Supraoptic Vasopressin Neurons Research Articles

Effects of bile duct ligation on the inhibitory control of supraoptic vasopressin neurons.

Dilutional hyponatremia due to increased plasma arginine vasopressin (AVP) is associated with liver cirrhosis. However, plasma AVP remains elevated despite progressive hypoosmolality. This study investigated changes to inhibitory control of supraoptic nucleus (SON) AVP neurons during liver cirrhosis. Experiments were conducted with adult male Sprague-Dawley rats. Bile duct ligation was used as a model of chronic liver cirrhosis. An adeno-associated virus containing a construct with an AVP promoter and either green fluorescent protein (GFP) or a ratiometric chloride indicator, ClopHensorN, was bilaterally injected into the SON of rats. After 2 weeks, rats received either BDL or sham surgery, and liver cirrhosis was allowed to develop for 4 weeks. In vitro, loose patch recordings of action potentials were obtained from GFP-labeled and unlabeled SON neurons in response to a brief focal application of the GABAA agonist muscimol (100 μM). Changes to intracellular chloride ([Cl]i) following muscimol application were determined by changes to the fluorescence ratio of ClopHensorN. The contribution of cation chloride cotransporters NKCC1 and KCC2 to changes in intracellular chloride was investigated using their respective antagonists, bumetanide (BU, 10 μM) and VU0240551 (10 μM). Plasma osmolality and hematocrit were measured as a marker of dilutional hyponatremia. The results showed reduced or absent GABAA -mediated inhibition in a greater proportion of AVP neurons from BDL rats as compared to sham rats (100% inhibition in sham vs. 47% in BDL, p = .001). Muscimol application was associated with increased [Cl]i in most cells from BDL as compared to cells from sham rats (χ2 = 30.24, p < .001). NKCC1 contributed to the impaired inhibition observed in BDL rats (p < .001 BDL - BU vs. BDL + BU). The results show that impaired inhibition of SON AVP neurons and increased intracellular chloride contribute to the sustained dilutional hyponatremia in liver cirrhosis.

Effects of salt‐loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+ /Cl- co-transporter2 (KCC2) and up-regulating Na+ /K+ /Cl- co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl- ]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl- ]i in response to focal application of the GABAA agonist muscimol (100μmolL-1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P<0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P<0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10μmolL-1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10μmolL-1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl- influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50μmolL-1 ) and protein kinase inhibitor (K252a) (100nmolL-1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl- ]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

OR06-4 Brain Derived Neurotrophic Factor in the Regulation of Supraoptic Vasopressin Neurons in Development of Hyponatremia

Dilutional hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in patients with liver failure. The current conventional therapies are palliative and frequently ineffective. The neural mechanisms causing dysregulation of AVP secretion are not completely understood. Based on previous studies, we hypothesis that inappropriate AVP release associated with liver failure is due to increased Brain Derived Neurotrophic Factor (BDNF) in the supraoptic nucleus (SON). BDNF diminishes GABAA inhibition in SON AVP neurons by increasing intracellular chloride ([Cl]i) through tyrosine receptor kinase B (TrkB) activation and downregulation of K+/Cl- co-transporter 2 (KCC2). This loss of inhibition could increase AVP secretion. To test this hypothesis, chronic bile duct ligated (BDL) rats, a model of dilutional hyponatremia associated with liver failure, were injected with shRNA to prevent increased BDNF in the SON. Adult male Sprague Dawley rats were bilaterally injected in the SON with AAV2- shRNA-BDNF-mCherry or AAV2- shRNA-SCR-mCherry. After two weeks, the rats had BDL or sham ligation surgery. Four weeks later, SON regions were collected by punches for western blot analysis. Fluid accumulation, liver and body weight were recorded to verify if the rats develop liver failure. Blood samples were collected to measure plasma osmolality, hematocrit, and circulating AVP concentration. Data were analyzed by one-way ANOVA with Bonferroni comparisons. The BDL rats had significant increases in liver to body weight ratio (p< 0.05; 6-9) compared to sham rats indicating liver failure and ascites with bile duct ligation. BDL rats with scrambled injections (BDL SCR) developed hyponatremia with significant decrease in plasma osmolality and hematocrit along with significant increase in plasma AVP concentration (all p< 0.05; 6-9) compared to sham groups. This is the first study to show that phosphorylation of TrkB (pTrkBY515) is significantly increased along with significant decrease in phosphorylation of KCC2 (pKCC2S940) in BDL SCR rats compared to the sham rats (p< 0.05; 6-8). Knockdown of BDNF in the SON of BDL rats (BDL shBDNF) prevented the rats from developing hyponatremia and caused significant increase in plasma osmolality and hematocrit compared to BDL SCR rats (p< 0.05; 6-9). The BDL shBDNF rats had significant (p< 0.05; 6-9) decreases in plasma AVP concentration compared to BDL SCR rats. shBDNF injections in the SON of BDL rats significantly blocked the increase in TrkB phosphorylation and decrease in KCC2 phosphorylation (p< 0.05;6-8) associated with bile duct ligation. mCherry expression was used to verify the specificity of stereotaxic injections. Rats that did not had successful virus injections in the SON were analyzed separately. The results indicate that BDNF produced in the SON contributes to increased AVP secretion and hyponatremia during liver cirrhosis.

Intracellular Chloride Regulation of Supraoptic Vasopressin Neurons during Salt Loading

Salt loading (SL) increases intracellular chloride concentration [Cl]i, impairing GABAA inhibition of arginine vasopressin (AVP) neurons in the supraoptic nucleus (SON) of hypothalamus. But the regulatory mechanisms leading to increased [Cl]i is not completely understood. Based on previous studies, we hypothesize that SL activates tyrosine receptor kinase B (TrkB) and downregulates K+/Cl− co‐transporter 2 (KCC2) membrane expression. Downregulation of KCC2 decreases the efflux of chloride ion causing increase in [Cl]i in SON AVP neurons. In this study, we have combined virally mediated ClopHensorN, a relatively new ratiometric chloride imaging technique with capillary based Simple Western technology to record changes in [Cl]i and specifically detect KCC2 protein expression in individual SON AVP neurons.Adult male Sprague Dawley rats were bilaterally injected in the SON with rAAV2‐0VP1‐ClopHensorN. The ClopHensorN (Addgene Plasmid #50758 from Colin Akerman) was packaged in an AAV2 vector with a AVP specific promotor (Addgene Plasmid #40868 from Harold Gainer). After 2 weeks, the rats were given either water or 2% NaCl to drink for 7 days. At the end of the protocol, the rats were anesthetized with inactin and their SONs were dissociated. The cells were plated on coverslips and placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. ClopHensorN positive neurons were tested for decrease or increase in [Cl]i to focal application of GABAA agonist muscimol (100uM). After imaging, individual neurons were collected by aspirating into a patch pipette to verify KCC2 and ß‐Actin protein expression. The standard matrix (12–230kDa) in Protein Simple Wes was used to identify and quantitate very low concentration of protein from single neuron. Data were analyzed by Chi‐squared test and one‐way ANOVA with Bonferroni comparisons.Muscimol application to SL SONs either significantly increased chloride efflux (p&lt;0.05;13/20) or did not change chloride flux (7/20). TrkB antagonist (AnA,50uM) significantly blocked the chloride effluxes. SONs from euhydrated rats showed muscimol induced chloride influx (p&lt;0.05;11/16). KCC2 antagonist (VU 0240551,10uM) significantly blocked the chloride influxes in euhydrated rats. SON AVP neurons that responded to pharmacological inhibitors during chloride imaging were viable and expressed KCC2 co‐transporter and ß‐Actin. Neurons that did not respond during chloride imaging did not had KCC2 and ß‐Actin protein expression. Salt loading increases [Cl]i in SON AVP neurons through TrKB‐KCC2 dependent mechanism.Support or Funding InformationSupported by R01 HL119458 and 18PRE34060035This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High salt loading increases brain derived neurotrophic factor in supraoptic vasopressin neurones.

High salt loading (SL) is associated with inappropriate arginine vasopressin (AVP) release and increased mean arterial pressure. Previous work has shown that chronic high salt intake impairs baroreceptor inhibition of rat AVP neurones through brain-derived neurotrophic factor (BDNF) dependent activation of tyrosine receptor kinase B (TrkB) and down-regulation of K+/Cl- co-transporter KCC2. This mechanism diminishes the GABAA inhibition of AVP neurones in the supraoptic nucleus (SON) by increasing intracellular chloride. However, the source of BDNF leading to this ionic plasticity is unknown. In the present study, we used adeno-associated viral vectors with short hairpin RNA against BDNF to test whether SON is the source of BDNF contributing to increased AVP release and elevated mean arterial pressure in high salt loaded rats. Virally mediated BDNF knockdown (shBDNF) in the SON of salt loaded rats significantly blocked the increases in BDNF mRNA and AVP heterogeneous RNA expression. The observed increase in the activation of TrkB receptor during salt loading is consistent with previous studies. Western blot analysis of SON punches revealed that tyrosine phosphorylation of TrkB (pTrkBY515) was significantly decreased in salt shBDNF rats compared to the salt scrambled (SCR) rats. Injections of shBDNF in the SON also significantly prevented the increase in plasma AVP concentration associated with salt loading. However, the salt loading induced increase in mean arterial pressure was not decreased with BDNF knockdown in the SON. Average daily fluid intake and urine output were significantly elevated in both salt SCR and salt shBDNF rats compared to the euhydrated controls. Daily average urine sodium concentration was significantly higher in shBDNF injected salt rats than the other groups. These findings indicate that BDNF produced in the SON contributes to the increased AVP secretion during high salt loading but not with respect to the subsequent increase in mean arterial pressure.

Virally Mediated ClopHensorN Chloride Imaging in the Supraoptic Vasopressin Neurons

A number of physiological manipulations in rats, including 2% salt loading (SL), alter chloride homeostasis effecting GABAA mediated inhibition in vasopressin (VP) neurons in the supraoptic nucleus (SON). Brain Derived Neurotrophic Factor (BDNF) and tyrosine receptor kinase B (TrkB) contribute to changes in chloride homeostasis in SON associated with SL. However, it can be difficult to measure these changes using traditional patch clamp electrophysiology. In this study, we used ClopHensorN (Addgene Plasmid #50758 from Colin Akerman), a relatively new technique for ratiometric intracellular chloride [Cl]i imaging to measure changes in the responses of acutely dissociated VP SON neurons to the GABAA receptor agonist muscimol following a 7‐day 2% SL protocol. ClopHensorN was packaged in an AAV2 vector with a VP specific promotor (Addgene Plasmid #40868 from Harold Gainer). Adult male Sprague Dawley rats (250–300 g b w) were anesthetized with isoflurane and bilaterally injected in the SON (300 nl) with rAAV2‐0VP1‐ClopHensorN. After two weeks of recovery, the rats were given either water or 2% NaCl to drink for 7 days. At the end of the protocol, the rats were anesthetized with inactin (100 mg/kg IP) and decapitated. The brains were removed and the SONs isolated for mechanical and chemical dissociation. The cells were plated on coverslips that were placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. Some brains (injected with a VP reporter, AAV2‐p2.OVPI‐eGFP) were sectioned in a vibratome and live 300 um sections containing the SONs were used for electrophysiological recording in order to confirm the ClopHensorN findings. ClopHensorN positive cells were tested for changes in 445/556nm fluorescence ratio indicating decreases or increase in [Cl]i after focal application of muscimol (100uM). To confirm the function of ClopHensorN, transfected cells were dialyzed with micropipettes containing either a high [Cl] (130 mM CsCl) or 0 [Cl] (140 mM KGlu). Dialysis with CsCl produced a decrease and KGlu produced an increase in ClopHensorN fluorescence consistent with Cl− influx and efflux, respectively. In experiments with muscimol on SON neurons from SL rats, 6 out of 10 cells showed significant increases in chloride efflux while 4 out of 10 had no change in chloride flux. TrKB antagonist incubation significantly blocked muscimol induced chloride efflux in SL rats. We also saw significant increase in [Cl]i in 5 of 9 cells from euhydrated rats with muscimol application and this was unaffected by TrkB antagonism. Using loose patch voltage clamp recordings, neurons from euhydrated rats (6/6) were inhibited by muscimol (100 uM). Neurons from SL rats were either unresponsive to muscimol (5/7) or responded with a rapid and robust increase in activity (2/7) followed by a period inactivity (characteristic of depolarization block). Results with ClopHensorN are consistent with electrophysiology recordings suggesting that it is a viable alternative to electrophysiological recordings. The ClopHensorN's ability measure chloride ion dynamics could make it an important tool.Support or Funding InformationSupported by R01 HL119458This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apoptosis of supraoptic AVP neurons is involved in the development of central diabetes insipidus after hypophysectomy in rats

BackgroundIt has been reported that various types of axonal injury of hypothalamo-neurohypophyseal tract can result in degeneration of the magnocellular neurons (MCNs) in hypothalamus and development of central diabetes insipidus (CDI). However, the mechanism of the degeneration and death of MCNs after hypophysectomy in vivo is still unclear. This present study was aimed to disclose it and to figure out the dynamic change of central diabetes insipidus after hypophysectomy.ResultsThe analysis on the dynamic change of daily water consumption (DWC), daily urine volume(DUV), specific gravity of urine(USG) and plasma vasopressin concentration showed that the change pattern of them was triphasic and neuron counting showed that the degeneration of vasopressin neurons began at 10 d, aggravated at 20 d and then stabilized at 30 d after hypophysectomy. There was marked upregulation of cleaved Caspase-3 expression of vasopressin neurons in hypophysectomy rats. A "ladder" pattern of migration of DNA internucleosomal fragments was detected and apoptotic ultrastructure was found in these neurons. There was time correlation among the occurrence of diabetes insipidus, the changes of plasma vasopressin concentration and the degeneration of vasopressin neurons after hypophysectomy.ConclusionThis study firstly demonstrated that apoptosis was involved in degeneration of supraoptic vasopressin neurons after hypophysectomy in vivo and development of CDI. Our study on time course and correlations among water metabolism, degeneration and apoptosis of vasopressin neurons suggested that there should be an efficient therapeutic window in which irreversible CDI might be prevented by anti-apoptosis.

Lesion of the perinuclear zone attenuates cardiac sensitivity of vasopressinergic supraoptic neurons.

Discrete stretch of the caval-atrial junction decreases the activity of vasopressin-secreting neurons in the supraoptic nucleus (SON). The perinuclear zone (PNZ) of the SON is necessary for inhibition of vasopressin neurons following an increase in blood pressure. To determine whether the PNZ is necessary for cardiopulmonary regulation of vasopressin neurons, male rats received three unilateral injections of the excitotoxin ibotenic acid (n = 9) or phosphate-buffered saline vehicle (n = 10) into the PNZ. Extracellular activity of antidromically identified phasic vasopressin neurons in the ipsilateral SON was recorded. Of the 26 neurons recorded from vehicle-injected rats 26 were inhibited by an increase in blood pressure and 22 of those neurons were sensitive to caval-atrial distension. Of the neurons recorded from PNZ-lesion rats, only 12 of 29 were inhibited by an increase in blood pressure (P < 0.05), and only 11 neurons were sensitive to caval-atrial stretch (P < 0.05). Functional lesion of the PNZ significantly attenuates both arterial and cardiopulmonary baroreceptor-mediated inhibition of supraoptic vasopressin neurons, suggesting that the PNZ is a necessary component of both pathways.

Baroreceptor sensitivity of rat supraoptic vasopressin neurons involves noncholinergic neurons in the DBB.

Previous studies suggest that cholinergic neurons in the diagonal band of Broca (DBB) participate in the baroreceptor-mediated inhibition of phasic vasopressin neurons in the supraoptic nucleus (SON). To test this hypothesis, extracellular recordings were obtained from putative vasopressin SON neurons of anesthetized rats injected with the cholinergic immunotoxin 192 IgG-saporin (0.8 microg/microl) in the DBB. Baroreceptor sensitivity of neurons was tested with brief phenylephrine-induced (10 microg/10 microl iv) increases in blood pressure of at least 40 mmHg. In rats injected with vehicle or unconjugated saporin, 19 of 21 and 18 of 20 phasic neurons, respectively, were inhibited by increased blood pressure. In rats injected with 192 IgG-saporin, which significantly reduced the number of choline acetyltransferase (ChAT)-positive DBB neurons, 33 of 36 phasic neurons were inhibited. Normal rats and rats with DBB saporin injections received rhodamine bead injections into the perinuclear zone (PNZ) to retrogradely label DBB neurons, and their brains were stained for ChAT. ChAT-positive DBB neurons were not retrogradely labeled from the PNZ. Together, these results indicate that the pathway relaying baroreceptor information to the SON involves noncholinergic DBB neurons.

Nitric Oxide Modulation of Supraoptic Oxytocin and Vasopressin Neurons Involves Potentiation of Gabaergic Synaptic Inputs.

P41 There is growing evidence that nitric oxide (NO) in the central nervous system functions as a local neuromodulator in water balance and osmotic regulation. However, the precise mechanisms by which these actions are mediated are still poorly understood. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in the supraoptic (SON) and paraventricular (PVN) nuclei. nNOS expression is up-regulated in conditions known to stimulate oxytocin (OT) and vasopressin (VP) hormone release, such as salt loading and water deprivation. Since several studies have shown that NO mainly decreases the excitability of OT and VP neurons, NO may function as a local inhibitory feedback on activated SON neurons. In the present study we combined in vivo and in vitro electrophysiological studies to investigate whether NO-induced inhibition of SON neurons is mediated by activation of GABAergic inputs in the SON. In vivo retrodialysis of the NO-donor SNP into the SON inhibited the firing activity of both OT and VP neurons. This inhibition was largely blocked by retrodialysis of the GABA A receptor antagonist bicuculline. In order to understand the cellular mechanisms linking NO and GABAergic systems in the SON, in vitro patch clamp recordings of GABA A synaptic currents were obtained from immunochemically identified neurons in hypothalamic slices. The frequency and amplitude of miniature GABA A synaptic currents in both OT and VP neurons were significantly increased by SNP. These results suggest that NO inhibition of neuronal excitability of OT and VP neurons is largely mediated by pre- and postsynaptic potentiation of GABAergic synaptic activity in the SON. Thus, the proposed NO-GABAergic inhibitory feedback in the SON might constitute an important mechanism by which NO regulates osmotic and fluid balance homeostasis.

Role of the locus ceruleus in baroreceptor regulation of supraoptic vasopressin neurons in the rat.

The goal of this study was to identify the source of baroreceptor-related noradrenergic innervation of the diagonal band of Broca (DBB). Male Sprague-Dawley rats underwent sinoaortic denervation (SAD, n = 13) or sham SAD surgery (n = 13). We examined Fos expression produced by baroreceptor activation and dopamine-beta-hydroxylase immunofluorescence in hindbrain regions that contain noradrenergic neurons. Baroreceptors were stimulated by increasing blood pressure >40 mmHg with phenylephrine (10 microgram. kg(-1). min(-1) iv) in sham SAD and SAD rats. Controls were infused with 0.9% saline. Only the locus ceruleus (LC) demonstrated a baroreceptor-dependent increase in Fos immunoreactivity in dopamine-beta-hydroxylase-positive neurons. In a second experiment, normal rats received rhodamine-labeled microsphere injections in the DBB (n = 12) before phenylephrine or vehicle infusion. In these experiments, only the LC consistently contained Fos-positive cells after phenylephrine infusion that were retrogradely labeled from the DBB. Finally, we lesioned the LC with ibotenic acid and obtained extracellular recordings from identified vasopressin neurons in the supraoptic nucleus. LC lesions significantly reduced the number of vasopressin neurons that were inhibited by acute baroreceptor stimulation. Together, these results suggest that noradrenergic neurons in the LC participate in the baroreflex activation of the DBB and may thus be important in the baroreflex inhibition of vasopressin-releasing neurons in the supraoptic nucleus.

Differential effects of the neurosteroid pregnenolone sulphate on oxytocin and vasopressin neurones in vitro.

Pregnenolone sulphate (PS) is a neurosteroid, synthesised within the brain, which has been reported to alter glutamatergic transmission primarily through allosteric interaction with NMDA receptors[1]. Previous work from this laboratory has shown that PS enhances NMDA-induced phasic firing in supraoptic vasopressin (VP) neurones[2]. The present study extends these initial findings by: (i) examining the effect of PS (100 µM) on firing evoked by the natural ligand, glutamate, in both supraoptic oxytocin (OT) and VP neurones and (ii) comparing the action of PS on NMDA- and AMPA-induced firing in these two cell types.

Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation.

1. To evaluate the implication of taurine in the physiology of supraoptic neurones, we (i) investigated the agonist properties of taurine on glycine and GABAA receptors of supraoptic magnocellular neurones acutely dissociated from adult rats, using whole-cell voltage clamp, (ii) studied the effects of taurine and strychnine in vivo by extracellular recordings of supraoptic vasopressin neurones in anaesthetized rats, and (iii) measured the osmolarity-dependent release of endogenous taurine from isolated supraoptic nuclei by HPLC. 2. GABA, glycine and taurine evoked rapidly activating currents that all reversed close to the equilibrium potential for Cl-, indicating activation of Cl(-)-selective channels. Glycine-activated currents were reversibly blocked by strychnine (IC50 of 35 nM with 100 microM glycine), but were unaffected by the GABAA antagonist gabazine (1-3 microM). GABA-activated currents were reversibly antagonized by 3 microM gabazine, but not by strychnine (up to 1 microM). 3. Responses to 1 mM taurine were blocked by strychnine but not by gabazine and showed no additivity with glycine-induced currents, indicating selective activation of glycine receptors. Responses to 10 mM taurine were partially antagonized by gabazine, the residual current being blocked by strychnine. Thus, taurine is also a weak agonist of GABAA receptors. 4. In the presence of gabazine, taurine activated glycine receptors with an EC50 of 406 microM. Taurine activated at most 70% of maximal glycine currents, suggesting that it is a partial agonist of glycine receptors. 5. In vivo, locally applied strychnine (300 nM) increased and taurine (1 mM) decreased the basal electrical activity of vasopressin neurones in normally hydrated rats. The effect of strychnine was markedly more pronounced in water-loaded rats. 6. Taurine, which is concentrated in supraoptic glial cells, could be released from isolated supraoptic nuclei upon hyposmotic stimulation. Decreases in osmolarity of 15 and 30% specifically enhanced basal release of taurine by 42 and 124%, respectively. 7. We conclude that supraoptic neurones express high amounts of glycine receptors, of which taurine may be regarded as a major natural agonist. We postulate that taurine, which can be released in hyposmotic situations, acts on glycine receptors to exert an inhibitory control on magnocellular neurones during alterations of body fluid homeostasis, implicating an active participation of glial cells in this neuroendocrine regulatory loop.

The neurosteroid pregnenolone sulphate enhances NMDA-induced phasic firing of vasopressin neurones in the rat supraoptic nucleus

The effect of the neurosteroid pregnenolone sulphate (PS) on N-methyl- d-aspartate (NMDA)-induced phasic firing of supraoptic vasopressin (VP) neurones was studied in rat hypothalamic slices in vitro. In VP neurones which were induced to fire phasically by continuous perifusion with NMDA (9–30 μM), addition of 100 μM PS to the incubation medium significantly increased overall spike frequency, with a rise in both proportion of time active and intraburst firing rate. A similar effect was seen during picrotoxin block of GABAergic transmission. No significant change in NMDA-induced phasic firing was observed with 100 μM dehydroepiandrosterone sulphate. VP neurones became silent in the absence of NMDA, and under these conditions PS had no effect. In conclusion, PS increases NMDA-induced phasic firing in VP neurones, providing a mechanism whereby this neurosteroid may participate in the regulation of VP secretion.

Estrogen modifies the baroreceptor-induced responses of supraoptic vasopressin neurons in the ovariectomized rat

Effects of estrogen (E2) and estrogen plus progesterone (EP) on the baroreceptor-induced response of the spontaneous discharge activity in the supraoptic (SON) vasopressin (AVP) neurons were examined in ovariectomized (OVX) female rats. An increase or a decrease in arterial blood pressure induced by intravenous injection of phenylephrine or nitroprusside, resulted in an inhibition or an acceleration of the discharge activity in the identified AVP neurons. The slopes that were derived from a least squares regression line for the arterial blood pressure and baroreceptor-induced responses of AVP neurons in both OVX + E2 and OVX + EP rats were statistically smaller ( P < 0.05) than that in the control OVX rat. These results suggest a functional relationship between ovarian endocrine and autonomic functions in the female rat.

Regulation of spontaneous phasic firing of rat supraoptic vasopressin neurones in vivo by glutamate receptors.

1. Vasopressin-secreting neurones in the rat hypothalamic supraoptic nucleus display patterned spontaneous phasic activity, which is apparently maintained in vivo through yet unidentified neurotransmitter system(s). The present investigation used extracellular recording techniques in anaesthetized Long-Evans rats to evaluate whether the neurotransmitter mechanism underlying phasic firing is provided via a family of ionotropic glutamate receptors. 2. N-Methyl-D-aspartate (NMDA) reliably evoked bursts of activity in twenty-seven of twenty-eight phasic neurones. Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and kainate also elicited pronounced excitations in twenty-one of twenty-one and and fourteen of fifteen phasic cells, respectively. 3. A rapid blockade of on-going phasic activity was consistently induced following brief applications of both NMDA and non-NMDA receptor antagonists; extended application of antagonists resulted in prolonged silent periods, during which phasic activity failed to recur for minutes. Neither saline nor a cholecystokinin receptor antagonist influenced cell firing. 4. In contrast to putative vasopressin cells, application of NMDA receptor ligands did not affect the spontaneous activity in most putative oxytocin-secreting neurones, whereas kainate and AMPA potently excited seven of nine and four of five putative oxytocin cells, respectively. 5. These results imply that the maintenance of spontaneous phasic discharges in vivo in supraoptic vasopressin-secreting neurones requires tonic synaptic activation involving both NMDA and non-NMDA glutamate receptors. In putative oxytocin-secreting neurones, spontaneous firing appears to be predominantly regulated by non-NMDA receptors. Glutamatergic innervations may be in a unique position to influence the genesis of patterned electrical activity in supraoptic vasopressin neurones.

GABA receptor mediation of median preoptic nucleus‐evoked inhibition of supraoptic neurosecretory neurones in rat.

1. This study evaluated the influence of focal electrical and chemical microstimulation in the median preoptic nucleus (MnPO) on the excitability of putative vasopressin and oxytocin neurones recorded in the supraoptic nucleus of urethane- or pentobarbitone-anaesthetized rats. 2. In vasopressin neurones, single 1 Hz stimulation reduced the excitability of 120/139 cells. Trains of repetitive 5-30 Hz stimulation, or microinfusion of glutamate into the MnPO, similarly induced a cessation in spontaneous phasic or continuous firing in 17/18 and 17/20 vasopressin neurones, respectively. In 20/21 cells, locally applied bicuculline (100 microM) attenuated MnPO-evoked depressant responses whereas strychnine (100 microM) and timolol (20 microM) were without effect on 5/5 vasopressin neurones. In three cells, bicuculline applications were associated with marked increases in MnPO-evoked excitations. 3. In oxytocin neurones, single-pulse (1 Hz) electrical stimulation in MnPO evoked an increase in the excitability in 51/59 cells. However, in 6/7 oxytocin cells tested, glutamate microinfusions into MnPO induced prolonged suppression in firing. During trains of stimuli (5-30 Hz), 26/44 cells displayed an initial increase in firing associated with the first few impulses but this was then replaced by suppression of activity; another ten cells displayed excitation alone, and eight cells demonstrated only suppression. The depressant responses evoked during trains of MnPO stimulation were blocked by 100 microM bicuculline (6/6 cells tested) whereas strychnine was ineffective (2/2 cells tested). 4. These results suggest that the MnPO provides a mainly depressant influence on supraoptic vasopressin and oxytocin neurones, perhaps through the activation of postsynaptic GABAA receptors.

N-methyl- d-aspartate receptor antagonist ketamine selectively attenuates spontaneous phasic activity of supraoptic vasopressin neurons in vivo

Supraoptic neurosecretory neurons express a prominent N-methyl- d-aspartate receptor system. Recent in vitro evidence reveals that N-methyl- d-aspartate receptor activation dramatically alters the spontaneous discharge patterns of supraoptic neurons. In this study we evaluate whether N-methyl- d-aspartate receptors in vivo contribute to the development of characteristic phasic discharge patterns displayed by vasopressin-secreting neurons. Intravenous administration of ketamine hydrochloride, a non-competitive N-methyl- d-aspartate receptor antagonist, was used to examine whether N-methyl- d-aspartate receptor blockade influences patterned spontaneous discharge observed in supraoptic neuro-secretory neurons. Extracellular recordings were obtained from identified hypothalamic supraoptic neurons in pentobarbital-anaesthetized Long-Evans rats. Systemic administration of ketamine (≤1.5 mg/kg) potently suppressed spontaneous phasic discharge in 16/19 putative vasopressin-secreting cells. The ketamine-induced blockade was dose dependent, fully reversible and was associated with the complete blockade of activity evoked by local pressure application of N-methyl- d-aspartate, but not the activity evoked by α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptor agonists (6/6 cells). Ketamine had no detectable effect on threshold or shape of antidromic action potentials. By comparison, the activity in 9/10 continuously active neurons (putative oxytocin-secreting) was unaffected by administration of identical doses of ketamine. These data suggest that N-methyl- d-aspartate receptors play an important role in regulating the onset and maintenance of spontaneous phasic activity patterns displayed by rat supraoptic vasopressin neurons in vivo.

Inhibition of supraoptic vasopressin neurones following systemic clonidine.

Experiments were undertaken in urethane-anaesthetized rats to investigate the effects of systemic clonidine on the firing of supraoptic vasopressin (VP) neurones, which were identified by their characteristic phasic activity pattern. Injection of clonidine (50 micrograms/kg i.v.) reduced the firing of all 16 VP neurones tested, and their overall mean activity decreased from 5.09 +/- 1.01 to 1.63 +/- 0.64 spikes/second (P < 0.02). In VP cells which were already firing phasically before clonidine, the inhibition resulted in complete quiescence. In VP cells which were originally continuously active, the inhibition resulted in a switch to phasic activity. This inhibitory effect, which was prevented by prior injection of the alpha-2 antagonist idazoxan (0.5 mg/kg), had a mean duration of 11.8 +/- 1.8 min. Subsequent experiments revealed that i.v. clonidine (50 micrograms/kg) caused a transient rise in blood pressure, but this had a shorter time-course and was unlikely to account for the prolonged neuronal inhibition. It was concluded that systemic clonidine acts centrally to suppress the activity of hypothalamic VP neurones, thereby explaining the fall in plasma VP levels found in previous studies.

Induction of c- fos immunoreactivity in tyrosine hydroxylase and phenylethanolamine- N-methyltransferase immunoreactive neurons of the medulla oblongata of the rat after phosphate-buffered saline load in the urethane-anaesthetized rat

We have studied the induction of c- fos immunoreactivity (c- fos IR) in catecholaminergic and vasopressinergic neurons after repeated phosphate-buffered saline (PBS) loading or after repeated elicitation of the baroreceptor reflex via repeated infusion of the vasoconstrictor agent l-phenylephrine. About 75% and 30%, respectively, of the tyrosine-hydroxylase immunoreactive (IR) cell bodies of the ventral noradrenaline (NA) A1/adrenaline (A) C1 and dorsal NA A2/A C2 areas and 60% and 30% respectively, of the phenylethanolamine N-methyltransferase IR nerve cells of the adrenaline C1 and C2 areas and 25% of the vasopressin (VP) IR neurons of the supraoptic (SO) nucleus developed nuclear c- fos IR after repeated PBS loading. This phenomenon remained unaltered by the repeated elicitation of the baroreceptor reflex. These results suggest that the activation of volume receptors promotes homeostatic responses via activation of early genes in subsets of central medullary noradrenaline and adrenaline neurons and SO VP neurons of the urethane-anesthetized rat.