Response of vasopressin and tyrosine hydroxylase expressing neurons of the rat supraoptic nucleus to chronic osmotic stimulation]

This study has evaluated the dynamic of intracellular vasopressin and tyrosine hydroxylase contents in the neuron cell bodies in the supraoptic nucleus and in the axons of the posterior lobe in rats drinking 2% NaCl for 1, 2, and 3 weeks. The number of vasopressin-immunoreactive neurons increased by the end of the second week of osmotic stimulation that might be explained by the onset of vasopressin synthesis in the neurons which do not synthesize this neurohormone under normal physiological conditions. The concentration of vasopressin fell down continuously during the first two weeks of salt-loading, apparently, due to predominance of the vasopressin release over its synthesis. Over the third week of salt-loading, the intracellular concentration of vasopressin was not changed significantly suggesting the establishment of the dynamic equilibrium between the vasopressin synthesis and release. The number of tyrosine hydroxylase-immunoreactive neurons and the amount of tyrosine hydroxylase in cell bodies and the large axonal swellings, Herring bodies, increased gradually showing that the rate of tyrosine hydroxylase synthesis prevailed over that of its enzymatic degradation. Thus, the chronic stimulation of vasopressin neurons is accompanied by a number of the adaptive reactions; the most important is related to the onset of vasopressin and tyrosine hydroxylase synthesis in the neurons which do not synthetize both of them under normal conditions.     

Effect on vasopressin release of microinjection of cholinergic agonists into the rat supraoptic nucleus.

It is likely that central cholinergic pathways to the paraventricular and supraoptic nuclei participate in the control of vasopressin release. We have shown previously that this is due, in part, to activation of muscarinic, but not nicotinic, receptors in the paraventricular nucleus. There is, however, reason to believe that this cholinergic effect in the supraoptic nucleus may be the result of activation of nicotinic receptors. To test this possibility, we have studied in conscious unrestrained rats the effect of microinjection of muscarinic and nicotinic agonists into the supraoptic nucleus on vasopressin release, mean arterial blood pressure, and heart rate. Under ether anesthesia, a stainless steel guide cannula was placed in the supraoptic nucleus 5-7 days before the experiment, and femoral, arterial, and venous catheters were implanted 1 day before the experiment. Microinjection of nicotine into the supraoptic nucleus at doses of 1 and 10 micrograms resulted in transient increases in the plasma vasopressin concentration that were 7-fold and 11-fold greater, respectively, than control values at 3 min. There were also small transient increases in mean arterial blood pressure, but heart rate was unchanged. The microinjection of 2 and 20 ng of oxotremorine, a muscarinic agonist, into the supraoptic nucleus had no effect on the plasma vasopressin concentration, mean arterial blood pressure, or heart rate. These doses of oxotremorine were previously shown to have potent stimulatory effects on vasopressin release when microinjected into the paraventricular nucleus. These findings suggest that the central cholinergic stimulation of vasopressin release is due, in part, to activation of muscarinic receptors in the paraventricular nucleus and nicotinic receptors in the supraoptic nucleus.     

Effect of vasopressin on changes in the behavioral reactions of rats induced by psychotropic substances

The effect was studied of arginyl-8-vasopressin (AVP) on changes in rats behavioural reactions, elicited by reserpine, haloperidol, aminazine, amitriptyline or nialamide. It has been shown that AVP administered intraperitoneally in a dose of 0.001 mg/kg, eliminates the deficit of elaboration of conditioned reaction of active avoidance produced by psychotropic drugs, without influencing the motor activity reduction developed after administration of these substances.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

Summary The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toadBufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to the serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toad Bufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to th serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Activity of vasopressin neurons in the human supraoptic nucleus: estrogen inhibitory effect

Activity of magnocellular vasopressin (VP) neurons in the human hypothalamus is sex- and age-dependent as judged from the size of the Golgi apparatus, neuronal size and VP mRNA levels. These parameters are significantly higher in young (< or = 50 years old) men than in young women and are markedly increased in postmenopausal women compared to premenopausal women. This data suggest an inhibitory effect of estrogens on metabolic activity of VP neurons in the human supraoptic nucleus (2SON), which is likely to be mediated via estrogen receptor (ER) beta. Estrogens were shown to mediate their inhibitory effect via ER beta. It is expressed to a much higher degree in the SON of young women than in other groups, whereas estrogen receptor alpha, that mediates stimulatory effects of estrogens, is present in a small proportion of SON neurons. In addition, estrogens inhibit p75 neurotrophin receptor expression in VP cells. In conclusion, we discuss the inhibitory role of estrogens in functional activity of human VP neurons, which is most probably mediated directly via ER beta and indirectly by p75 neurotrophin receptor.     

Influence of substance P on the behavioral changes induced by haloperidol in rats

Locomotor activity and grooming behavior of rats were recorded for a period of 30 min following intraventricular injections of substance P(SP) in doses of 0.60 and 2.50 microgram/rat. The lower dose of the peptide significantly increased locomotion for 10 min and time spent grooming for 25 min. The effects of the same two doses of SP on the hypokinesia induced by various pharmacological treatments modifying catecholaminergic systems were then examined. SP did not affect the behavioral depression produced by alpha-methyl-para-tyrosine (250 mg/kg), FLA-63 (25 mg/kg) and phenoxybenzamine (20 mg/kg). However, SP, in dose of 0.60 microgram/rat, systematically reversed the decrease in locomotor activity induced by a relatively small dose of haloperidol, 0.1 mg/kg. The dame dose of the peptide significantly counteracted the rigidity but not the hypokinesia and catalepsy resulting from the previous administration of a higher dose of haloperidol, 3 mg/kg. The results support the hypothesis that SP may exert direct or indirect function in motor behavior, possible via a modulatory action on brain dopaminergic systems.     

Ca2+ homeostasis,Ca2+ signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca²⁺ homeostasis and provide for Ca²⁺ signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca²⁺ clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca²⁺ homeostatic molecules on cytosolic Ca²⁺ ([Ca²⁺]_i) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca²⁺]_i dynamics. When bathing the cells in a Na⁺-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca²⁺-ATPase (PMCA), La³⁺, all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca²⁺]_i transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca²⁺ transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca²⁺ homeostatic pathways, the Na⁺/Ca²⁺ exchanger, the endoplasmic reticulum Ca²⁺ pump, the plasmalemmal Ca²⁺ pump and mitochondria, are complementary in actively clearing Ca²⁺ from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca²⁺]_i dynamics; (iii) there is (are) Ca²⁺ clearance mechanism(s) distinct from the four outlined above; and (iv) Ca²⁺ homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.     

雌激素对电刺激诱发杏仁核多巴胺释放的影响

用快速周期伏安法（FCV）测定了去卵巢（OVX）雌鼠,经雌激素处理的去卵巢雌鼠及正常雌鼠的中央杏仁核（CAN）的多巴胺（DA）释放,并应用放射免疫技术检测了大鼠在不同处理条件下血清雌二醇（E2）含量,结果表明,雌激素处理的OVX组大鼠的杏仁核DA释放量均高于对照OVX组大鼠,并随苯甲酸雌二醇（EB）注射剂量的增加,DN释放量及血清E2含量迹增加,有明显的量－效关系,提示雌激素可能是调节大鼠CAN区DA释放量的重要因素。     

Quantitativein situ hybridization to measure single-cell changes in vasopressin and oxytocin mRNA levels after osmotic stimulation

1. The measurement of cellular mRNA content by quantitative in situ hybridization is a valuable approach to the study of gene expression in brain since this tissue exhibits a high degree of phenotypic heterogeneity. 2. The cellular content of vasopressin and oxytocin mRNA in hypothalamo-neurohypophysial system neurons was altered by maintaining rats for 24 hr on 2% sodium chloride water. 3. Statistical and graphical techniques were then used to analyze cell by cell how mRNA levels were altered as a result of osmotic stimulation. We propose that the negative binomial probability distribution is a suitable model to describe how mRNA content varies across a defined cell population. For both measures of oxytocin and vasopressin mRNA levels, maximum-likelihood estimation indicated that this model adequately described empirical findings obtained from rats drinking tap water or salt water. 4. Both graphical and statistical analyses suggested how the defined neural system responds to osmotic stimulation: mRNA content was altered as a multiplicative function of "initial state." The utility and limitations of the quantitative approach are discussed.     

Calbindin D28k and calretinin in oxytocin and vasopressin neurons of the rat supraoptic nucleus

The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca2+]i. Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.     

Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality

Magnocellular neurons of the hypothalamo-neurohypophysial system play a fundamental role in the maintenance of body homeostasis by secreting vasopressin and oxytocin in response to systemic osmotic perturbations. During chronic hyperosmolality, vasopressin and oxytocin mRNA levels increase twofold, whereas, during chronic hyposmolality, these mRNA levels decrease to 10-20% of that of normoosmolar control animals. To determine what other genes respond to these osmotic perturbations, we have analyzed gene expression during chronic hyper- versus hyponatremia. Thirty-seven cDNA clones were isolated by differentially screening cDNA libraries that were generated from supraoptic nucleus tissue punches from hyper- or hyponatremic rats. Further analysis of 12 of these cDNAs by in situ hybridization histochemistry confirmed that they are osmotically regulated. These cDNAs represent a variety of functional classes and include cytochrome oxidase, tubulin, Na(+)-K(+)-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated, synaptic glycoprotein, regulator of GTPase stimulation, and gene for oligodendrocyte lineage-myelin basic proteins. This analysis therefore suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the supraoptic nucleus.     

Coincident detection of CSF Na+ and osmotic pressure in osmoregulatory neurons of the supraoptic nucleus

Behavioral and neuroendocrine responses underlying systemic osmoregulation are under the concerted control of centrally located osmoreceptors and cerebrospinal fluid (CSF) Na+ concentration ([Na+]) detectors. Although the process underlying osmoreception is understood, the mechanism by which [Na+] is detected and integrated with cellular information derived from osmoreceptors is unknown. Here, we show that shifts in extracellular [Na+] ([Na+]0) cause proportional changes in the relative Na+ permeability of mechanosensitive cation channels responsible for signal transduction in the osmosensory neurons of the supraoptic nucleus. This effect causes the generation of Na+ specific receptor potentials under isotonic conditions and modulates osmoreceptor potentials and electrical responsiveness during osmotic perturbation. These results provide a cellular basis for Na+-sensing and for the coordinated detection of CSF [Na+] and osmolality in central osmoregulatory neurons.     

Ca(2+) homeostasis, Ca(2+) signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca(2+) homeostasis and provide for Ca(2+) signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca(2+) clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca(2+) homeostatic molecules on cytosolic Ca(2+) ([Ca(2+)](i)) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca(2+)](i) dynamics. When bathing the cells in a Na(+)-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca(2+)-ATPase (PMCA), La(3+), all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca(2+)](i) transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca(2+) transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca(2+) homeostatic pathways, the Na(+)/Ca(2+) exchanger, the endoplasmic reticulum Ca(2+) pump, the plasmalemmal Ca(2+) pump and mitochondria, are complementary in actively clearing Ca(2+) from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca(2+)](i) dynamics; (iii) there is (are) Ca(2+) clearance mechanism(s) distinct from the four outlined above; and (iv) Ca(2+) homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.     

Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction.     

Oxytocin actions within the supraoptic and paraventricular nuclei: differential effects on peripheral and intranuclear vasopressin release

In response to forced swimming (FS), AVP is released somato-dendritically within the supraoptic nucleus (SON) and paraventricular nucleus (PVN), but not from neurohypophyseal terminals into blood. Together with AVP, oxytocin (OXT) is released within the SON and PVN. Here, we studied the role of intra-SON and intra-PVN OXT in the regulation of local AVP release and into the blood in male rats. Within the SON, bilateral retrodialysis of an OXT receptor antagonist (OXT-A) increased local AVP release in response to FS [60 s, 21 degrees C, vehicle twofold, not significant (ns); OXT-A: 15-fold increase, P < 0.05] without significantly affecting basal AVP release. In addition, local OXT-A elevated plasma AVP secretion under basal conditions (twofold increase, P < 0.05) without further elevation after FS. Within the PVN, exposure to FS elevated local AVP release, reaching significance only in the OXT-A group (vehicle: 1.4-fold, ns; OXT-A: 1.6-fold increase, P = 0.050). Bilateral OXT-A into the PVN did not affect peripheral AVP secretion either under basal or stress conditions. Basal ACTH concentrations tended to be elevated by local OXT-A within the PVN (1.7-fold increase, P = 0.076). In contrast, the swim-induced ACTH secretion was attenuated after retrodialysis of OXT-A within both the SON (at 5 min) and PVN (at 15 min) (P < 0.05 both) compared with vehicle. The results demonstrate a receptor-mediated effect of OXT within the SON and PVN on local and neurohypophyseal AVP release, which depends upon the activity conditions. Further, while exerting an inhibitory effect on hypothalamo-pituitary-adrenal axis activity under basal conditions, hypothalamic OXT is essential for an adequate acute ACTH response.     

Effect of acute and chronic cardiac denervation on osmotic release of vasotocin in chickens

Hypo- and hyperosmotic NaCl were infused intravenously to examine osmotic release of arginine vasotocin (AVT) in anesthetized, acutely cardiac-denervated chickens and in conscious, chronically denervated birds. Mean arterial blood pressure was consistently higher in denervated compared to sham-operated chickens but heart rates were similar in experimental and control groups. Plasma AVT concentrations (pAVT) were significantly higher than controls in acutely, but not chronically, denervated chickens. The slope of the regression line relating pAVT to plasma sodium concentration was higher in denervated birds indicating that removal of cardiac receptor activity increases the osmotic sensitivity of the AVT system. The results suggest that cardiac end-net receptor activity may participate in the regulation of blood pressure and can modulate the release of antidiuretic hormone in the chicken.