Organum Vasculosum Research Articles

Appearance of the Organum Vasculosum of the Lamina Terminalis on Contrast-enhanced MR Imaging.

Purpose:Circumventricular organs (CVOs) lack a blood brain barrier and are also called “brain windows”. Among CVOs, the organum vasculosum of the lamina terminalis (OVLT) is an osmotic regulator involved in the release of vasopressin. In a previous study of healthy subjects, it was reported that contrast enhancement in the OVLT can be recognized in only 34% of 3 Tesla thin slice contrast-enhanced T1-weighted images. The purpose of this study was to evaluate the leakage of gadolinium contrast from the OVLT in healthy subjects using heavily T2-weighted three dimensional-fluid attenuated inversion recovery (3D-FLAIR) (HF) imaging.Methods:Eight healthy male subjects were included in this study. A standard dose (0.1 mmol/kg) of gadoteridol was intravenously administered. Magnetic resonance cisternography (MRC) and HF were obtained before and 0.5, 1.5, 3, 4.5 and 6 h after the injection. Enhancement of the OVLT including the surrounding cerebral spinal fluid (CSF) was measured by manually drawing a rectangular ROI centered on the OVLT. The ROI was copied to the HF image and the signal intensity was measured. The signal intensity ratio (SIR) was obtained by dividing the signal intensity value of the OVLT ROI by that of the midbrain.Results:The differences between the mean SIR at pre-contrast and those at 0.5, 1.5, 3, 4.5, and 6 h were significant (P < 0.05). The mean SIR at 0.5 h was higher than those at all other time points (P < 0.05).Conclusion:Using HF imaging, enhancement around the OVLT was observed in all subjects at 0.5 h after intravenous administration of single dose gadoteridol.

Abstract 083: Neurons in the Organum Vasculosum of the Lamina Terminalis Contribute to Salt-sensitive Hypertension

Excess dietary salt intake raises plasma and cerebrospinal fluid NaCl concentrations to elevate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). Changes in extracellular NaCl concentrations are sensed by neurons in the organum vasculosum of the lamina terminalis (OVLT) - a circumventricular organ that lacks a complete blood-brain barrier. The purpose of the present study was to investigate the hypothesis that salt-sensitive hypertension was mediated, in part, by an elevated activity of OVLT neurons. Dahl-Salt-Sensitive or Sprague-Dawley rats (8-10 weeks) were fed 0.5% or 4.0% NaCl diets for 3-4 weeks. First, in vivo single-unit recordings demonstrate the discharge of OVLT neurons in Dahl-Salt-Sensitive rats was higher after a 4.0% versus 0.5% NaCl diet (4.1±0.4 Hz vs 1.9±0.3 Hz, n=6 per group, P&lt;0.05). OVLT neuronal discharge of Sprague-Dawley rats was not different after a 4.0% or 0.5% NaCl diet (2.1±0.4 Hz vs 1.7±0.3 Hz, n=6-9 per group, P&gt;0.5). In a second set of experiments, injection of hypertonic NaCl (1.0M NaCl, 20nL) into the OVLT produced significantly greater increases in lumbar SNA (131±6% vs 116±3%, n=4 per group, P&lt;0.05) and mean ABP (14±2 vs 8±2 mmHg, n=4 per group, P&lt;0.05) of Dahl-Salt-Sensitive rats fed 4.0% versus 0.5% NaCl respectively. Sprague-Dawley rats fed 4.0% versus 0.5% NaCl exhibited responses of smaller magnitude for both lumbar SNA (115±4 vs 108±3%, n=4 per group, P&lt;0.05) and mean ABP (9±2 vs 6±2 mmHg, n=4 per group, P&lt;0.05). Interestingly, the duration of the response was much longer in Dahl-Salt-Sensitive versus Sprague-Dawley rats (data not shown). Finally, inhibition of neuronal activity by injection of the GABA agonist muscimol (5mM, 20nL) into the OVLT produced a significantly greater fall in lumbar SNA (-25±4% vs -11±3%, n=4 per group, P&lt;0.05) and mean ABP (-19±4 vs -6±2 mmHg, n=4 per group, P&lt;0.05) of Dahl-Salt-Sensitive rats fed 4.0% versus 0.5% NaCl, respectively. Injection of muscimol into the OVLT of Sprague-Dawley rats did not significantly affect SNA or mean ABP. Collectively, these findings suggest a high salt diet increases the activity of OVLT neurons to elevate SNA and ABP in salt-sensitive hypertension.

Abstract 116: Potent Hypertensive Actions of Angiotensin-sensitive Neurons Within the Lamina Terminalis

It is accepted that activation of angiotensin type 1a receptors (AT1a) within the CNS elevates blood pressure by influencing sympathetic outflow and vasopressin (VP) secretion; however, the neuronal circuits mediating these effects are not completely understood. The present studies characterize the structure and function of AT1a neurons residing in the median preoptic nucleus (MnPO) and the organum vasculosum of the lamina terminalis (OVLT), thereby evaluating their potential role in blood pressure control. Using male mice that express Cre-recombinase prior to the STOP codon of the AT1a gene, initial studies combined genetic reporting with in situ hybridization to reveal that AT1a neurons in the MnPO and OVLT are largely excitatory (87±4% express vesicular glutamate transporter 2). Subsequently, AT1a-Cre mice were delivered a Cre-inducible adeno-associated virus to induce expression of channelrhodopsin-2 (ChR2) and enhanced yellow fluorescent protein (eYFP) specifically within AT1a neurons of the MnPO/OVLT (AAV-ChR2-eYFP; n = 4). Control mice were delivered AAV-eYFP (n = 4). Analysis of eYFP immunofluorescence revealed that neurons within the MnPO/OVLT that express AT1a send projections to the paraventricular nucleus of the hypothalamus (PVN; an area involved in sympathetic outflow and VP secretion) that appear to synapse onto VP synthesizing neurons. To evaluate the functionality of this connection, we optogenetically stimulated AT1a neurons in the region while recording cardiovascular parameters in anesthetized mice. Ten-minutes of optogenetic stimulation (473 nM;15 Hz; 20 ms pulse width; 60 s ON/OFF) robustly elevated systolic blood pressure in AAV-ChR2-eYFP mice relative to AAV-eYFP controls. This effect was rapid in its onset (34 ± 9 vs. -2 ± 4 mmHg at 5 min, p &lt; 0.05) but persisted for the entire 50 min of cardiovascular recording (45 ± 11 vs. -3 ± 7 mmHg at 50 min, p &lt; 0.05). Intriguingly, the optogenetic stimulation also resulted in 62% increase in Fos induction in AVP neurons within the PVN relative to AAV-eYFP controls (86 ± 5 vs. 52 ± 2 %; p&lt;0.01). Collectively, these results suggest that excitation of AT1a neurons in the MnPO/OVLT recruits autonomic and neuroendocrine responses that promote robust and sustained increases in blood pressure.

Differential effects of relaxin-3 and a selective relaxin-3 receptor agonist on food and water intake and hypothalamic neuronal activity in rats

The neuropeptide relaxin-3 (RLN3) binds with high affinity to its cognate receptor, relaxin-family peptide receptor 3 (RXFP3), and with lower affinity to RXFP1, the cognate receptor for relaxin. Intracerebroventricular (icv) administration of RLN3 in rats strongly increases food and water intake and alters the activity of the hypothalamic-pituitary-adrenal (HPA) and gonadal (HPG) axes, but the relative involvement of RXFP3 and RXFP1 in these effects is not known. Therefore, the effects of icv administration of equimolar (1.1 nmol) amounts of RLN3 and the RXFP3-selective agonist RXFP3-A2 on food and water intake, plasma levels of corticosterone, testosterone, and oxytocin and c-fos mRNA expression in key hypothalamic regions in male rats were compared. Food intake was increased by both RLN3 and RXFP3-A2, but the orexigenic effects of RXFP3-A2 were significantly stronger than RLN3, 30 and 60min after injection. Water intake and plasma corticosterone and testosterone levels were significantly increased by RLN3, but not by RXFP3-A2. Conversely, RXFP3-A2 but not RLN3 decreased oxytocin plasma levels. RLN3, but not RXFP3-A2, increased c-fos mRNA levels in the parvocellular (PVNp) and magnocellular (PVNm) paraventricular and supraoptic (SON) hypothalamic nuclei, in the ventral medial preoptic area (MPAv), and in the organum vasculosum of the lamina terminalis (OVLT). A significant increase in c-fos mRNA expression was induced in the perifornical lateral hypothalamic area (LHApf) by RLN3 and RXFP3-A2. These results suggest that RXFP1 is involved in the RLN3 stimulation of water intake and activation of the HPA and HPG axes. The reduced food intake stimulation by RLN3 compared to RXFP3-A2 may relate to activation of both orexigenic and anorexigenic circuits by RLN3.

The use of siRNA as a pharmacological tool to assess a role for the transcription factor NF-IL6 in the brain under in vitro and in vivo conditions during LPS-induced inflammatory stimulation.

Studies with NF-IL6-deficient mice indicate that this transcription factor plays a dual role during systemic inflammation with pro- and anti-inflammatory capacities. Here, we aimed to characterize the role of NF-IL6 specifically within the brain. In this study, we tested the capacity of short interfering (si) RNA to silence the inflammatory transcription factor nuclear factor-interleukin 6 (NF-IL6) in brain cells under in vitro and in vivo conditions. In cells of a mixed neuronal and glial primary culture from the rat area postrema (AP), short interfering RNA (siRNA) directed against NF-IL6 strongly reduced basal and lipopolysaccharide (LPS)-induced nuclear immunoreactivity of this transcription factor, with the strongest effect on astrocytes. The siRNA did not exert inflammatory effects in the primary culture as confirmed by unaltered levels of IL-6 in supernatants. In vivo, intracerebroventricular (i.c.v.) injections of fluorochrome labelled siRNA caused its appearance in relevant brain structures for fever induction pathways such as the vascular organ of lamina terminalis, the subfornical organ, the median preoptic nucleus (MnPO) and the AP in several cell types, including microglial cells. However, i.c.v. injections of siRNA per se caused signs of fever, anorexia and reduced locomotor activity, i.e. sickness behavior. This approach was, thus, not suitable to characterize the role NF-IL6 in the brain in vivo, namely during experimentally induced systemic inflammation.

Hypertonic NaCl versus osmotic stimuli: distinct OVLT neurones can sense the difference to control sympathetic outflow and blood pressure.

Despite large fluctuations in salt and water intake, mammals are able to maintain electrolyte concentrations, particularly sodium (Na+) as the major determinant of osmolality of the extracellular fluid (ECF), within narrow physiological limits (∼300 mOsm kg−1). The two distinct sensory systems monitoring Na+ concentration and osmolality in the hypothalamic area of the brain are located outside the blood–brain barrier and are therefore known as circumventricular organs (CVOs). The osmosensitive neurones of CVOs are located in the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT). Direct projections from these areas to hypothalamic nuclei containing neurosecretory magnocellular neurones promote the release of water-conserving hormones such as arginine vasopressin (McKinley et al. 2001). Interestingly, these neurones also project to smaller parvocellular neurones located within other brain structures, such as the paraventricular nucleus of the hypothalamus (PVN) that modulate sympathetic nerve activity (SNA), an important mechanism in blood pressure (BP) homeostasis. The distinct physiological responses to maintain fluid balance triggered by infusion of hypertonic sodium chloride (NaCl) and equi-osmolar solutions have been debated for decades. McKinley and colleagues (1978) have shown that a peripheral osmoreceptor mechanism was most likely to be responsible for the thirst and antidiuresis induced by intracarotid infusion of hypertonic NaCl or sucrose in sheep. However, they also found support for a periventricular Na+ receptor mechanism because of differential drinking responses to intracerebroventricular (i.c.v.) administration of hypertonic NaCl or sucrose. The SFO seems to be the primary locus of NaCl sensing for the control of salt-intake behaviour at the central level, identified as a sensory mechanism that relies on activation of a Na+ channel (Nax); it is not osmosensing (Hyiama et al. 2004). The SFO is also involved in mediating the central Na+-induced pressor response, since administration of NaCl-rich artificial cerebrospinal fluid (aCSF) into the SFO caused an increase in BP, which was blunted by an angiotensin II type 1 (AT1) receptor antagonist at the same site. However, equi-osmotic infusion of manitol did not change BP, indicating that the pressor response is Na+ specific and not due to osmolality or volume effects (Tiruneh et al. 2013). Not only SFO, but also OVLT neurones are capable of detecting extracellular NaCl concentration and altering the sympathetic nerve discharge in order increase BP (Kinsman et al. 2017b). However, to date, no study has been able to show whether distinct cellular processes of osmosensitive neurones can differentiate between hyperosmolality versus hypertonic NaCl stimulus to selectively control SNA and BP. In this issue of The Journal of Physiology, Kinsman and co-workers (2017a) addressed this important issue by performing an elegant in vivo neurophysiological study. They determined how subsets of OVLT neurones respond differentially to hypertonic NaCl versus osmolality and subsequently regulate the sympathetic nervous system. More precisely, they showed that local injection of hypertonic saline into the OVLT produced an increase in SNA and BP, whereas an equi-osmotic stimulus of mannitol/sorbitol solutions had no effect. Moreover, these authors combined in vitro whole-cell recordings to demonstrate that stimulation with hypertonic saline caused the discharge frequency of OVLT neurones to increase, unlike stimulation with the equi-osmotic mannitol solution. These findings by Kinsman and colleagues (2017a) open several interesting perspectives for further investigation regarding the functional significance of distinct cellular responses of Na+ and osmosensitive OVLT neurones: (i) What are the physiological mechanisms, phenotype and neurotransmitters released by the functionally distinct OVLT neurones? (ii) How do these differences translate to hypertonic NaCl and osmotically driven sympathetic responses and BP control? (iii) Do salt-sensitive rats (e.g. Dahl-Salt) show alterations in OVLT neuronal populations that control hydroelectrolytic balance, sympathetic hyperactivity and hypertension in response to high salt diet? (iv) It would be interesting to determine the contribution of the SFO compared to the OVLT to the sympathetic responses to hypertonic saline and osmolality challenges. Understanding the characteristics of the CVO neurones relating to control of body fluid and cardiovascular homeostasis in response to distinct osmotic stimuli will require a range of techniques from anatomical, molecular, biochemical and pharmaco/opto-genetic tools, and this paper by Kinsman and co-workers (2017a) is a great kick-off in elucidating the complex hierarchy and mechanisms of the osmoregulatory signalling network at the CNS level. None declared. V.R.A. is supported by The National Council for Scientific and Technological Development (CNPq) Research Fellow, and The State of Sao Paulo Research Funding Agency (FAPESP) no. 2016-21991-3.

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus

AbstractNeurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS‐expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS‐immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα‐immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFPVglut2, EYFPVgat, and GFPGad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.

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

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. 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 (5μl/10min) of 1.0m NaCl produced a significantly greater increase in lumbar sympathetic nerve activity (SNA), adrenal SNA and ABP than equi-osmotic sorbitol (2.0osmoll-1 ). Second, OVLT microinjection (20nl) of 1.0m 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 (+7.5mm) and mannitol (+15mm). 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 0.5m 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.

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus.

Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS-expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS-immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα-immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFPVglut2 , EYFPVgat , and GFPGad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.

The effects of experimental gestational hypertension on maternal blood pressure and fluid intake and pre-weanling hypothalamic neuronal activity

To examine the fetal programming effects of maternal hypertension, natriophilia and hyperreninemia [experimentally induced in rats by partial inter-renal aortic ligature (PAL) prior to mating] fos immunoreactivity was studied in 6-day-old offspring of PAL and control mothers. The purposes of the present set of experiments were twofold. The first was to characterize the effects of PAL on the mother's arterial blood pressure and intake of salt (1.8% NaCl solution) and water over the course of gestation. Second, was to study the pattern of neuronal activation in key brain areas of 6-day-old offspring treated with the dipsogen isoproterenol that were from PAL and control mothers. Beta-adrenergic receptor agonist-treated pups allowed the determination whether there were neuroanatomical correlates within the neural substrates controlling thirst and the enhanced water intake evidenced by the isoproterenol treated pups of PAL mothers. Hydromineral ingestive behavior along with blood pressure and heart rate of PAL (M-PAL) and control (M-sPAL) dams throughout gestation was studied. Higher salt and water intakes along with blood pressures and heart rates were found during gestation and lactation in the M-PAL group. Maternal PAL evoked significantly increased isoproterenol-elicited Fos staining in brain regions (e.g. subfornical organ, organum vasculosum of the lamina terminalis, supraoptic nucleus, hypothalamic paraventricular nucleus and median preoptic nucleus) of 6-day-old pups, which is the age of animals shown enhanced thirst responses in PAL offspring. These results indicate that PAL is compatible with pregnancy, producing a sustained increase in blood pressure and heart rate, along with increased water and salt intake. The present study demonstrates that the neural substrates involved in cardiovascular homeostasis and fluid balance in adult rats are responsive in six-day-old rats, and can be altered by fetal programming.

Interleukin-18 and its receptor are expressed in gonadotropin-releasing hormone neurons of mouse and rat forebrain

Interleukin-18 (IL-18) is a pro-inflammatory cytokine and an important mediator of peripheral inflammation and host immune response. IL-18 functions through its binding with the IL-18 receptor (IL-18R), which consists of two chains, an IL-18-binding α chain (IL-18Rα) and a signaling β chain. IL-18 and IL-18R are expressed in the brain; however, limited information is available on IL-18R expression and the role of IL-18 in neurosecretory cells. In the present study, we used immunohistochemical techniques to investigate the distribution of IL-18Rα and IL-18 in the hypothalamus of male mice and rats. IL-18Rα-positive and IL-18-positive perikarya and fibers were found scattered throughout the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the organum vasculosum of the laminae terminalis, the preoptic area, and the anterior hypothalamic area. It is well known that gonadotropin-releasing hormone (GnRH) neuronal somata and/or fibers are found in these regions. Therefore, we performed double-label immunofluorescence for IL-18Rα/IL-18 and GnRH. IL-18Rα was expressed in approximately 60% of GnRH-immunopositive perikarya, and IL-18 was distributed in all GnRH-immunopositive perikarya. These observations suggest that IL-18 exerts direct effects upon the GnRH neuron via IL-18Rα and acts on GnRH neurons through an autocrine or paracrine pathway.

The effect of sodium depletion with or without concomitant dehydration on Fos immunoreactivity in the different subregions of the subfornical organ and organum vasculosum of the lamina terminalis of the rat

In rats, each of the two forebrain sensory circumventricular organs, the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT) can be categorised anatomically and functionally into subregions. The main divisions of the SFO are its outer shell and its ventromedial core, while the OVLT has a lateral zone, dorsal cap and a central vascular plexus. The aim of these experiments was to determine the effect of Na depletion for 24 hours by furosemide (20 mg/kg s.c.) treatment and low Na diet on rats (4 per group) with or without water available to drink on Fos immunoreactivity (Fos‐IR) in the subregions of the SFO and OVLT. We also examined the effect after 2 hours on these responses of correcting the Na deficit by drinking either isotonic 0.15M or hypertonic 0.3M NaCl. After these treatments, rats were killed by Na pentobarbitone (100 mg/kg i.p.) and standard immunohistochemical methods used to identify Fos in cell nuclei of CVO neurons. Whereas control untreated rats on low Na diet for 24 h exhibited minimal Fos‐IR in any subregions of the SFO and OVLT, rats subjected to sodium depletion with water available to drink exhibited many Fos‐labelled cells in the SFO that were almost exclusively confined to its ventromedial core, while in the OVLT, Fos expression was observed predominantly in the lateral zone with little Fos‐IR in the dorsal cap. Fos‐IR was not observed in the supraoptic nucleus (SON), signifying that the rats were not dehydrated. Treatment with the angiotensin AT1 receptor antagonist valsartan (5 mg/kg s.c. × 2 doses) inhibited the Fos response in the ventromedial core of the SFO. If water was withheld from Na‐depleted rats, some Fos‐IR was now observed in the outer shell, although most Fos‐IR was still observed in the ventromedial core; in the OVLT, Fos‐IR was observed in both its dorsal cap and lateral zone and the SON expressed Fos‐IR. Correction of the Na‐deficit in water‐replete but Na‐depleted rats by drinking 0.15M NaCl reduced the intensity, but did not abolish the Fos‐IR in the ventromedial core of the SFO or the lateral zone of the OVLT by 2 h, whereas water‐replete, Na‐depleted rats that drank hypertonic 0.3M NaCl exhibited additional Fos‐IR in the outer shell region of the SFO and in the dorsal cap of the OVLT as well as the less intense Fos‐IR in the ventromedial core of SFO and lateral zone of OVLT. These data confirm that the ventromedial core of SFO and lateral zone of the OVLT are activated by loss of body Na, whereas neurons in the outer shell of the SFO and dorsal cap of OVLT are more attuned to loss of body water and hypertonicity.Support or Funding InformationSupported by G. Harold and Leila Y. Mathers Charitable Foundation

Preserving brain function in a comatose patient with septic hyperpyrexia (41.6\xa0\xb0C): a case report

BackgroundPyrexia is a physiological response through which the immune system responds to infectious processes. Hyperpyrexia is known to be neurodegenerative leading to brain damage. Some of the neurotoxic effects of hyperpyrexia on the brain include seizures, decreased cognitive speed, mental status changes, coma, and even death. In the clinical management of hyperpyrexia, the goal is to treat the underlying cause of elevated temperature and prevent end organ damage.Case presentationThis case illustrates a 39-year-old white American man referred from another medical facility where he had undergone an upper gastrointestinal tract diagnostic procedure which became complicated by blood aspiration and respiratory distress. During hospitalization, he developed a core body temperature of 41.6 °C (106.9 °F) leading to cognitive decline and coma with a Glasgow Coma Score of 3. Levetiracetam and amantadine were utilized effectively for preserving and restoring neurocognitive function. Prior studies have shown that glutamate levels can increase during an infectious process. Glutamate is an excitatory neurotransmitter that is utilized by the organum vasculosum laminae terminalis through the neuronal excitatory system and causes an increase in body temperature which can lead to hyperpyrexia. Similar to neurogenic fevers, hyperpyrexia can lead to neurological decline and irreversible cognitive dysfunction. Inhibition of the glutamate aids a decrease in excitatory states, and improves the brain’s regulatory mechanism, including temperature control. To further improve cognitive function, dopamine levels were increased with a dopamine agonist.ConclusionsWe propose that a combination of levetiracetam and amantadine may provide neuroprotective and neurorestorative properties when administered during a period of hyperpyrexia accompanied by any form of mental status changes, particularly if there is a decline in Glasgow Coma Score.

Topological and histological description of preoptic area and hypothalamus in cardinal tetra Paracheirodon axelrodi (Characiformes: Characidae)

ABSTRACT Topological and histological descriptions of the preoptic area and hypothalamus of the cardinal tetra Paracheirodon axelrodi were performed. Standard histological paraffin sections were used and stained with Nissl technique, and plastic sections for high-resolution optic microscopy (HROM). The preoptic area showed some differences related to the location of the magnocellular preoptic nucleus (PM) and the size of the neurons in this region, as they were the biggest in all the preoptic area. Additionally, within the preoptic area, the different structures that comprise the organum vasculosum of the lamina terminalis (OVLT) were identified and characterized. The hypothalamus could be subdivided in three regions - the ventral, the dorsal and the caudal hypothalamic regions - neuron morphology, size and staining pattern were similar in all of them.

Distinct neural mechanisms for the control of thirst and salt appetite in the subfornical organ

Body fluid conditions are continuously monitored in the brain to regulate thirst and salt-appetite sensations. Angiotensin II drives both thirst and salt appetite; however, the neural mechanisms underlying selective water- and/or salt-intake behaviors remain unknown. Using optogenetics, we show that thirst and salt appetite are driven by distinct groups of angiotensin II receptor type 1a-positive excitatory neurons in the subfornical organ. Neurons projecting to the organum vasculosum lamina terminalis control water intake, while those projecting to the ventral part of the bed nucleus of the stria terminalis control salt intake. Thirst-driving neurons are suppressed under sodium-depleted conditions through cholecystokinin-mediated activation of GABAergic neurons. In contrast, the salt appetite-driving neurons were suppressed under dehydrated conditions through activation of another population of GABAergic neurons by Nax signals. These distinct mechanisms in the subfornical organ may underlie the selective intakes of water and/or salt and may contribute to body fluid homeostasis.

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.

Firing Up in Anticipation

Firing Up in Anticipation

Clock-driven vasopressin neurotransmission mediates anticipatory thirst prior to sleep.

Circadian rhythms have evolved to anticipate and adapt animals to the constraints of the earth's 24-hour light cycle. Although the molecular processes that establish periodicity in clock neurons of the suprachiasmatic nucleus (SCN) are well understood, the mechanisms by which axonal projections from the central clock drive behavioural rhythms are unknown. Here we show that the sleep period in mice (Zeitgeber time, ZT0-12) is preceded by an increase in water intake promoted entirely by the central clock, and not motivated by physiological need. Mice denied this surge experienced significant dehydration near the end of the sleep period, indicating that this water intake contributes to the maintenance of overnight hydromineral balance. Furthermore, this effect relies specifically on the activity of SCN vasopressin (VP) neurons that project to thirst neurons in the OVLT (organum vasculosum lamina terminalis), where VP is released as a neurotransmitter. SCN VP neurons become electrically active during the anticipatory period (ZT21.5-23.5), and depolarize and excite OVLT neurons through the activation of postsynaptic VP V1a receptors and downstream non-selective cation channels. Optogenetic induction of VP release before the anticipatory period (basal period; ZT19.5-21.5) excited OVLT neurons and prompted a surge in water intake. Conversely, optogenetic inhibition of VP release during the anticipatory period inhibited the firing of OVLT neurons and prevented the corresponding increase in water intake. Our findings reveal the existence of anticipatory thirst, and demonstrate this behaviour to be driven by excitatory peptidergic neurotransmission mediated by VP release from central clock neurons.

Abstract 017: Optogenetic Activation of OVLT Neurons Stimulates Water Intake and Produces a Sympathetically-Mediated Increase in Arterial Blood Pressure

The organum vasculosum of the lamina terminalis (OVLT) plays a pivotal role in body fluid homeostasis and arterial blood pressure (ABP) regulation. The OVLT lacks a complete blood-brain-barrier and responds to an array of circulating factors such as NaCl and angiotensin II. Lesion of the anteroventral third ventricular region which includes the OVLT attenuates or reverses several forms of salt-sensitive hypertension. However, there is limited evidence to demonstrate that direct activation of OVLT neurons alters body fluid homeostasis or elevates ABP. To address this question, Male-Sprague-Dawley rats (300-350 g) received an injection of rAAV9-CamKII-hChR2(H134R)-EYFP (10 12 particles/mL, 200nL) into the OVLT. A fiber optic cannula (200μm) was implanted 300μm dorsal to OVLT. Approximately 2-3 week later, optogenetic activation of OVLT neurons (10ms pulse, 50% duty cycle, 30 min) produced frequency-dependent increases in water intake (1Hz: 1.0±0.5mL; 5Hz: 4.2±0.6mL; 10Hz: 8.0±1.8; 20Hz: 10.2±2.1mL, n=4, P&lt;0.05). In separate experiments, optogenetic activation of OVLT neurons produced a frequency-dependent increase in mean ABP (1Hz: 1±1 mmHg; 5Hz: 3±1mmHg; 10Hz: 7±1mmHg; 20Hz: 13±1mmHg, n=4, P&lt;0.05) and heart rate (1Hz: 3±6 bpm; 5Hz: 15±5bpm; 10Hz: 40±12 bpm; 20Hz: 62±14bpm, n=4, P&lt;0.05). Pretreatment with the vasopressin antagonist Manning Compound (10ug/kg, IV) did not affect these responses. However, pretreatment with the ganglionic blocker chlorisondamine (5mg/kg, IV) abolished the pressor (20Hz: 1±1 mmHg, P&lt;0.01) and tachycardic (20Hz: 4±7 bpm, P&lt;0.05) responses to activation of OVLT neurons. Finally, in vivo single-unit recordings demonstrate that optogenetic activation produced frequency-dependent increases in cell discharge of OVLT neurons responsive to either intracarotid injection of hypertonic NaCl (0.3M NaCl, 50μL over 10 s, n=6) or angiotensin II (100ng over 10s, n=3). Collectively, these data provide evidence that direct activation of OVLT neurons stimulates thirst and produces a sympathetically-mediated increase in ABP.

Abstract 072: Brain Epithelial Sodium Channels on Organum Vasculosum of the Lamina Terminalis Neurons Mediate Sympathoexcitatory Pressor Responses and Neuronal Excitation by Hypertonic NaCl

High dietary salt intake raises cerebrospinal fluid (CSF) [Na + ] in salt sensitive subjects to elevate sympathetic nerve activity (SNA), and arterial blood pressure (ABP). This occurs through excitation of NaCl-sensitive sites in the brain, including the organum vasculosum of the lamina terminalis (OVLT). Intriguingly, intracerebroventricular (ICV) pretreatment with benzamil (a non-voltage gated Na + channel blocker) also attenuates those systemic responses to central NaCl. Thus, I hypothesized that benzamil acts on NaCl-sensitive OVLT neurons to attenuate neuronal excitation and elevated SNA and ABP in response to hypertonic NaCl. To evaluate this hypothesis, lumbar SNA and ABP were measured in anesthetized adult rats in response to ICV infusion of 0.15, 0.5, and 1.0 M NaCl with and without prior OVLT microinjection of benzamil (5nmol per 20nL). ICV infusion of NaCl produced concentration-dependent increases in lumbar SNA (0.15M: 101±3%, 0.5M: 118±3%, 1.0M: 130±9% n=4 per group, P&lt;0.05) and mean ABP (0.15M: 1±1mmHg; 0.5M: 5±1mmHg, 1.0M: 12±2mmHg). OVLT microinjection of benzamil significantly attenuated the increase in lumbar SNA (0.15M: 100±2%, 0.5M: 108±2%, 1.0M: 115±3% n=4 per group, P&lt;0.05) and mean ABP (0.15M: 1±0mmHg; 0.5M: 2±1mmHg, 1.0M: 6±2mmHg). In a parallel set of experiments, in vitro whole-cell recordings of OVLT neurons in slices were performed to assess whether blockade of epithelial sodium channels (ENaC) attenuated NaCl-induced excitation. Bath application of +7.5mM NaCl increase action potential (AP) discharge of OVLT neurons (n=11) from 0.46±0.16 Hz to 1.09±0.26 Hz (P&lt;0.05). Subsequent addition of an ENaC selective concentration of benzamil (0.5μM) reversed AP discharge (0.75±0.24 Hz, P&lt;0.05). Isotonic 0.5μM benzamil did not significantly change NaCl-sensitive OVLT neuron (n=13) AP discharge from baseline (0.54±0.14 Hz to 0.60±0.16 Hz, P&gt;0.05). Collectively, this data indicates that elevations in CSF NaCl concentrations excite OVLT neurons via ENaC to elevate SNA and ABP.