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Individual neurons of the hypothalamic suprachiasmatic nuclei (SCN) contain an intracellular molecular clock that drives these neurons to exhibit day-night variation in excitability. The neuropeptide vasoactive intestinal polypeptide (VIP) and its cognate receptor, VPAC 2 , are synthesized by SCN neurons and this intercellular VIP-VPAC 2 receptor signal facilitates coordination of SCN neuronal activity and timekeeping. How the loss of VPAC 2 receptor signalling affects the electrophysiological properties and states of SCN neurons as well as their responses to excitatory inputs is unclear. Here we used patch-clamp electrophysiology and made recordings of SCN neurons in brain slices prepared from transgenic animals that do not express VPAC 2 receptors (Vipr2 -/- mice) as well as animals that do (Vipr2 +/+ mice). We report that while Vipr2 +/+ neurons exhibit coordinated day-night variation in their electrical state, Vipr2 -/- neurons lack this and instead manifest a range of states during both day and night. Further, at the population level, Vipr2 +/+ neurons vary the membrane threshold potential at which they start to fire action potentials from day to night, while Vipr2 -/- neurons do not. We provide evidence that Vipr2 -/- neurons lack a component of voltage-gated sodium currents that contribute to SCN neuronal excitability. Moreover, we determine that this aberrant temporal control of neuronal state and excitability alters neuronal responses to a neurochemical mimic of the light-input pathway to the SCN. These results highlight the critical role VIP-VPAC 2 receptor signalling plays in the temporal expression of individual neuronal states as well as appropriate ensemble activity and input gating of the SCN neural network., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Prokineticin 2 (Prok2) is a small protein expressed in a subpopulation of neurons in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals. Prok2 has been implicated as a candidate output molecule from the SCN to control multiple circadian rhythms. Genetic manipulation specific to Prok2-producing neurons would be a powerful approach to understanding their function. Here, we report the generation of Prok2-tTA knock-in mice expressing the tetracycline transactivator (tTA) specifically in Prok2 neurons and an application of these mice to in vivo recording of Ca 2+ rhythms in these neurons. First, the specific and efficient expression of tTA in Prok2 neurons was verified by crossing the mice with EGFP reporter mice. Prok2-tTA mice were then used to express a fluorescent Ca 2+ sensor protein to record the circadian Ca 2+ rhythm in SCN Prok2 neurons in vivo. Ca 2+ in these cells showed clear circadian rhythms in both light-dark and constant dark conditions, with their peaks around midday. Notably, the hours of high Ca 2+ nearly coincided with the rest period of the behavioral rhythm. These observations fit well with the predicted function of Prok2 neurons as a candidate output pathway of the SCN by suppressing locomotor activity during both daytime and subjective daytime., (© 2023. Springer Nature Limited.)

The suprachiasmatic nucleus (SCN) of the hypothalamus serves as the master circadian clock in mammals. Most SCN neurons express the inhibitory neurotransmitter GABA (gamma amino butyric acid) along with a peptide cotransmitter. Notably, the neuropeptides vasopressin (VP) and vasoactive intestinal peptide (VIP) define two prominent clusters within the SCN: those located in the ventral core (VIP) and those forming the dorsomedial "shell" of the nucleus (VP). Axons emerging from VP neurons in the shell are thought to mediate much of the SCN's output to other brain regions as well as VP release into the cerebrospinal fluid (CSF). Previous work has shown that VP release by SCN neurons is activity dependent and SCN VP neurons fire action potentials at a higher rate during the light phase. Accordingly, CSF VP levels are higher during daytime. Interestingly, the amplitude of the CSF VP rhythm is greater in males than females, suggesting the existence of sex differences in the electrical activity of SCN VP neurons. Here we investigated this hypothesis by performing cell-attached recordings from 1070 SCN VP neurons across the entire circadian cycle in both sexes of transgenic rats that express green fluorescent protein (GFP) driven by the VP gene promoter. Using an immunocytochemical approach we confirmed that >60% of SCN VP neurons display visible GFP. Recordings in acute coronal slices revealed that VP neurons display a striking circadian pattern of action potential firing, but the characteristics of this activity cycle differ in males and females. Specifically, neurons in males reached a significantly higher peak firing frequency during subjective daytime compared to females and the acrophase occurred ~1 h earlier in females. Peak firing rates in females were not significantly different at various phases of the estrous cycle., (© 2023 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

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.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker of the mammalian biological clock. Here, we provide a detailed protocol for long-term recording of calcium signals in SCN neurons of freely moving mice through a multichannel optical fiber recording system. This system can simultaneously collect calcium signals from up to seven animals. The calcium signals can be visualized by the appropriate software and code. This protocol can be used to explore the long-term response of SCN to external environmental stimulation. For complete details on the use and execution of this protocol, please refer to Zhai et al. (2022)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Disruptions to the circadian system alter reproductive capacity, particularly in females. Mice lacking the core circadian clock gene, Bmal1 , are infertile and have evidence of neuroendocrine disruption including the absence of the preovulatory luteinizing hormone (LH) surge and enhanced responsiveness to exogenous kisspeptin. Here, we explore the role of Bmal1 in suprachiasmatic nucleus (SCN) neuron populations known to project to the neuroendocrine axis. We generated four mouse lines using Cre/Lox technology to create conditional deletion of Bmal1 in arginine vasopressin ( Bmal1 fl/fl :Avp cre ), vasoactive intestinal peptide ( Bmal1 fl/fl :Vip cre ), both ( Bmal1 fl/fl :Avp cre +Vip cre ), and neuromedin-s ( Bmal1 fl/fl :Nms cre ) neurons. We demonstrate that the loss of Bmal1 in these populations has substantial effects on home-cage circadian activity and temperature rhythms. Despite this, we found that female mice from these lines demonstrated normal estrus cycles, fecundity, kisspeptin responsiveness, and inducible LH surge. We found no evidence of reproductive disruption in constant darkness. Overall, our results indicate that while conditional Bmal1 knockout in AVP, VIP, or NMS neurons is sufficient to disrupted locomotor activity, this disruption is insufficient to recapitulate the neuroendocrine reproductive effects of the whole-body Bmal1 knockout., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tonsfeldt, Cui, Lee, Walbeek, Brusman, Jin, Mieda, Gorman and Mellon.)

The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Viral-mediated gene augmentation offers tremendous promise for the treatment of inherited retinal diseases. The development of effective gene therapy requires an understanding of the vector's tissue-specific behavior, which may vary depending on serotype, route of delivery, or target species. Using an ex vivo organotypic explant system, we previously demonstrated that retinal tropism and transduction of adeno-associated virus type 2 (AAV2) vary significantly depending on serotype in human eyes. However, the ex vivo system has limited ability to assess route of ocular delivery, and relatively little literature exists on tropic differences between serotypes and routes of delivery in vivo . In this study, we demonstrate that retinal tropism and transduction efficiency of five different AAV2 serotypes (AAV2/1, AAV2/2, AAV2/6, AAV2/8, and AAV2/9) expressing enhanced green fluorescent protein driven by a cytomegalovirus promoter vary greatly depending on serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats. With subretinal delivery, all serotypes successfully transduced the retinal pigmented epithelium and outer nuclear layer (ONL), with AAV2/1 displaying the highest transduction efficiency and AAV2/2 and AAV2/6 showing lower ONL transduction. There was minimal transduction of the inner retina through subretinal delivery for any serotype. Tropism by suprachoroidal delivery mirrored that of subretinal delivery for all AAV serotypes but resulted in a wider distribution and greater ONL transduction. With intravitreal delivery, retinal transduction was seen primarily in the inner retina (retinal nerve fiber, ganglion cell, and inner nuclear layers) for AAV2/1 and AAV2/6, with AAV2/6 showing the highest transduction. When compared with data from human explant models, there are substantial differences in tropism and transduction that are important to consider when using rats as preclinical models for the development of ocular gene therapies for humans.

The suprachiasmatic nucleus (SCN) acts as a master pacemaker driving circadian behavior and physiology. Although the SCN is small, it is composed of many cell types, making it difficult to study the roles of particular cells. Here we develop bioluminescent circadian reporter mice that are Cre dependent, allowing the circadian properties of genetically defined populations of cells to be studied in real time. Using a Color-Switch PER2::LUCIFERASE reporter that switches from red PER2::LUCIFERASE to green PER2::LUCIFERASE upon Cre recombination, we assess circadian rhythms in two of the major classes of peptidergic neurons in the SCN: AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide). Surprisingly, we find that circadian function in AVP neurons, not VIP neurons, is essential for autonomous network synchrony of the SCN and stability of circadian rhythmicity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Circadian rhythms in Per1, PER2 expression and intracellular Ca 2+ were measured from a solitary SCN neuron or glial cell which was physically isolated from other cells. Dispersed cells were cultured on a platform of microisland (100-200 μm in diameter) in a culture dish. Significant circadian rhythms were detected in 57.1% for Per1 and 70.0% for PER2 expression. When two neurons were located on the same island, the circadian rhythms showed desynchronization, indicating a lack of oscillatory coupling. Circadian rhythms were also detected in intracellular Ca 2+ of solitary SCN neurons. The ratio of circadian positive neurons was significantly larger without co-habitant of glial cells (84.4%) than with it (25.0%). A relatively large fraction of SCN neurons generates the intrinsic circadian oscillation without neural or humoral networks. In addition, glial cells seem to interrupt the expression of the circadian rhythmicity of intracellular Ca 2+ under these conditions.

Transmembrane Ca 2+ influx is essential to the proper functioning of the central clock in the suprachiasmatic nucleus (SCN). In the rat SCN neurons, the clearance of somatic Ca 2+ following depolarization-induced Ca 2+ transients involves Ca 2+ extrusion via Na + /Ca 2+ exchanger (NCX) and mitochondrial Ca 2+ buffering. Here we show an important role of intracellular Na + in the regulation of [Ca 2+ ] i in these neurons. The effect of Na + loading on [Ca 2+ ] i was determined with the Na + ionophore monensin and the cardiac glycoside ouabain to block Na + /K + -ATPase (NKA). Ratiometric Na + and Ca 2+ imaging was used to measure the change in [Na + ] i and [Ca 2+ ] i , and cell-attached recordings to investigate the effects of monensin and ouabain on spontaneous firing. Our results show that in spite of opposite effects on spontaneous firing and basal [Ca 2+ ], both monensin and ouabain induced Na + loading, and increased the peak amplitude, slowed the fast decay rate, and enhanced the slow decay phase of 20 mM K + -evoked Ca 2+ transients. Furthermore, both ouabain and monensin preferentially enhanced nimodipine-insensitive Ca 2+ transients. Together, our results indicate that in the SCN neurons the NKA plays an important role in regulating [Ca 2+ ] i , in particular, associated with nimodipine-insensitive Ca 2+ channels.

Cryptochrome (CRY) plays an important role in the input of circadian clocks in various species, but gene copies in each species are evolutionarily divergent. Type I CRYs function as a photoreceptor molecule in the central clock, whereas type II CRYs directly regulate the transcriptional activity of clock proteins. Functions of other types of animal CRYs in the molecular clock remain unknown. The water flea Daphnia magna contains four Cry genes. However, it is still difficult to analyse these four genes. In this study, we took advantage of powerful genetic resources available from Drosophila to investigate evolutionary and functional differentiation of CRY proteins between the two species. We report differences in subcellular localisation of each D. magna CRY protein when expressed in the Drosophila clock neuron. Circadian rhythm behavioural experiments revealed that D. magna CRYs are not functionally conserved in the Drosophila molecular clock. These findings provide a new perspective on the evolutionary conservation of CRY, as functions of the four D. magna CRY proteins have diverse subcellular localisation levels. Furthermore, molecular clocks of D. magna have been evolutionarily differentiated from those of Drosophila. This study highlights the extensive functional diversity existing among species in their complement of Cry genes.

The mammalian suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker, integrating environmental input to align physiological and behavioral rhythms to local time cues. Approximately 10% of SCN neurons express vasoactive intestinal polypeptide (VIP); however, it is unknown how firing activity of VIP neurons releases VIP to entrain circadian rhythms. To identify physiologically relevant firing patterns, we optically tagged VIP neurons and characterized spontaneous firing over 3 days. VIP neurons had circadian rhythms in firing rate and exhibited two classes of instantaneous firing activity. We next tested whether physiologically relevant firing affected circadian rhythms through VIP release. We found that VIP neuron stimulation with high, but not low, frequencies shifted gene expression rhythms in vitro through VIP signaling. In vivo, high-frequency VIP neuron activation rapidly entrained circadian locomotor rhythms. Thus, increases in VIP neuronal firing frequency release VIP and entrain molecular and behavioral circadian rhythms. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Transmembrane Ca 2+ influx is critical for molecular rhythmicity, metabolic activity, and neuropeptide release in the central clock of the suprachiasmatic nucleus (SCN). We previously reported that both the Na + /Ca 2+ exchanger (NCX) and mitochondria play a role in regulating intracellular Ca 2+ homeostasis in the rat SCN neurons. Here we present evidence to show differential regulation by NCX and mitochondria of nimodipine-sensitive and -insensitive Ca 2+ influx., Methods: Ratiometric Ca 2+ imaging was used to measure change in [Ca 2+ ] i and patch clamp recordings to study spontaneous firing, membrane potential, and voltage-dependent Ca 2+ channels in neurons from reduced SCN slice preparations. Immunofluorescent staining was used to determine the distribution pattern of CaV1.2 and CaV1.3 and their colocalization with NCX1., Results: Ratiometric Ca 2+ imaging indicates that nimodipine (2 μM) blocked most of 20 (mM) K + -induced, but less so of 50 K + -induced, Ca 2+ rise. The nimodipine-sensitive 50 K + -induced Ca 2+ transient rose more rapidly but decayed similarly with the nimodipine-insensitive component, suggesting both components were extruded by NCX. Immunofluorescent stains showed the expression of both CaV1.2 and CaV1.3 and their colocalization with NCX1, whereas functional studies suggest that CaV1.2 mediated most of the nimodipine-sensitive Ca 2+ rise but had insignificant effect on spontaneous firing. After normalization relative to the Ca 2+ -free solution, nimodipine reduced ~ 65% of basal Ca 2+ influx, and TTX lowered it by ~ 35%, leaving ~ 25% basal Ca 2+ influx in the combined presence of TTX and nimodipine. With the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to inhibit mitochondrial Ca 2+ uptake, 20 K + -induced Ca 2+ transients became larger and slower, both in the absence and presence of nimodipine. FCCP markedly enhanced nimodipine-insensitive, but not nimodipine-sensitive, Ca 2+ transients, suggesting that mitochondria preferentially buffer nimodipine-insensitive Ca 2+ influx. Results from using CaV2 channel blockers further indicate that FCCP enhanced Ca 2+ transients mediated by N-, P/Q-, and the blocker cocktail-insensitive Ca 2+ channels., Conclusions: The differential regulation of transmembrane Ca 2+ influx by NCX and mitochondria suggests that Ca 2+ entry via different sources may be regulated differently to play different roles in SCN physiology.

In rodents, restricted food access to a limited period each day at a predictable time results in the appearance of food anticipatory activity (FAA). Two shorter periods of food access each day can result in two FAA bouts. In this study, we examine FAA under 12:12 and 18:6 photoperiods in mice (Mus musculus) with one or two food access periods per day and measure the activation of the suprachiasmatic, dorsomedial and arcuate nuclei by assaying Fos protein expression, while making use of tissue-type plasminogen activator knockout mice to assess the role of neural plasticity in adaptation to restricted feeding cycles. Long days were utilised to allow for temporal separation of two restricted feeding periods during the light phase. Mice fed twice per day generally divided FAA into two distinct bouts, with mice lacking tissue-type plasminogen activator showing reduced FAA. Increases in Fos expression in response to one restricted feeding period per day were seen in the dorsomedial and arcuate nuclei in both 12:12 and 18:6 conditions, with an increase seen in the SCN in only the 12:12 condition. These increases were eliminated or reduced in the two feeding time conditions (done in 18:6 only). Both activity patterns and Fos expression differed for single restricted feeding times between 18:6 and 12:12 photoperiods. Fos activation was lower during RF in 18:6 than 12:12 across all three brain regions, a pattern not reflective of changes in FAA. These data suggest that involvement of these regions in FAA may be influenced by photoperiodic context., (© 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Several reports have described excitatory GABA transmission in the suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. However, there is disagreement regarding the prevalence, timing, and neuronal location of excitatory GABA transmission in the SCN. Whether GABA is inhibitory or excitatory depends, in part, on the intracellular concentration of chloride ([Cl - ] i ). Here, using ratiometric Cl - imaging, we have investigated intracellular chloride regulation in AVP and VIP-expressing SCN neurons and found evidence suggesting that [Cl - ] i is higher during the day than during the night in both AVP+ and VIP+ neurons. We then investigated the contribution of the cation chloride cotransporters to setting [Cl - ] i in these SCN neurons and found that the chloride uptake transporter NKCC1 contributes to [Cl - ] i regulation in SCN neurons, but that the KCCs are the primary regulators of [Cl - ] i in SCN neurons. Interestingly, we observed that [Cl - ] i is differentially regulated between AVP+ and VIP+ neurons-a low concentration of the loop diuretic bumetanide had differential effects on AVP+ and VIP+ neurons, while blocking the KCCs with VU0240551 had a larger effect on VIP+ neurons compared to AVP+ neurons.