Organum Vasculosum Research Articles

Estradiol Decreases MAPKs phosphorylation in Response to Angiotensin II by Increasing MKP‐1 in the Hypothalamus, but not in the Lamina Terminalis

IntroductionThe angiotensin II (ANGII) plays an important role in the control of body fluids and estradiol (E2) modulates several actions of ANGII in the brain. Recently, our group showed that ERK1/2, JNK and p38MAPK are involved in sodium intake induced by ANGII in female rats. On the other hand, ANGII‐induced water intake requires JNK and p38MAPK, but not ERK1/2. In regarding to neurohypohysial hormone release, our group showed that ERK1/2 is involved in vasopressin (AVP) release but not in oxytocin (OT) release induced by ANGII. While ANGII‐induced OT release requires p38MAPK signaling. Therefore, herein the aim of study was evaluated whether E2 modulates MAPKs phosphorylation in response to ANGII as well MAPK phosphatase 1 (MKP‐1) which is responsible for inhibiting all the MAPKs (ERK1/2, JNK and p38MAPK) in the paraventricular (PVN) and supraoptic nuclei (SON) of hypothalamus, and in structures of lamina terminalis.Materials and MethodsWistar rats (~250g) were submitted to ovariectomy (OVX) and on the following day they were treated with estradiol cypionate (10μg/rat, sc, OVX+E2) or vehicle (corn oil, 0.1mL/rat, sc) for eight days. On the eighth day, the rats received an icv (lateral ventricle) injection of ANGII (25ng/2μL/rat) or vehicle (0.9% saline, 2μL/rat). After ten min of ANGII injection the animals were decapitated for brain collection for ERK1/2, JNK and p38MAPK total and phosphorylated, MKP‐1 and β‐Actin analyses by western blot. Data were analyzed using ANOVA two‐way, followed by Newman‐Keuls post‐test and the level of significance was set at 5%.ResultsThe treatment with E2 attenuated JNK and p38MAPK phosphorylation in response to ANGII in the lamina terminalis (E2 x ANGII interaction: F1,17=19.00, p<0.001; E2 x ANGII interaction: F1,29=6.1, p<0.05, respectively). In regarding to PVN and SON, it was observed that E2 attenuated ERK1/2 and p38MAPK phosphorylation in response to ANGII (E2 x ANGII interaction: F1,29=7.91, p<0.01; F1,26=4.1, p<0.05, respectively). E2, but not ANGII alone, was able to increase MKP‐1 expression in the PVN and SON (E2: F1,18=24.29, p<0.001), but not in the lamina terminalis.ConclusionThese results suggest that E2 dephosphorylates ERK1/2 and p38MAPK in response to ANGII by increasing MKP‐1 in the PVN and SON, but not in structures of lamina terminalis, such as subfornical organ, median preoptic nucleus and organum vasculosum of the lamina terminalis. E2 is able to dephosphorylate JNK and p38MAPK in the lamina terminalis in response to ANGII, however this effect does not involve MKP‐1.Support or Funding InformationFinancial support: FAPESP, CNPq and CAPES.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Whole body sodium depletion modifies AT1 mRNA expression and serotonin content in the dorsal raphe nucleus.

Angiotensin II (Ang II) acts on Ang II type 1 (AT1) receptors located in the organum vasculosum and subfornical organ (SFO) of the lamina terminalis as a main facilitatory mechanism of sodium appetite. The brain serotonin (5-HT) system with soma located in the dorsal raphe nucleus (DRN) provides a main inhibitory mechanism. In the present study, we first investigated the existence of Ang II AT1 receptors in serotonergic DRN neurones. Then, we examined whether whole body sodium depletion affects the gene expression of the AT1a receptor subtype and the presumed functional significance of AT1 receptors. Using confocal microscopy, we found that tryptophan hydroxylase-2 and serotonin neurones express AT1 receptors in the DRN. Immunofluorescence quantification showed a significant reduction in 5-HT content but no change in AT1 receptor expression or AT1/5-HT colocalisation in the DRN after sodium depletion. Whole body sodium depletion also significantly increased Agtr1a mRNA expression in the SFO and DRN. Oral treatment with the AT1 receptor antagonist losartan reversed the changes in Agtr1a expression in the SFO but not the DRN. Losartan injection into either the DRN or the mesencephalic aqueduct had no influence on sodium depletion-induced 0.3molL-1 NaCl intake. The results indicate the expression of Agtr1a mRNA in the DRN and SFO as a marker of sodium depletion. They also suggest that serotonergic DRN neurones are targets for Ang II. However, the function of their AT1 receptors remains elusive.

From sensory circumventricular organs to cerebral cortex: Neural pathways controlling thirst and hunger.

Much progress has been made during the past 30years with respect to elucidating the neural and endocrine pathways by which bodily needs for water and energy are brought to conscious awareness through the generation of thirst and hunger. One way that circulating hormones influence thirst and hunger is by acting on neurones within sensory circumventricular organs (CVOs). This is possible because the subfornical organ and organum vasculosum of the lamina terminalis (OVLT), the sensory CVOs in the forebrain, and the area postrema in the hindbrain lack a normal blood-brain barrier such that neurones within them are exposed to blood-borne agents. The neural signals generated by hormonal action in these sensory CVOs are relayed to several sites in the cerebral cortex to stimulate or inhibit thirst or hunger. The subfornical organ and OVLT respond to circulating angiotensin II, relaxin and hypertonicity to drive thirst-related neural pathways, whereas circulating amylin, leptin and possibly glucagon-like peptide-1 act at the area postrema to influence neural pathways inhibiting food intake. As a result of investigations using functional brain imaging techniques, the insula and anterior cingulate cortex, as well as several other cortical sites, have been implicated in the conscious perception of thirst and hunger in humans. Viral tracing techniques show that the anterior cingulate cortex and insula receive neural inputs from thirst-related neurones in the subfornical organ and OVLT, with hunger-related neurones in the area postrema having polysynaptic efferent connections to these cortical regions. For thirst, initially, the median preoptic nucleus and, subsequently, the thalamic paraventricular nucleus and lateral hypothalamus have been identified as likely sites of synaptic links in pathways from the subfornical organ and OVLT to the cortex. The challenge remains to identify the links in the neural pathways that relay signals originating in sensory CVOs to cortical sites subserving either thirst or hunger.

Leukemia Inhibitory Factor Represses GnRH Gene Expression via cFOS during Inflammation in Male Mice

Background: The mechanisms whereby neuroinflammation negatively affects neuronal function in the hypothalamus are not clear. Our previous study determined that obesity-mediated chronic inflammation elicits sex-specific impairment in reproductive function via reduction in spine density in gonadotropin-releasing hormone (GnRH) neurons. Neuroinflammation and subsequent decrease in GnRH neuron spine density was specific for male mice, while protection in females was independent of ovarian estrogens. Methods: To examine if neuroinflammation-induced cytokines can directly regulate GnRH gene expression, herein we examined signaling pathways and mechanisms in males in vivo and in GnRH-expressing cell line, GT1–7. Results: GnRH neurons express cytokine receptors, and chronic or acute neuroinflammation represses GnRH gene expression in vivo. Leukemia inhibitory factor (LIF) in particular represses GnRH expression in GT1–7 cells, while other cytokines do not. STAT3 and MAPK pathways are activated following LIF treatment, but only MAPK pathway, specifically p38α, is sufficient to repress the GnRH gene. LIF induces cFOS that represses the GnRH gene via the -1,793 site in the enhancer region. In vivo, following high-fat diet, cFOS is induced in GnRH neurons and neurons juxtaposed to the leaky blood brain barrier of the organum vasculosum of the lamina terminalis, but not in the neurons further away. Conclusion: Our results indicate that the increase in LIF due to neuroinflammation induces cFOS and represses the GnRH gene. Therefore, in addition to synaptic changes in GnRH neurons, neuroinflammatory cytokines directly regulate gene expression and reproductive function, and the specificity for neuronal targets may stem from the proximity to the fenestrated capillaries.

Patterns of preoptic-hypothalamic neuronal activation and LH secretion in female sheep following the introduction and withdrawal of novel males.

The neuroendocrine response of female sheep to a novel male involves neural activation in the hypothalamus. However, if males are removed, the gonadotrophic signal declines, so the neural activity is likely to change. We examined Fos-immunoreactive (IR) cells in hypothalamic tissues from seasonally anovulatory female sheep exposed to males for 2 or 6h, or for 2h followed by 4h isolation from males. Control females were killed in the absence of male exposure. Male introduction increased LH secretion in all females; male removal was associated with a reduction only in mean and basal LH concentrations. Females exposed to males for 2h had more Fos-IR cells in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH) and organum vasculosum of the lamina terminalis (OVLT) than control females. Fos-IR cells in the preoptic area (POA) were only greater than in control females after 6h exposure to a male. Removal of males decreased the number of Fos-IR cells in the ARC, VMH and OVLT, but not in the POA. Thus, hypothalamic neural activation and LH secretion in female sheep are stimulated by males and decline after male removal. However, activation in the POA persists after removal and may explain the incomplete decline in the LH response.

Sodium Is Detected by the OVLT to Regulate Sympathetic Tone

Hypernatremia is known to elicit a rise in sympathetic tone and blood pressure. In this issue of Neuron, Nomura etal. (2018) now show that this is mediated via the organum vasculosum laminae terminalis (OVLT). Na+ activates OVLT neurons via a paracrine mechanism involving sodium channel Nax expressed by astrocytes and the ependyma.

Na+] Increases in Body Fluids Sensed by Central Nax Induce Sympathetically Mediated Blood Pressure Elevations via H+-Dependent Activation of ASIC1a

Increases in sodium concentrations ([Na+]) in body fluids elevate blood pressure (BP) by enhancing sympathetic nerve activity (SNA). However, the mechanisms by which information on increased [Na+] is translated to SNA have not yet been elucidated. We herein reveal that sympathetic activation leading to BP increases is not induced by mandatory high salt intakes or the intraperitoneal/intracerebroventricular infusions of hypertonic NaCl solutions in Nax-knockout mice in contrast to wild-type mice. We identify Nax channels expressed in specific glial cells in the organum vasculosum lamina terminalis (OVLT) as the sensors detecting increases in [Na+] in body fluids and show that OVLT neurons projecting to the paraventricular nucleus (PVN) are activated via acid-sensing ion channel 1a (ASIC1a) by H+ ions exported from Nax-positive glial cells. The present results provide an insight into the neurogenic mechanisms responsible for salt-induced BP elevations.

Abstract 107: Neurons in the Organum Vasculosum of the Lamina Terminalis Sense NaCl and Angiotensin II to Regulate Sympathetic Nerve Activity and Arterial Blood Pressure

The organum vasculosum of the lamina terminalis (OVLT) contains specialized neurons that sense changes in extracellular NaCl concentrations to regulate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). However, OVLT neurons also abundantly express angiotensin II (AngII)-type I receptors. Therefore, we performed a series of in vitro and in vivo studies in adult male Sprague-Dawley rats to determine whether OVLT neurons sense both NaCl and AngII to impact SNA and ABP. First, in vitro whole-cell recordings revealed the majority of OVLT neurons (66%, 23/35) showed an increased discharge frequency to both +7.5mM NaCl (1.1±0.2 to 2.2±0.3Hz, P<0.001) and 100pM AngII (1.3±0.3 to 2.2±0.3Hz, P<0.001). Second, in vivo single-unit recordings demonstrate the majority of OVLT neurons (61%, 11/18) displayed an increase in cell discharge to intracarotid injection of 0.5M NaCl (3.6±0.6 to 9.0±1.6Hz, P<0.01) and 200ng AngII (3.2±0.7 to 9.2±1.9Hz, P<0.01). Third, optogenetic inhibition of OVLT neurons (rAAV2-CaMKII-eNpHR3.0-mCherry, 5x10 12 particles/mL, 561nm, 10mW) attenuated thirst responses to IV infusion of 2M NaCl (1.25mL/30min; Laser OFF: 6.1±0.5mL vs ON: 2.0±0.7mL, P<0.05) and AngII (Laser OFF 7.2±0.7mL vs ON: 2.2±1.2mL; n=5 per group, P<0.05). Fourth, OVLT injection (20nL) of 0.5M NaCl or 10pmol AngII increased SNA (121±6% or 117±5%, n=4/group; P<0.05 respectively) and mean ABP (7±1 or 9±2mmHg, n=4/group; P<0.05 respectively). To determine whether chronic activation of OVLT neurons produces hypertension, rats received an OVLT injection of rAAV2-hSyn-HM3D(Gq)-mCherry (3x10 12 particles/mL, 50nL) and given access to 0.1% NaCl chow and 0.9% NaCl drinking solution. After 4 weeks, daily administration of clozapine-N-oxide to the drinking solution (3mg/kg/day) significantly increased 24-h fluid intake (baseline: 60±10mL vs Day 7: 178±29mL; n=6, P<0.01) and 24-h mean ABP (baseline: 97±2mmHg vs Day 7: 108±1 mmHg; n=6, P<0.05). Ganglionic blockade with hexamethonium (30mg/kg, ip) produced a greater fall in mean ABP at Day 7 (baseline: -38±6 mmHg vs Day 7: -53±5 mmHg, P<0.05). These findings suggest OVLT neurons sense both NaCl and AngII to regulate body fluid homeostasis and SNA to produce a sympathetically-mediated hypertension.

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.

Upregulation of hypothalamic arginine vasopressin by peripherally administered furosemide in transgenic rats expressing arginine vasopressin-enhanced green fluorescent protein.

Furosemide, which is used worldwide as a diuretic agent, inhibits sodium reabsorption in the Henle's loop, resulting in diuresis and natriuresis. Arginine vasopressin (AVP) is synthesized in the supraoptic nucleus (SON), paraventricular nucleus (PVN), and suprachiasmatic nucleus (SCN) of the hypothalamus. The synthesis AVP in the magnocellular neurons of SON and PVN physiologically regulated by plasma osmolality and blood volume and contributed water homeostasis by increasing water reabsorption in the collecting duct. Central AVP dynamics after peripheral administration of furosemide remain unclear. Here, we studied the effects of intraperitoneal (i.p.) administration of furosemide (20 mg/kg) on hypothalamic AVP by using transgenic rats expressing AVP-enhanced green fluorescent protein (eGFP) under the AVP promoter. The i.p. administration of furosemide did not affect plasma osmolality in the present study; however, eGFP in the SON and magnocellular divisions of the PVN (mPVN) were significantly increased after furosemide administration compared to the control. Immunohistochemical analysis revealed Fos-like immunoreactivity (IR) in eGFP-positive neurons in the SON and mPVN 90 min after i.p. administration of furosemide, and AVP heteronuclear (hn) RNA and eGFP mRNA levels were significantly increased. These furosemide-induced changes were not observed in the suprachiasmatic AVP neurons. Furthermore, furosemide induced a remarkable increase in Fos-IR in the organum vasculosum laminae terminals (OVLT), median preoptic nucleus (MnPO), subfornical organ (SFO), locus coeruleus (LC), nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVLM) after i.p. administration of furosemide. In conclusion, we were able to visualize and quantitatively evaluate AVP-eGFP synthesis and neuronal activations after peripheral administration of furosemide, using the AVP-eGFP transgenic rats. The results of this study may provide new insights into the elucidation of physiological mechanisms underlying body fluid homeostasis induced by furosemide. This article is protected by copyright. All rights reserved.

Lesion of the OVLT markedly attenuates chronic DOCA-salt hypertension in rats.

Lesions of the anteroventral third ventricle (AV3V region) are known to prevent many forms of experimental hypertension, including mineralocorticoid [deoxycorticosterone acetate (DOCA)-salt] hypertension in the rat. However, AV3V lesions include the organum vasculosum of the lamina terminalis (OVLT), portions of the median preoptic nucleus, and efferent fibers from the subfornical organ (SFO), thereby limiting the ability to define the individual contribution of these structures to the prevention of experimental hypertension. Having previously reported that the SFO does not play a significant role in the development of DOCA-salt hypertension, the present study was designed to test the hypothesis that the OVLT is necessary for DOCA-salt hypertension in the rat. In uninephrectomized OVLT-lesioned (OVLTx; n = 6) and sham-operated ( n = 4) Sprague-Dawley rats consuming a 0.1% NaCl diet and 0.9% NaCl drinking solution, 24-h mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg sc per rat). No differences in control MAP were observed between groups. The chronic pressor response to DOCA was attenuated in OVLTx rats such that MAP increased to 133 ± 3 mmHg in sham-operated rats by day 21 of DOCA compared with 120 ± 4 mmHg (means ± SE) in OVLTx rats. These results support the hypothesis that the OVLT is an important brain site of action for the pathogenesis of DOCA-salt hypertension in the rat.

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 partthrough 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.

Role of the OVLT‐gut microbiome axis on the development of DOCA‐salt hypertension

Limited studies suggest an association of gut microbiome changes with certain forms of hypertension, but a cause/effect relationship has yet to be fully defined. The deoxycorticosterone acetate (DOCA)‐salt model of hypertension in rats is known to have a neurogenic component linked to increased sympathetic nervous system activity. Indeed, we have recently shown the hypertensive response in DOCA‐treated rats requires the organum vasculosum of the lamina terminalis (OVLT), a circumventricular organ that has direct connections to the paraventricular nucleus (PVN) to increase sympathetic activity. Furthermore, celiac ganglionectomy also supports a role of splanchnic sympathetic innervation to the gut in the hypertensive response to DOCA. Taken together, we hypothesize an OVLT‐sympathetic‐gut (OSG) microbiome axis is involved in the development of hypertension. Having previously reported that the OVLT plays a significant role in the development of DOCA‐salt hypertension, the present study was designed to test the hypothesis that the OVLT mediates changes in the gut microbiome associated with concomitant hypertension. In uninephrectomized OVLT lesioned (OVLTx; n = 5) and SHAM (n = 2) Sprague‐Dawley rats consuming a 2% NaCl diet and 0.9% NaCl drinking solution, 24‐hour mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg/rat; SQ). Microbiome analyses were performed on regional gut samples at the end of the study and compared to normal rats (n=2). The chronic pressor response to DOCA was attenuated in OVLTx rats such that MAP increased to 153±4 mmHg in SHAM rats by Day 21 of DOCA compared to 127±5 mmHg in OVLTx rats. Moreover, DOCA hypertension in SHAM rats was associated with decreased levels of Lactobacillus species in the jejunum and ileum, and increased Escherichia levels in the cecum compared with normal rats. However, these changes in bacterial genera were mitigated and offset by lesion of the OVLT and these bacterial populations in OVLT lesioned rats with attenuated hypertension more closely resembled those in normal control rats. We conclude that DOCA‐salt hypertension is associated with specific microbiome changes in the gut, and the attenuated hypertensive effects of DOCA‐salt in OVLT lesioned rats is mediated in part through counteracting changes in these bacterial populations.Support or Funding InformationUniversity of Minnesota Grant‐In‐Aid # 107426This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurons in the Organum Vasculosum of the Lamina Terminalis Sense Both Angiotensin II and NaCl to Regulate Thirst

Angiotensin II (AngII) and NaCl are sensed by hypothalamic circumventricular organs to subsequently regulate body fluid homeostasis and blood pressure. AngII predominantly acts within the subfornical organ (SFO), whereas NaCl is sensed by neurons in the organum vasculosum of the lamina terminalis (OVLT). However, OVLT neurons also abundantly express AngII‐type I receptors. Therefore, we performed a series of in vitro and in vivo experiments in adult male Sprague‐Dawley rats to determine whether OVLT neurons sense both AngII and NaCl to impact body fluid homeostasis. First, in vitro whole‐cell recordings were performed on OVLT neurons in the presence of kynurenic acid (1mM) and bicuculline (20μM) to assess discharge responses to +7.5 mM NaCl (3 min) versus 100pM AngII (90s) in a randomized order. The majority of OVLT neurons (66%, 23/35) demonstrated an increase in discharge frequency to both AngII (1.31±0.25Hz to 2.19±0.31Hz; p<0.001) and hypertonic NaCl (1.11±0.18Hz to 2.16±0.27 Hz; p<0.001). Smaller proportions of OVLT neurons were either excited by NaCl alone (3/35), AngII alone (4/35), neither stimulus (3/35), or were inhibited by NaCl (2/35). In a second set of experiments, in vivo single‐unit recordings of OVLT neurons were performed to assess discharge responses to intracarotid injection (50uL over 15 s) of hypertonic NaCl (0.5M NaCl) or AngII (200ng). Half of OVLT neurons (50%, 6/12) displayed an increase in discharge to intracarotid injection of AngII (3.2±0.7 to 9.2±1.9 Hz, P<0.01) and 0.5M NaCl (3.6±0.6 to 9.0±1.6 Hz, P<0.01). The remainder of OVLT neurons showed an increase in discharge to hypertonic NaCl only (n=1) or AngII only (5/12). In a final set of experiments, we employed an optogenetic approach to determine the extent by which inhibition of OVLT neurons would attenuate thirst stimulated by NaCl versus AngII. rAAV2‐CamKII‐eNPHR3.0‐mCherry (5×1012 particles/mL, 50nL) was injected into the OVLT, and an optical ferrule (200um OD) was implanted 300um dorsal to the injection site. Approximately 4 weeks later, optogenetic inhibition of OVLT neurons (561nm, 10mW) significantly reduced water intake in response to intravenous infusion (1.25 mL per 30 min) of 2M NaCl (control: 5.1±0.9 mL vs laser: 1.6±0.9 mL; n=4, P<0.01). In addition, optogenetic inhibition of OVLT neurons also attenuated hypotension‐induced thirst produced by intravenous injection of 25 mg/kg diazoxide (control: 4.5±0.8 mL vs laser: 1.3±0.7 mL; n=4, P<0.01). These findings suggest that individual OVLT neurons can sense both NaCl and AngII to functionally regulate thirst to both stimuli.Support or Funding InformationNIH R01 HL113270 and AHA Established Investigator AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identifying ‘angiotensin sensitive’ neurons in the lamina terminalis that coordinate endocrine, cardiovascular and behavioral responses mediating body fluid homeostasis

Body fluid homeostasis is controlled by endocrine, cardiovascular and behavioral responses that maintain blood osmolality and perfusion pressure at optimal levels. Angiotensin type 1a receptors (AT1a) in the brain are known to coordinate these responses but the underlying neuronal circuits remain unclear. This study investigates the structure and function of AT1a neurons in the median preoptic nucleus (MnPO) and the organum vasculosum of the lamina terminalis (OVLT) to better understand their role in body fluid homeostasis. Using male mice that express Cre‐recombinase prior to the STOP codon of the AT1a gene (i.e., AT1a‐Cre mice), initial studies combined genetic reporting with in situ hybridization to reveal that neurons in the MnPO and OVLT that express AT1a are largely glutamatergic. 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. Neuroanatomical and in vitro electrophysiological studies revealed that AT1a neurons in the MnPO/OVLT send dense glutamatergic projections into the paraventricular nucleus of the hypothalamus that synapse onto vasopressin (AVP)‐synthesizing neurons. To evaluate the functionality of this connection, we optogenetically stimulated AT1a‐expressing neurons in the MnPO/OVLT and recorded cardiovascular parameters in anesthetized mice. Relative to control mice given virus that only expressed eYFP, 10 min of optogenetic stimulation robustly elevated systolic blood pressure in mice expressing ChR2. This effect was rapid in its onset (34 ± 9 vs. −2 ± 4 mmHg at 5 min, p < 0.05) but persisted for the entire 50 min of cardiovascular recording (45 ± 11 vs. −3 ± 7 mmHg at 50 min, p < 0.05). Intriguingly, optogenetic stimulation elicited a significant increase in Fos induction in AVP neurons within the PVN relative to controls. To evaluate behavioral responses, AT1a neurons in the MnPO/OVLT were optogenetically stimulated while conscious freely moving mice had access to both water and 0.3M NaCl. Relative to controls, mice delivered ChR2 showed a significant increase in both water and sodium consumption. Collectively, the results suggest that AT1a neurons in the MnPO/OVLT comprise a neuronal circuit that mediates endocrine, cardiovascular and behavioral responses that maintain body fluid homeostasis.Support or Funding InformationNIH: HL122494 (EGK), MH104641(CJF), HL125805 and AHA GRNT33660969 (AdK)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Centrally administered Ang‐(1‐7) via Mas receptors reverses psychosocial stress‐induced cardiovascular responses in mice with muscular dystrophy

Muscular dystrophy is a devastating disease of muscle weakness, atrophy and dilated cardiomyopathy (DCM). We have demonstrated that angiotensin II (Ang II)‐dependent autonomic dysfunction precedes and predicts development of DCM in sarcoglycan delta deficient (Sgcd−/−) mice, an established model of muscular dystrophy [Exp Physiol 2015]. More recently, we found that Sgcd−/− mice exhibit depression‐like behaviors and are susceptible to psychosocial stress‐induced bradycardia, hypotension and arrhythmias at older ages (>45 weeks of age) [FASEB J 2017]. Thus, we hypothesized that central administration of angiotensin‐(1‐7) in older Sgcd−/− mice protects against Ang II‐mediated cardiovascular abnormalities during acute psychosocial stress challenge. Mean blood pressure (MAP) and heart rate (HR) were measured by telemetry in control C57BL6 (n=3–5) and Sgcd−/− (n=3–8) mice during social defeat (~15 min exposure to intruder mouse). At 40±3 weeks of age, mice were randomly assigned to one of the four experimental groups – (i) vehicle (saline); (ii) losartan (Ang II‐type 1 receptor antagonist, 1 mcg/kg/hr); (iii) Ang‐(1‐7) (100 ng/kg/hr); and (iv) Ang‐(1‐7)+Mas antagonist A779 (100 ng/kg/hr and 0.5 mcg/kg/hr, respectively). Drugs were administered ICV (catheter connected to osmotic minipump) for 8 weeks. Stressed Sgcd−/− mice exhibit an increase in AT1R‐binding (receptor autoradiography) in subfornical organ, organum vasculosum laminae terminalis, and paraventricular nucleus of hypothalamus. Social defeat produced profound bradycardia and hypotension in Sgcd−/− mice (see Table). Interestingly, while these drastic effects on MAP and HR in Sgcd−/− mice were modestly attenuated by losartan, the effects were completely abolished by Ang‐(1‐7) (P<0.05 vs. vehicle) and remained unaltered by Ang‐(1‐7)+A779 (NS vs. vehicle). Moreover, the increased plasma corticosterone (+86%) and norepinephrine (+62%) in stressed Sgcd−/− mice were normalized by Ang‐(1‐7) (P<0.05 vs. control).We conclude that Ang‐(1‐7) acting centrally via Mas receptors abrogates cardiovascular response to psychosocial stress and is likely a more effective treatment strategy than Ang II‐type 1 receptor antagonists in muscular dystrophy.Support or Funding InformationAHA GIA17GRNT33670004This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The origins of the circumventricular organs.

The circumventricular organs (CVOs) are specialised neuroepithelial structures found in the midline of the brain, grouped around the third and fourth ventricles. They mediate the communication between the brain and the periphery by performing sensory and secretory roles, facilitated by increased vascularisation and the absence of a blood-brain barrier. Surprisingly little is known about the origins of the CVOs (both developmental and evolutionary), but their functional and organisational similarities raise the question of the extent of their relationship. Here, I review our current knowledge of the embryonic development of the seven major CVOs (area postrema, median eminence, neurohypophysis, organum vasculosum of the lamina terminalis, pineal organ, subcommissural organ, subfornical organ) in embryos of different vertebrate species. Although there are conspicuous similarities between subsets of CVOs, no unifying feature characteristic of their development has been identified. Cross-species comparisons suggest that CVOs also display a high degree of evolutionary flexibility. Thus, the term 'CVO' is merely a functional definition, and features shared by multiple CVOs may be the result of homoplasy rather than ontogenetic or phylogenetic relationships.

Development of Circumventricular Organs in the Mirror of Zebrafish Enhancer-Trap Transgenics.

The circumventricular organs (CVOs) are small structures lining the cavities of brain ventricular system. They are associated with the semitransparent regions of the blood-brain barrier (BBB). Hence it is thought that CVOs mediate biochemical signaling and cell exchange between the brain and systemic blood. Their classification is still controversial and development not fully understood largely due to an absence of tissue-specific molecular markers. In a search for molecular determinants of CVOs we studied the green fluorescent protein (GFP) expression pattern in several zebrafish enhancer trap transgenics including Gateways (ET33-E20) that has been instrumental in defining the development of choroid plexus. In Gateways the GFP is expressed in regions of the developing brain outside the choroid plexus, which remain to be characterized. The neuroanatomical and histological analysis suggested that some previously unassigned domains of GFP expression may correspond to at least six other CVOs–the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), paraventricular organ (PVO), pineal (epiphysis), area postrema (AP) and median eminence (ME). Two other CVOs, parapineal and subcommissural organ (SCO) were detected in other enhancer-trap transgenics. Hence enhancer-trap transgenic lines could be instrumental for developmental studies of CVOs in zebrafish and understanding of the molecular mechanism of disease such a hydrocephalus in human. Their future analysis may shed light on general and specific molecular mechanisms that regulate development of CVOs.

The Forebrain Thirst Circuit Drives Drinking through Negative Reinforcement

The brain transforms the need for water into the desire to drink, but how this transformation is performed remains unknown. Here we describe the motivational mechanism by which the forebrain thirst circuit drives drinking. We show that thirst-promoting subfornical organ neurons are negatively reinforcing and that this negative-valence signal is transmitted along projections to the organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus (MnPO). We then identify molecularly defined cell types within the OVLT and MnPO that are activated by fluid imbalance and show that stimulation of these neurons is sufficient to drive drinking, cardiovascular responses, and negative reinforcement. Finally, we demonstrate that the thirst signal exits these regions through at least three parallel pathways and show that these projections dissociate the cardiovascular and behavioral responses to fluid imbalance. These findings reveal a distributed thirst circuit that motivates drinking by the common mechanism of drive reduction.

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.