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The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro . The AVP and OT, secreted somato-dendritically ( i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca 2+ signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca 2+ ] i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca 2+ ] i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca 2+ ] i dynamics. In rats dehydrated for 3 or 5 days almost 90% of neurones displayed spontaneous [Ca 2+ ] i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs . about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca 2+ signals, their remarkable intrinsic in vivo physiological properties in an isolated condition. [ABSTRACT FROM AUTHOR]

Isolated supraoptic neurones generate spontaneous [Ca 2+ ] i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca 2+ , Na + ), Na + /Ca 2+ exchanger (NCX), intracellular Ca 2+ release channels (InsP 3 Rs and RyRs), Ca 2+ storage organelles, plasma membrane Ca 2+ pump and intracellular signal transduction cascades into spontaneous Ca 2+ activity. While removal of extracellular Ca 2+ or incubation with non-specific voltage-gated Ca 2+ channel (VGCC) blocker Cd 2+ suppressed the oscillations, neither Ni 2+ nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca 2+ ] i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca 2+ ] i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na + or application of an inhibitor of the reverse operation mode of Na + /Ca 2+ exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca 2+ ] i activity. Exposure of neurones to Ca 2+ mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K + current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca 2+ channels and the NCX fluctuating between forward and reverse modes. [ABSTRACT FROM AUTHOR]

Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 ( Trpv1 )-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1 , although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca 2+ concentration ([Ca 2+ ] i ) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca 2+ ] i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca 2+ ] i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca 2+ ] i elevation when the temperature was increased to 36 °C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules. [ABSTRACT FROM AUTHOR]

Abstract: 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 (50mM KCl)-induced [Ca2+]i transients. The release of AVP was stimulated by 50mM 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. [ABSTRACT FROM AUTHOR]