Developmental Patterning of Peptide Transcription in the Central Circadian Clock in Both Sexes

Introduction: Neuropeptide signaling modulates the function of central clock neurons in the suprachiasmatic nucleus (SCN) during development and adulthood. Arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) are expressed early in SCN development, but the precise timing of transcriptional onset has been difficult to establish due to age-related changes in the rhythmic expression of each peptide. Methods: To provide insight into spatial patterning of peptide transcription during SCN development, we used a transgenic approach to define the onset of Avp and Vip transcription. Avp-Cre or Vip-Cre males were crossed to Ai9+/+ females, producing offspring in which the fluorescent protein tdTomato (tdT) is expressed at the onset of Avp or Vip transcription. Spatial patterning of Avp-tdT and Vip-tdT expression was examined at critical developmental time points spanning mid-embryonic age to adulthood in both sexes. Results: We find that Avp-tdT and Vip-tdT expression is initiated at different developmental time points in spatial subclusters of SCN neurons, with developmental patterning that differs by sex. Conclusions: These data suggest that SCN neurons can be distinguished into further subtypes based on the developmental patterning of neuropeptide expression, which may contribute to regional and/or sex differences in cellular function in adulthood

Long Days Enhance Recognition Memory and Increase Insulin-like Growth Factor 2 in the Hippocampus

Light improves cognitive function in humans; however, the neurobiological mechanisms underlying positive effects of light remain unclear. One obstacle is that most rodent models have employed lighting conditions that cause cognitive deficits rather than improvements. Here we have developed a mouse model where light improves cognitive function, which provides insight into mechanisms underlying positive effects of light. To increase light exposure without eliminating daily rhythms, we exposed mice to either a standard photoperiod or a long day photoperiod. Long days enhanced long-term recognition memory, and this effect was abolished by loss of the photopigment melanopsin. Further, long days markedly altered hippocampal clock function and elevated transcription of Insulin-like Growth Factor2 (Igf2). Up-regulation of Igf2 occurred in tandem with suppression of its transcriptional repressor Wilm’s tumor1. Consistent with molecular de-repression of Igf2, IGF2 expression was increased in the hippocampus before and after memory training. Lastly, long days occluded IGF2-induced improvements in recognition memory. Collectively, these results suggest that light changes hippocampal clock function to alter memory, highlighting novel mechanisms that may contribute to the positive effects of light. Furthermore, this study provides insight into how the circadian clock can regulate hippocampus-dependent learning by controlling molecular processes required for memory consolidation

Seasonal Plasticity in GABA\u3csup\u3eA\u3c/sup\u3e Signaling is Necessary for Restoring Phase Synchrony in the Master Circadian Clock Network

Annual changes in the environment threaten survival, and numerous biological processes in mammals adjust to this challenge via seasonal encoding by the suprachiasmatic nucleus (SCN). To tune behavior according to day length, SCN neurons display unified rhythms with synchronous phasing when days are short, but will divide into two sub-clusters when days are long. The transition between SCN states is critical for maintaining behavioral responses to seasonal change, but the mechanisms regulating this form of neuroplasticity remain unclear. Here we identify that a switch in chloride transport and GABAA signaling is critical for maintaining state plasticity in the SCN network. Further, we reveal that blocking excitatory GABAA signaling locks the SCN into its long day state. Collectively, these data demonstrate that plasticity in GABAA signaling dictates how clock neurons interact to maintain environmental encoding. Further, this work highlights factors that may influence susceptibility to seasonal disorders in humans

Reduced VIP Expression Affects Circadian Clock Function in VIP-IRES-CRE Mice (JAX 010908)

Circadian rhythms are programmed by the suprachiasmatic nucleus (SCN), which relies on neuropeptide signaling to maintain daily timekeeping. Vasoactive intestinal polypeptide (VIP) is critical for SCN function, but the precise role of VIP neurons in SCN circuits is not fully established. To interrogate their contribution to SCN circuits, VIP neurons can be manipulated specifically using the DNA-editing enzyme Cre recombinase. Although the Cre transgene is assumed to be inert by itself, we find that VIP expression is reduced in both heterozygous and homozygous adult VIP-IRES-Cre mice (JAX 010908). Compared with wild-type mice, homozygous VIP-Cre mice display faster reentrainment and shorter free-running period but do not become arrhythmic in constant darkness. Consistent with this phenotype, homozygous VIP-Cre mice display intact SCN PER2::LUC rhythms, albeit with altered period and network organization. We present evidence that the ability to sustain molecular rhythms in the VIP-Cre SCN is not due to residual VIP signaling; rather, arginine vasopressin signaling helps to sustain SCN function at both intracellular and intercellular levels in this model. This work establishes that the VIP-IRES-Cre transgene interferes with VIP expression but that loss of VIP can be mitigated by other neuropeptide signals to help sustain SCN function. Our findings have implications for studies employing this transgenic model and provide novel insight into neuropeptide signals that sustain daily timekeeping in the master clock

Somatostatin Regulates Central Clock Function and Circadian Responses to Light

Daily and annual changes in light are processed by central clock circuits that control the timing of behavior and physiology. The suprachiasmatic nucleus (SCN) in the anterior hypothalamus processes daily photic inputs and encodes changes in day length (i.e., photoperiod), but the SCN circuits that regulate circadian and photoperiodic responses to light remain unclear. Somatostatin (SST) expression in the hypothalamus is modulated by photoperiod, but the role of SST in SCN responses to light has not been examined. Our results indicate that SST signaling regulates daily rhythms in behavior and SCN function in a manner influenced by sex. First, we use cell-fate mapping to provide evidence that SST in the SCN is regulated by light via de novo Sst activation. Next, we demonstrate that Sst  -/- mice display enhanced circadian responses to light, with increased behavioral plasticity to photoperiod, jetlag, and constant light conditions. Notably, lack of Sst  -/- eliminated sex differences in photic responses due to increased plasticity in males, suggesting that SST interacts with clock circuits that process light differently in each sex. Sst  -/- mice also displayed an increase in the number of retinorecipient neurons in the SCN core, which express a type of SST receptor capable of resetting the molecular clock. Last, we show that lack of SST signaling modulates central clock function by influencing SCN photoperiodic encoding, network after-effects, and intercellular synchrony in a sex-specific manner. Collectively, these results provide insight into peptide signaling mechanisms that regulate central clock function and its response to light

Organic Cation Transporter 3 and the Dopamine Transporter Differentially Regulate Catecholamine Uptake in the Basolateral Amygdala and Nucleus Accumbens

Regional alterations in kinetics of catecholamine uptake are due in part to variations in clearance mechanisms. The rate of clearance is a critical determinant of the strength of catecholamine signaling. Catecholamine transmission in the nucleus accumbens core (NAcc) and basolateral amygdala (BLA) is of particular interest due to involvement of these regions in cognition and motivation. Previous work has shown that catecholamine clearance in the NAcc is largely mediated by the dopamine transporter (DAT), but clearance in the BLA is less DAT‐dependent. A growing body of literature suggests that organic cation transporter 3 (OCT3) also contributes to catecholamine clearance in both regions. Consistent with different clearance mechanisms between regions, catecholamine clearance is more rapid in the NAcc than in the BLA, though mechanisms underlying this have not been resolved. We compared the expression of DAT and OCT3 and their contributions to catecholamine clearance in the NAcc and BLA. We found DAT protein levels were ~ 4‐fold higher in the NAcc than in the BLA, while OCT3 protein expression was similar between the two regions. Immunofluorescent labeling of the two transporters in brain sections confirmed these findings. Ex vivo voltammetry demonstrated that the magnitude of catecholamine release was greater, and the clearance rate was faster in the NAcc than in the BLA. Additionally, catecholamine clearance in the BLA was more sensitive to the OCT3 inhibitor corticosterone, while clearance in the NAcc was more cocaine sensitive. These distinctions in catecholamine clearance may underlie differential effects of catecholamines on behavioral outputs mediated by these regions

Arginine Vasopressin Modulates Circadian Timekeeping in a Sex-Dependent Manner

Daily rhythms in behavior and physiology are programmed by the circadian system, which is a collection of endogenous biological clocks that allow organisms to anticipate recurring daily environmental events relevant for survival. The suprachiasmatic nucleus (SCN) acts as the central clock coordinating downstream tissues to the environment, and SCN signaling is critical for circadian timekeeping. Arginine vasopressin (AVP) is produced by a subset of SCN neurons, which is transduced by the V1A and V1B receptors. V1A and V1B are expressed in the SCN itself, suggesting AVP signaling modulates central clock function through mechanisms that remain unclear. To gain deeper insight into the role of AVP signaling in the central clock, here I tested that AVP is necessary and sufficient to modulate SCN function using pharmacological and genetic approaches. First, I used V1 antagonists to test that AVP signaling modulates SCN molecular rhythms in vitro. V1 antagonism lengthened period and altered phase relationships among SCN neurons in a region-specific manner, suggesting differential roles for V1A and V1B receptors. Second, I used an AVP deficient mouse model to test how AVP signaling modulates circadian rhythms in locomotor behavior. Loss of AVP signaling increased the period, imprecision, and plasticity of behavioral rhythms in a manner influenced by sex, indicating that AVP circuits are sexually dimorphic. Last, I used V1 receptor agonists to test that AVP signaling is sufficient to reset the SCN clock in vitro. V1 agonists reset the SCN molecular clock, with larger responses in females. Further, I found that both Gαq and Gαs signaling are necessary for the V1-induced SCN resetting, which indicates that AVP interacts with other SCN neuropeptides to modulate central clock function. Overall, this work provides novel insights by revealing AVP signaling alters SCN period, phase, and precision by interacting with other signals in a manner that varies by sex

Vasopressin Resets the Central Circadian Clock in a Manner Influenced by Sex and Vasoactive Intestinal Polypeptide Signaling

Background/Aims: Circadian rhythms in behavior and physiology are programmed by the suprachiasmatic nucleus (SCN) of the hypothalamus. A subset of SCN neurons produce the neuropeptide arginine vasopressin (AVP), but it remains unclear whether AVP signaling influences the SCN clock directly. Methods: Here, we test that AVP signaling acting through V1A and V1B receptors influences molecular rhythms in SCN neurons. V1 receptor agonists were applied ex vivo to PERIOD2::LUCIFERASE SCN slices, allowing for real-time monitoring of changes in molecular clock function. Results: V1A/B agonists reset the phase of the SCN molecular clock in a time-dependent manner, with larger magnitude responses by the female SCN. Further, we found evidence that both Gαq and Gαs signaling pathways interact with V1A/B-induced SCN resetting, and that this response requires vasoactive intestinal polypeptide (VIP) signaling. Conclusions: Collectively, this work indicates that AVP signaling resets SCN molecular rhythms in conjunction with VIP signaling and in a manner influenced by sex. This highlights the utility of studying clock function in both sexes and suggests that signal integration in central clock circuits regulates emergent properties important for the control of daily rhythms in behavior and physiology

Vasopressin Regulates Daily Rhythms and Circadian Clock Circuits in a Manner Influenced by Sex

Arginine vasopressin (AVP) is a neurohormone that alters cellular physiology through both endocrine and synaptic signaling. Circadian rhythms in AVP release and other biological processes are driven by the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Loss of vasopressin signaling alters circadian behavior, but the basis of these effects remains unclear. Here we investigate the role of AVP signaling in circadian timekeeping by analyzing behavior and SCN function in a novel AVP-deficient mouse model. Consistent with previous work, loss of AVP signaling increases water consumption and accelerates recovery to simulated jetlag. We expand on these results to show that loss of AVP increases period, imprecision and plasticity of behavioral rhythms under constant darkness. Interestingly, the effect of AVP deficiency on circadian period was influenced by sex, with loss of AVP lengthening period in females but not males. Examining SCN function directly with ex vivo bioluminescence imaging of clock protein expression, we demonstrate that loss of AVP signaling modulates the period, precision, and phase relationships of SCN neurons in both sexes. This pattern of results suggests that there are likely sex differences in downstream targets of the SCN. Collectively, this work indicates that AVP signaling modulates circadian circuits in a manner influenced by sex, which provides new insight into sexual dimorphisms in the regulation of daily rhythms

Developmental patterning of peptide transcription in the central circadian clock in both sexes

IntroductionNeuropeptide signaling modulates the function of central clock neurons in the suprachiasmatic nucleus (SCN) during development and adulthood. Arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) are expressed early in SCN development, but the precise timing of transcriptional onset has been difficult to establish due to age-related changes in the rhythmic expression of each peptide.MethodsTo provide insight into spatial patterning of peptide transcription during SCN development, we used a transgenic approach to define the onset of Avp and Vip transcription. Avp-Cre or Vip-Cre males were crossed to Ai9+/+ females, producing offspring in which the fluorescent protein tdTomato (tdT) is expressed at the onset of Avp or Vip transcription. Spatial patterning of Avp-tdT and Vip-tdT expression was examined at critical developmental time points spanning mid-embryonic age to adulthood in both sexes.ResultsWe find that Avp-tdT and Vip-tdT expression is initiated at different developmental time points in spatial subclusters of SCN neurons, with developmental patterning that differs by sex.ConclusionsThese data suggest that SCN neurons can be distinguished into further subtypes based on the developmental patterning of neuropeptide expression, which may contribute to regional and/or sex differences in cellular function in adulthood