/C, C/HF, and HF/HF offspring were altered, with increased feeding bouts and activity during the day and increased food intake but reduced activity at night. Gene expression patterns and levels of Clock, Bmal1, Per2, and Cry2 in the SCN and Npy and Pomc in the ARC were altered in HF diet-exposed offspring. The altered expression of hypothalamic molecular clock components and appetite genes, together with changes in activity and feeding rhythms, could be contributing to offspring obesity.

Degeneration of the Suprachiasmatic Nucleus in an Alzheimer's Disease Mouse Model Monitored by in vivo Magnetic Resonance Relaxation Measurements and Immunohistochemistry.

In Alzheimer's disease (AD), disturbances in the circadian rhythm and sleep-wake cycle are frequently observed. Both are controlled by the master clock: the suprachiasmatic nucleus (SCN), which was reported in postmortem studies of AD subjects to be compromised. However, the influence of age and gender on the biophysical integrity and subtle microstructural changes of SCN and mechanistic connections between SCN dysfunction and AD progression in vivo remain to be explored. In the present study, we utilized state-of-the-art in vivo magnetic resonance relaxation measurements in combination with immunohistochemistry to follow microstructural changes in SCN of the Tg2576 mouse model of AD. Longitudinal monitoring of in vivo T2 relaxation with age shows significant shortening of T2 values in the SCN of transgenic mice and more substantially in female transgenic than aged-matched controls. Multiexponential T2 analysis detected a unique long T2 component in SCN of transgenic mice which was absent in wild-type mice. Immunohistochemical examination revealed significantly elevated numbers of activated astrocytes and an increase in the astrocyte to neuron ratio in SCN of transgenic compared to wild-type mice. This increase was more substantial in female than in male transgenic mice. In addition, low GABA production in SCN of transgenic mice was detected. Our results offer a brief appraisal of SCN dysfunction in AD and demonstrate that inflammatory responses may be an underlying perpetrator for the changes in circadian rhythmicity and sleep disturbance in AD and could also be at the root of marked sex disparities observed in AD subjects.

Novel Rbfox2 isoforms associated with alternative exon usage in rat cortex and suprachiasmatic nucleus.

Transcriptome diversity in adult neurons is partly mediated by RNA binding proteins (RBPs), including the RBFOX factors. RBFOX3/NeuN, a neuronal maturity marker, is strangely depleted in suprachiasmatic nucleus (SCN) neurons, and may be compensated by a change in Rbfox2 expression. In this study, we found no superficial changes in Rbfox2 expression in the SCN, but mRNA population analysis revealed a distinct SCN transcript profile that includes multiple novel Rbfox2 isoforms. Of eleven isoforms in SCN and cerebral cortex that exhibit exon variation across two protein domains, we found a 3-fold higher abundance of a novel ('-12-40') C-terminal domain (CTD)-variant in the SCN. This isoform embraces an alternative reading frame that imparts a 50% change in CTD protein sequence, and functional impairment of exon 7 exclusion activity in a RBFOX2-target, the L-type calcium channel gene, Cacna1c. We have also demonstrated functional correlates in SCN gene transcripts; inclusion of Cacna1c exon 7, and also exclusion of both NMDA receptor gene Grin1 exon 4, and Enah exon 12, all consistent with a change in SCN RBFOX activity. The demonstrated regional diversity of Rbfox2 in adult brain highlights the functional adaptability of this RBP, enabling neuronal specialization, and potentially responding to disease-related neuronal dysfunction.

Na⁺/Ca²âº Exchanger 2 (NCX2) in the Circadian Clock of the Rat Suprachiasmatic Nucleus: Colocalization with Neuropeptides and Daily Profiles of Gene Expression and Protein Levels.

The plasmalemmal Na⁺/Ca²âº changer (NCX) regulates intracellular Ca²âº by exchanging 3 Na⁺ for 1 Ca²âº in either the Ca²âº exit or Ca²âº entry mode. All three NCX isoforms NCX1, NCX2, and NCX3 are expressed in the rat brain, with isoform-specific differential distribution. In the central clock of suprachiasmatic nucleus (SCN), intracellular Ca²âº controls the circadian release of major neuropeptides, which are the arginine vasopressin (AVP), vasoactive intestinal peptide (VIP) and gastrin releasing peptide (GRP), and the NCX, most likely NCX1, rapidly clears depolarization-induced somatic Ca²âº influx. However, the role of NCX2 in the SCN remains unknown. This study aimed to investigate the colocalization of NCX2 with neuropeptides and daily expression profiles of NCX2 in mRNA and protein levels. Consistent with the restricted distribution of NCX2 in the retinorecipient ventral SCN, the immunostaining results showed colocalization of NCX2 with VIP, GRP and VIP/GRP in the ventral SCN, but not with AVP in the dorsal SCN, or markers for astrocyte and major input pathways. Importantly, the presynaptic marker Bassoon was found to colocalize with NCX2/GRP and NCX2/ VIP, indicating localization of both VIP/NCX2 and GRP/NCX2 at the presynaptic sites. Furthermore, real-time PCR and western blotting revealed no day-night difference in NCX2 mRNA and protein levels, in contrast to a robust circadian rhythm in the expression of clock genes Per1 and Per2. Together the results suggest a role of NCX2 in the regulation of the release of VIP and GRP.

The synchronization of neuronal oscillators determined by the directed network structure of the suprachiasmatic nucleus under different photoperiods.

The main function of the principal clock located in the suprachiasmatic nucleus (SCN) of mammals is synchronizing the body rhythms to the 24 h light-dark cycle. Additionally, the SCN is able to adapt to the photoperiod of the cycle which varies among seasons. Under the long photoperiod (LP), the synchronization degree of the SCN neurons is lower than that under the photoperiod (SP). In the present study, a potential explanation is given for this phenomenon. We propose that the asymmetrical coupling between the light-signal-sensitive part (the ventralateral part, abbreviation: VL) and the light-signal-insensitive part (the dorsalmedial part, abbreviation: DM) of the SCN plays a role in the synchronization degree, which is reflected by the ratio of the number of the directed links from the VL neurons to the DM neurons to the total links of both directions between the VL and the DM. The ratio is assumed to characterize the directed network structure under different photoperiods, which is larger under the SP and smaller under the LP. We found that with the larger ratio in the situation of the SP, the synchronization degree is higher. Our finding may shed new light on the asymmetrical coupling between the VL and the DM, and the network structure of the SCN.

Chronic Alcohol Consumption in Rats Leads to Desynchrony in Diurnal Rhythms and Molecular Clocks.

BACKGROUND: Circadian rhythms are essential for adapting to the environment. Chronic alcohol consumption often leads to sleep and circadian disruptions, which may impair the life quality of individuals with alcohol use disorders and contribute to the morbidity associated with alcoholism. METHODS: We used a pair-feeding liquid diet alcohol exposure protocol (6 weeks duration) in PER1::LUC transgenic rats to examine the effects of chronic alcohol exposure on: (i) diurnal rhythms of core body temperature and locomotor activity, (ii) plasma corticosterone (CORT) concentrations, and (iii) rhythms of ex vivo Period1 (Per1) expression in the suprachiasmatic nucleus (SCN), pituitary, and adrenal glands. We followed multiple circadian outputs not only to examine individual components, but also to assess the relative phase relationships among rhythms. RESULTS: We found that chronic alcohol consumption: (i) reduced 24-hour body temperature and locomotor activity counts in the dark period, (ii) advanced the acrophase of diurnal rhythms of body temperature and locomotor activity, (iii) abolished the phase difference between temperature and activity rhythms, (iv) blunted and advanced the diurnal CORT rhythm, and (v) advanced Per1 expression in the adrenal and pituitary glands but not in the SCN. We found that chronic alcohol altered the phase relationships among diurnal rhythms and between the central (SCN) and peripheral (adrenal and pituitary) molecular clocks. CONCLUSIONS: Our findings suggest that desynchrony among internal rhythms is an important and overlooked aspect of alcohol-induced circadian disruptions. The misalignment of phases among rhythms may compromise normal physiological functions and put individuals with chronic alcohol use at greater risk for developing other physical and mental health issues. How this desynchrony occurs and the extent to which it participates in alcohol-related pathologies requires further investigation.

Age-related changes in neurochemical components and retinal projections of rat intergeniculate leaflet.

Aging leads to several anatomical and functional deficits in circadian timing system. In previous works, we observed morphological alterations with age in hypothalamic suprachiasmatic nuclei, one central component of this system. However, there are few data regarding aging effects on other central components of this system, such as thalamic intergeniculate leaflet (IGL). In this context, we studied possible age-related alterations in neurochemical components and retinal projections of rat IGL. For this goal, young (3 months), adult (13 months), and aged (23 months) Wistar rats were submitted to an intraocular injection of neural tracer, cholera toxin subunit b (CTb), 5 days before a tissue fixation process by paraformaldehyde perfusion. Optical density measurements and cell count were performed at digital pictures of brain tissue slices processed by immunostaining for glutamic acid decarboxylase (GAD), enkephalin (ENK), neuropeptide Y (NPY) and CTb, characteristic markers of IGL and its retinal terminals. We found a significant age-related loss in NPY immunoreactive neurons, but not in immunoreactivity to GAD and ENK. We also found a decline of retinal projections to IGL with age. We conclude aging impairs both a photic environmental clue afferent to IGL and a neurochemical expression which has an important modulatory circadian function, providing strong anatomical correlates to functional deficits of the aged biological clock.

Delivery of pineal melatonin to the brain and SCN: role of canaliculi, cerebrospinal fluid, tanycytes and Virchow-Robin perivascular spaces.

Historically, the direct release of pineal melatonin into the capillary bed within the gland has been accepted as the primary route of secretion. Herein, we propose that the major route of melatonin delivery to the brain is after its direct release into the cerebrospinal fluid (CSF) of the third ventricle (3V). Melatonin concentrations in the CSF are not only much higher than in the blood, also, there is a rapid nocturnal rise at darkness onset and precipitous decline of melatonin levels at the time of lights on. Because melatonin is a potent free radical scavenger and antioxidant, we surmise that the elevated CSF levels are necessary to combat the massive free radical damage that the brain would normally endure because of its high utilization of oxygen, the parent molecule of many toxic oxygen metabolites, i.e., free radicals. Additionally, the precise rhythm of CSF melatonin provides the master circadian clock, the suprachiasmatic nucleus, with highly accurate chronobiotic information regarding the duration of the dark period. We predict that the discharge of melatonin directly into the 3V is aided by a number of epithalamic structures that have heretofore been overlooked; these include interpinealocyte canaliculi and evaginations of the posterodorsal 3V that directly abut the pineal. Moreover, the presence of tanycytes in the pineal recess and/or a discontinuous ependymal lining in the pineal recess allows melatonin ready access to the CSF. From the ventricles melatonin enters the brain by diffusion and by transport through tanycytes. Melatonin-rich CSF also circulates through the aqueduct and eventually into the subarachnoid space. From the subarachnoid space surrounding the brain, melatonin penetrates into the deepest portions of the neural tissue via the Virchow-Robin perivascular spaces from where it diffuses into the neural parenchyma. Because of the high level of pineal-derived melatonin in the CSF, all portions of the brain are better shielded from oxidative stress resulting from toxic oxygen derivatives.

Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus.

Mammalian circadian rhythm is maintained by the suprachiasmatic nucleus (SCN) via an intricate set of neuropeptides and other signaling molecules. In this work, peptidomic analyses from two times of day were examined to characterize variation in SCN peptides using three different label-free quantitation approaches: spectral count, spectra index and SIEVE. Of the 448 identified peptides, 207 peptides were analyzed by two label-free methods, spectral count and spectral index. There were 24 peptides with significant (adjusted p-value < 0.01) differential peptide abundances between daytime and nighttime, including multiple peptides derived from secretogranin II, cocaine and amphetamine regulated transcript, and proprotein convertase subtilisin/kexin type 1 inhibitor. Interestingly, more peptides were analyzable and had significantly different abundances between the two time points using the spectral count and spectral index methods than with a prior analysis using the SIEVE method with the same data. The results of this study reveal the importance of using the appropriate data analysis approaches for label-free relative quantitation of peptides. The detection of significant changes in so rich a set of neuropeptides reflects the dynamic nature of the SCN and the number of influences such as feeding behavior on circadian rhythm. Using spectral count and spectral index, peptide level changes are correlated to time of day, suggesting their key role in circadian function.

Hypothalamic neurosecretory and circadian vasopressinergic neuronal systems in the blind cone-rod homeobox knockout mouse (Crx-/-) and the 129sv wild-type mouse.

Vasopressin (AVP) is both a neuroendocrine hormone located in magnocellular neurosecretory neurons of the hypothalamus of mammals but also a neurotransmitter/neuromodulator in the parvocellular suprachiasmatic nucleus (SCN). The SCN is the endogenous clock of the brain and exhibits a prominent circadian AVP rhythm. We have in this study of the brown 129sv mouse and the visual blind cone-rod homeobox gene knock out mouse (Crx(-/-) ) with degeneration of the retinal rods and cones, but a preserved non-image forming optic system, studied the temporal Avp expression in both the neurosecretory magnocellular and parvocellular vasopressinergic systems in both genotypes. We here present a detailed mapping of all classical hypothalamopituitary and accessory magnocellular nuclei and neurons in the hypothalamus by use of immunohistochemistry and in situ hybridization in both genotypes. Semiquantitative in situ hybridization revealed a very high expression of Avp mRNA in all the magnocellular nuclei compared with a much lower level in the parvocellular suprachiasmatic nucleus. In a series of mice killed every 4 hours, the Avp mRNA expression in the SCN showed a significant daily rhythm with a zenith at late day time and nadir during the dark in both the Crx(-/-) and the wild type mouse. None of the magnocellular neurosecretory neurons exhibited a diurnal vasopressin expression. Light stimulation of both genotypes during the dark period did not change the Avp expression in the SCN. This shows that Avp expression in the mouse SCN is independent of Crx-regulated photoreceptor systems.

Quantitative peptidomics for discovery of circadian-related peptides from the rat suprachiasmatic nucleus.

In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, 10 endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥ 30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.

Spatiotemporal distribution of vasoactive intestinal polypeptide receptor 2 in mouse suprachiasmatic nucleus.

Vasoactive intestinal polypeptide (VIP) signaling is critical for circadian rhythms. For example, the expression of VIP and its main receptor, VPAC2R, is necessary for maintaining synchronous daily rhythms among neurons in the suprachiasmatic nucleus (SCN), a master circadian pacemaker in animals. Where and when VPAC2R protein is expressed in the SCN and other brain areas has not been examined. Using immunohistochemistry, we characterized a new antibody and found that VPAC2R was highly enriched in the SCN and detectable at low levels in many brain areas. Within the SCN, VPAC2R was circadian, peaking in the subjective morning, and abundantly expressed from the rostral to caudal margins with more in the dorsomedial than ventrolateral area. VPAC2R was found in nearly all SCN cells including neurons expressing either VIP or vasopressin (AVP). SCN neurons mainly expressed VPAC2R in their somata and dendrites, not axons. Finally, constant light increased VIP and AVP expression, but not VPAC2R. We conclude that the circadian clock, not the ambient light level, regulates VPAC2R protein localization. These results are consistent with VPAC2R playing a role in VIP signaling at all times of day, broadly throughout the brain and in all SCN cells.

Disrupted circadian rhythms in a mouse model of schizophrenia.

Sleep and circadian rhythm disruption has been widely observed in neuropsychiatric disorders including schizophrenia [1] and often precedes related symptoms [2]. However, mechanistic basis for this association remains unknown. Therefore, we investigated the circadian phenotype of blind-drunk (Bdr), a mouse model of synaptosomal-associated protein (Snap)-25 exocytotic disruption that displays schizophrenic endophenotypes modulated by prenatal factors and reversible by antipsychotic treatment [3, 4]. Notably, SNAP-25 has been implicated in schizophrenia from genetic [5-8], pathological [9-13], and functional studies [14-16]. We show here that the rest and activity rhythms of Bdr mice are phase advanced and fragmented under a light/dark cycle, reminiscent of the disturbed sleep patterns observed in schizophrenia. Retinal inputs appear normal in mutants, and clock gene rhythms within the suprachiasmatic nucleus (SCN) are normally phased both in vitro and in vivo. However, the 24 hr rhythms of arginine vasopressin within the SCN and plasma corticosterone are both markedly phase advanced in Bdr mice. We suggest that the Bdr circadian phenotype arises from a disruption of synaptic connectivity within the SCN that alters critical output signals. Collectively, our data provide a link between disruption of circadian activity cycles and synaptic dysfunction in a model of neuropsychiatric disease.

Transportin 1 in the mouse brain: appearance in regions of neurogenesis, cerebrospinal fluid production/sensing, and circadian clock.

Transportin1 (Tnpo1) is a carrier protein belonging to the importin-ß family, which transports substrates between the cytoplasm and the nucleus. To gain insight into the role of Tnpo1 gene in the brain, we investigated the localization of Tnpo1-, Tnpo2-, and Tnpo3-expressing cells by in situ hybridization histochemistry. Tnpo1 mRNA-positive cells were distributed throughout the brain from the olfactory bulb to the medulla oblongata. The cells in the subventricular zone of the lateral ventricle, where neurogenesis occurs even in the adult, and its progeny neurons in the granular cells of the olfactory bulb and the islands of Calleja were strongly labeled. It is also noteworthy that cerebrospinal fluid (CSF)-generating epithelial cells in the choroid plexus and CSF-contacting and -sensing circumventricular organs, including organum vasculosum lamina terminalis, subfornical organ, and subcommissural organ, expressed high amounts of Tnpo1. The strongest signals were found in the suprachiasmatic nucleus (SCN), where the biological clock resides, which prompted us to examine the circadian characteristics of Tnpo1. Under constant-dark conditions, the circadian expression profiles of Tnpo1 mRNA in the SCN showed a peak in the subjective night and a trough in the subjective day. Tnpo2 and Tnpo3 showed similar patterns of expression, except in the choroids plexus, the subventricular zone, and the SCN, where the expression was notably weaker. These findings suggest that Tnpo1 is involved in a variety of functions in the adult brain, including neurogenesis, CSF production and sensing, and circadian rhythms.

Cholecystokinin (CCK)-expressing neurons in the suprachiasmatic nucleus: innervation, light responsiveness and entrainment in CCK-deficient mice.

The suprachiasmatic nucleus (SCN) is the principal pacemaker driving circadian rhythms of physiology and behaviour. Neurons within the SCN express both classical and neuropeptide transmitters which regulate clock functions. Cholecyctokinin (CCK) is a potent neurotransmitter expressed in neurons of the mammalian SCN, but its role in circadian timing is not known. In the present study, CCK was demonstrated in a distinct population of neurons located in the shell region of the SCN and in a few cells in the core region. The CCK neurons did not express vasopressin or vasoactive intestinal peptide. However, CCK-containing processes make synaptic contacts with both groups of neurons and some CCK cell bodies were innervated by VIPergic neurons. The CCK neurons received no direct input from the three major pathways to the SCN, and the CCK neurons were not light-responsive as evaluated by induction of cFOS, and did not express the core clock protein PER1. Accordingly, CCK-deficient mice showed normal entrainment and had similar τ, light-induced phase shift and negative masking behaviour as wild-type animals. In conclusion, CCK signalling seems not to be involved directly in light-induced resetting of the clock or in regulating core clock function. The expression of CCK in a subpopulation of neurons, which do not belonging to either the VIP or AVP cells but which have synaptic contacts to both cell types and reverse innervation of CCK neurons from VIP neurons, suggests that the CCK neurons may act in non-photic regulation within the clock and/or, via CCK projections, mediate clock information to hypothalamic nuclei.

Suprachiasmatic nucleus: cell autonomy and network properties.

The suprachiasmatic nucleus (SCN) is the primary circadian pacemaker in mammals. Individual SCN neurons in dispersed culture can generate independent circadian oscillations of clock gene expression and neuronal firing. However, SCN rhythmicity depends on sufficient membrane depolarization and levels of intracellular calcium and cAMP. In the intact SCN, cellular oscillations are synchronized and reinforced by rhythmic synaptic input from other cells, resulting in a reproducible topographic pattern of distinct phases and amplitudes specified by SCN circuit organization. The SCN network synchronizes its component cellular oscillators, reinforces their oscillations, responds to light input by altering their phase distribution, increases their robustness to genetic perturbations, and enhances their precision. Thus, even though individual SCN neurons can be cell-autonomous circadian oscillators, neuronal network properties are integral to normal function of the SCN.

Mass spectrometry-based discovery of circadian peptides.

A significant challenge to understanding dynamic and heterogeneous brain systems lies in the chemical complexity of secreted intercellular messengers that change rapidly with space and time. Two solid-phase extraction collection strategies are presented that relate time and location of peptide release with mass spectrometric characterization. Here, complex suites of peptide-based cell-to-cell signaling molecules are characterized from the mammalian suprachiasmatic nucleus (SCN), site of the master circadian clock. Observed SCN releasates are peptide rich and demonstrate the co-release of established circadian neuropeptides and peptides with unknown roles in circadian rhythms. Additionally, the content of SCN releasate is stimulation specific. Stimulation paradigms reported to alter clock timing, including electrical stimulation of the retinohypothalamic tract, produce releasate mass spectra that are notably different from the spectra of compounds secreted endogenously over the course of the 24-h cycle. In addition to established SCN peptides, we report the presence of proSAAS peptides in releasates. One of these peptides, little SAAS, exhibits robust retinohypothalamic tract-stimulated release from the SCN, and exogenous application of little SAAS induces a phase delay consistent with light-mediated cues regulating circadian timing. These mass spectrometry-based analyses provide a new perspective on peptidergic signaling within the SCN and demonstrate that the integration of secreted compounds with information relating time and location of release generates new insights into intercellular signaling in the brain.

Live imaging of altered period1 expression in the suprachiasmatic nuclei of Vipr2-/- mice.

Vasoactive intestinal polypeptide and its receptor, VPAC(2), play important roles in the functioning of the brain's circadian clock in the suprachiasmatic nuclei (SCN). Mice lacking VPAC(2) receptors (Vipr2(-/-)) show altered circadian rhythms in locomotor behavior, neuronal firing rate, and clock gene expression, however, the nature of molecular oscillations in individual cells is unclear. Here, we used real-time confocal imaging of a destabilized green fluorescent protein (GFP) reporter to track the expression of the core clock gene Per1 in live SCN-containing brain slices from wild-type (WT) and Vipr2(-/-) mice. Rhythms in Per1-driven GFP were detected in WT and Vipr2(-/-) cells, though a significantly lower number and proportion of cells in Vipr2(-/-) slices expressed detectable rhythms. Further, Vipr2(-/-) cells expressed significantly lower amplitude oscillations than WT cells. Within each slice, the phases of WT cells were synchronized whereas cells in Vipr2(-/-) slices were poorly synchronized. Most GFP-expressing cells, from both genotypes, expressed neither vasopressin nor vasoactive intestinal polypeptide. Pharmacological blockade of VPAC(2) receptors in WT SCN slices partially mimicked the Vipr2(-/-) phenotype. These data demonstrate that intercellular communication via the VPAC(2) receptor is important for SCN neurons to sustain robust, synchronous oscillations in clock gene expression.

Circadian expression of 86- and 84-kDa heat shock proteins in the mouse suprachiasmatic nucleus.

Circadian rhythm pervades in many aspects of the biological processes including basic cellular functions. Here we examined the circadian gene expression of two forms of 90 kDa heat shock proteins referred to HSP86 and HSP84 in the mouse suprachiasmatic nucleus, the circadian center. In both light-dark, and constant dark conditions, Hsp86 mRNA showed an overt circadian rhythm showing a peak at (subjective) night and a trough at (subjective) day. Hsp84 mRNA also showed the similar expression profile, but the amplitude was weaker. These results indicate that gene expression of molecular chaperone such as Hsp86 and Hsp84 are regulated by the circadian clock.