RESUMEN
Homeostatic challenges may alter the drive for social interaction. The neural activity that prompts this motivation remains poorly understood. In the present study, we identify direct projections from the hypothalamic supraoptic nucleus to the cortico-amygdalar nucleus of the lateral olfactory tract (NLOT). Dual in situ hybridization with probes for pituitary adenylate cyclase-activating polypeptide (PACAP), as well as vesicular glutamate transporter (VGLUT)1, VGLUT2, V1a and V1b, revealed a population of vasopressin-receptive PACAPergic neurons in NLOT layer 2 (NLOT2). Water deprivation (48 h, WD48) increased sociability compared to euhydrated subjects, as assessed with the three-chamber social interaction test (3CST). Fos expression immunohistochemistry showed NLOT and its main efferent regions had further increases in rats subjected to WD48 + 3CST. These regions strongly expressed PAC1 mRNA. Microinjections of arginine vasopressin (AVP) into the NLOT produced similar changes in sociability to water deprivation, and these were reduced by co-injection of V1a or V1b antagonists along with AVP. We conclude that, during challenge to water homeostasis, there is a recruitment of a glutamatergic-multi-peptidergic cooperative circuit that promotes social behavior.
Asunto(s)
Neocórtex , Núcleo Supraóptico , Humanos , Ratas , Animales , Núcleo Supraóptico/metabolismo , Arginina Vasopresina/metabolismo , Bulbo Olfatorio , Neocórtex/metabolismo , Ratas Wistar , Vasopresinas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Conducta Social , Homeostasis , Agua/metabolismoRESUMEN
Impairment of social behaviour is a hallmark of emotional disorders, with increased avoidance of social contact. In rats, the 24 h maternal deprivation (DEP) paradigm is used to understand the impact of extreme neglect on neurodevelopment. Due to the distinct immediate effects of DEP on postnatal days (PND) 3 (DEP3) or 11 (DEP11), in the present study we investigated the long-term effects of DEP at these ages on anxiety-like behaviour, by recording the visits and time spent in the centre part of the open-field, social investigation of a confined, same-sex, unfamiliar animal, basal and post-social test corticosterone plasma levels and the immunoreactivity to oxytocin in the paraventricular (PVN) and supraoptic nuclei of the hypothalamus (SON). Whole litters were distributed into control (CTL), DEP3 or DEP11 groups and behavioural tests and biological samples were collected between PNDs 40 and 45 in males and females. There were no differences in the exploration of the central part of the open field or on the time investigating the unfamiliar rat. However, the percent increase in post-test corticosterone secretion from baseline was greater for both DEP3 male and female subgroups than their CTL and DEP11 counterparts. DEP3 females showed more oxytocin staining than DEP11 counterparts in magnocellular neurons of the SON and PVN. These results suggest that DEP at the ages chosen does not alter social investigation, although it results in distinct neurobiological outcomes, depending on the developmental phase when it is imposed.
Asunto(s)
Corticosterona , Privación Materna , Animales , Femenino , Masculino , Oxitocina , Núcleo Hipotalámico Paraventricular , Ratas , Conducta Social , Núcleo SupraópticoRESUMEN
Vasopressinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei express oestrogen receptor (ER)ß and receive afferent projections from osmosensitive neurones that express ERα. However, which subtype of these receptors mediates the effects of oestradiol on vasopressin (AVP) secretion induced by hydromineral challenge has not yet been demonstrated in vivo. Moreover, AVP secretion induced by hyperosmolality is known to involve activation of TRPV1 (transient receptor potential vanilloid, member 1) in magnocellular neurones, although whether oestradiol modulates expression of this receptor is unknown. Thus, the present study aimed to clarify the mechanisms involved in the modulation exerted by oestradiol on AVP secretion, specifically investigating the involvement of ERß, ERα and TRPV1 receptors in response to water deprivation (WD). We observed that treatment with an ERß agonist potentiated AVP secretion and vasopressinergic neuronal activation induced by WD. This increase in AVP secretion induced by WD was reversed by an ERß antagonist. By contrast to ERß, the ERα agonist did not alter plasma AVP concentrations or activation of AVP neurones in the SON and PVN. Additionally, Fos expression in the subfornical organ was not altered by the ERα agonist. TRPV1 mRNA expression was increased by WD in the SON, although this response was not altered by any treatment. The results of the present study suggest that ERß mediates the effects of oestradiol on AVP secretion in response to WD, indicating that the effects of oestradiol occur directly in AVP neurones without affecting TRPV1.
Asunto(s)
Estradiol/farmacología , Receptor beta de Estrógeno/fisiología , Neuronas/fisiología , Vasopresinas/fisiología , Privación de Agua/fisiología , Animales , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Femenino , Concentración Osmolar , Núcleo Hipotalámico Paraventricular/química , Núcleo Hipotalámico Paraventricular/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Wistar , Elastómeros de Silicona , Núcleo Supraóptico/química , Núcleo Supraóptico/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/fisiología , Vasopresinas/análisis , Vasopresinas/sangreRESUMEN
NEW FINDINGS: What is the central question of this study? The central goal of this study was to understand the effects of central angiotensin-(1-7) on basal and osmotically stimulated water intake in rats. What is the main finding and its importance? This study demonstrated that central administration of angiotensin-(1-7) did not induce thirst in basal conditions but increased water intake after osmotic stimulation, such as water deprivation and salt loading. These results indicate a new function for this peptide, which, in turn, allows for future research on the mechanisms through which angiotensin-(1-7) influences osmotic thirst. Angiotensin-(1-7) [Ang-(1-7)] is generated by type 2 angiotensin-converting enzyme (ACE2) and binds to the MAS receptor. Although it is well known that Ang-(1-7) functionally antagonizes the effects of the classical renin-angiotensin system in several situations, the role of Ang-(1-7) in hydromineral homeostasis is not clear. The aim of this study was to assess the role of Ang-(1-7) on neuroendocrine responses to hyperosmolality in rats. Male Wistar rats were divided into the following three groups: control; 24 h of water deprivation (WD); and 24 h of salt loading (SL; 1.8% NaCl). Intracerebroventricular (i.c.v.) injections of Ang-(1-7) or vehicle were given to assess water intake and plasma concentration of vasopressin. Additionally, the brains from control and WD groups were collected to evaluate gene expression in the subfornical organ (SFO), paraventricular nucleus (PVN) and supraoptic nucleus (SON). It was found that i.c.v. Ang-(1-7) did not change water and salt intake in control rats; however, Ang-(1-7) increased water intake after WD and SL, with no change in salt intake. Plasma vasopressin was not changed by i.c.v. Ang-(1-7) in control or WD rats. Moreover, WD increased Mas gene expression in the SON and PVN, with no changes in Ace2 mRNA levels. In conclusion, Ang-(1-7) increases thirst after osmotic stimuli, indicating that a previous sensitization to its action is necessary. This finding is consistent with the increased Mas gene expression in the PVN and SON after water deprivation.
Asunto(s)
Angiotensina I/administración & dosificación , Ingestión de Líquidos/efectos de los fármacos , Presión Osmótica , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Fragmentos de Péptidos/administración & dosificación , Órgano Subfornical/efectos de los fármacos , Núcleo Supraóptico/efectos de los fármacos , Sed/efectos de los fármacos , Enzima Convertidora de Angiotensina 2 , Animales , Inyecciones Intraventriculares , Masculino , Núcleo Hipotalámico Paraventricular/metabolismo , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Cloruro de Sodio/administración & dosificación , Órgano Subfornical/metabolismo , Núcleo Supraóptico/metabolismo , Regulación hacia Arriba , Vasopresinas/sangre , Privación de AguaRESUMEN
The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to the neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula and other brain regions in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA). The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS), consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptor mRNAs were not detected, using the same method. Water-deprivation (WD) for 24 h, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze (EPM) test, and this effect was mimicked by bilateral microinfusion of AVP into the CeA. Anxious behavior induced by either WD or AVP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of CeA inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala.
Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas/farmacología , Ansiedad/metabolismo , Arginina Vasopresina/metabolismo , Núcleo Amigdalino Central , Glutamato Descarboxilasa/metabolismo , Hipotálamo , Neuronas , Receptores de Vasopresinas/metabolismo , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas/administración & dosificación , Ansiedad/inducido químicamente , Conducta Animal , Núcleo Amigdalino Central/citología , Núcleo Amigdalino Central/metabolismo , Modelos Animales de Enfermedad , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/metabolismo , Hipotálamo/citología , Hipotálamo/metabolismo , Masculino , Privación Materna , Neuronas/citología , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/metabolismo , Ratas Wistar , Núcleo Supraóptico/citología , Núcleo Supraóptico/metabolismo , Privación de AguaRESUMEN
Water deprivation (WD) induces changes in plasma volume and osmolality, which in turn activate several responses, including thirst, the activation of the renin-angiotensin system (RAS) and vasopressin (AVP) and oxytocin (OT) secretion. These systems seem to be influenced by oestradiol, as evidenced by the expression of its receptor in brain areas that control fluid balance. Thus, we investigated the effects of oestradiol treatment on behavioural and neuroendocrine changes of ovariectomized rats in response to WD. We observed that in response to WD, oestradiol treatment attenuated water intake, plasma osmolality and haematocrit but did not change urinary volume or osmolality. Moreover, oestradiol potentiated WD-induced AVP secretion, but did not alter the plasma OT or angiotensin II (Ang II) concentrations. Immunohistochemical data showed that oestradiol potentiated vasopressinergic neuronal activation in the lateral magnocellular PVN (PaLM) and supraoptic (SON) nuclei but did not induce further changes in Fos expression in the median preoptic nucleus (MnPO) or subfornical organ (SFO) or in oxytocinergic neuronal activation in the SON and PVN of WD rats. Regarding mRNA expression, oestradiol increased OT mRNA expression in the SON and PVN under basal conditions and after WD, but did not induce additional changes in the mRNA expression for AVP in the SON or PVN. It also did not affect the mRNA expression of RAS components in the PVN. In conclusion, our results show that oestradiol acts mainly on the vasopressinergic system in response to WD, potentiating vasopressinergic neuronal activation and AVP secretion without altering AVP mRNA expression.
Asunto(s)
Deshidratación/fisiopatología , Estradiol/uso terapéutico , Estrógenos/uso terapéutico , Neuronas/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Supraóptico/efectos de los fármacos , Desequilibrio Hidroelectrolítico/prevención & control , Animales , Arginina Vasopresina/agonistas , Arginina Vasopresina/análisis , Arginina Vasopresina/metabolismo , Conducta Animal/efectos de los fármacos , Deshidratación/terapia , Ingestión de Líquidos/efectos de los fármacos , Terapia de Reemplazo de Estrógeno , Femenino , Fluidoterapia , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Ovariectomía/efectos adversos , Núcleo Hipotalámico Paraventricular/metabolismo , Núcleo Hipotalámico Paraventricular/patología , Área Preóptica/efectos de los fármacos , Área Preóptica/metabolismo , Área Preóptica/patología , Ratas Wistar , Órgano Subfornical/efectos de los fármacos , Órgano Subfornical/metabolismo , Órgano Subfornical/patología , Núcleo Supraóptico/metabolismo , Núcleo Supraóptico/patología , Núcleo Vestibular Lateral/efectos de los fármacos , Núcleo Vestibular Lateral/metabolismo , Núcleo Vestibular Lateral/patología , Desequilibrio Hidroelectrolítico/sangre , Desequilibrio Hidroelectrolítico/etiología , Desequilibrio Hidroelectrolítico/fisiopatologíaRESUMEN
Previous studies from our laboratory have shown that methysergide, a serotonergic antagonist, injected into the lateral parabrachial nucleus (LPBN) combined with a pre-load of 2 M NaCl, given by gavage, induces 0.3 M NaCl intake. The mechanisms involved in this paradoxical behavior are still unknown. In the present work, we investigated the effect of serotonergic blockade into the LPBN on hindbrain and hypothalamic activity, gastric emptying and arterial blood pressure in cell-dehydrated rats. Methysergide plus 2 M NaCl infused intragastrically or intravenously promoted 0.3 M NaCl intake in two-bottle tests. In cell-dehydrated rats with no access to fluids, methysergide compared to vehicle increased Fos immunoreactivity in the medial nucleus of the solitary tract, area postrema and non-oxytocinergic cells of the ventral portion of the hypothalamic paraventricular nucleus (PVN). There was no alteration in the number of neurons double-labeled for Fos-ir and oxytocin in the PVN and supraoptic nuclei. There was also no alteration in plasma oxytocin and vasopressin, or arterial pressure. In rats cell-dehydrated by i.v. 2 M NaCl, methysergide also did not change the amount of an intragastric load of 0.3 M NaCl retained in the stomach or intestine. The results suggest that methysergide injected into the LPBN of cell-dehydrated rat does not alter primary inhibitory signals that control sodium intake. The inhibitory signals blocked by methysergide in the LPBN possibly originated from activation of brain osmoreceptors, second order visceral/hormonal signals or a combination of both.
Asunto(s)
Deshidratación/metabolismo , Vaciamiento Gástrico/efectos de los fármacos , Metisergida/farmacología , Núcleos Parabraquiales/efectos de los fármacos , Solución Salina Hipertónica , Antagonistas de la Serotonina/farmacología , Animales , Área Postrema/efectos de los fármacos , Área Postrema/metabolismo , Presión Arterial/efectos de los fármacos , Presión Arterial/fisiología , Modelos Animales de Enfermedad , Vaciamiento Gástrico/fisiología , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxitocina/metabolismo , Núcleos Parabraquiales/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas Sprague-Dawley , Solución Salina Hipertónica/administración & dosificación , Cloruro de Sodio Dietético/administración & dosificación , Núcleo Solitario/efectos de los fármacos , Núcleo Solitario/metabolismo , Núcleo Supraóptico/efectos de los fármacos , Núcleo Supraóptico/metabolismo , Vasopresinas/sangreRESUMEN
Cholinergic activation of the medial septal area (MSA) with carbachol produces thirst, natriuresis, antidiuresis and pressor response. In the brain, hydrogen peroxide (H2O2) modulates autonomic and behavioral responses. In the present study, we investigated the effects of the combination of carbachol and H2O2 injected into the MSA on water intake, renal excretion, cardiovascular responses and the activity of vasopressinergic and oxytocinergic neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Furthermore, the possible modulation of carbachol responses by H2O2 acting through K+ATP channels was also investigated. Male Holtzman rats (280-320 g) with stainless steel cannulas implanted in the MSA were used. The pre-treatment with H2O2 in the MSA reduced carbachol-induced thirst (7.9±1.0, vs. carbachol: 13.2±2.0 ml/60 min), antidiuresis (9.6±0.5, vs. carbachol: 7.0±0.8 ml/120 min,), natriuresis (385±36, vs. carbachol: 528±46 µEq/120 min) and pressor response (33±5, vs. carbachol: 47±3 mmHg). Combining H2O2 and carbachol into the MSA also reduced the number of vasopressinergic neurons expressing c-Fos in the PVN (46.4±11.2, vs. carbachol: 98.5±5.9 c-Fos/AVP cells) and oxytocinergic neurons expressing c-Fos in the PVN (38.5±16.1, vs. carbachol: 75.1±8.5 c-Fos/OT cells) and in the SON (57.8±10.2, vs. carbachol: 102.7±7.4 c-Fos/OT cells). Glibenclamide (K+ATP channel blocker) into the MSA partially reversed H2O2 inhibitory responses. These results suggest that H2O2 acting through K+ATP channels in the MSA attenuates responses induced by cholinergic activation in the same area.
Asunto(s)
Carbacol/farmacología , Fármacos del Sistema Nervioso Central/farmacología , Agonistas Colinérgicos/farmacología , Peróxido de Hidrógeno/farmacología , Tabique del Cerebro/efectos de los fármacos , Animales , Presión Arterial/efectos de los fármacos , Presión Arterial/fisiología , Catéteres de Permanencia , Diuresis/efectos de los fármacos , Diuresis/fisiología , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Líquidos/fisiología , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Canales KATP/metabolismo , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Oxitocina/metabolismo , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas Sprague-Dawley , Tabique del Cerebro/fisiología , Núcleo Supraóptico/efectos de los fármacos , Núcleo Supraóptico/fisiología , Sed/efectos de los fármacos , Sed/fisiología , Vasopresinas/metabolismoRESUMEN
Nitric oxide (NO) and carbon monoxide (CO) are diffusible gas messengers in the brain. Previously, we have shown their independent involvement in central fluid/electrolyte homeostasis control. In the present study, we investigated a possible functional interaction between NO/CO in the regulation of vasopressin (VP) and oxytocin (OT) magnocellular neurosecretory cells (MNCs) activity in euhydrated (EU) and dehydrated [48-h water-deprived (48WD)] rats. Using brain slices from EU and 48WD rats, we measured, by immunohistochemistry, the expression of neuronal NO synthase (nNOS, which synthesises NO) and haeme-oxygenase (HO-1, which synthesises CO) in the hypothalamic supraoptic nucleus (SON). In addition, we used patch-clamp electrophysiology to investigate whether regulation of SON MNC firing activity by endogenous CO was dependent on NO bioavailability and GABAergic inhibitory synaptic function. We found a proportion of OT and VP SON MNCs in EU rats to co-express both of HO-1 and nNOS (33.2 ± 2.9% and 15.3 ± 1.4%, respectively), which was increased in 48WD rats (55.5 ± 0.9% and 21.0 ± 1.7%, respectively, P < 0.05 for both). Inhibition of endogenous HO activity [chromium mesoporphyrin IX chloride (CrMP) 20 µm] induced MNC membrane hyperpolarisation and decreased firing activity, and these effects were blunted by previous blockade of endogenous NOS activity (l-NAME, 2 mm) or blockade of inhibitory GABA function [Picrotoxin (Sigma-Aldrich, St Louis, MO, USA), 50 µm]. No significant changes in SON NO bioavailability (4,5 diaminofluorescein diacetate fluorescence) were observed after CrMP treatment. Taken together, our results support a state-dependent functional inter-relationship between NO and CO in MNCs, in which CO acts as an excitatory gas molecule, whose effects are largely dependent on interactions with the inhibitory SON signals NO and GABA.
Asunto(s)
Monóxido de Carbono/metabolismo , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Núcleo Supraóptico/metabolismo , Privación de Agua/fisiología , Animales , Hemo Oxigenasa (Desciclizante)/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo I/metabolismo , Oxitocina/metabolismo , Ratas , Ratas Wistar , Núcleo Supraóptico/citología , Vasopresinas/metabolismoRESUMEN
Feeding increases plasma osmolality and ovarian steroids may influence the balance of fluids. Vasopressin (AVP) neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) express estrogen receptor type ß (ERß), but not estrogen receptor type α (ERα). The circumventricular organs express ERα and project efferent fibers to the PVN and SON. Our aim was to assess whether interactions exist between food state-related osmolality changes and the action of estrogen on AVP neuron activity and estrogen receptor expression. We assessed plasma osmolality and AVP levels; fos-coded protein (FOS)- and AVP-immunoreactivity (-IR) and FOS-IR and ERα-IR in the median preoptic nucleus (MnPO) and organ vasculosum lamina terminalis (OVLT) in estrogen-primed and unprimed ovariectomized rats under the provision of ad libitum food, 48h of fasting, and subsequent refeeding with standard chow or sodium-free food. Refeeding with standard chow increased plasma osmolality and AVP as well as the co-expression of FOS-IR/AVP-IR in the PVN and SON. These responses were not altered by estrogen, with the exception of the decreases in FOS-IR/AVP-IR in the lateral PVN. During refeeding, estrogen modulates only a subpopulation of AVP neurons in the lateral PVN. FOS-ERα co-expression in the ventral median preoptic nucleus (vMnPO) was reduced by estrogen and increased after refeeding with standard chow following fasting. It appears that estrogen may indirectly modulate the activity of AVP neurons, which are involved in the mechanism affected by hyperosmolality-induced refeeding after fasting. This indirect action of estrogen can be at least in part via ERα in the vMnPO.
Asunto(s)
Ingestión de Alimentos/fisiología , Estrógenos/metabolismo , Ayuno/fisiología , Neuronas/fisiología , Sodio en la Dieta , Vasopresinas/metabolismo , Animales , Análisis Químico de la Sangre , Receptor alfa de Estrógeno/metabolismo , Estrógenos/administración & dosificación , Femenino , Núcleo Hipotalámico Paraventricular/fisiología , Área Preóptica/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas Wistar , Sodio en la Dieta/administración & dosificación , Núcleo Supraóptico/fisiologíaRESUMEN
The phenotypic differentiation between oxytocin (OT)- and vasopressin (VP)-secreting magnocellular neurosecretory cells (MNCs) from the supraoptic nucleus is relevant to understanding how several physiological and pharmacological challenges affect their electrical activity. Although the firing patterns of OT and VP neurons, both in vivo and in vitro, may appear different from each other, much is assumed about their characteristics. These assumptions make it practically impossible to obtain a confident phenotypic differentiation based exclusively on the firing patterns. The presence of a sustained outward rectifying potassium current (SOR) and/or an inward rectifying hyperpolarization-activated current (IR), which are presumably present in OT neurons and absent in VP neurons, has been used to distinguish between the two types of MNCs in the past. In this study, we aimed to analyze the accuracy of the phenotypic discrimination of MNCs based on the presence of rectifying currents using comparisons with the molecular phenotype of the cells, as determined by single-cell RT-qPCR and immunohistochemistry. Our results demonstrated that the phenotypes classified according to the electrophysiological protocol in brain slices do not match their molecular counterparts because vasopressinergic and intermediate neurons also exhibit both outward and inward rectifying currents. In addition, we also show that MNCs can change the relative proportion of each cell phenotype when the system is challenged by chronic hypertonicity (70% water restriction for 7 days). We conclude that for in vitro preparations, the combination of mRNA detection and immunohistochemistry seems to be preferable when trying to characterize a single MNC phenotype.
Asunto(s)
Potenciales de Acción/fisiología , Neuronas/metabolismo , Oxitocina/metabolismo , ARN Mensajero/metabolismo , Núcleo Supraóptico/metabolismo , Vasopresinas/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Dieta , Expresión Génica , Masculino , Microtomía , Neuronas/clasificación , Neuronas/citología , Neuronas/efectos de los fármacos , Oxitocina/genética , Técnicas de Placa-Clamp , Fenotipo , ARN Mensajero/genética , Ratas , Ratas Wistar , Análisis de la Célula Individual , Sodio en la Dieta/farmacología , Núcleo Supraóptico/citología , Núcleo Supraóptico/efectos de los fármacos , Técnicas de Cultivo de Tejidos , Vasopresinas/genética , Privación de AguaRESUMEN
The blockade of central nitric oxide (NO) signaling modifies the thermoregulatory and metabolic adjustments that occur during exercise, thereby impairing physical performance. However, the brain areas involved in this response remain unknown. Nitrergic neurons are present in the hypothalamic areas that are activated during exercise and participate in autonomic and neuroendocrine responses, such as, the hypothalamic paraventricular nucleus (PVN) and the supraoptic nucleus (SON). To investigate whether brain NO signaling affects thermoregulation during exercise through the activation of hypothalamic neurons, rats underwent acute submaximal treadmill exercise (18 mmin(-1), 5% inclination) until fatigue received an intracerebroventricular injection of 1.43 µmol Nω-nitro-l-arginine metil ester (L-NAME), a nitric oxide synthase inhibitor, or saline (SAL). Skin tail temperature (Tsk) and internal body temperature (Ti) were continuously recorded and c-Fos expression was determined in the PVN and the SON. L-NAME treatment reduced physical performance by 48%, which was positively correlated with tail vasodilation capacity, which was reduced by 28%, and negatively correlated with heat storage rate (HSR), which was increased by 38%. Physical exercise until fatigue increased the number of c-Fos-immunoreactive (ir) neurons in the PVN and the SON. L-NAME-treatment significantly reduced the exercise-induced c-Fos expression in the PVN, whereas it had no effect in the SON. Interestingly, the number of c-Fos-ir neurons in the PVN was closely correlated with physical performance and inversely associated with HSR. Thus, the inhibition of central NO attenuates neuronal activation induced by exercise in the PVN, impairs the autonomic regulation of heat dissipation, and anticipates the fatigue. Brain NO seems to play a role in exercise performance through the regulation of neuronal activation in the PVN, but not in the SON, although the SON neurons are also activated by running exercise. Moreover, this role in performance mediated by neuronal activation in the PVN can be related with the improvement of thermoregulatory adjustments that occur during exercise.
Asunto(s)
Regulación de la Temperatura Corporal/fisiología , Fatiga/metabolismo , Neuronas/metabolismo , Óxido Nítrico/fisiología , Núcleo Hipotalámico Paraventricular/fisiología , Condicionamiento Físico Animal/fisiología , Animales , Regulación de la Temperatura Corporal/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Neuronas/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Wistar , Núcleo Supraóptico/efectos de los fármacos , Núcleo Supraóptico/metabolismo , Núcleo Supraóptico/fisiologíaRESUMEN
It has been shown that the endocannabinoid system is involved in the neurohypophyseal hormone secretion produced by exposure to several different stimuli; however, the influence of this system on neuroendocrine responses during lactation is unclear. Therefore, the aim of our study was to investigate the influence of an acute peripheral administration of WIN55,212-2 (cannabinoid receptor agonist) on behavioral and neuroendocrine responses during lactation. On day 6 of lactation, female rats were treated with vehicle or WIN55,212-2 30 min before the start of our experiments. To evaluate maternal behavior, the pups were returned to their home cages to the side of the cage opposite the previous nest, and the resulting behavior of the lactating rats was recorded for the next 30 min. Aggressive behavior was evaluated for 10 min following the placement of an intruder male rat in the home cage. The plasma level of oxytocin and the amount of milk consumption by the pups were evaluated 15 min after the onset of suckling. In addition, double-labelled c-Fos/oxytocin neurons in the medial magnocellular subdivision of the paraventricular nucleus and in the supraoptic nucleus were quantified for each lactating rat. The results show that WIN decreased maternal care, decreased aggressive behaviors, suppressed maternal anxiolysis, decreased plasma oxytocin levels and milk consumption by pups and decreased activation of oxytocinergic neurons in hypothalamic nuclei. Our results indicate that the changes in the behavioral responses of lactating rats treated with WIN maybe can be related to disruption in the neuroendocrine control of oxytocin secretion.
Asunto(s)
Benzoxazinas/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Lactancia/efectos de los fármacos , Conducta Materna/efectos de los fármacos , Morfolinas/farmacología , Naftalenos/farmacología , Neuronas/efectos de los fármacos , Oxitocina/metabolismo , Agresión/efectos de los fármacos , Agresión/fisiología , Animales , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Femenino , Inmunohistoquímica , Lactancia/fisiología , Masculino , Conducta Materna/fisiología , Neuronas/fisiología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Wistar , Núcleo Supraóptico/efectos de los fármacos , Núcleo Supraóptico/fisiologíaRESUMEN
Physiological evidence indicates that the supraoptic nucleus (SON) is an important region for integrating information related to homeostasis of body fluids. Located bilaterally to the optic chiasm, this nucleus is composed of magnocellular neurosecretory cells (MNCs) responsible for the synthesis and release of vasopressin and oxytocin to the neurohypophysis. At the cellular level, the control of vasopressin and oxytocin release is directly linked to the firing frequency of MNCs. In general, we can say that the excitability of these cells can be controlled via two distinct mechanisms: 1) the intrinsic membrane properties of the MNCs themselves and 2) synaptic input from circumventricular organs that contain osmosensitive neurons. It has also been demonstrated that MNCs are sensitive to osmotic stimuli in the physiological range. Therefore, the study of their intrinsic membrane properties became imperative to explain the osmosensitivity of MNCs. In addition to this, the discovery that several neurotransmitters and neuropeptides can modulate their electrical activity greatly increased our knowledge about the role played by the MNCs in fluid homeostasis. In particular, nitric oxide (NO) may be an important player in fluid balance homeostasis, because it has been demonstrated that the enzyme responsible for its production has an increased activity following a hypertonic stimulation of the system. At the cellular level, NO has been shown to change the electrical excitability of MNCs. Therefore, in this review, we focus on some important points concerning nitrergic modulation of the neuroendocrine system, particularly the effects of NO on the SON.
Asunto(s)
Neuronas/fisiología , Sistemas Neurosecretores/fisiología , Óxido Nítrico/fisiología , Oxitocina/metabolismo , Núcleo Supraóptico/fisiología , Vasopresinas/metabolismo , Potenciales de Acción/fisiología , Animales , Guanilato Ciclasa/metabolismo , Humanos , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Ratas , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
Physiological evidence indicates that the supraoptic nucleus (SON) is an important region for integrating information related to homeostasis of body fluids. Located bilaterally to the optic chiasm, this nucleus is composed of magnocellular neurosecretory cells (MNCs) responsible for the synthesis and release of vasopressin and oxytocin to the neurohypophysis. At the cellular level, the control of vasopressin and oxytocin release is directly linked to the firing frequency of MNCs. In general, we can say that the excitability of these cells can be controlled via two distinct mechanisms: 1) the intrinsic membrane properties of the MNCs themselves and 2) synaptic input from circumventricular organs that contain osmosensitive neurons. It has also been demonstrated that MNCs are sensitive to osmotic stimuli in the physiological range. Therefore, the study of their intrinsic membrane properties became imperative to explain the osmosensitivity of MNCs. In addition to this, the discovery that several neurotransmitters and neuropeptides can modulate their electrical activity greatly increased our knowledge about the role played by the MNCs in fluid homeostasis. In particular, nitric oxide (NO) may be an important player in fluid balance homeostasis, because it has been demonstrated that the enzyme responsible for its production has an increased activity following a hypertonic stimulation of the system. At the cellular level, NO has been shown to change the electrical excitability of MNCs. Therefore, in this review, we focus on some important points concerning nitrergic modulation of the neuroendocrine system, particularly the effects of NO on the SON.
Asunto(s)
Animales , Humanos , Ratas , Neuronas/fisiología , Sistemas Neurosecretores/fisiología , Óxido Nítrico/fisiología , Oxitocina , Núcleo Supraóptico/fisiología , Vasopresinas , Potenciales de Acción/fisiología , Guanilato Ciclasa/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
Increases in plasma osmolality enhance nitric oxide (NO) levels in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and modulate the secretion of both vasopressin (VP) and oxytocin (OT). In this paper, we describe the effects of hypertonicity on the electrical properties of MNCs by focusing on the nitrergic modulation of their activity in this condition. Membrane potentials were measured using the patch clamp technique, in the presence of both glutamatergic and GABAergic neurotransmission blockers, in coronal brain slices of male Wistar rats. The recordings were first made under a control condition (295 mosm/kg H2O), then in the presence of a hypertonic stimulus (330 mosm/kg H2O) and, finally, with a hypertonic stimulus plus 500 µM L-Arginine or 100 µM N-nitro-L-Arginine methyl ester hydrochloride (L-NAME). Hypertonicity per se increased the firing frequency of the neurons. L-Arginine prevented the increase in fire frequency induced by hypertonic stimulus, and L-NAME (inhibitor of nitric oxide synthase) induced an additional increase in frequency when applied together with the hypertonic solution. Moreover, L-Arginine hyperpolarizes the resting potential and decreases the peak value of the after-hyperpolarization; both effects were blocked by L-NAME and hypertonicity and/or L-NAME reduced the time constant of the rising phase of the after-depolarization. These results demonstrate that an intrinsic nitrergic system is part of the mechanisms controlling the excitability of MNCs of the SON when the internal fluid homeostasis is disturbed.
Asunto(s)
Núcleo Basal de Meynert/metabolismo , Neuronas/metabolismo , Óxido Nítrico/biosíntesis , Concentración Osmolar , Núcleo Supraóptico/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Arginina/farmacología , Núcleo Basal de Meynert/citología , Fenómenos Electrofisiológicos/fisiología , Inhibidores Enzimáticos/farmacología , Soluciones Hipertónicas/farmacología , Soluciones Hipotónicas/farmacología , Técnicas In Vitro , Cinética , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Núcleo Supraóptico/citologíaRESUMEN
The nucleus of the solitary tract (NTS) is the primary site of visceral afferents to the central nervous system. In the present study, we investigated the effects of lesions in the commissural portion of the NTS (commNTS) on the activity of vasopressinergic neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, plasma vasopressin, arterial pressure, water intake, and sodium excretion in rats with plasma hyperosmolality produced by intragastric 2 M NaCl (2 ml/rat). Male Holtzman rats with 15-20 days of sham or electrolytic lesion (1 mA; 10 s) of the commNTS were used. CommNTS lesions enhanced a 2 M NaCl intragastrically induced increase in the number of vasopressinergic neurons expressing c-Fos in the PVN (28 ± 1, vs. sham: 22 ± 2 c-Fos/AVP cells) and SON (26 ± 4, vs. sham: 11 ± 1 c-Fos/AVP cells), plasma vasopressin levels (21 ± 8, vs. sham: 6.6 ± 1.3 pg/ml), pressor responses (25 ± 7 mmHg, vs. sham: 7 ± 2 mmHg), water intake (17.5 ± 0.8, vs. sham: 11.2 ± 1.8 ml/2 h), and natriuresis (4.9 ± 0.8, vs. sham: 1.4 ± 0.3 meq/1 h). The pretreatment with vasopressin antagonist abolished the pressor response to intragastric 2 M NaCl in commNTS-lesioned rats (8 ± 2.4 mmHg at 10 min), suggesting that this response is dependent on vasopressin secretion. The results suggest that inhibitory mechanisms dependent on commNTS act to limit or counterbalance behavioral, hormonal, cardiovascular, and renal responses to an acute increase in plasma osmolality.
Asunto(s)
Presión Sanguínea/fisiología , Ingestión de Líquidos/fisiología , Riñón/fisiología , Núcleo Solitario/metabolismo , Desequilibrio Hidroelectrolítico/metabolismo , Animales , Masculino , Concentración Osmolar , Oxitocina , Núcleo Hipotalámico Paraventricular/citología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Núcleo Supraóptico/citología , VasopresinasRESUMEN
We report changes in plasma arginine vasopressin (AVP) and oxytocin (OT) concentrations evoked by the microinjection of l-glutamate (l-glu) into the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) of unanesthetized rats, as well as which local mechanisms are involved in their mediation. l-Glu microinjection (10 nmol/100 nl) into the SON increased the circulating levels of both AVP and OT. The AVP increases were blocked by local pretreatment with the selective non-N-methyl-d-aspartate (NMDA) receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (2 nmol/100 nl), but it was not affected by pretreatment with the NMDA-receptor antagonist LY235959 (2 nmol/100 nl). The OT response to l-glu microinjection into the SON was blocked by local pretreatment with either NBQX or LY235959. Furthermore, the administration of either the non-NMDA receptor agonist (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) (5 nmol/100 nl) or NMDA receptor agonist NMDA (5 nmol/100 nl) into the SON had no effect on OT baseline plasma levels, but when both agonists were microinjected together these levels were increased. l-Glu microinjection into the PVN did not change circulating levels of either AVP or OT. However, after local pretreatment with LY235959, the l-glu microinjection increased plasma levels of the hormones. The l-glu microinjection into the PVN after the local treatment with NBQX did not affect the circulating AVP and OT levels. Therefore, results suggest the AVP release from the SON is mediated by activation of non-NMDA glutamate receptors, whereas the OT release from this nucleus is mediated by an interaction of NMDA and non-NMDA receptors. The present study also suggests an inhibitory role for NMDA receptors in the PVN on the release of AVP and OT.
Asunto(s)
Oxitocina/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Núcleo Supraóptico/metabolismo , Vasopresinas/metabolismo , Animales , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Masculino , Microinyecciones , N-Metilaspartato/farmacología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Núcleo Supraóptico/efectos de los fármacosRESUMEN
Increased plasma osmolality by food intake evokes augmentation of plasma oxytocin (OT). Ovarian steroids may also influence the balance of body fluids by acting on OT neurones. Our aim was to determine if estrogen influences the activity of OT neurones in paraventricular nucleus (PVN) and supraoptic nucleus (SON) under different osmotic situations. Ovariectomized rats (OVX) were treated with either estradiol (E(2)) or vehicle and were divided into three groups: group I was fed ad libitum, group II underwent 48âh of fasting, and group III was refed after 48âh of fasting. On the day of the experiment, blood samples were collected to determine the plasma osmolality and OT. The animals were subsequently perfused, and OT/FOS immunofluorescence analysis was conducted on neurones in the PVN and the SON. When compared to animals which were fasted or fed ad libitum, the plasma osmolality of refed animals was higher, regardless of whether they were treated with vehicle or E(2). We observed neural activation of OT cells in vehicle- or E(2)-treated OVX rats refed after 48âh of fasting, but not in animals fed ad libitum or in animals that only underwent 48âh of fasting. Finally, the percentage of neurones that co-expressed OT and FOS was lower in both the PVN and the SON of animals treated with E(2) and refed, when compared to vehicle-treated animals. These results suggest that E(2) may have an inhibitory effect on OT neurones and may modulate the secretion of OT in response to the increase of osmolality induced by refeeding.
Asunto(s)
Ingestión de Alimentos , Estradiol/metabolismo , Hipotálamo Anterior/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Oxitocina/metabolismo , Equilibrio Hidroelectrolítico , Animales , Femenino , Hipotálamo Anterior/citología , Proteínas del Tejido Nervioso/sangre , Neuronas/citología , Concentración Osmolar , Ovariectomía , Oxitocina/sangre , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Wistar , Núcleo Supraóptico/citología , Núcleo Supraóptico/metabolismo , Transmisión SinápticaRESUMEN
Neuronal nitric oxide synthase (nNOS) has been reported to be up-regulated in the hypothalamic supraoptic nucleus (SON) during dehydration which in turn could increase nitric oxide (NO) production and consequently affect arginine vasopressin (AVP) secretion. The anteroventral third ventricle (AV3V) region has strong afferent connections with the SON. Herein we describe our analysis of the effects of an AV3V lesion on AVP secretion, and c-fos and nNOS expression in the SON following dehydration. Male Wistar rats had their AV3V region electrolytically lesioned or were sham operated. After 21 days they were submitted to dehydration or left as controls (euhydrated). Two days later, one group was anaesthetized, perfused and the brains were processed for Fos protein and nNOS immunohistochemistry (IHC). Another group was decapitated, the blood collected for hematocrit, osmolality, serum sodium and AVP plasma level analysis. The brains were removed for measurement of neurohypophyseal AVP content, and the SON was punched out and processed for nNOS detection by western blotting. The AV3V lesion reduced AVP plasma levels and c-fos expression in the SON following dehydration (P<0.05). Western blotting revealed an up-regulation of nNOS in the SON of control animals following dehydration, whereas such up-regulation was not observed in AV3V-lesioned rats (P<0.05). We conclude that the AV3V region plays a role in regulating the expression of nNOS in the SON of rats submitted to dehydration, and thus may affect the local nitric oxide production and the secretion of vasopressin.