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.

NaCl and osmolarity produce different responses in organum vasculosum of the lamina terminalis neurons, sympathetic nerve activity and blood pressure.

KEY POINTS: Changes in extracellular osmolarity stimulate thirst and vasopressin secretion through a central osmoreceptor; however, central infusion of hypertonic NaCl produces a greater sympathoexcitatory and pressor response than infusion of hypertonic mannitol/sorbitol. Neurons in the organum vasculosum of the lamina terminalis (OVLT) sense changes in extracellular osmolarity and NaCl. In this study, we discovered that intracerebroventricular infusion or local OVLT injection of hypertonic NaCl increases lumbar sympathetic nerve activity, adrenal sympathetic nerve activity and arterial blood pressure whereas equi-osmotic mannitol/sorbitol did not alter any variable. In vitro whole-cell recordings demonstrate the majority of OVLT neurons are responsive to hypertonic NaCl or mannitol. However, hypertonic NaCl stimulates a greater increase in discharge frequency than equi-osmotic mannitol. Intracarotid or intracerebroventricular infusion of hypertonic NaCl evokes a greater increase in OVLT neuronal discharge frequency than equi-osmotic sorbitol. Collectively, these novel data suggest that subsets of OVLT neurons respond differently to hypertonic NaCl versus osmolarity and subsequently regulate body fluid homeostasis. These responses probably reflect distinct cellular mechanisms underlying NaCl- versus osmo-sensing. ABSTRACT: Systemic or central infusion of hypertonic NaCl and other osmolytes readily stimulate thirst and vasopressin secretion. In contrast, central infusion of hypertonic NaCl produces a greater increase in arterial blood pressure (ABP) than equi-osmotic mannitol/sorbitol. Although these responses depend on neurons in the organum vasculosum of the lamina terminalis (OVLT), these observations suggest OVLT neurons may sense or respond differently to hypertonic NaCl versus osmolarity. The purpose of this study was to test this hypothesis in Sprague-Dawley rats. First, intracerebroventricular (icv) infusion (5 µl/10 min) of 1.0 m NaCl produced a significantly greater increase in lumbar sympathetic nerve activity (SNA), adrenal SNA and ABP than equi-osmotic sorbitol (2.0 osmol l-1 ). Second, OVLT microinjection (20 nl) of 1.0 m NaCl significantly raised lumbar SNA, adrenal SNA and ABP. Equi-osmotic sorbitol did not alter any variable. Third, in vitro whole-cell recordings demonstrate that 50% (18/36) of OVLT neurons display an increased discharge to both hypertonic NaCl (+7.5 mm) and mannitol (+15 mm). Of these neurons, 56% (10/18) displayed a greater discharge response to hypertonic NaCl vs mannitol. Fourth, in vivo single-unit recordings revealed that intracarotid injection of hypertonic NaCl produced a concentration-dependent increase in OVLT cell discharge, lumbar SNA and ABP. The responses to equi-osmotic infusions of hypertonic sorbitol were significantly smaller. Lastly, icv infusion of 0.5 m NaCl produced significantly greater increases in OVLT discharge and ABP than icv infusion of equi-osmotic sorbitol. Collectively, these findings indicate NaCl and osmotic stimuli produce different responses across OVLT neurons and may represent distinct cellular processes to regulate thirst, vasopressin secretion and autonomic function.

Effects of prostaglandin E2 on cells cultured from the rat organum vasculosum laminae terminalis and median preoptic nucleus.

The time course of the induction of enzymes responsible for the formation of prostaglandin E2 (PGE2) after an inflammatory insult, in relation to the concomitant febrile response, suggests that peripherally generated PGE2 is involved in the induction of the early phase of fever, while centrally produced PGE2 exerts pyrogenic capacities during the later stages of fever within the hypothalamic median preoptic nucleus (MnPO). The actions of peripherally derived PGE2 on the brain might occur at the level of the organum vasculosum laminae terminalis (OVLT), which lacks a tight blood-brain barrier and is implicated in fever, while the effects of PGE2 within the MnPO might interfere with glutamatergic neurotransmission within a recently characterized central efferent pathway for the activation of cold-defence reactions. Using the fura-2 ratio imaging technique we, therefore, measured changes of the intracellular Ca(2+)-concentration in primary neuroglial microcultures of rat OVLT and MnPO stimulated with PGE2 and/or glutamate. In cultures from the OVLT, as opposed to those derived from the MnPO, substantial numbers of neurons (8% of 385), astrocytes (19% of 645) and microglial cells (28% of 43) directly responded to PGE2 with a transient increase of intracellular Ca(2+). The most pronounced effect of PGE2 on cells from MnPO microcultures was its modulatory influence on the strength of glutamate-induced Ca(2+)-signals. In 72 out of 512 neurons and in 105 out of 715 astrocytes PGE2 significantly augmented glutamate-induced Ca(2+)-signals. About 30% of these neurons were GABAergic. These observations are in agreement with putative roles of peripheral PGE2 as a directly acting circulating agent at the level of the OVLT, and of central MnPO-intrinsic PGE2 as an enhancer of glutamatergic neurotransmission, which causes disinhibition of thermogenic heat production, a crucial component for the manifestation of fever. In microcultures from both brain sites investigated incubation with PGE2 significantly reduced the lipopolysaccharide-induced release of cytokines (tumor necrosis factor-α and interleukin-6) into the supernatant. PGE2, thus, seems to be involved in a negative feed-back loop to limit the strength of the brain inflammatory process and to play a dual role with pro- as well as anti-inflammatory properties.

The OVLT initiates the fall in arterial pressure evoked by high dose lipopolysaccharide: evidence that dichotomous, dose-related mechanisms mediate endotoxic hypotension.

This study tested the hypothesis that lipopolysaccharide (LPS) lowers arterial pressure through two different mechanisms depending on the dose. Previously, we found that a low hypotensive dose of LPS (1mg/kg) lowers arterial pressure by activating vagus nerve afferents. Here we report that hypotension evoked by high dose LPS (15mg/kg) can be prevented by injecting lidocaine into the OVLT but not by vagotomy or inactivation of the NTS. The hypotension produced by both LPS doses was correlated with elevated extracellular norepinephrine concentrations in the POA and prevented by blocking alpha-adrenergic receptors. Thus, initiation of endotoxic hypotension is dose-related, mechanistically.

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.