Suprachiasmatic Nucleus Cells Research Articles

GABAA receptor subunit composition regulates circadian rhythms in rest–wake and synchrony among cells in the suprachiasmatic nucleus

The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) produces robust daily rhythms including rest-wake. SCN neurons synthesize and respond to γ-aminobutyric acid (GABA), but its role remains unresolved. We tested the hypothesis that γ2- and δ-subunits of the GABAA receptor in the SCN differ in their regulation of synchrony among circadian cells. We used two approaches: 1) shRNA to knock-down (KD) the expression of either γ2 or δ subunits in the SCN or 2) knock-in mice harboring a point mutation in the M2 domains of the endogenous GABAA γ2 or δ subunits. KD of either γ2 or δ subunits in the SCN increased daytime running and reduced nocturnal running by reducing their circadian amplitude by a third. Similarly, δ subunit knock-in mice showed decreased circadian amplitude, increased duration of daily activity, and decreased total daily activity. Reduction, or mutation of either γ2 or δ subunits halved the synchrony among, and amplitude of, circadian SCN cells as measured by firing rate or expression of the PERIOD2 protein, in vitro. Surprisingly, overexpression of the γ2 subunit rescued these phenotypes following KD or mutation of the δ subunit, and overexpression of the δ subunit rescued deficiencies due to γ2 subunit KD or mutation. We conclude that γ2 and δ GABAA receptor subunits play similar roles in maintaining circadian synchrony in the SCN and amplitude of daily rest-wake rhythms, but that modulation of their relative densities can change the duration and amplitude of daily activities.

The role of N6-methyladenosine RNA methylation in the crosstalk of circadian clock and neuroinflammation in rodent suprachiasmatic nuclei.

N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark that regulates the fate of RNA molecules. Recent studies have revealed a bidirectional interaction between m6A modification and the circadian clock. However, the precise temporal dynamics of m6A global enrichment in the central circadian pacemaker have not been fully elucidated. Our study investigates the relationship between FTO demethylase and molecular clocks in primary cells of the suprachiasmatic nucleus (SCN). In addition, we examined the effects of lipopolysaccharide (LPS) on Fto expression and the role of FTO in LPS-induced reactive oxygen species (ROS) production in primary SCN cell culture. We observed circadian rhythmicity in the global m6A levels, which mirrored the rhythmic expression of the Fto demethylase. Silencing FTO using siRNA reduced the mesor of Per2 rhythmicity in SCN primary cells and extended the period of the PER2 rhythm in SCN primary cell cultures from PER2::LUC mice. When examining the immune response, we discovered that exposure to LPS upregulated global m6A levels while downregulating Fto expression in SCN primary cell cultures. Interestingly, we found a loss of circadian rhythmicity in Fto expression following LPS treatment, indicating that the decrease of FTO levels may contribute to m6A upregulation without directly regulating its circadian rhythm. To explore potential protective mechanisms against neurotoxic inflammation, we examined ROS production following LPS treatment in SCN primary cell cultures pretreated with FTO siRNA. We observed a time-dependent pattern of ROS induction, with significant peak at 32 h but not at 20 h after synchronization. Silencing the FTO demethylase abolished ROS induction following LPS exposure, supporting the hypothesis that FTO downregulation serves as a protective mechanism during LPS-induced neuroinflammation in SCN primary cell cultures.

Diverse genetic alteration dysregulates neuropeptide and intracellular signalling in the suprachiasmatic nuclei.

In mammals, intrinsic 24 h or circadian rhythms are primarily generated by the suprachiasmatic nuclei (SCN). Rhythmic daily changes in the transcriptome and proteome of SCN cells are controlled by interlocking transcription-translation feedback loops (TTFLs) of core clock genes and their proteins. SCN cells function as autonomous circadian oscillators, which synchronize through intercellular neuropeptide signalling. Physiological and behavioural rhythms can be severely disrupted by genetic modification of a diverse range of genes and proteins in the SCN. With the advent of next generation sequencing, there is unprecedented information on the molecular profile of the SCN and how it is affected by genetically targeted alteration. However, whether the expression of some genes is more readily affected by genetic alteration of the SCN is unclear. Here, using publicly available datasets from recent RNA-seq assessments of the SCN from genetically altered and control mice, we evaluated whether there are commonalities in transcriptome dysregulation. This was completed for four different phases across the 24 h cycle and was augmented by Gene Ontology Molecular Function (GO:MF) and promoter analysis. Common differentially expressed genes (DEGs) and/or enriched GO:MF terms included signalling molecules, their receptors, and core clock components. Finally, examination of the JASPAR database indicated that E-box and CRE elements in the promoter regions of several commonly dysregulated genes. From this analysis, we identify differential expression of genes coding for molecules involved in SCN intra- and intercellular signalling as a potential cause of abnormal circadian rhythms.

The circadian clock in the choroid plexus drives rhythms in multiple cellular processes under the control of the suprachiasmatic nucleus

Choroid plexus (ChP), the brain structure primarily responsible for cerebrospinal fluid production, contains a robust circadian clock, whose role remains to be elucidated. The aim of our study was to [1] identify rhythmically controlled cellular processes in the mouse ChP and [2] assess the role and nature of signals derived from the master clock in the suprachiasmatic nuclei (SCN) that control ChP rhythms. To accomplish this goal, we used various mouse models (WT, mPer2Luc, ChP-specific Bmal1 knockout) and combined multiple experimental approaches, including surgical lesion of the SCN (SCNx), time-resolved transcriptomics, and single cell luminescence microscopy. In ChP of control (Ctrl) mice collected every 4 h over 2 circadian cycles in darkness, we found that the ChP clock regulates many processes, including the cerebrospinal fluid circadian secretome, precisely times endoplasmic reticulum stress response, and controls genes involved in neurodegenerative diseases (Alzheimer’s disease, Huntington’s disease, and frontotemporal dementia). In ChP of SCNx mice, the rhythmicity detected in vivo and ex vivo was severely dampened to a comparable extent as in mice with ChP-specific Bmal1 knockout, and the dampened cellular rhythms were restored by daily injections of dexamethasone in mice. Our data demonstrate that the ChP clock controls tissue-specific gene expression and is strongly dependent on the presence of a functional connection with the SCN. The results may contribute to the search for a novel link between ChP clock disruption and impaired brain health.

FRI277 Temporal Coordination Of Female Reproductive Axis Activity By Vasoactive Intestinal Polypeptide (VIP) Expressing Cells In The Suprachiasmatic Nucleus

Abstract Disclosure: C. Phumsatitpong: None. S.R. Bever: None. S.L. Hu: None. M.C. Ambrose: None. L.J. Kriegsfeld: None. Circadian rhythms orchestrated by a master clock in the suprachiasmatic nucleus (SCN) are essential for ovulation in spontaneously-ovulating rodents. SCN signaling integrates with estradiol positive feedback to generate the preovulatory luteinizing hormone (LH) surge. Additionally, subordinate clocks in reproductively-relevant SCN targets temporally gate responsiveness to upstream signaling to coordinate ovulation with the active time of day. We have previously shown that the SCN communicates directly to kisspeptin (Kp) and RFamide-related peptide-3 (RFRP-3) neurons to temporally balance key stimulatory and inhibitory input to GnRH neurons required for LH surge generation. Because VIP-expressing neurons in the SCN have been implicated in the LH surge, we employed a cell-specific chemogenetic approach to determine the functional role of this cell phenotype in female reproductive axis regulation required for surge generation. We hypothesized that temporal gating by subordinate cellular clocks will prevent morning stimulation of VIP-expressing cells from increasing Kp and GnRH, and decreasing RFRP-3, cellular activity. In the afternoon, we expected inhibition of VIP-expressing cells to decrease Kp and GnRH, and increase RFRP-3, cellular activity. Specifically, female VIP-Cre mice were stereotaxically injected with either AAV-hM3Dq or hM4Di to express activating (3Dq) or inhibitory (4Di) DREADDs in VIP-expressing SCN cells. Following recovery, mice were injected clozapine n-oxide (CNO) either in the morning, prior to the LH surge (3Dq experiment), or the afternoon, around the time of the LH surge (4Di experiment). Brains were collected to examine neuronal activation in Kp, GnRH, and RFRP-3 neurons using FOS immunofluorescence as a marker. Morning stimulation of SCN VIP neurons decreased RFRP-3 neuronal activation, whereas Kp and GnRH neurons were unaffected at this time, suggesting a temporal gating mechanism at GnRH and/or Kp cells prevents premature ovulation. In contrast, afternoon inhibition of SCN VIP-expressing neurons led to an increase in neuronal activation in RFRP-3 neurons concomitant with a decrease in GnRH and Kp neurons, suggesting that VIP stimulation is required to remove RFRP-3 inhibition on the GnRH and Kp systems. We confirmed this possibility by localizing afternoon injections of CNO to RFRP-3 cells, which led to comparable decreases in GnRH and Kp cellular activity. These data indicate the importance of SCN VIP neurons in the temporally coordinated disinhibition of RFRP-3 neuronal activity required for stimulation of GnRH and Kp neurons around the time of LH surge. Supported by NIH Grant HD-050470. Presentation: Friday, June 16, 2023

Regulation of CRE-Dependent Transcriptional Activity in a Mouse Suprachiasmatic Nucleus Cell Line.

We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.

Prenatal androgen treatment impairs the suprachiasmatic nucleus arginine-vasopressin to kisspeptin neuron circuit in female mice.

Polycystic ovary syndrome (PCOS) is associated with elevated androgen and luteinizing hormone (LH) secretion and with oligo/anovulation. Evidence indicates that elevated androgens impair sex steroid hormone feedback regulation of pulsatile LH secretion. Hyperandrogenemia in PCOS may also disrupt the preovulatory LH surge. The mechanisms through which this might occur, however, are not fully understood. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) convey hormonal cues to gonadotropin-releasing hormone (GnRH) neurons. In rodents, the preovulatory surge is triggered by these hormonal cues and coincident timing signals from the central circadian clock in the suprachiasmatic nucleus (SCN). Timing signals are relayed to GnRH neurons, in part, via projections from SCN arginine-vasopressin (AVP) neurons to RP3VKISS1 neurons. Because rodent SCN cells express androgen receptors (AR), we hypothesized that these circuits are impaired by elevated androgens in a mouse model of PCOS. In prenatally androgen-treated (PNA) female mice, SCN Ar expression was significantly increased compared to that found in prenatally vehicle-treated mice. A similar trend was seen in the number of Avp-positive SCN cells expressing Ar. In the RP3V, the number of kisspeptin neurons was preserved. Anterograde tract-tracing, however, revealed reduced SCNAVP neuron projections to the RP3V and a significantly lower proportion of RP3VKISS1 neurons with close appositions from SCNAVP fibers. Functional assessments showed, on the other hand, that RP3VKISS1 neuron responses to AVP were maintained in PNA mice. These findings indicate that PNA changes some of the neural circuits that regulate the preovulatory surge. These impairments might contribute to ovulatory dysfunction in PNA mice modeling PCOS.

Nested calcium dynamics support daily cell unity and diversity in the suprachiasmatic nuclei of free-behaving mice.

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus host the circadian pacemaker that synchronizes mammalian rhythms with the day-night cycle. SCN neurons are intrinsically rhythmic, thanks to a conserved cell-autonomous clock mechanism. In addition, circuit-level emergent properties confer a unique degree of precision and robustness to SCN neuronal rhythmicity. However, the multicellular functional organization of the SCN is not yet fully understood. Indeed, although SCN neurons are well-coordinated, experimental evidences indicate that some neurons oscillate out of phase in SCN explants, and possibly to a larger extent in vivo. Here, to tackle this issue we used microendoscopic Ca2+ i imaging and investigated SCN rhythmicity at a single cell resolution in free-behaving mice. We found that SCN neurons in vivo exhibited fast Ca2+ i spikes superimposed upon slow changes in baseline Ca2+ i levels. Both spikes and baseline followed a time-of-day modulation in many neurons, but independently from each other. Daily rhythms in basal Ca2+ i were highly coordinated, while spike activity from the same neurons peaked at multiple times of the light cycle, and unveiled clock-independent coactivity in neuron subsets. Hence, fast Ca2+ i spikes and slow changes in baseline Ca2+ i levels highlighted how multiple individual activity patterns could articulate within the temporal unity of the SCN cell network in vivo, and provided support for a multiplex neuronal code in the circadian pacemaker.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Oak extracts modulate circadian rhythms of clock gene expression in vitro and wheel-running activity in mice.

In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which coordinates the circadian rhythm and controls locomotor activity rhythms. In addition to SCN cells, the peripheral tissues and embryonic fibroblasts also have clock genes, such as Per1/2 and Bmal1, which generate the transcriptional-translational feedback loop to produce an approximately 24-h cycle. Aging adversely affects the circadian clock system and locomotor functions. Oak extract has been reported to improve age-related physiological changes. However, no study has examined the effect of oak extract on the circadian clock system. We examined the effects of oak extract and its metabolites (urolithin A [ULT] and ellagic acid [EA]) on clock gene expression rhythms in mouse embryonic fibroblasts (MEFs) and SCN. Furthermore, locomotor activity rhythm was assessed in young and aged mice. Chronic treatment with EA and ULT delayed the phase of PER2::LUC rhythms in SCN explants, and ULT prolonged the period of PER2::LUC rhythms in MEFs in a dose-dependent manner and increased the amplitude of PER2::LUC rhythms in MEFs, though only at low concentrations. Acute treatment with ULT affected the phase of PER2::LUC rhythms in MEFs depending on the concentration and timing of the treatment. In addition, oak extract prolonged the activity time of behavioral rhythms in old mice and tended to increase daily wheel-running revolutions in both young and old mice. These results suggest that oak extract is a novel modulator of the circadian clock in vitro and in vivo. The online version contains supplementary material available at 10.1007/s41105-021-00365-2.

Retina-attached slice recording reveals light-triggered tonic GABA signaling in suprachiasmatic nucleus

Light is a powerful external cue modulating the biological rhythm of internal clock neurons in the suprachiasmatic nucleus (SCN). GABA signaling in SCN is critically involved in this process. Both phasic and tonic modes of GABA signaling exist in SCN. Of the two modes, the tonic mode of GABA signaling has been implicated in light-mediated synchrony of SCN neurons. However, modulatory effects of external light on tonic GABA signalling are yet to be explored. Here, we systematically characterized electrophysiological properties of the clock neurons and determined the spatio-temporal profiles of tonic GABA current. Based on the whole-cell patch-clamp recordings from 76 SCN neurons, the cells with large tonic GABA current (>15 pA) were more frequently found in dorsal SCN. Moreover, tonic GABA current in SCN was highly correlated with the frequency of spontaneous inhibitory postsynaptic current (sIPSC), raising a possibility that tonic GABA current is due to spill-over from synaptic release. Interestingly, tonic GABA current was inversely correlated with slice-to-patch time interval, suggesting a critical role of retinal light exposure in intact brain for an induction of tonic GABA current in SCN. To test this possibility, we obtained meticulously prepared retina-attached SCN slices and successfully recorded tonic and phasic GABA signaling in SCN neurons. For the first time, we observed an early-onset, long-lasting tonic GABA current, followed by a slow-onset, short-lasting increase in the phasic GABA frequency, upon direct light-illumination of the attached retina. This result provides the first evidence that external light cue can directly trigger both tonic and phasic GABA signaling in SCN cell. In conclusion, we propose tonic GABA as the key mediator of external light in SCN.

Zfhx3-mediated genetic ablation of the SCN abolishes light entrainable circadian activity while sparing food anticipatory activity

SummaryCircadian rhythms persist in almost all organisms and are crucial for maintaining appropriate timing in physiology and behaviour. Here, we describe a mouse mutant where the central mammalian pacemaker, the suprachiasmatic nucleus (SCN), has been genetically ablated by conditional deletion of the transcription factor Zfhx3 in the developing hypothalamus. Mutants were arrhythmic over the light-dark cycle and in constant darkness. Moreover, rhythms of metabolic parameters were ablated in vivo although molecular oscillations in the liver maintained some rhythmicity. Despite disruptions to SCN cell identity and circuitry, mutants could still anticipate food availability, yet other zeitgebers - including social cues from cage-mates - were ineffective in restoring rhythmicity although activity levels in mutants were altered. This work highlights a critical role for Zfhx3 in the development of a functional SCN, while its genetic ablation further defines the contribution of SCN circuitry in orchestrating physiological and behavioral responses to environmental signals.

The Circadian Clock Is Sustained in the Thyroid Gland of VIP Receptor 2 Deficient Mice.

VIP/VPAC2-receptor signaling is crucial for functioning of the circadian clock in the suprachiasmatic nucleus (SCN) since the lack results in disrupted synchrony between SCN cells and altered locomotor activity, body temperature, hormone secretion and heart rhythm. Endocrine glands, including the thyroid, show daily oscillations in clock gene expression and hormone secretion, and SCN projections target neurosecretory hypothalamic thyroid-stimulating hormone (TSH)-releasing hormone cells. The aim of the study was to gain knowledge of mechanisms important for regulation of the thyroid clock by evaluating the impact of VIP/VPAC2-receptor signaling. Quantifications of mRNAs of three clock genes (Per1, Per2 and Bmal1) in thyroids of wild type (WT) and VPAC2-receptor deficient mice were done by qPCR. Tissues were taken every 4th h during 24-h 12:12 light-dark (LD) and constant darkness (DD) periods, both genders were used. PER1 immunoreactivity was visualized on sections of both WT and VPAC2 lacking mice during a LD cycle. Finally, TSH and the thyroid hormone T4 levels were measured in the sera by commercial ELISAs. During LD, rhythmic expression of all three mRNA was found in both the WT and knockout animals. In VPAC2-receptor knockout animals, the amplitudes were approximately halved compared to the ones in the WT mice. In the WT, Per1 mRNA peaked around “sunset”, Per2 mRNA followed with approximately 2 h, while Bmal1 mRNA was in antiphase with Per1. In the VPAC2 knockout mice, the phases of the mRNAs were advanced approximately 5 h compared to the WT. During DD, the phases of all the mRNAs were identical to the ones found during LD in both groups of mice. PER1 immunoreactivity was delayed compared to its mRNA and peaked during the night in follicular cells of both the thyroid and parathyroid glands in the WT animals. In WT animals, TSH was high around the transition to darkness compared to light-on, while T4 did not change during the 24 h cycle. In conclusion, sustained and identical rhythms (phases and amplitudes) of three clock genes were found in VPAC2 deficient mice during LD and DD suggesting high degree of independence of the thyroid clock from the master SCN clock.

Modelling the functional roles of synaptic and extra-synaptic γ-aminobutyric acid receptor dynamics in circadian timekeeping.

In the suprachiasmatic nucleus (SCN), γ-aminobutyric acid (GABA) is a primary neurotransmitter. GABA can signal through two types of GABAA receptor subunits, often referred to as synaptic GABAA (gamma subunit) and extra-synaptic GABAA (delta subunit). To test the functional roles of these distinct GABAA in regulating circadian rhythms, we developed a multicellular SCN model where we could separately compare the effects of manipulating GABA neurotransmitter or receptor dynamics. Our model predicted that blocking GABA signalling modestly increased synchrony among circadian cells, consistent with published SCN pharmacology. Conversely, the model predicted that lowering GABAA receptor density reduced firing rate, circadian cell fraction, amplitude and synchrony among individual neurons. When we tested these predictions, we found that the knockdown of delta GABAA reduced the amplitude and synchrony of clock gene expression among cells in SCN explants. The model further predicted that increasing gamma GABAA densities could enhance synchrony, as opposed to increasing delta GABAA densities. Overall, our model reveals how blocking GABAA receptors can modestly increase synchrony, while increasing the relative density of gamma over delta subunits can dramatically increase synchrony. We hypothesize that increased gamma GABAA density in the winter could underlie the tighter phase relationships among SCN cells.

NPAS4 regulates the transcriptional response of the suprachiasmatic nucleus to light and circadian behavior

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals and is entrained byenvironmental light. However, the molecular basis of the response of the SCN to light is not fully understood. We used RNA/chromatin immunoprecipitation/single-nucleus sequencing with circadian behavioral assays to identify mouse SCN cell types and explore their responses to light. We identified three peptidergic cell types that responded to light in the SCN: arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), and cholecystokinin (CCK). In each cell type, light-responsive subgroups were enriched for expression of neuronal Per-Arnt-Sim (PAS) domain protein 4 (NPAS4) target genes. Further, mice lacking Npas4 had a longer circadian period under constant conditions, a damped phase response curve to light, and reduced light-induced gene expression in the SCN. Our data indicate that NPAS4 is necessary for normal transcriptional responses to light in the SCN and critical for photic phase-shifting of circadian behavior.

Comparative Immune-reactivity Patterns of Arginine Vasotocin (AVT) and Melatonin Receptors (Mel1a & Mel1b) in Hypothalamic Regions of Male Japanese Quail Coturnix coturnix japonica : Possible Role in Water-Electrolyte Balance

Photoperiod influences circulatory Melatonin (Mel) and hypothalamic functions via retino-hypothalamic tract. However, interrelation between Mel receptors and Arginine Vasotocin (AVT), a water-electrolyte balancing hormone receptor expression in hypothalamic regions of avian brain has never been explored. We noted the expression pattern of two Mel receptors (Mel1a & Mel1b) along with AVT, in terms of neuronal immuno-positivity, in hypothalamic region of Japanese quail under different photoperiodic conditions with/without melatonin treatment. This is interesting and equally important because both Mel and AVT levels are regulated by light/dark cycle. Confocal imaging revealed specific regional localization of Mel1a/Mel1b in Supra-Chiasmatic Nucleus (SCN), Supra-Optic Nucleus (SON) and Para-Ventricular Nucleus (PVN), the intensity of which was dependent on the photoperiodic condition (long day, LD or short day, SD) and melatonin treatment. Mel1a/Mel1b was mostly co-localized along with AVT. Mel1b was abundant in hypothalamic regions in contrast to the Mel1a as reported in mammals. Mel1a immune-positivity was detected in SCN and SON regions of brain. Compared to control birds, a high intensity of Mel1a immunoreactivity was found in hypothalamic regions of birds under short photoperiod (SD, 8h L: 16h D) after Mel treatment. Further, Mel1b immunopositivity was high only in birds exposed to long days (LD, 16h L: 8h D). In SCN, abundant Mel1a and AVT immunoreative cells were found in Mel pretreated and SDexposed birds compared to LD-exposed ones. Mel1a and AVT immunoreative cells were less in PVN of both SD and LD exposed birds. Our data of co-localization of Mel receptor(s) along with AVT in hypothalamic regions (exposed to short or long days with/without melatonin administration) strongly suggest a role for Mel along with AVT in water-electrolyte balance of birds which is important during long duration nuptial migration/flight.

Roles of Neuropeptides, VIP and AVP, in the Mammalian Central Circadian Clock.

In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Individual SCN cells exhibit intrinsic oscillations, and their circadian period and robustness are different cell by cell in the absence of cellular coupling, indicating that cellular coupling is important for coherent circadian rhythms in the SCN. Several neuropeptides such as arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) are expressed in the SCN, where these neuropeptides function as synchronizers and are important for entrainment to environmental light and for determining the circadian period. These neuropeptides are also related to developmental changes of the circadian system of the SCN. Transcription factors are required for the formation of neuropeptide-related neuronal networks. Although VIP is critical for synchrony of circadian rhythms in the neonatal SCN, it is not required for synchrony in the embryonic SCN. During postnatal development, the clock genes cryptochrome (Cry)1 and Cry2 are involved in the maturation of cellular networks, and AVP is involved in SCN networks. This mini-review focuses on the functional roles of neuropeptides in the SCN based on recent findings in the literature.

Modulation of single cell circadian response to NMDA by diacylglycerol lipase inhibition reveals a role of endocannabinoids in light entrainment of the suprachiasmatic nucleus

Suprachiasmatic nucleus (SCN) of the hypothalamus is the master clock that drives circadian rhythms in physiology and behavior and adjusts their timing to external cues. Neurotransmitter glutamate and glutamatergic receptors sensitive to N-methyl-d-aspartate (NMDA) play a dual role in the SCN by coupling astrocytic and neuronal single cell oscillators and by resetting their phase in response to light. Recent reports suggested that signaling by endogenous cannabinoids (ECs) participates in both of these functions. We have previously shown that ECs, such as 2-arachidonoylglycerol (2-AG), act via CB1 receptors to affect the SCN response to light-mimicking NMDA stimulus in a time-dependent manner. We hypothesized that this ability is linked to the circadian regulation of EC signaling. We demonstrate that circadian clock in the rat SCN regulates expression of 2-AG transport, synthesis and degradation enzymes as well as its receptors. Inhibition of the major 2-AG synthesis enzyme, diacylglycerol lipase, enhanced the phase delay and lowered the amplitude of explanted SCN rhythm in response to NMDAR activation. Using microscopic PER2 bioluminescence imaging, we visualized how individual single cell oscillators in different parts of the SCN respond to the DAGL inhibition/NMDAR activation and shape response of the whole pacemaker. Additionally, we present strong evidence that the zero amplitude behavior of the SCN in response to single NMDA stimulus in the middle of subjective night is the result of a loss of rhythm in individual SCN cells. The paper provides new insights into the modulatory role of endocannabinoid signaling during the light entrainment of the SCN.

Zfhx3-Mediated Genetic Ablation of the Mouse SCN Abolishes Light Entrainable Circadian Activity While the Food Entrainable Oscillator Remains Intact

Circadian rhythms persist in almost all organisms and are crucial for maintaining appropriate timing in physiology and behaviour. Here, we describe a mouse mutant where the central mammalian pacemaker, the suprachiasmatic nucleus (SCN), has been genetically ablated by conditional deletion of the transcription factor Zfhx3 in the developing hypothalamus. Mutants were arrhythmic over the light-dark cycle and in constant darkness. Moreover, rhythms of metabolic parameters were reduced or ablated in vivo although molecular oscillations in the liver maintained some rhythmicity. Despite disruptions to SCN cell identity and circuitry, mutants could still entrain to food availability, yet other zeitgebers - including social cues from cage-mates - were ineffective in restoring rhythmicity. This work highlights, for the first time, a critical role for Zfhx3 in the development of a functional SCN, while its genetic ablation further defines the contribution of SCN circuitry in orchestrating physiological and behavioural responses to environmental signals.

Role of G-Substrate in the NO/cGMP/PKG Signal Transduction Pathway for Photic Entrainment of the Hamster Circadian Clock.

The mammalian circadian clock at the hypothalamic suprachiasmatic nuclei (SCN) entrains biological rhythms to the 24-h cyclic environment, by encoding light-dark transitions in SCN neurons. Light pulses induce phase shifts in the clock and in circadian rhythms; photic signaling for circadian phase advances involves a nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway, increasing the expression of Period (Per) genes. Effectors downstream of PKG remain unknown. Here we investigate the role of G-substrate (GS), a PKG substrate, in the hamster SCN. GS and phosphorylated G-substrate (p-GS) were present in a subset of SCN cells. Moreover, GS phosphorylation (p-GS/GS ratio) increased in SCN homogenates after light pulses delivered at circadian time (CT) 18 and intraperitoneal treatment with sildenafil, an inhibitor of phosphodiesterase 5 (a cGMP-specific phosphodiesterase). On the other hand, intracerebroventricular treatment with the PKG inhibitor KT5823, reduced photic phosphorylation of GS to basal levels. Since p-GS could act as a protein phosphatase 2 A (PP2A) inhibitor, we demonstrated physical interaction between p-GS and PP2A in SCN homogenates, and also a light-pulse dependent decrease of PP2A activity. Intracerebroventricular treatment with okadaic acid, a PP2A inhibitor, increased the magnitude of light-induced phase advances of locomotor rhythms. We provide evidence on the physiological phosphorylation of GS as a new downstream effector in the NO/cGMP/PKG photic pathway in the hamster SCN, including its role as a PP2A inhibitor.