Sex-specific differences in the circadian pattern of action potential firing by rat suprachiasmatic nucleus vasopressin neurons.

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.

Sodium regulates clock time and output via an excitatory GABAergic pathway.

The suprachiasmatic nucleus (SCN) serves as the body's master circadian clock that adaptively coordinates changes in physiology and behaviour in anticipation of changing requirements throughout the 24-h day-night cycle1-4. For example, the SCN opposes overnight adipsia by driving water intake before sleep5,6, and by driving the secretion of anti-diuretic hormone7,8 and lowering body temperature9,10 to reduce water loss during sleep11. These responses can also be driven by central osmo-sodium sensors to oppose an unscheduled rise in osmolality during the active phase12-16. However, it is unknown whether osmo-sodium sensors require clock-output networks to drive homeostatic responses. Here we show that a systemic salt injection (hypertonic saline) given at Zeitgeber time 19-a time at which SCNVP (vasopressin) neurons are inactive-excited SCNVP neurons and decreased non-shivering thermogenesis (NST) and body temperature. The effects of hypertonic saline on NST and body temperature were prevented by chemogenetic inhibition of SCNVP neurons and mimicked by optogenetic stimulation of SCNVP neurons in vivo. Combined anatomical and electrophysiological experiments revealed that osmo-sodium-sensing organum vasculosum lamina terminalis (OVLT) neurons expressing glutamic acid decarboxylase (OVLTGAD) relay this information to SCNVP neurons via an excitatory effect of γ-aminobutyric acid (GABA). Optogenetic activation of OVLTGAD neuron axon terminals excited SCNVP neurons in vitro and mimicked the effects of hypertonic saline on NST and body temperature in vivo. Furthermore, chemogenetic inhibition of OVLTGAD neurons blunted the effects of systemic hypertonic saline on NST and body temperature. Finally, we show that hypertonic saline significantly phase-advanced the circadian locomotor activity onset of mice. This effect was mimicked by optogenetic activation of the OVLTGAD→ SCNVP pathway and was prevented by chemogenetic inhibition of OVLTGAD neurons. Collectively, our findings provide demonstration that clock time can be regulated by non-photic physiologically relevant cues, and that such cues can drive unscheduled homeostatic responses via clock-output networks.

Depolarizing GABA Transmission Restrains Activity-Dependent Glutamatergic Synapse Formation in the Developing Hippocampal Circuit.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mature brain but has the paradoxical property of depolarizing neurons during early development. Depolarization provided by GABAA transmission during this early phase regulates neural stem cell proliferation, neural migration, neurite outgrowth, synapse formation, and circuit refinement, making GABA a key factor in neural circuit development. Importantly, depending on the context, depolarizing GABAA transmission can either drive neural activity or inhibit it through shunting inhibition. The varying roles of depolarizing GABAA transmission during development, and its ability to both drive and inhibit neural activity, makes it a difficult developmental cue to study. This is particularly true in the later stages of development when the majority of synapses form and GABAA transmission switches from depolarizing to hyperpolarizing. Here, we addressed the importance of depolarizing but inhibitory (or shunting) GABAA transmission in glutamatergic synapse formation in hippocampal CA1 pyramidal neurons. We first showed that the developmental depolarizing-to-hyperpolarizing switch in GABAA transmission is recapitulated in organotypic hippocampal slice cultures. Based on the expression profile of K+-Cl- co-transporter 2 (KCC2) and changes in the GABA reversal potential, we pinpointed the timing of the switch from depolarizing to hyperpolarizing GABAA transmission in CA1 neurons. We found that blocking depolarizing but shunting GABAA transmission increased excitatory synapse number and strength, indicating that depolarizing GABAA transmission can restrain glutamatergic synapse formation. The increase in glutamatergic synapses was activity-dependent but independent of BDNF signaling. Importantly, the elevated number of synapses was stable for more than a week after GABAA inhibitors were washed out. Together these findings point to the ability of immature GABAergic transmission to restrain glutamatergic synapse formation and suggest an unexpected role for depolarizing GABAA transmission in shaping excitatory connectivity during neural circuit development.

Detection of activity-dependent vasopressin release from neuronal dendrites and axon terminals using sniffer cells.

Our understanding of neuropeptide function within neural networks would be improved by methods allowing dynamic detection of peptide release in living tissue. We examined the usefulness of sniffer cells as biosensors to detect endogenous vasopressin (VP) release in rat hypothalamic slices and from isolated neurohypophyses. Human embryonic kidney cells were transfected to express the human V1a VP receptor (V1aR) and the genetically encoded calcium indicator GCaMP6m. The V1aR couples to Gq11, thus VP binding to this receptor causes an increase in intracellular [Ca2+] that can be detected by a rise in GCaMP6 fluorescence. Dose-response analysis showed that VP sniffer cells report ambient VP levels >10 pM (EC50 = 2.6 nM), and this effect could be inhibited by the V1aR antagonist SR 49059. When placed over a coverslip coated with sniffer cells, electrical stimulation of the neurohypophysis provoked a reversible, reproducible, and dose-dependent increase in VP release using as few as 60 pulses delivered at 3 Hz. Suspended sniffer cells gently plated over a slice adhered to the preparation and allowed visualization of VP release in discrete regions. Electrical stimulation of VP neurons in the suprachiasmatic nucleus caused significant local release as well as VP secretion in distant target sites. Finally, action potentials evoked in a single magnocellular neurosecretory cell in the supraoptic nucleus provoked significant VP release from the somatodendritic compartment of the neuron. These results indicate that sniffer cells can be used for the study of VP secretion from various compartments of neurons in living tissue. NEW & NOTEWORTHY The specific functional roles of neuropeptides in neuronal networks are poorly understood due to the absence of methods allowing their real-time detection in living tissue. Here, we show that cultured "sniffer cells" can be engineered to detect endogenous release of vasopressin as an increase in fluorescence.

Activation of organum vasculosum neurons and water intake in mice by vasopressin neurons in the suprachiasmatic nucleus.

Previous studies have shown that mice housed under 12:12 h light-dark conditions display a pronounced increase in water intake during a 2-hour anticipatory period (AP) near the end of their active period (Zeitgeber Time ZT; ZT21.5-ZT23.5) compared to the preceding basal period (BP, ZT19.5-ZT21.5). This increased water intake during the AP is not associated with physiological stimuli for thirst, such as food intake, hyperosmolality, hyperthermia, or hypovolemia. Denying mice the water intake supplement during the AP causes them to be dehydrated at wake time. These observations suggest that this form of thirst may be driven by the circadian clock and serve to mitigate the dehydrating effect of absence of water intake during sleep. Here we review recent findings showing that this behavior is mediated by vasopressin (VP) containing neurons in the suprachiasmatic nucleus (SCN). SCN VP neurons project to the organum vasculosum lamina terminalis (OVLT) where the activity dependent release of VP causes excitation of thirst-promoting neurons. SCN VP neurons increase their electrical activity during the AP and the resultant release of VP causes an increase in the action potential firing rate of OVLT neurons. Experiments involving optogenetic control of VP release from the axon terminals of SCN neurons indicate that this network mechanism is necessary and sufficient to mediate pre-sleep water intake in mice. These findings provide insight into the output mechanisms that are used by the central clock to generate circadian rhythms, and reveal that the regulation of water intake contributes to osmoregulatory homeostasis during sleep. This article is protected by copyright. All rights reserved.

The neural basis of homeostatic and anticipatory thirst.

Water intake is one of the most basic physiological responses and is essential to sustain life. The perception of thirst has a critical role in controlling body fluid homeostasis and if neglected or dysregulated can lead to life-threatening pathologies. Clear evidence suggests that the perception of thirst occurs in higher-order centres, such as the anterior cingulate cortex (ACC) and insular cortex (IC), which receive information from midline thalamic relay nuclei. Multiple brain regions, notably circumventricular organs such as the organum vasculosum lamina terminalis (OVLT) and subfornical organ (SFO), monitor changes in blood osmolality, solute load and hormone circulation and are thought to orchestrate appropriate responses to maintain extracellular fluid near ideal set points by engaging the medial thalamic-ACC/IC network. Thirst has long been thought of as a negative homeostatic feedback response to increases in blood solute concentration or decreases in blood volume. However, emerging evidence suggests a clear role for thirst as a feedforward adaptive anticipatory response that precedes physiological challenges. These anticipatory responses are promoted by rises in core body temperature, food intake (prandial) and signals from the circadian clock. Feedforward signals are also important mediators of satiety, inhibiting thirst well before the physiological state is restored by fluid ingestion. In this Review, we discuss the importance of thirst for body fluid balance and outline our current understanding of the neural mechanisms that underlie the various types of homeostatic and anticipatory thirst.

Central and peripheral roles of vasopressin in the circadian defense of body hydration.

Vasopressin is a neuropeptide synthesized by specific subsets of neurons within the eye and brain. Studies in rats and mice have shown that vasopressin produced by magnocellular neurosecretory cells (MNCs) that project to the neurohypophysis is released into the blood circulation where it serves as an antidiuretic hormone to promote water reabsorption from the kidney. Moreover vasopressin is a neurotransmitter and neuromodulator that contributes to time-keeping within the master circadian clock (i.e. the suprachiasmatic nucleus, SCN) and is also used as an output signal by SCN neurons to direct centrally mediated circadian rhythms. In this chapter, we review recent cellular and network level studies in rodents that have provided insight into how circadian rhythms in vasopressin mediate changes in water intake behavior and renal water conservation that protect the body against dehydration during sleep.