NaX Channel Is a Physiological [Na+] Detector in Oxytocin- and Vasopressin-Releasing Magnocellular Neurosecretory Cells of the Rat Supraoptic Nucleus.

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.

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.

Synaptic control of rat magnocellular neurosecretory cells by warm-sensing neurons in the organum vasculosum lamina terminalis.

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.

Mechanism and function of phasic firing in vasopressin-releasing magnocellular neurosecretory cells.

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.

High dietary salt amplifies osmoresponsiveness in vasopressin-releasing neurons.

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.

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.

Trpv4 Mediates Hypotonic Inhibition of Central Osmosensory Neurons via Taurine Gliotransmission.

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.

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.

Role of Vasopressin in Rat Models of Salt-Dependent Hypertension.

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 GABAA 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. 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.

eIF2α phosphorylation controls thermal nociception.

A response to environmental stress is critical to alleviate cellular injury and maintain cellular homeostasis. Eukaryotic initiation factor 2 (eIF2) is a key integrator of cellular stress responses and an important regulator of mRNA translation. Diverse stress signals lead to the phosphorylation of the α subunit of eIF2 (Ser51), resulting in inhibition of global protein synthesis while promoting expression of proteins that mediate cell adaptation to stress. Here we report that eIF2α is instrumental in the control of noxious heat sensation. Mice with decreased eIF2α phosphorylation (eIF2α+/S51A) exhibit reduced responses to noxious heat. Pharmacological attenuation of eIF2α phosphorylation decreases thermal, but not mechanical, pain sensitivity, whereas increasing eIF2α phosphorylation has the opposite effect on thermal nociception. The impact of eIF2α phosphorylation (p-eIF2α) on thermal thresholds is dependent on the transient receptor potential vanilloid 1. Moreover, we show that induction of eIF2α phosphorylation in primary sensory neurons in a chronic inflammation pain model contributes to thermal hypersensitivity. Our results demonstrate that the cellular stress response pathway, mediated via p-eIF2α, represents a mechanism that could be used to alleviate pathological heat sensation.

Adult NG2-Glia Are Required for Median Eminence-Mediated Leptin Sensing and Body Weight Control.

While leptin is a well-known regulator of body fat mass, it remains unclear how circulating leptin is sensed centrally to maintain energy homeostasis. Here we show that genetic and pharmacological ablation of adult NG2-glia (also known as oligodendrocyte precursors), but not microglia, leads to primary leptin resistance and obesity in mice. We reveal that NG2-glia contact the dendritic processes of arcuate nucleus leptin receptor (LepR) neurons in the median eminence (ME) and that these processes degenerate upon NG2-glia elimination, which explains the consequential attenuation of these neurons' molecular and electrical responses to leptin. Our data therefore indicate that LepR dendrites in the ME represent the principal conduits of leptin's anorexigenic action and that NG2-glia are essential for their maintenance. Given that ME-directed X-irradiation confirmed the pharmacological and genetically mediated ablation effects on body weight, our findings provide a rationale for the known obesity risk associated with cranial radiation therapy.

Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling.

Astrocytes are specialized and heterogeneous cells that contribute to central nervous system function and homeostasis. However, the mechanisms that create and maintain differences among astrocytes and allow them to fulfill particular physiological roles remain poorly defined. We reveal that neurons actively determine the features of astrocytes in the healthy adult brain and define a role for neuron-derived sonic hedgehog (Shh) in regulating the molecular and functional profile of astrocytes. Thus, the molecular and physiological program of astrocytes is not hardwired during development but, rather, depends on cues from neurons that drive and sustain their specialized properties.

Effects of Peritoneal Sepsis on Rat Central Osmoregulatory Neurons Mediating Thirst and Vasopressin Release.

Sepsis is a life-threatening condition caused by the systemic inflammatory response to a bacterial infection. Although much is known about the cellular and molecular changes that characterize the peripheral inflammatory response to sepsis, almost nothing is known of the neuronal changes that cause associated perturbations in the central control of homeostasis. Osmoregulation is one of the key homeostatic systems perturbed during sepsis. In healthy subjects, systemic hypertonicity normally excites osmoreceptor neurons in the organum vasculosum laminae terminalis (OVLT), which then activates downstream neurons that induce a parallel increase in water intake and arginine vasopressin (AVP) secretion to promote fluid expansion and maintain blood pressure. However, recent studies have shown that the early phase of sepsis is associated with increased AVP levels and suppressed thirst. Here we examined the electrophysiological properties of OVLT neurons and magnocellular neurosecretory cells (MNCs) in acute in vitro preparations obtained from rats subjected to sham surgery or cecal ligation and puncture (CLP). We found that the intrinsic excitability of OVLT neurons was not affected significantly 18-24 h after CLP. However, OVLT neurons in CLP rats were hyperpolarized significantly compared with shams. Moreover, a reduced proportion of these cells displayed spontaneous electrical activity and osmoresponsiveness in septic animals. In contrast, the osmoresponsiveness of MNCs was only attenuated by CLP, and a larger proportion of these neurons displayed spontaneous electrical activity in septic animals. These results suggest that acute sepsis disrupts centrally mediated osmoregulatory reflexes through differential effects on the properties of neurons in the OVLT and supraoptic nucleus. SIGNIFICANCE STATEMENT: Sepsis is a life-threatening condition caused by the systemic inflammatory response to bacterial infection. Although the early phase of sepsis features impaired thirst and enhanced vasopressin release, the basis for these defects is unknown. Here, we show that cecal ligation and puncture (CLP) in rats impairs the osmoresponsiveness of neurons in the organum vasculosum lamina terminalis (OVLT; which drives thirst) and attenuates that of neurosecretory neurons in the supraoptic nucleus (SON; which secrete oxytocin and vasopressin). Notably, we found that OVLT neurons are hyperpolarized and electrically silenced. In contrast, CLP increased the proportion of SON neurons displaying spontaneous electrical activity. Therefore, CLP affects the properties of osmoregulatory neurons in a manner that can affect systemic osmoregulation.

ΔN-TRPV1: A Molecular Co-detector of Body Temperature and Osmotic Stress.

Thirst and antidiuretic hormone secretion occur during hyperthermia or hypertonicity to preserve body hydration. These vital responses are triggered when hypothalamic osmoregulatory neurons become depolarized by ion channels encoded by an unknown product of the transient receptor potential vanilloid-1 gene (Trpv1). Here, we show that rodent osmoregulatory neurons express a transcript of Trpv1 that mediates the selective translation of a TRPV1 variant that lacks a significant portion of the channel's amino terminus (ΔN-TRPV1). The mRNA transcript encoding this variant (Trpv1dn) is widely expressed in the brains of osmoregulating vertebrates, including the human hypothalamus. Transfection of Trpv1dn into heterologous cells induced the expression of ion channels that could be activated by either hypertonicity or by heating in the physiological range. Moreover, expression of Trpv1dn rescued the osmosensory and thermosensory responses of single hypothalamic neurons obtained from Trpv1 knockout mice. ΔN-TRPV1 is therefore a co-detector of core body temperature and fluid tonicity.

Anatomical organization of the rat organum vasculosum laminae terminalis.

The organum vasculosum of the laminae terminalis (OVLT) is a circumventricular organ located along the ventral part of the anterior wall of the third ventricle. Because it lacks a complete blood-brain barrier (BBB), blood-borne signals detected in the OVLT provide the brain with information from the periphery and contribute to the generation of centrally mediated responses to humoral feedback and physiological stressors. Experimental studies on the rat OVLT are hindered by a poor understanding of its precise anatomical dimensions and cellular organization. In this study, we use histological techniques to characterize the spatial outline of the rat OVLT and to examine the location of neurons, astrocytes, tanycytes, and ependymocytes within its confines. Our data reveal that OVLT neurons are embedded in a dense network of tanycyte processes. Immunostaining against the neuronal marker NeuN revealed that neurons are distributed throughout the OVLT, except for a thick midline septum, which comprises densely packed cells of unknown function or lineage. Moreover, the most ventral aspect of the OVLT is devoid of neurons and is occupied by a dense network of glial cell processes that form a thick layer between the neurons and the pial surface on the ventral aspect of the nucleus. Lastly, combined detection of NeuN and c-Fos protein following systemic injection of hypertonic NaCl revealed that neurons responsive to this stimulus are located along the entire midline core of the OVLT, extending from its most anterior ventral aspect to the more caudally located "dorsal cap" region.