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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, VPAC2, are synthesized by SCN neurons and this intercellular VIP‐VPAC2 receptor signal facilitates coordination of SCN neuronal activity and timekeeping. How the loss of VPAC2 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 VPAC2 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‐VPAC2 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. [ABSTRACT FROM AUTHOR]

Temporal control of brain and behavioral states emerges as a consequence of the interaction between circadian and homeostatic neural circuits. This interaction permits the daily rhythm of sleep and wake, regulated in parallel by circadian cues originating from the suprachiasmatic nuclei (SCN) and arousal-promoting signals arising from the orexin-containing neurons in the tuberal hypothalamus (TH). Intriguingly, the SCN circadian clock can be reset by arousal-promoting stimuli while activation of orexin/hypocretin neurons is believed to be under circadian control, suggesting the existence of a reciprocal relationship. Unfortunately, since orexin neurons are themselves activated by locomotor promoting cues, it is unclear how these two systems interact to regulate behavioral rhythms. Here mice were placed in conditions of constant light, which suppressed locomotor activity, but also revealed a highly pronounced circadian pattern in orexin neuronal activation. Significantly, activation of orexin neurons in the medial and lateral TH occurred prior to the onset of sustained wheel-running activity. Moreover, exposure to a 6 h dark pulse during the subjective day, a stimulus that promotes arousal and phase advances behavioral rhythms, activated neurons in the medial and lateral TH including those containing orexin. Concurrently, this stimulus suppressed SCN activity while activating cells in the median raphe. In contrast, dark pulse exposure during the subjective night did not reset SCN-controlled behavioral rhythms and caused a transient suppression of neuronal activation in the TH. Collectively these results demonstrate, for the first time, pronounced circadian control of orexin neuron activation and implicate recruitment of orexin cells in dark pulse resetting of the SCN circadian clock.

Vasoactive intestinal polypeptide and its receptor, VPAC2, play important roles in the functioning of the brain’s circadian clock in the suprachiasmatic nuclei (SCN). Mice lacking VPAC2 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 VPAC2 receptors in WT SCN slices partially mimicked the Vipr2− /− phenotype. These data demonstrate that intercellular communication via the VPAC2 receptor is important for SCN neurons to sustain robust, synchronous oscillations in clock gene expression. [ABSTRACT FROM AUTHOR]

Individual neurons in the suprachiasmatic nuclei (SCN) contain an intracellular molecular clock and use intercellular signaling to synchronize their timekeeping activities so that the SCN can coordinate brain physiology and behavior. The neuropeptide vasoactive intestinal polypeptide (VIP) and its VPAC2 receptor form a key component of intercellular signaling systems in the SCN and critically control cellular coupling. Targeted mutations in either the intracellular clock or intercellular neuropeptide signaling mechanisms, such as VIP-VPAC2 signaling, can lead to desynchronization of SCN neuronal clocks and loss of behavioral rhythms. An important goal in chronobiology is to develop interventions to correct deficiencies in circadian timekeeping. Here we show that extended exposure to constant light promotes synchrony among SCN clock cells and the expression of ~24 h rhythms in behavior in mice in which intercellular signaling is disrupted through loss of VIP-VPAC2 signaling. This study highlights the importance of SCN synchrony for the expression of rhythms in behavior and reveals how non-invasive manipulations in the external environment can be used to overcome neurochemical communication deficits in this important brain system. [ABSTRACT FROM AUTHOR]