Osmosensory neurons detect hypertonic stress when ΔN-TRPV1 channels are activated through a push force delivered by microtubules during cell shrinking, and this process requires an essential yet unknown contribution from actin filaments. Here, we show that the actin cortex of these neurons feature fenestrations that allow for the formation of pits that magnify the osmotically induced displacement of the plasma membrane compared to that expected from uniform shrinking. Furthermore, we found that many N-terminal variant of the transient receptor potential vanilloid 1 (ΔN-TRPV1) channels attached to microtubules are aligned with fenestrae and such sites undergo greater hypertonicity-induced displacement than predicted by geometrically uniform cell shrinking. These results indicate that actin filaments contribute to the establishment of nanoscale architecture at sites which may optimize osmosensory transduction.

The maintenance of extracellular fluid (ECF) osmolality and sodium concentration ([Na+]o) near optimal "set point" values sustains physiological functions and prevents pathological states such as hypo- and hypernatremia. The peptide hormones vasopressin (antidiuretic hormone) and oxytocin (a natriuretic hormone in rats) play key roles in this process. These hormones are synthesized by hypothalamic magnocellular neurosecretory cells (MNCs) that project to the neurohypophysis and are released into the systemic circulation in response to rises in ECF osmolality or [Na+]o. These homeostatic responses are highly sensitive. For example, vasopressin release is elicited by an increase in ECF osmolality as small as ≥1%. The osmotic and sodium-dependent control of vasopressin and oxytocin release at the neurohypophysis is directly regulated by the electrical activity of MNCs. This regulation involves an array of mechanisms that include synaptic inputs from the brain and periphery, the effects of chemicals released by glial cells, and intrinsic sensory properties of MNCs. These overlapping mechanisms may offer an important degree of redundancy for the homeostatic control of vasopressin and oxytocin release and contribute to the high sensitivity of these responses. Recent work has shown that the intrinsic sodium sensitivity and osmosensitivity of MNCs play an important role in the control of these neurons in vivo. This review provides an update of our current understanding of the molecular and cellular mechanisms that contribute to the cell-autonomous sensory properties of MNCs.

Neuron-glia interactions play a central role in regulating synaptic transmission and neuronal excitability. The structural plasticity of astrocytes is associated with numerous physiological and pathological conditions; however, the mechanism underlying this process remains unknown. To examine the basis for structural astrocyte plasticity, we used the classic example of the loss of astrocytic processes that takes place in the rat hypothalamic magnocellular system during chronic high-salt intake. We discovered that a high-salt diet triggers a local accumulation of reactive microglia around vasopressin-secreting neurons but not in other brain areas. Microglia phagocytose astrocytic processes, reducing astrocytic coverage of vasopressin neurons. The pruning of astrocytic processes impairs synaptic glutamate clearance, enabling activation of extrasynaptic glutamate NMDA receptors and increasing the activity of vasopressin neurons. Inhibiting microglia-mediated astrocyte pruning attenuates the increased neuronal activity and vasopressin-dependent hypertensive phenotype of rats fed a high-salt diet. Thus, microglia orchestrate neuron-glia interactions and regulate neuronal activity through astrocyte pruning.

Central autonomic and endocrine dysfunctions following traumatic brain injury (TBI) are believed to involve the hypothalamus; however, underlying mechanisms are unknown. Although chronic deficits might be caused by irreversible tissue damage, various neuroendocrine and autonomic symptoms are only observed transiently, suggesting they might result from a temporary alteration in the activity of hypothalamic neurons. We therefore examined if a mouse model of mild TBI could induce reversible autonomic phenotypes and cause acute changes in c-Fos expression within corresponding regions of the hypothalamus. Adult C57Bl/6 male mice were lightly anesthetized with isoflurane and subjected to TBI by lateral head impact using a Gothenburg impactor. Mice treated the same way, but without the head impact served as controls (shams). We monitored body weight and core body temperature by infrared thermography and performed immunohistochemistry against c-Fos in various regions of the hypothalamus. We determined that a projectile velocity of 9 m/s significantly delayed recovery from the anesthesia without inducing skull fractures and signs of discomfort disappeared within 3 h, as assessed by grimace scale. Compared with shams, TBI mice displayed a rapid decrease in core body temperature which resolved within 48 h. Daily body weight gain was also significantly lower in TBI mice on the day following injury but recovered thereafter. c-Fos analysis revealed a significantly higher density of c-Fos-positive cells in the paraventricular nucleus and a significantly lower density in the median preoptic nucleus and medial preoptic area. We conclude that mild TBI induced by a single lateral head impact in mice at 9 m/s produces acute and reversible symptoms associated with hypothalamic dysfunction accompanied by significant changes in c-Fos expression within relevant areas of the hypothalamus. These findings support the hypothesis that a temporary alteration of neuronal activity may underlie the expression of reversible central autonomic and neuroendocrine symptoms.

The Scn7A gene encodes NaX, an atypical noninactivating Na+ channel, whose expression in sensory circumventricular organs is essential to maintain homeostatic responses for body fluid balance. However, NaX has also been detected in homeostatic effector neurons, such as vasopressin (VP)-releasing magnocellular neurosecretory cells (MNCVP) that secrete VP (antidiuretic hormone) into the bloodstream in response to hypertonicity and hypernatremia. Yet, the physiological relevance of NaX expression in these effector cells remains unclear. Here, we show that rat MNCVP in males and females is depolarized and excited in proportion with isosmotic increases in [Na+]. These responses were caused by an inward current resulting from a cell-autonomous increase in Na+ conductance. The Na+-evoked current was unaffected by blockers of other Na+-permeable ion channels but was significantly reduced by shRNA-mediated knockdown of Scn7A expression. Furthermore, reducing the density of NaX channels selectively impaired the activation of MNCVP by systemic hypernatremia without affecting their responsiveness to hypertonicity in vivo These results identify NaX as a physiological Na+ sensor, whose expression in MNCVP contributes to the generation of homeostatic responses to hypernatremia.SIGNIFICANCE STATEMENT In this study, we provide the first direct evidence showing that the sodium-sensing channel encoded by the Scn7A gene (NaX) mediates cell-autonomous sodium detection by MNCs in the low millimolar range and that selectively reducing the expression of these channels in MNCs impairs their activation in response to a physiologically relevant sodium stimulus in vitro and in vivo These data reveal that NaX operates as a sodium sensor in these cells and that the endogenous sensory properties of osmoregulatory effector neurons contribute to their homeostatic activation in vivo.

Clinical studies have shown that hyponatremia can be induced by the ingestion of the recreational drug MDMA (3,4-Methyl​enedioxy​methamphetamine, ecstasy) when combined with a water intake (Baggot et al., 2016). However, the mechanisms by which MDMA induces hyponatremia is unknown. Hyponatremia is a life-threatening condition that causes rapid swelling of the brain and is commonly encountered in critical care medicine (emergency) (Woods et al., 1993 & Kirch, Bigelow, Weinberger, 1985). Under the Controlled Drugs and Substances Act (CDSA), MDMA is a schedule 1 drug (illegal) and consumption usually occurs at dance parties or raves. Nonetheless, despite MDMA’s scheduling, the Multidisciplinary Association for Psychedelic Studies (MAPS) has headed the movement toward using MDMA for assisted psychotherapy for PTSD (Sessa, Higbed, & Nutt, 2019). In summary, with a prevalence of 3.5% of young adults 18-25 years old using MDMA in the United States in 2014 (Betzler, Viohl, & Romanczuk-Seiferth, 2017), along with the mainstream marketing of MDMA for PTSD, combined with very serious and potentially deathly side effects makes this substance worthwhile to investigate. Findings could not only aid in MDMA associated hyponatremia but could also provide the foundation clinically for fluid regulation during MDMA assisted psychotherapy. Clinically, ingestion of MDMA has been shown to cause an increase in the hormones vasopressin and oxytocin (Wolf et al., 2006). Therefore, we examined cFos activity in male rats, we show that there is an increase in both cFos activation of vasopressin as well as oxytocin neurons of the supra optic nucleus. Next, we wanted to test the notion that MDMA causes an increase in thirst. Our data shows that an injection of MDMA does not increase drinking behavior. Combatting the conventional thinking that MDMA causes an increase in thirst. Interestingly, in line with the human literature, when you combine MDMA with a gastric water load, it is sufficient to drop serum sodium levels to hyponatremic levels i.e. <135mmol/L. Indicating that a water load combined with MDMA is necessary for MDMA induced hyponatremia. Conventionally, MDMA has been shown to increase the amount of serotonin within the synaptic cleft, via the inhibition of uptake via the serotonin transporter (Oeri, 2021). To determine if MDMA is working via the release of 5HT in a temperature-controlled environment we utilized whole cell patch clamp slice electrophysiology of male double transgenic Wistar rats enabling fluorescent identification of vasopressin and oxytocin neurons. Interestingly, at a bath temperature of a constant 30°C, a bath application of 5HT was sufficient in causing an enhancement of action potential firing, and this could be blocked by a 5HT receptor antagonist. Moreover, a bath application of MDMA also caused an increase in action potential firing, which too could be blocked by a 5HT receptor antagonist. CIHR. HBHL, McGill Faculty of Medicine This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Increases in core body temperature cause secretion of vasopressin (vasopressin, antidiuretic hormone) to promote water reabsorption and blunt water losses incurred through homeostatic evaporative cooling. Subtypes of transient receptor potential vanilloid (Trpv) channels have been shown to contribute to the intrinsic regulation of vasopressin‐releasing magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). However, MNCs in vivo can also be excited by local heating of the adjacent preoptic area, indicating they receive thermosensory information from other areas. Here, we investigated whether neurons in the organum vasculosum lamina terminalis (OVLT) contribute to this process using in vitro electrophysiological approaches in male rats. We found that the majority of OVLT neurons are thermosensitive in the physiological range (36–39°C) and that this property is retained under conditions blocking synaptic transmission. A subset of these neurons could be antidromically activated by electrical stimulation in the SON. Whole cell recordings from SON MNCs revealed that heating significantly increases the rate of spontaneous excitatory postsynaptic currents (sEPCSs), and that this response is abolished by lesions targeting the OVLT, but not by bilateral lesions placed in the adjacent preoptic area. Finally, local heating of the OVLT caused a significant excitation of MNCs in the absence of temperature changes in the SON, and this effect was blocked by inhibitors of ionotropic glutamate receptors. These findings indicate that the OVLT serves as an important thermosensory nucleus and contributes to the activation of MNCs during physiological heating.

High dietary salt increases arterial pressure partly through activation of magnocellular neurosecretory cells (MNCVP) that secrete the antidiuretic and vasoconstrictor hormone vasopressin (VP) into the circulation. Here, we show that the intrinsic and synaptic excitation of MNCVP caused by hypertonicity are differentially potentiated in two models of salt-dependent hypertension in rats. One model combined salty chow with a chronic subpressor dose of angiotensin II (AngII-salt), the other involved replacing drinking water with 2% NaCl (salt loading, SL). In both models, we observed a significant increase in the quantal amplitude of EPSCs on MNCVP. However, model-specific changes were also observed. AngII-salt increased the probability of glutamate release by osmoreceptor afferents and increased overall excitatory network drive. In contrast, SL specifically increased membrane stiffness and the intrinsic osmosensitivity of MNCVP. These results reveal that dietary salt increases the excitability of MNCVP through effects on the cell-autonomous and synaptic osmoresponsiveness of MNCVP.

Magnocellular neurosecretory cells that release vasopressin (MNCVP ) from axon terminals in the neurohypophysis display a unique pattern of action potential firing termed phasic firing. Under basal conditions, only a small proportion of MNCVP display spontaneous phasic firing. However, acute and chronic conditions that stimulate vasopressin release, such as hemorrhage and dehydration, greatly enhance the number of MNCVP that fire phasically. Phasic firing optimizes VP neurosecretion at axon terminals by allowing action potential broadening to promote calcium-dependent frequency-facilitation, at the same time as preventing the secretory fatigue caused by spike inactivation that occurs during prolonged continuous stimulation. This review provides an update on our mechanistic understanding of these processes and highlights important gaps in our knowledge that must be addressed in future experiments.

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 signalling. 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.

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 cycle 1 – 4 . For example, the SCN opposes overnight adipsia by driving water intake before sleep 5 , 6 , and by driving the secretion of anti-diuretic hormone 7 , 8 and lowering body temperature 9 , 10 to reduce water loss during sleep 11 . These responses can also be driven by central osmo-sodium sensors to oppose an unscheduled rise in osmolality during the active phase 12 – 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 SCN VP (vasopressin) neurons are inactive—excited SCN VP 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 SCN VP neurons and mimicked by optogenetic stimulation of SCN VP neurons in vivo. Combined anatomical and electrophysiological experiments revealed that osmo-sodium-sensing organum vasculosum lamina terminalis (OVLT) neurons expressing glutamic acid decarboxylase (OVLT GAD ) relay this information to SCN VP neurons via an excitatory effect of γ-aminobutyric acid (GABA). Optogenetic activation of OVLT GAD neuron axon terminals excited SCN VP neurons in vitro and mimicked the effects of hypertonic saline on NST and body temperature in vivo. Furthermore, chemogenetic inhibition of OVLT GAD 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 OVLT GAD → SCN VP pathway and was prevented by chemogenetic inhibition of OVLT GAD 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.

The maintenance of hydromineral homeostasis requires bidirectional detection of changes in extracellular fluid osmolality by primary osmosensory neurons (ONs) in the organum vasculosum laminae terminalis (OVLT). Hypertonicity excites ONs in part through the mechanical activation of a variant transient receptor potential vanilloid-1 channel (dn-Trpv1). However, the mechanism by which local hypotonicity inhibits ONs in the OVLT remains unknown. Here, we show that hypotonicity can reduce the basal activity of dn-Trpv1 channels and hyperpolarize acutely isolated ONs. Surprisingly, we found that mice lacking dn-Trpv1 maintain normal inhibitory responses to hypotonicity when tested in situ. In the intact setting, hypotonicity inhibits ONs through a non-cell-autonomous mechanism that involves glial release of the glycine receptor agonist taurine through hypotonicity activated anion channels (HAAC) that are activated subsequent to Ca2+ influx through Trpv4 channels. Our study clarifies how Trpv4 channels contribute to the inhibition of OVLT ONs during hypotonicity in situ.

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.

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.

Circadian rhythms have evolved to produce cyclic changes in metabolism or behavior that adapt the body to changing requirements throughout the 24 h light/dark cycle. Here, we highlight recent advances in our understanding of the mechanisms that maintain body hydration and highlight the contribution of the central clock to the generation of endocrine and behavioral rhythms that contribute significantly to this process.

Median preoptic neurons modulate water intake and the urge to drink

Purpose of Review Dietary salt intake increases both plasma sodium and osmolality and therefore increases vasopressin (VP) release from the neurohypophysis. Although this effect could increase blood pressure by inducing fluid reabsorption and vasoconstriction, acute activation of arterial baroreceptors inhibits VP neurons via GABA(A) receptors to oppose high blood pressure. Here we review recent findings demonstrating that this protective mechanism fails during chronic high salt intake in rats.Recent Findings Two recent studies showed that chronic high sodium intake causes an increase in intracellular chloride concentration in VP neurons. This effect causes GABAA receptors to become excitatory and leads to the emergence of VP-dependent hypertension. One study showed that the increase in intracellular chloride was provoked by a decrease in the expression of the chloride exporter KCC2 mediated by local secretion of brain-derived neurotrophic factor and activation of TrkB receptors.Summary Prolonged high dietary salt intake can cause pathological plasticity in a central homeostatic circuit that controls VP secretion and thereby contribute to peripheral vasoconstriction and hypertension.

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.