Integration of Hypernatremia and Angiotensin II by the Organum Vasculosum of the Lamina Terminalis Regulates Thirst.

The organum vasculosum of the lamina terminalis (OVLT) contains NaCl-sensitive neurons to regulate thirst, neuroendocrine function, and autonomic outflow. The OVLT also expresses the angiotensin II (AngII) type1 receptor, and AngII increases Fos expression in OVLT neurons. The present study tested whether individual OVLT neurons sensed both NaCl and AngII to regulate thirst and body fluid homeostasis. A multifaceted approach, including in vitro whole-cell patch recordings, in vivo single-unit recordings, and optogenetic manipulation of OVLT neurons, was used in adult, male Sprague Dawley rats. First, acute intravenous infusion of hypertonic NaCl or AngII produced anatomically distinct patterns of Fos-positive nuclei in the OVLT largely restricted to the dorsal cap versus vascular core, respectively. However, in vitro patch-clamp recordings indicate 66% (23 of 35) of OVLT neurons were excited by bath application of both hypertonic NaCl and AngII. Similarly, in vivo single-unit recordings revealed that 52% (23 of 44) of OVLT neurons displayed an increased discharge to intracarotid injection of both hypertonic NaCl and AngII. In marked contrast to Fos immunoreactivity, neuroanatomical mapping of Neurobiotin-filled cells from both in vitro and in vivo recordings revealed that NaCl- and AngII-responsive neurons were distributed throughout the OVLT. Next, optogenetic excitation of OVLT neurons stimulated thirst but not salt appetite. Conversely, optogenetic inhibition of OVLT neurons attenuated thirst stimulated by hypernatremia or elevated AngII but not hypovolemia. Collectively, these findings provide the first identification of individual OVLT neurons that respond to both elevated NaCl and AngII concentrations to regulate thirst and body fluid homeostasis.SIGNIFICANCE STATEMENT Body fluid homeostasis requires the integration of neurohumoral signals to coordinate behavior, neuroendocrine function, and autonomic function. Extracellular NaCl concentrations and the peptide hormone angiotensin II (AngII) are two major neurohumoral signals that regulate body fluid homeostasis. Herein, we present the first compelling evidence that individual neurons located in the organum vasculosum of the lamina terminalis detect both NaCl and AngII. Furthermore, optogenetic interrogations demonstrate that these neurons play a pivotal role in the regulation of thirst stimulated by NaCl and AngII. These novel observations lay the foundation for future investigations for how such inputs as well as others converge onto unique organum vasculosum of the lamina terminalis neurons to coordinate body fluid homeostasis and contribute to disorders of fluid balance.

Organum Vasculosum of the Lamina Terminalis Detects NaCl to Elevate Sympathetic Nerve Activity and Blood Pressure.

High-salt diet elevates NaCl concentrations in the cerebrospinal fluid to increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. The organum vasculosum of the lamina terminalis (OVLT) resides along the rostral wall of the third ventricle, lacks a complete blood-brain barrier, and plays a pivotal role in body fluid homeostasis. Therefore, the present study used a multifaceted approach to examine whether OVLT neurons of Sprague-Dawley rats are intrinsically sensitive to changes in extracellular NaCl concentrations and mediate the sympathoexcitatory responses to central NaCl loading. Using in vitro whole-cell recordings, step-wise increases in extracellular NaCl concentrations (2.5-10 mmol/L) produced concentration-dependent excitation of OVLT neurons. Additionally, these excitatory responses were intrinsic to OVLT neurons because hypertonic NaCl evoked inward currents, despite pharmacological synaptic blockade. In vivo single-unit recordings demonstrate that the majority of OVLT neurons (72%, 13/19) display concentration-dependent increases in neuronal discharge to intracarotid (50 µL/15 s) or intracerebroventricular infusion (5 µL/10 minutes) of hypertonic NaCl. Microinjection of hypertonic NaCl (30 nL/60 s) into the OVLT, but not adjacent areas, increased lumbar SNA, adrenal SNA, and arterial blood pressure in a concentration-dependent manner. Renal SNA decreased and splanchnic SNA remained unaffected. Finally, local inhibition of OVLT neurons with the GABAA receptor agonist muscimol (24 nL/10 s) significantly attenuated the sympathoexcitatory and pressor responses to intracerebroventricular infusion of 0.5 mol/L or 1.0 mol/L NaCl. Collectively, these findings indicate that OVLT neurons detect changes in extracellular NaCl concentrations to selectively alter SNA and raise arterial blood pressure.

High fat diet attenuates glucose-dependent facilitation of 5-HT3 -mediated responses in rat gastric vagal afferents.

KEY POINTS: Glucose regulates the density and function of 5-HT3 receptors on gastric vagal afferent neurones. Diet-induced obesity compromises the excitability and responsiveness of vagal afferents. In this study, we assessed whether exposure to a high fat diet (HFD) compromises the glucose-dependent modulation of 5-HT responses in gastric vagal afferents prior to the development of obesity. We show that HFD does not alter the response of gastric vagal afferent nerves and neurones to 5-HT but attenuates the ability of glucose to amplify 5-HT3-induced responses. These results suggest that glucose-dependent vagal afferent signalling is compromised by relatively short periods of exposure to HFD well in advance of the development of obesity or glycaemic dysregulation. Glucose regulates the density and function of 5-HT3 receptors on gastric vagal afferent neurones. Since diet-induced obesity attenuates the responsiveness of gastric vagal afferents to several neurohormones, the aim of the present study was to determine whether high fat diet (HFD) compromises the glucose-dependent modulation of 5-HT responses in gastric vagal afferents prior to the development of obesity. Rats were fed control or HFD (14% or 60% kilocalories from fat, respectively) for up to 8 weeks. Neurophysiological recordings assessed the ability of 5-HT to increase anterior gastric vagal afferent nerve (VAN) activity in vivo before and after acute hyperglycaemia, while electrophysiological recordings from gastric-projecting nodose neurones assessed the ability of glucose to modulate the 5-HT response in vitro. Immunocytochemical studies determined alterations in the neuronal distribution of 5-HT3 receptors. 5-HT and cholecystokinin (CCK) induced dose-dependent increases in VAN activity in all rats; HFD attenuated the response to CCK, but not 5-HT. The 5-HT-induced response was amplified by acute hyperglycaemia in control, but not HFD, rats. Similarly, although 5-HT induced an inward current in both control and HFD gastric nodose neurones in vitro, the 5-HT response and receptor distribution was amplified by acute hyperglycaemia only in control rats. These data suggest that, while HFD does not affect the response of gastric-projecting vagal afferents to 5-HT, it attenuates the ability of glucose to amplify 5-HT effects. This suggests that glucose-dependent vagal afferent signalling is compromised by short periods of exposure to HFD well in advance of obesity or glycaemic dysregulation.

Cerebrospinal Fluid Hypernatremia Elevates Sympathetic Nerve Activity and Blood Pressure via the Rostral Ventrolateral Medulla.

Elevated NaCl concentrations of the cerebrospinal fluid increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. Neurons of the rostral ventrolateral medulla (RVLM) play a pivotal role in the regulation of SNA and receive mono- or polysynaptic inputs from several hypothalamic structures responsive to hypernatremia. Therefore, the present study investigated the contribution of RVLM neurons to the SNA and pressor response to cerebrospinal fluid hypernatremia. Lateral ventricle infusion of 0.15 mol/L, 0.6 mol/L, and 1.0 mol/L NaCl (5 µL/10 minutes) produced concentration-dependent increases in lumbar SNA, adrenal SNA, and arterial blood pressure, despite no change in splanchnic SNA and a decrease in renal SNA. Ganglionic blockade with chlorisondamine or acute lesion of the lamina terminalis blocked or significantly attenuated these responses, respectively. RVLM microinjection of the gamma-aminobutyric acid (GABAA) agonist muscimol abolished the sympathoexcitatory response to intracerebroventricular infusion of 1 mol/L NaCl. Furthermore, blockade of ionotropic glutamate, but not angiotensin II type 1, receptors significantly attenuated the increase in lumbar SNA, adrenal SNA, and arterial blood pressure. Finally, single-unit recordings of spinally projecting RVLM neurons revealed 3 distinct populations based on discharge responses to intracerebroventricular infusion of 1 mol/L NaCl: type I excited (46%; 11/24), type II inhibited (37%; 9/24), and type III no change (17%; 4/24). All neurons with slow conduction velocities were type I cells. Collectively, these findings suggest that acute increases in cerebrospinal fluid NaCl concentrations selectively activate a discrete population of RVLM neurons through glutamate receptor activation to increase SNA and arterial blood pressure.

Osmoregulatory thirst in mice lacking the transient receptor potential vanilloid type 1 (TRPV1) and/or type 4 (TRPV4) receptor.

Recent studies suggest the ability of the central nervous system to detect changes in osmolality is mediated by products of the genes encoding the transient receptor potential vanilloid-1 (TRPV1) or vanilloid-4 (TRPV4) channel. The purpose of the present study was to determine whether deletion of TRPV1 and/or TRPV4 channels altered thirst responses to cellular dehydration in mice. Injection of 0.5 or 1.0 M NaCl produced dose-dependent increases in cumulative water intakes of wild-type (WT), TRPV1-/-, TRPV4-/-, and TRPV1-/-V4-/- mice. However, there were no differences in cumulative water intakes between WT versus any other strain despite similar increases in plasma electrolytes and osmolality. Similar results were observed after injection of hypertonic mannitol. This was a consistent finding regardless of the injection route (intraperitoneal vs. subcutaneous) or timed access to water (delayed vs. immediate). There were also no differences in cumulative intakes across strains after injection of 0.15 M NaCl or during a time-controlled period (no injection). Chronic hypernatremia produced by sole access to 2% NaCl for 48 h also produced similar increases in water intake across strains. In a final set of experiments, subcutaneous injection of 0.5 M NaCl produced similar increases in the number of Fos-positive nuclei within the organum vasculosum of the lamina terminalis and median preoptic nucleus across strains but significantly smaller number in the subfornical organ of WT versus TRPV1-/-V4-/- mice. Collectively, these findings suggest that TRPV1 and/or TRPV4 channels are not the primary mechanism by which the central nervous system responds to cellular dehydration during hypernatremia or hyperosmolality to increase thirst.

Dietary salt intake exaggerates sympathetic reflexes and increases blood pressure variability in normotensive rats.

Previous studies have reported that chronic increases in dietary salt intake enhance sympathetic nerve activity and arterial blood pressure (ABP) responses evoked from brain stem nuclei of normotensive, salt-resistant rats. The purpose of the present study was to determine whether this sensitization results in exaggerated sympathetic nerve activity and ABP responses during activation of various cardiovascular reflexes and also increases ABP variability. Male Sprague-Dawley rats were fed 0.1% NaCl chow (low), 0.5% NaCl chow (medium), 4.0% NaCl chow (high) for 14 to 17 days. Then, the animals were prepared for recordings of lumbar, renal, and splanchnic sympathetic nerve activity and ABP. The level of dietary salt intake directly correlated with the magnitude of sympathetic nerve activity and ABP responses to electrical stimulation of sciatic afferents or intracerebroventricular infusion of 0.6 mol/L or 1.0 mol/L NaCl. Similarly, there was a direct correlation between the level of dietary salt intake and the sympathoinhibitory responses produced by acute volume expansion and stimulation of the aortic depressor nerve or cervical vagal afferents. In contrast, dietary salt intake did not affect the sympathetic and ABP responses to chemoreflex activation produced by hypoxia or hypercapnia. Chronic lesion of the anteroventral third ventricle region eliminated the ability of dietary salt intake to modulate these cardiovascular reflexes. Finally, rats chronically instrumented with telemetry units indicate that increased dietary salt intake elevated blood pressure variability but not mean ABP. These findings indicate that dietary salt intake works through the forebrain hypothalamus to modulate various centrally mediated cardiovascular reflexes and increase blood pressure variability.