Melatonin inhibits the substance P-induced secretion of vasopressin and oxytocin from the rat hypothalamo-neurohypophysial system: in vitro studies

The aim of the present investigations was to study the influence of substance P (a member of a family of peptides known as tachykinins) on basal and K(+)-evoked vasopressin (AVP) and oxytocin (OT) release from rat hypothalamo-neurohypophysial system in vitro as well as to determine whether this effect of substance P is sensitive to melatonin.The present results show that substance P stimulates basal AVP and OT release from isolated hypothalamo-neurohypophysial system, when used at the concentrations of 10(-6) and 10(-7)M/l. At the concentration of 10(-9)M/l, however, substance P was found to stimulate the in vitro secretion of AVP, but not that of OT. Melatonin diminished basal release of AVP; it also significantly inhibited the substance P-stimulated secretion of AVP and OT. K(+)-evoked release of the neurohypophysial hormones was not further modified by either substance P or melatonin.The present results show that the stimulatory effect of substance P on basal release of AVP and OT from rat hypothalamo-neurohypophysial system in vitro is sensitive to inhibitory influence of melatonin.     

Luteinizing hormone-releasing hormone and function of the magnocellular vasopressinergic system

Mechanisms by which luteinizing hormone-releasing hormone (LHRH) affects vasopressin secretion were investigated using the isolated rat hypothalamo-neurohypophysial explants. LHRH in a concentration of 4 x 10(-7)M inhibited both the basal and K(+)-stimulated vasopressin release from explants isolated from euhydrated rats. When, however, the tissue was obtained from animals previously salt-loaded, the inhibitory effect of LHRH was completely abolished, thus implying a decrease in the sensitivity to LHRH. LHRH did not affect vasopressin secretion under conditions of generalized blockade of synaptic inputs by 15 mM MgSO(4), suggesting the indirect action of this neurohormone on the hypothalamic magnocellular system.It is concluded that LHRH may play the role of a neuromodulator of vasopressinergic neurones in the rat.     

Neurokinin A inhibits oxytocin and GABA release from the posterior pituitary by stimulating nitric oxide synthase

Neurokinin A (NKA) is a tachykinin that participates in the control of neuroendocrine functions. The posterior pituitary lobe (PP) contains abundant nitric oxide synthase (NOS), suggesting that nitric oxide (NO) may play a role in controlling the release of neuropeptides and neurotransmitters. In the present project, we investigated the in vitro effect of NKA on oxytocin release from hypothalamic explants and PP of male rats and the possible involvement of NO in the action of NKA. Since NKA inhibits gamma-aminobutyric acid (GABA) release from PP, we also examined the role of NO in the effect of NKA on basal and K(+)-evoked GABA release. NKA (10(-7)-10(-5) M) significantly decreased oxytocin release from PP, whereas it did not affect its release from hypothalamic explants. The inhibitory effect of NKA on oxytocin release from PP was completely blocked by the NOS inhibitors N(G)-monomethyl-L-arginine (L-NMMA, 0.5 mM) or N(G)-nitro-L-arginine-methyl-ester (L-NAME, 1 mM). Sodium nitroprusside (0.5 mM), an NO releaser, had no effect on basal GABA release but significantly decreased K(+)-evoked GABA release. L-NMMA (0.3 mM) and L-NAME (0.5 mM) increased K(+)-evoked GABA release, indicating that NO plays an inhibitory role in GABA release from PP. The inhibition in both basal and K(+)-evoked GABA release induced by NKA (10(-7) M) was reduced by L-NAME (1 mM). Also, NKA (10(-7) M) increased NO synthesis as measured by [(14)C] citrulline production. Considered all together, our data indicate that NO may mediate the inhibitory effect of NKA on the release of both oxytocin and GABA from PP.     

Cerebral metabolic and vasopressin and oxytocin responses during osmotic stimulation in conscious rats

Intravenous infusion of hypertonic saline increased plasma [Na (+) ] and osmolality and induced a short-latency drinking response. These changes were associated with increased glucose utilization in the supraoptic and paraventricular nuclei and neural lobe, and decreases in the medial septum and nucleus ambiguus. The increases in glucose utilization were more accentuated in the supraoptic nuclei than in paraventricular nuclei, indicating that they are more sensitive to osmotic stimulation than the paraventricular nuclei. In association with enhanced activity in the hypothalamo-neurohypophysial system, plasma vasopressin and oxytocin concentrations increased, with a preferential increase of oxytocin over vasopressin. The hormonal contents in the neural lobe were not depleted by the osmotic stimulus despite the large increases of their concentrations in the plasma.     

Oxytocin in Brattleboro Rats: Increased Synthesis is Contrasted by Blunted Intrahypothalamic Release From Supraoptic Nucleus Neurones

Adult male Brattleboro rats were used to investigate the impact of the congenital absence of vasopressin on the release pattern of oxytocin (OXT) within the hypothalamic supraoptic nucleus (SON) in response to a 10-min forced swimming session and osmotic stimulation. Both immunohistochemical and in situ hybridisation data suggest that vasopressin-deficient animals have more oxytocin-synthesising neurones in the SON than homozygous wild-type controls. Unexpectedly, both forced swimming and peripheral osmotic stimulation resulted in a blunted release profile of oxytocin within the SON of vasopressin-deficient rats compared to controls. A similar intranuclear OXT response to direct osmotic stimulation of the SON by retrodialysis with hypertonic Ringer's solution in both genotypes confirmed the capability of SON neurones to locally release oxytocin in vasopressin-deficient rats, indicating an altered processing of information originating from multisynaptic inputs rather than a deficit in release capacity. Taken together with data obtained in previous studies, the present findings provide evidence suggesting that autocrine and paracrine signalling of magnocellular neurones differs within the paraventricular nucleus and the SON. Thus, significant alterations in intra-SON oxytocin mRNA levels cannot easily be extrapolated to intranuclear release profiles and the local signal intensity of this neuropeptide after physiological stimulation.     

Interleukin-1beta release in the supraoptic nucleus area during osmotic stimulation requires neural function

Interleukin (IL)-1beta is present throughout the magnocellular neuroendocrine system and co-depletes with oxytocin and vasopressin from the neural lobe during salt-loading. To examine whether IL-1beta is released from the dendrites/soma of magnocellular neurones during osmotic stimulation, microdialysis adjacent to the supraoptic nucleus (SON) in conscious rats was combined with immunocapillary electrophoresis and laser-induced fluorescence detection to quantify cytokine in 5-min dialysates collected before (0-180 min; basal), and after (180-240 min), hypertonic saline injected s.c. (1.5 m NaCl). Osmotic release of IL-1beta was compared after inhibiting local voltage-gated channels for Na+ (tetrodotoxin) and Ca2+ (cadmium and nickel) or by reducing intracellular Ca2+ stores (thapsigargin). Immunohistochemistry combined with microdialysis was used to localise cytokine sources (IL-1beta+) and microglia (OX-42+). Under conditions of microdialysis, the basal release of IL-1beta+ in the SON area was measurable and stable (pg/ml; mean +/- SEM) from 0-60 min (2.2 +/- 0.06), 60-120 min (2.32 +/- 0.05) and 120-180 min (2.33 +/- 0.06), likely originating locally from activated microglia (OX42+; IL-1beta+; ameboid, hypertrophied) and magnocellular neurones expressing IL-1beta. In response to osmotic stimulation, IL-1beta increased progressively in dialysates of the SON area by a mechanism dependent on intracellular Ca2+ stores sensitive to thapsigargin and, similar to dendritic secretion of oxytocin and vasopressin, required local voltage-gated Na+ and Ca2+ channels for activation by osmoregulatory pathways from the forebrain. During osmotic stimulation, neurally dependent release of IL-1beta in the SON area likely upregulates osmosensitive cation currents on magnocellular neurones (observed in vitro by others), to facilitate dendritic release of neurohypophysial hormones.     

Effects of glucagon-like peptide-1 (7-36) amide on neurohypophysial hormone secretion induced by acute hyperosmotic challenge

This study was designed to investigate possible effects of glucagon-like peptide-1 (7-36) amide on the vasopressin and oxytocin release induced by acute peripheral or central osmotic stimulation. In the first series of experiments, rats were injected intraperitoneally with the isotonic (0.15 M) or hypertonic (1.5 M) NaCl solution and then, intracerebroventricularly, with either 1 microg glucagon-like peptide-1 (7-36) amide dissolved in 5 microl of isotonic saline or with the vehicle only. In the second study, 1 microg glucagon-like peptide-1 (7-36) amide, dissolved in isotonic or hypertonic (0.6 M) saline, was injected into the cerebroventricular system. Control rats were treated with isotonic or hypertonic saline only. All the animals were decapitated 10 min after the intracerebroventricular injection. Glucagon-like peptide-1 (7-36) amide enhanced significantly the basal secretion of vasopressin and oxytocin. Moreover, this peptide increased additionally the release of both neurohypophysial hormones stimulated previously by peripheral osmotic challenge. On the other hand, the peptide increased the oxytocin but not vasopressin secretion brought about by an intracerebroventricular injection of hypertonic saline thus suggesting that the central osmotic stimulation decreases the sensitivity of vasopressin neurons to glucagon-like peptide-1 (7-36) amide. It is concluded that glucagon-like peptide-1 (7-36) amide may affect the secretory activity of the hypothalamo-neurohypophysial system under acute osmotic challenge.     

Allopregnanolone and induction of endogenous opioid inhibition of oxytocin responses to immune stress in pregnant rats

In virgin rats, systemic administration of interleukin (IL)-1β (i.e. to mimic infection), increases oxytocin secretion and the firing rate of oxytocin neurones in the supraoptic nucleus (SON). However, in late pregnancy, stimulated oxytocin secretion is inhibited by an endogenous opioid mechanism, preserving the expanded neurohypophysial oxytocin stores for parturition and minimising the risk of preterm labour. Central levels of the neuroactive metabolite of progesterone, allopregnanolone, increase during pregnancy and allopregnanolone acting on GABA(A) receptors on oxytocin neurones enhances inhibitory transmission. In the present study, we tested whether allopregnanolone induces opioid inhibition of the oxytocin system in response to IL-1β in late pregnancy. Inhibition of 5α-reductase (an allopregnanolone-synthesising enzyme) with finasteride potentiated IL-1β-evoked oxytocin secretion in late pregnant rats, whereas allopregnanolone reduced the oxytocin response in virgin rats. IL-1β increased the number of magnocellular neurones in the SON and paraventricular nucleus (PVN) expressing Fos (an indicator of neuronal activation) in virgin but not pregnant rats. In immunoreactive oxytocin neurones in the SON and PVN, finasteride increased IL-1β-induced Fos expression in pregnant rats. Conversely, allopregnanolone reduced the number of magnocellular oxytocin neurones activated by IL-1β in virgin rats. Treatment with naloxone (an opioid antagonist) greatly enhanced the oxytocin response to IL-1β in pregnancy, and finasteride did not enhance this effect, indicating that allopregnanolone and the endogenous opioid mechanisms do not act independently. Indeed, allopregnanolone induced opioid inhibition over oxytocin responses to IL-1β in virgin rats. Thus, in late pregnancy, allopregnanolone induces opioid inhibition over magnocellular oxytocin neurones and hence on oxytocin secretion in response to immune challenge. This mechanism will minimise the risk of preterm labour and prevent the depletion of neurohypophysial oxytocin stores, which are required for parturition.     

Expression of corticotropin releasing factor (CRF), urocortin and CRF type 1 receptors in hypothalamic-hypophyseal systems under osmotic stimulation

The expression of corticotropin releasing factor (CRF) and urocortin in hypothalamic magnocellular neurones increases in response to osmotic challenge. To gain a better understanding of the physiological roles of CRF and urocortin in fluid homeostasis, CRF, urocortin and CRF type 1 receptor (CRFR-1) gene expression was examined in the hypothalamic-hypophyseal system usingin situ and double-label in situ hybridization following chronic salt loading. CRFR-1 expression was further examined by immunohistochemistry and receptor binding. Ingestion of hypertonic saline by Sprague-Dawley rats for 7 days induced CRF mRNA exclusively in the oxytocin neurones of the magnocellular paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but induced CRFR-1 mRNA in both oxytocin and vasopressin-containing magnocellular neurones. Hypertonic saline treatment also increased urocortin mRNA expression in the PVN and the SON. In the SON, urocortin was localized to vasopressin and oxytocin neurones but was rarely seen in CRF-positive cells. Changes in CRFR-1 mRNA expression in magnocellular neurones by hypertonic saline treatment were accompanied by changes in CRFR-1 protein levels and receptor binding. Hypertonic saline treatment increased CRFR-1-like immunoreactivity in the magnocellular PVN and SON, and decreased it in the parvocellular PVN. CRF receptor binding in the PVN and SON was also increased in response to osmotic stimulation. Finally, hypertonic saline treatment increased CRFR-1 mRNA, CRFR-1-like immunoreactivity and CRF receptor binding in the intermediate pituitary. These results demonstrate that the increase in the expression of CRF and urocortin message in magnocellular neurones induced by salt loading is accompanied by an increase in CRF receptor levels and binding in the hypothalamus and intermediate pituitary. Thus, CRF and urocortin may exert modulatory effects locally within magnocellular neurones as well as at the pituitary gland in response to osmotic stimulation.     

Opioids and Coupling of the Anterior Peri-Third Ventricular Input to Oxytocin Neurones in Anaesthetized Pregnant Rats

In the pregnant rat the osmotic drive to oxytocin neurones is reduced and oxytocin secretion itself is inhibited by endogenous opioids. Coupling of the anterior peri-third ventricular input pathway, involved in osmoregulation, to magnocellular oxytocin neurones was studied in urethane-anaesthetized virgin and 21 day pregnant rats using electrical stimulation of the region anterior and ventral to the third cerebral ventricle (AV3V region) to drive the oxytocin neurones, and giving naloxone to prevent the action of any endogenous opioids on the system. Trains ofstimuli (0.5 mA, 1 ms pulses, 10 s on 10 s off, at either 10 Hz or 25 Hz for 10 or 2 min respectively) were given at 20 or 30 min intervals via an electrode stereotaxically-implanted in the AV3V region, and femoral arterial blood plasma samples collected immediately before and after each stimulation were radioimmunoassayed for oxytocin concentration. The first (control) AV3V stimulation increased plasma oxytocin concentration reproducibly and similarly in virgin and 21-day pregnant rats. Naloxone administered 10 min before the second stimulus increased basal plasma oxytocin concentration in virgin and pregnant rats and increased the oxytocin secretory response to 25 Hz AV3V stimulation in virgin but not pregnant rats, and the response was significantly greater in virgin rats. Naloxone reveals oxytocin secretion unrestrained by endogenous opioids, therefore it appears that there is an opioid-independent reduction in the excitatory coupling of the AV3V input to oxytocin neurones which may explain the reduced osmoresponsiveness of oxytocin neurones at the end of pregnancy. In other experiments, morphine (μ-opioid agonist; at 1 & 5 mg/kg 5 min before the second and third stimulation periods respectively) or U50,488 (κ-opioid agonist; 0.5 and 2.5 mg/kg respectively) was given to test the opioid sensitivity of the oxytocin neurone response to stimulation of the AV3V input. Morphine did not significantly affect the oxytocin secretory response to 25 Hz AV3V stimulation in either virgin or pregnant rats but there was a significant dose related inhibition of the oxytocin secretory response to 10 Hz stimulation in both groups. U50,488 also inhibited the oxytocin secretory response to 25 Hz AV3V stimulation in a dose-related manner in both virgin and pregnant rats. The inhibitory effects of morphine and U50,488 were not different between virgin and pregnant rats; this indicates that central opioid interactions with the AV3V input to oxytocin neurones are not changed in pregnancy. The initial similar oxytocin secretory response to AV3V stimulation in pregnant and virgin rats seems to be a product of a reduced effectiveness of the excitatory AV3V input to oxytocin neurones in late pregnancy and the previously established reduced posterior pituitary sensitivity to inhibitory endogenous opioids.     

Effects of ions and ionic channel activators or blockers on release of alpha-MSH from perifused rat hypothalamic slices.

The involvement of sodium and chloride ions in the process of alpha-melanocyte-stimulating hormone (a-MSH) release from hypothalamic neurons was investigated using perifused rat hypothalamic slices. Three different stimuli were found to increase a-MSH release from hypothalamic slices: high K+ concentration (50 mM), veratridine (50 microM), and the Na+/K(+)-ATPase inhibitor ouabain (1 mM). Spontaneous or K(+)-evoked a-MSH release was insensitive to the specific Na+ channel blocker tetrodotoxin (TTX; 1.5 microM) and to the blocker of K+ channels tetraethylammonium (TEA; 30 mM) or 4-aminopyridine (4-AP; 4 mM). In contrast, blockage of ouabain-sensitive Na+/K(+)-ATPase increased the resting level of a-MSH and caused a dramatic potentiation of K(+)-evoked a-MSH release. The Na+ channel activator veratridine (50 microM) triggered a-MSH release. This stimulatory effect was blocked by TTX and prolonged by TEA application, indicating the occurrence of voltage-sensitive Na+ and K+ channels on a-MSH neurons. Replacement of Na+ by impermeant choline ions from 95 to 60 mM did not alter K(+)-evoked a-MSH release. Conversely, dramatic reduction of the external Na+ concentration to 16 mM caused a robust increase of a-MSH secretion from hypothalamic neurons, likely through activation of the Na+/Ca2+ exchange system. These data indicate that the depolarizing effect of K+ results from direct activation of voltage-operated Ca2+ channels. The lack of effect of TEA on basal a-MSH release prompted us to investigate the possible involvement of chloride ions in the regulation of the spontaneous activity of a-MSH neurons. Substitution of Cl- for impermeant acetate ions did not affect basal or K(+)-evoked a-MSH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticotropin-releasing factor type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic stimulation

In rats, acute stress substantially increases corticotropin-releasing factor (CRF) type 1 receptor (CRFR-1) mRNA expression in the paraventricular nucleus (PVN) and osmotic stimulation induces both CRF and CRFR-1 mRNA in magnocellular PVN and supraoptic nucleus (SON). However, these phenomena have not been analysed in other species. We compared CRF and CRFR-1 expression in rat and mouse hypothalamus. Male C57BL/6 mice and Wistar rats were exposed to acute restraint stress for 3 h, or to hypertonic saline ingestion for 7 days. Restraint stress increased CRF and c-fos mRNA expression in both rat and mouse PVN. CRFR-1 mRNA was barely detectable in controls, whereas restraint stress substantially increased CRFR-1 mRNA in rat PVN, but not in mouse. Hypertonic saline ingestion induced CRF mRNA in magnocellular PVN and SON of the rat, but did not alter CRF mRNA levels in mouse hypothalamus. CRFR-1 mRNA was also induced in magnocellular PVN and SON of the rat in response to osmotic stimulation, but not in mouse. Immunohistochemistry demonstrated that CRFR-1-like immunoreactivity (ir) was distributed within parvocellular and magnocellular PVN of mouse and rat. CRFR-1-ir in rat PVN was increased by acute stress and osmotic stimulation. By contrast, these treatments did not alter CRFR-1-ir in mouse PVN. Combined immunohistochemistry and in situ hybridization revealed that CRFR-1-ir was most frequently colocalized to CRF in mouse PVN, whereas only a small percentage of oxytocin and vasopressin-producing cells coexpressed CRFR-1-ir. These results indicate that (i) by contrast to rats, neither acute stress nor osmotic stimulation induces CRFR-1 mRNA expression in the mouse PVN; (ii) osmotic stimulation does not alter CRF mRNA expression in parvocellular and magnocellular neurones of mouse PVN; and (iii) acute stress increases c-fos and CRF mRNA to a similar degree in mouse and rat PVN. Thus, differences may exist between mouse and rat in the regulation of CRF and CRFR-1 gene expression in hypothalamus following stress and osmotic stimulation.     

Glial Cells: Indispensable Partners of Hypothalamic Magnocellular Neurones

The hypothalamo-neurohypophysial system is comprised of magnocellular neurones that synthesise the neuropeptides oxytocin or vasopressin. As neurohormones, these peptides intervene in the regulation of vital functions such as parturition, lactation, osmotic and cardiovascular regulation. The release of these peptides in the general circulation depends on the electrical activity of their parent neurones, which in turn is regulated by the activity of their afferent inputs conveying distinct information. Thus, in view of the diversity of information governing the activity of magnocellular neurones, it is crucial that the system adapts the appropriate release of oxytocin and vasopressin upon physiological demand. Until recently, it was considered that only neurones could provide such adaptation and regulation. However, a third partner of the synapse, the astrocyte, has been shown to provide further control. Astrocytic processes are in proximity of the magnocellular neurones and their synapses, well positioned to detect and modulate synaptic signals. For instance, astrocytes detect a synaptic signal owing to their diverse neurotransmitter/neuropeptide receptors. In addition, they release a variety of neuroactive substances (i.e. gliotransmitters), which in turn modulate synaptic activity. An important gliotransmitter is the amino acid, d -serine, which, together with glutamate, activates NMDA receptors. Once activated, NMDA receptors govern the weight of individual inputs on magnocellular neurones and thus the impact of distinct types of information on neuronal activity. As reviewed here, numerous observations show that astrocytes must be considered as key elements in the functioning of the hypothalamo-neurohypophysial system.     

Regulation of rat APJ receptor messenger ribonucleic acid expression in magnocellular neurones of the paraventricular and supraopric nuclei by osmotic stimuli

The novel apelin receptor (APJ receptor, APJR) has a restricted expression in the central nervous system suggestive of an involvement in the regulation of body fluid homeostasis. The endogenous ligand for APJR, apelin, is also highly concentrated in regions that are involved in the control of drinking behaviour. While the physiological roles of APJR and apelin are not fully known, apelin has been shown to stimulate drinking behaviour in rats and to have a regulatory effect on vasopressin release from magnocellular neurones of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. To determine the role of APJR in the regulation of water balance, this study examined the effects of osmotic stimulation on the expression of APJR mRNA in the magnocellular PVN (mPVN) and SON of salt-loaded and water-deprived rats. Intake of 2% NaCl and water deprivation for 48 h induced expression of APJR mRNA in the mPVN and SON. Using dual-label in situ hybridization histochemistry, we also investigated whether APJR is colocalized within vasopressin neurones in control, salt-loaded and water-deprived rats. APJR mRNA was found to colocalize with a small population of vasopressin-containing magnocellular neurones in control and water-deprived rats. Salt-loading resulted in an increased colocalization of APJR and vasopressin mRNAs in the SON. These data verify a role for APJ receptors in body fluid regulation and suggest a role for apelin in the regulation of vasopressin-containing neurones via a local autocrine/paracrine action of the peptide.     

Impaired somatodendritic responses to pituitary adenylate cyclase-activating polypeptide (PACAP) of supraoptic neurones in PACAP type I -receptor deficient mice

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.     

Excitatory effect of dopamine on oxytocin and vasopressin reflex releases in the rat

The involvement of dopamine in the release of oxytocin and vasopressin was investigated in lactating rats during suckling or after changes in plasma osmolality. The effects of intraventricular injections of dopamine, agonists and antagonists, were tested on electrical unit activity of oxytocinergic or vasopressinergic cells in the paraventricular nucleus, on intramammary pressure (index of oxytocin release) and diuresis (index of vasopressin release).In urethane-anaesthetized lactating suckled rats, dopamine (1 μg), apomorphine (2.5 and 5 μg) facilitated the established milk-ejection reflex, increasing the frequency and the amplitude of neurosecretory bursts of oxytocinergic cells. They also triggered the reflex in lactating rats without milk-ejections during suckling. The small doses injected were in no way such as to induce an acceleration in firing rate of oxytocinergic cells or an increase in mammary pressure.In alcohol-loaded rats, during water diuresis, dopamine (2 μg) and apomorphine (5 μg) activated the depressed vasopressinergic cells and inhibited diuresis. These facilitatory effects were progressive, reaching a maximum 10–15 min after injection.Haloperidol (5 μg) and α-flupentixol (10 μg) had an inhibitory effect on both types of neurosecretory cells in urethane-anaesthetized rats. They prevented the reflex activation of oxytocinergic cells induced by suckling and of vasopressinergic cells after a hyperosmotic stimulus (1 ml i.p 9% NaCl solution). These inhibitory effects were not of the ‘all-or-none’ type.So, we can postulate that dopamine regulates the reflex release of oxytocin and vasopressin in the hypothalamus. On the one hand, dopamine permits and controls the periodic activation of oxytocinergic cells as long as the mothers are being suckled. On the other hand, it modulates the activity of vasopressinergic cells whenever the plasma osmolality changes.     

Oestradiol potentiates hormone secretion and neuronal activation in response to hypertonic extracellular volume expansion in ovariectomised rats

Secretion of vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) is an essential mechanism for the maintenance of hydromineral homeostasis. Secretion of these hormones is modulated by several circulating factors, including oestradiol. However, it remains unclear how oestradiol exerts this modulation. In the present study we investigated the participation of oestradiol in the secretion of VP, OT and ANP and in activation of vasopressinergic and oxytocinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to extracellular volume expansion (EVE). For this purpose, ovariectomised (OVX) rats treated for 7 days with vehicle (corn oil, 0.1 ml/rat, OVX+O group) or oestradiol (oestradiol cypionate, 10 μg/kg, OVX+E group) were subjected to either 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.) EVE. Blood samples were collected for plasma VP, OT and ANP determination. Another group of rats was subjected to cerebral perfusion, and brain sections were processed for c‐Fos‐VP and c‐Fos‐OT double‐labelling immunohistochemistry. In OVX+O rats, we observed that both isotonic and hypertonic EVE increased plasma OT and ANP concentrations, although no changes were observed in VP secretion. Oestradiol replacement did not alter hormonal secretion in response to isotonic EVE, but it increased VP secretion and potentiated plasma OT and ANP concentrations in response to hypertonic EVE. Immunohistochemical data showed that, in the OVX+O group, hypertonic EVE increased the number of c‐Fos‐OT and c‐Fos‐VP double‐labelled neurones in the PVN and SON. Oestradiol replacement did not alter neuronal activation in response to isotonic EVE, but it potentiated vasopressinergic and oxytocinergic neuronal activation in the medial magnocellular PVN (PaMM) and SON. Taken together, these results suggest that oestradiol increases the responsiveness of vasopressinergic and oxytocinergic magnocellular neurones in the PVN and SON in response to osmotic stimulation.     

Chronic hypoosmolality induces a selective decrease in magnocellular neurone soma and nuclear size in the rat hypothalamic supraoptic nucleus

The magnocellular neurones of the hypothalamo-neurohypophysial system (HNS) play a vital role in the maintenance of body homeostasis by regulating oxytocin (OT) and vasopressin (VP) secretion from the posterior pituitary. During hyperosmolality, OT and VP mRNA levels are known to increase by approximately two-fold, whereas during chronic hypoosmolality, OT and VP mRNA levels decrease to approximately 10-20% of basal levels. In these studies, we evaluated changes in cell size associated with these physiological conditions. Cell and nuclear sizes of neurones in the supraoptic nucleus (SON), the nucleus of the lateral olfactory tract (LOT) and the medial habenular nucleus (MHB) were measured from neurones identified by in situ hybridization histochemistry for beta(III)-tubulin mRNA, and measurements were made from OT and AVP magnocellular neurones in the SON after phenotypic identification by immunohistochemistry. Under hypoosmolar conditions, the cell and nuclear sizes of OT and VP magnocellular neurones decreased to approximately 60% of basal values, whereas cell and nuclear sizes of OT and VP neurones in hyperosmolar rats increased to approximately 170% of basal values. In contrast, neither hyperosmolality, nor hypoosmolality significantly affected cell and nuclear sizes in the LOT and MHB. These results confirm previous studies that showed that magnocellular neurones increase cell size in response to hyperosmolar conditions and, for the first time, demonstrate a marked decrease in cell size in the SON in response to chronic hypoosmolar conditions. These dramatic changes in cell and nuclear size directly parallel changes in OT and VP gene expression in the magnocellular neurones of the SON and, consequently, are consistent with the pronounced bidirectional changes in gene expression and cellular activity found during these osmotic perturbations. Our results therefore support the concept of global alterations in the synthetic activity of magnocellular OT and AVP neurones in response to extracellular osmolality.     

Median Preoptic Neurones Projecting to the Supraoptic Nucleus are Sensitive to Haemodynamic Changes as well as to Rise in Plasma Osmolality in Rats

Extracellular single unit activity was recorded from 73 neurones in the median preoptic nucleus (MnPO), identified by antidromic activation as projecting to the supraoptic nucleus (SON) area in urethane-anaesthetized male rats. Thirteen of 73 identified MnPO neurones were silent, and 44 of 60 spontaneously active MnPO neurones were tested for their responses to electrical stimulation of the nucleus tractus solitarius (NTS). The cells were divided into 4 groups according to their responses; those which were excited orthodromically (OD⁺; n = 15), those which were unresponsive (UN; n = 21), those which were inhibited orthodromically (OD⁻; n=4), those which showed initial inhibition followed by excitation (OD⁺ n = 4). Some of these neurones were further tested for their responses to haemorrhage and/or increase in blood pressure produced by intravenous administration of the oe-agonist, phenylephrine, and/or to hyperosmotic stimulation produced by intraperitoneal injection of 1.5 M NaCl. Six out of 10 OD⁺ cells were excited by haemorrhage, 6 out of 11 OD⁺ cells were inhibited by phenylephrine, and 5 out of 9 OD⁺ cells were excited by hypertonic saline. On the other hand the UN cells tended to be unresponsive to each type of stimulus. Three out of 7 OD⁺ cells were excited by both haemorrhage and hypertonic saline, and 3 out of 8 OD⁺ cells were inhibited by phenylephrine and excited by hypertonic saline. The results may suggest that MnPO neurones which receive afferent input from the NTS may be sensitive not only to haemodynamic change but also to change in plasma osmotic pressure and that such population of MnPO neurones may integrate a part of the haemodynamic and osmotic information and contribute to the control of neurohypophysial hormone release.