Temporal dissociation between sodium depletion and sodium appetite appearance: Involvement of inhibitory and stimulatory signals.

Our aim was to analyze the participation of inhibitory and stimulatory signals in the temporal dissociation between sodium depletion (SD) induced by peritoneal dialysis (PD) and the appearance of sodium appetite (SA), particularly 2h after PD, when the rats are hypovolemic/natremic but SA is not evident. We investigated the effects of bilateral injections of the serotonin (5-HT) receptor antagonist, methysergide, into the lateral parabrachial nucleus (LPBN) on hypertonic NaCl and water intake 2h vs. 24h after PD. We also studied plasma renin activity (PRA) and aldosterone (ALDO) concentration 2h vs. 24h after PD. Additionally, we combined the analysis of brain Fos immunoreactivity (Fos-ir) with the detection of double immunoreactivity in 5HT and oxytocinergic (OT) cells 2h after PD. Bilateral LPBN injections of methysergide (4µg/200nl at each site) increased NaCl intake when tested 2h after PD compared to controls. We found a significant increase in PRA and ALDO concentration after PD but no differences between 2 and 24h after PD. We also found for the first time a significant increase 2h after PD in the number of Fos-ir neurons in the brainstem nuclei that have been shown to be involved in the inhibition of SA. In summary, the results show that 5HT-mechanisms in the LPBN modulate sodium intake during the delay of SA when the renin angiotensin aldosterone system (RAAS) is increased. In addition, the activation of brainstem areas previously associated with the satiety phase of SA is in part responsible for the temporal dissociation between SD and behavioral arousal.

Mapping and signaling of neural pathways involved in the regulation of hydromineral homeostasis.

Several forebrain and brainstem neurochemical circuitries interact with peripheral neural and humoral signals to collaboratively maintain both the volume and osmolality of extracellular fluids. Although much progress has been made over the past decades in the understanding of complex mechanisms underlying neuroendocrine control of hydromineral homeostasis, several issues still remain to be clarified. The use of techniques such as molecular biology, neuronal tracing, electrophysiology, immunohistochemistry, and microinfusions has significantly improved our ability to identify neuronal phenotypes and their signals, including those related to neuron-glia interactions. Accordingly, neurons have been shown to produce and release a large number of chemical mediators (neurotransmitters, neurohormones and neuromodulators) into the interstitial space, which include not only classic neurotransmitters, such as acetylcholine, amines (noradrenaline, serotonin) and amino acids (glutamate, GABA), but also gaseous (nitric oxide, carbon monoxide and hydrogen sulfide) and lipid-derived (endocannabinoids) mediators. This efferent response, initiated within the neuronal environment, recruits several peripheral effectors, such as hormones (glucocorticoids, angiotensin II, estrogen), which in turn modulate central nervous system responsiveness to systemic challenges. Therefore, in this review, we shall evaluate in an integrated manner the physiological control of body fluid homeostasis from the molecular aspects to the systemic and integrated responses.

Mapping and signaling of neural pathways involved in the regulation of hydromineral homeostasis

Several forebrain and brainstem neurochemical circuitries interact with peripheral neural and humoral signals to collaboratively maintain both the volume and osmolality of extracellular fluids. Although much progress has been made over the past decades in the understanding of complex mechanisms underlying neuroendocrine control of hydromineral homeostasis, several issues still remain to be clarified. The use of techniques such as molecular biology, neuronal tracing, electrophysiology, immunohistochemistry, and microinfusions has significantly improved our ability to identify neuronal phenotypes and their signals, including those related to neuron-glia interactions. Accordingly, neurons have been shown to produce and release a large number of chemical mediators (neurotransmitters, neurohormones and neuromodulators) into the interstitial space, which include not only classic neurotransmitters, such as acetylcholine, amines (noradrenaline, serotonin) and amino acids (glutamate, GABA), but also gaseous (nitric oxide, carbon monoxide and hydrogen sulfide) and lipid-derived (endocannabinoids) mediators. This efferent response, initiated within the neuronal environment, recruits several peripheral effectors, such as hormones (glucocorticoids, angiotensin II, estrogen), which in turn modulate central nervous system responsiveness to systemic challenges. Therefore, in this review, we shall evaluate in an integrated manner the physiological control of body fluid homeostasis from the molecular aspects to the systemic and integrated responses.

Oxytocin in the central amygdaloid nucleus modulates the neuroendocrine responses induced by hypertonic volume expansion in the rat.

The present study investigated the involvement of the oxytocinergic neurones that project into the central amygdala (CeA) in the control of electrolyte excretion and hormone secretion in unanaesthetised rats subjected to acute hypertonic blood volume expansion (BVE; 0.3 M NaCl, 2 ml/100 g of body weight over 1 min). Oxytocin and vasopressin mRNA expression in the paraventricular (Pa) and supraoptic nucleus (SON) of the hypothalamus were also determined using the real time-polymerase chain reaction and in situ hybridisation. Male Wistar rats with unilaterally implanted stainless steel cannulas in the CeA were used. Oxytocin (1 µg/0.2 µl), vasotocin, an oxytocin antagonist (1 µg/0.2 µl) or vehicle was injected into the CeA 20 min before the BVE. In rats treated with vehicle in the CeA, hypertonic BVE increased urinary volume, sodium excretion, plasma oxytocin (OT), vasopressin (AVP) and atrial natriuretic peptide (ANP) levels and also increased the expression of OT and AVP mRNA in the Pa and SON. In rats pre-treated with OT in the CeA, previously to the hypertonic BVE, there were further significant increases in plasma AVP, OT and ANP levels, urinary sodium and urine output, as well as in gene expression (AVP and OT mRNA) in the Pa and SON compared to BVE alone. Vasotocin reduced sodium, urine output and ANP levels, although no changes were observed in plasma AVP and OT levels or in the expression of the AVP and OT genes in both hypothalamic nuclei. The results of the present study suggest that oxytocin in the CeA exerts a facilitatory role in the maintenance of hydroelectrolyte balance in response to changes in extracellular volume and osmolality.

Moxonidine into the lateral parabrachial nucleus reduces renal and hormonal responses to cell dehydration.

The deactivation of the inhibitory mechanisms with injections of moxonidine (α2-adrenoceptor/imidazoline receptor agonist) into the lateral parabrachial nucleus (LPBN) increases hypertonic NaCl intake by intra- or extracellular dehydrated rats. In the present study, we investigated the changes in the urinary sodium and volume, sodium balance, and plasma vasopressin and oxytocin in rats treated with intragastric (i.g.) 2 M NaCl load (2 ml/rat) combined with injections of moxonidine into the LPBN. Male Holtzman rats (n=5-12/group) with stainless steel cannulas implanted bilaterally into LPBN were used. Bilateral injections of moxonidine (0.5 nmol/0.2 µl) into the LPBN decreased i.g. 2 M NaCl-induced diuresis (4.6±0.7 vs. vehicle: 7.4±0.6 ml/120 min) and natriuresis (1.65±0.29 vs. vehicle: 2.53±0.17 mEq/120 min), whereas the previous injection of the α2-adrenoceptor antagonist RX 821002 (10 nmol/0.2 µl) into the LPBN abolished the effects of moxonidine. Moxonidine injected into the LPBN reduced i.g. 2 M NaCl-induced increase in plasma oxytocin and vasopressin (14.6±2.8 and 2.2±0.3 vs. vehicle: 25.7±7 and 4.3±0.7 pg/ml, respectively). Moxonidine injected into the LPBN combined with i.g. 2 M NaCl also increased 0.3 M NaCl intake (7.5±1.7 vs. vehicle: 0.5±0.2 mEq/2 h) and produced positive sodium balance (2.3±1.4 vs. vehicle: -1.2±0.4 mEq/2 h) in rats that had access to water and NaCl. The present results show that LPBN α2-adrenoceptor activation reduces renal and hormonal responses to intracellular dehydration and increases sodium and water intake, which facilitates sodium retention and body fluid volume expansion.

Characterization of Kiss1 neurons using transgenic mouse models.

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or ß-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction.

GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats.

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 microL) or bicuculline (1.6 nmol/0.2 microL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 +/- 0.3 vs vehicle: 4.8 +/- 0.2 and hypertonic: 4.0 +/- 0.7 vs vehicle: 8.7 +/- 0.6 microEq.100 g-1.40 min-1); urinary volume after hypertonic BVE (83.8 +/- 10 vs vehicle: 255.6 +/- 16.5 microL.100 g-1.40 min-1); plasma OT levels (isotonic: 15.3 +/- 0.6 vs vehicle: 19.3 +/- 1 and hypertonic: 26.5 +/- 2.6 vs vehicle: 48 +/- 3 pg/mL), and ANP levels (isotonic: 97 +/- 12.8 vs vehicle: 258.3 +/- 28.1 and hypertonic: 160 +/- 14.6 vs vehicle: 318 +/- 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.

GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 µL) or bicuculline (1.6 nmol/0.2 µL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 ± 0.3 vs vehicle: 4.8 ± 0.2 and hypertonic: 4.0 ± 0.7 vs vehicle: 8.7 ± 0.6 µEq·100 g-1·40 min-1); urinary volume after hypertonic BVE (83.8 ± 10 vs vehicle: 255.6 ± 16.5 µL·100 g-1·40 min-1); plasma OT levels (isotonic: 15.3 ± 0.6 vs vehicle: 19.3 ± 1 and hypertonic: 26.5 ± 2.6 vs vehicle: 48 ± 3 pg/mL), and ANP levels (isotonic: 97 ± 12.8 vs vehicle: 258.3 ± 28.1 and hypertonic: 160 ± 14.6 vs vehicle: 318 ± 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.

The effect of adrenalectomy on Fos expression in vasopressinergic and oxytocinergic neurons in response to stress in the rat.

This study evaluated the responses of vasopressin (AVP) and oxytocin (OT) neurons to alterations in hypothalamo-pituitary axis activity by adrenalectomy (ADX) or after restraint stress compared with basal conditions. Wistar male rats were perfuse-fixed by cardiac perfusion under anesthesia 3 h, 1, 3 and 14 days after ADX or Sham surgery. Coronal hypothalamic sections were used for evaluation of Fos, AVP and OT expression by immunohistochemistry. Under basal conditions and after stress, Fos-AVP double labeling showed no difference in the magnocellular subdivisions of the paraventricular nuclei (PVN) or in the supraoptic nuclei (SON), suggesting that the magnocellular AVP system is unlikely to contribute to ACTH secretion after restraint in both Sham and ADX rats. Fos-AVP double labeling in the parvocellular medial paraventricular nucleus (PaMP) in ADX groups was increased after 3 h in basal conditions, and in all periods after restraint stress. There were no differences between Sham and ADX groups in Fos-OT double labeling in any subdivision of the PVN; however, in the SON, the number of Fos-OT double labeled cells was increased at all time-points after stress in the ADX group. Fos expression was increased in the PaMP after 3 h and after restraint stress in the Sham and ADX groups, especially in the ADX group. In conclusion, Fos expression in different cell populations of the PVN can be differentially regulated by short- and long-term absence of glucocorticoid negative feedback and also by stress-related excitatory and/or inhibitory neural inputs. The Fos-AVP double labeling findings in the PaMP also indicate a minor participation of these vasopressinergic neurons in the regulation of the HPA axis after ADX.

Glucocorticoid modulation of neuronal activity and hormone secretion induced by blood volume expansion.

The present study evaluated the involvement of glucocorticoid in the activation of vasopressinergic and oxytocinergic neurons of hypothalamic nuclei and plasma levels of vasopressin (AVP), oxytocin (OT), atrial natriuretic peptide (ANP) and corticosterone (CORT) in response to both isotonic and hypertonic blood volume expansion (BVE). Rats were subjected to isotonic (0.15 M NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 M NaCl, 2 ml/100 g b.w., i.v.) BVE with or without pre-treatment with dexamethasone (1 mg/kg, i.p.). Results showed that isotonic BVE increased OT, ANP and CORT, and decreased AVP plasma levels. On the other hand, hypertonic BVE enhanced AVP, ANP, OT, and CORT plasma concentrations. Both hypertonic and isotonic BVE induced an increase in the number of Fos-OT double-labeled magnocellular neurons in the PVN and SON. Pre-treatment with dexamethasone reduced OT secretion, as well as Fos-OT immunoreactive neurons in response to both isotonic and hypertonic BVE. We also observed that dexamethasone pre-treatment had no effect on AVP secretion in response to hypertonic BVE, although this effect was associated with a blockade of Fos expression in the vasopressinergic magnocellular neurons in the PVN and SON. In conclusion, these data suggest that, not only the rapid OT release from storages, but also the oxytocinergic cellular activation induced by BVE are modulated by glucocorticoids. However, this pattern of response was not observed for AVP cells, suggesting that dexamethasone is not likely to influence rapid release of AVP but seems to modulate the activation of these neurons in response to hypertonic BVE.