Voltage-induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals.

Depolarisation-secretion coupling is assumed to be dependent only on extracellular calcium ([Ca2+ ]o ). Ryanodine receptor (RyR)-sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca2+ ]i ) that are voltage-dependent. We hypothesised that voltage-elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage-gated calcium channels and/or from such voltage-sensitive ryanodine-mediated calcium stores. Increases in [Ca2+ ]i upon depolarisation in the presence of voltage-gated calcium channel blockers, or in the absence of [Ca2+ ]o , still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca2+ ]o , there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K+ in 0 [Ca2+ ]o . Furthermore, such depolarisations lead to increases in [Ca2+ ]i . Pre-incubation with BAPTA-AM resulted in > 50% inhibition of peptide secretion elicited by high K+ in 0 [Ca2+ ]o . Nifedipine but not nicardipine inhibited both the high K+ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage-induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation-induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine-sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.

Advances in the neurophysiology of magnocellular neuroendocrine cells.

Hypothalamic magnocellular neuroendocrine cells have unique electrical properties and a remarkable capacity for morphological and synaptic plasticity. Their large somatic size, their relatively uniform and dense clustering in the supraoptic and paraventricular nuclei, and their large axon terminals in the neurohypophysis make them an attractive target for direct electrophysiological interrogation. Here, we provide a brief review of significant recent findings in the neuroplasticity and neurophysiological properties of these neurones that were presented at the symposium "Electrophysiology of Magnocellular Neurons" during the 13th World Congress on Neurohypophysial Hormones in Ein Gedi, Israel in April 2019. Magnocellular vasopressin (VP) neurones respond directly to hypertonic stimulation with membrane depolarisation, which is triggered by cell shrinkage-induced opening of an N-terminal-truncated variant of transient receptor potential vanilloid type-1 (TRPV1) channels. New findings indicate that this mechanotransduction depends on actin and microtubule cytoskeletal networks, and that direct coupling of the TRPV1 channels to microtubules is responsible for mechanical gating of the channels. Vasopressin neurones also respond to osmostimulation by activation of epithelial Na+ channels (ENaC). It was shown recently that changes in ENaC activity modulate magnocellular neurone basal firing by generating tonic changes in membrane potential. Both oxytocin and VP neurones also undergo robust excitatory synapse plasticity during chronic osmotic stimulation. Recent findings indicate that new glutamate synapses induced during chronic salt loading express highly labile Ca2+ -permeable GluA1 receptors requiring continuous dendritic protein synthesis for synapse maintenance. Finally, recordings from the uniquely tractable neurohypophysial terminals recently revealed an unexpected property of activity-dependent neuropeptide release. A significant fraction of the voltage-dependent neurohypophysial neurosecretion was found to be independent of Ca2+ influx through voltage-gated Ca2+ channels. Together, these findings provide a snapshot of significant new advances in the electrophysiological signalling mechanisms and neuroplasticity of the hypothalamic-neurohypophysial system, a system that continues to make important contributions to the field of neurophysiology.

Purinergic receptor types in the hypothalamic-neurohypophysial system.

Many different types of purinergic receptors are present in the Hypothalamic-Neurohypophysial System (HNS), which synthesizes and releases vasopressin and oxytocin. The specific location of purinergic receptor subtypes has important functional repercussions for neuronal activity and synaptic output. Yet, until the advent of receptor KOs, this had been hindered by the low selectivity of the available pharmacological tools. The HNS offers an excellent opportunity to differentiate the functional properties of these purinergic receptors in cell bodies vs. terminals of the same physiological system. P2X2, P2X3, P2X4 and P2X7 receptors are present in vasopressin terminals while oxytocin terminals exclusively express the P2X7 subtype. The latter is not functional in the cell bodies of the HNS. These purinergic receptor subtypes are permeable to sodium vs. calcium in varying amounts and this could play an important role in the release of vasopressin vs. oxytocin during bursting activity. Endogenous ATP and its metabolite, adenosine, have autocrine and paracrine modulatory effects on the release of these neuropeptides during physiological stimulation. Finally, we hypothesize that during such action potential bursts, ATP potentiates the release of vasopressin but not of oxytocin, and that adenosine, via A1 receptors, inhibits the release of both neuropeptides. This article is protected by copyright. All rights reserved.

Peripheral vascular reactivity and serum BDNF responses to aerobic training are impaired by the BDNF Val66Met polymorphism.

Besides neuronal plasticity, the neurotrophin brain-derived neurotrophic factor (BDNF) is also important in vascular function. The BDNF has been associated with angiogenesis through its specific receptor tropomyosin-related kinase B (TrkB). Additionally, Val66Met polymorphism decreases activity-induced BDNF. Since BDNF and TrkB are expressed in vascular endothelial cells and aerobic exercise training can increase serum BDNF, this study aimed to test the hypotheses: 1) Serum BDNF levels modulate peripheral blood flow; 2) The Val66Met BDNF polymorphism impairs exercise training-induced vasodilation. We genotyped 304 healthy male volunteers (Val66Val, n = 221; Val66Met, n = 83) who underwent intense aerobic exercise training on a running track three times/wk for 4 mo. We evaluated pre- and post-exercise training serum BDNF and proBDNF concentration, heart rate (HR), mean blood pressure (MBP), forearm blood flow (FBF), and forearm vascular resistance (FVR). In the pre-exercise training, BDNF, proBDNF, BDNF/proBDNF ratio, FBF, and FVR were similar between genotypes. After exercise training, functional capacity (V̇o2 peak) increased and HR decreased similarly in both groups. Val66Val, but not Val66Met, increased BDNF (interaction, P = 0.04) and BDNF/proBDNF ratio (interaction, P < 0.001). Interestingly, FBF (interaction, P = 0.04) and the FVR (interaction, P = 0.01) responses during handgrip exercise (HG) improved in Val66Val compared with Val66Met, even with similar responses of HR and MBP. There were association between BDNF/proBDNF ratio and FBF (r = 0.64, P < 0.001) and FVR (r = -0.58, P < 0.001) during HG exercise. These results show that peripheral vascular reactivity and serum BDNF responses to exercise training are impaired by the BDNF Val66Met polymorphism and such responsiveness is associated with serum BDNF concentrations in healthy subjects.

Ethanol Effect on BK Channels is Modulated by Magnesium.

BACKGROUND: Alcoholics have been reported to have reduced levels of magnesium in both their extracellular and intracellular compartments. Calcium-dependent potassium channels (BK) are known to be one of ethanol (EtOH)'s better known molecular targets. METHODS: Using outside-out patches from hippocampal neuronal cultures, we examined the consequences of altered intracellular Mg(2+) on the effects that EtOH has on BK channels. RESULTS: We find that the effect of EtOH is bimodally influenced by the Mg(2+) concentration on the cytoplasmic side. More specifically, when internal Mg(2+) concentrations are ≤200 µM, EtOH decreases BK activity, whereas it increases activity when Mg(2+) is at 1 mM. Similar results are obtained when using patches from HEK cells expressing only the α-subunit of BK. When patches are made with the actin destabilizer cytochalasin D present on the cytoplasmic side, the potentiation caused by EtOH becomes independent of the Mg(2+) concentration. Furthermore, in the presence of the actin stabilizer phalloidin, EtOH causes inhibition even at Mg(2+) concentrations of 1 mM. CONCLUSIONS: Internal Mg(2+) can modulate the EtOH effects on BK channels only when there is an intact, internal actin interaction with the channel, as is found at synapses. We propose that the EtOH-induced decrease in cytoplasmic Mg(2+) observed in frequent/chronic drinkers would decrease EtOH's actions on synaptic (e.g., actin-bound) BK channels, producing a form of molecular tolerance.

Elimination of influences of the ACTN3 R577X variant on oxygen uptake by endurance training in healthy individuals.

AIM: To study the relationship between the ACTN3 R577X polymorphism and oxygen uptake (VO2) before and after exercise training. METHODS: Police recruits (N=206, 25±4 y) with RR (n=75), RX (n=97), and XX (n=33) genotypes were selected. After baseline measures, they underwent 18 wk of running endurance training. Peak VO2 was obtained by cardiopulmonary exercise testing. RESULTS: Baseline body weight was not different among genotypes. At baseline, XX individuals displayed higher VO2 at anaerobic threshold, respiratory compensation point, and exercise peak than did RR individuals (P<.003). Endurance training significantly increased VO2 at anaerobic threshold, respiratory compensation point, and exercise peak (P<2×10(-6)), but the differences between XX and RR were no longer observed. Only relative peak VO2 exercise remained higher in XX than in RR genotype (P=.04). In contrast, the increase in relative peak VO2 was greater in RR than in XX individuals (12% vs 6%; P=.02). CONCLUSION: ACTN3 R577X polymorphism is associated with VO2. XX individuals have greater aerobic capacity. Endurance training eliminates differences in peak VO2 between XX and RR individuals. These findings suggest a ceiling-effect phenomenon, and, perhaps, trained individuals may not constitute an adequate population to explain associations between phenotypic variability and gene variations.

Functional ryanodine receptors in the membranes of neurohypophysial secretory granules.

Highly localized Ca(2+) release events have been characterized in several neuronal preparations. In mouse neurohypophysial terminals (NHTs), such events, called Ca(2+) syntillas, appear to emanate from a ryanodine-sensitive intracellular Ca(2+) pool. Traditional sources of intracellular Ca(2+) appear to be lacking in NHTs. Thus, we have tested the hypothesis that large dense core vesicles (LDCVs), which contain a substantial amount of calcium, represent the source of these syntillas. Here, using fluorescence immunolabeling and immunogold-labeled electron micrographs of NHTs, we show that type 2 ryanodine receptors (RyRs) are localized specifically to LDCVs. Furthermore, a large conductance nonspecific cation channel, which was identified previously in the vesicle membrane and has biophysical properties similar to that of an RyR, is pharmacologically affected in a manner characteristic of an RyR: it is activated in the presence of the RyR agonist ryanodine (at low concentrations) and blocked by the RyR antagonist ruthenium red. Additionally, neuropeptide release experiments show that these same RyR agonists and antagonists modulate Ca(2+)-elicited neuropeptide release from permeabilized NHTs. Furthermore, amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Collectively, our findings suggest that RyR-dependent syntillas could represent mobilization of Ca(2+) from vesicular stores. Such localized vesicular Ca(2+) release events at the precise location of exocytosis could provide a Ca(2+) amplification mechanism capable of modulating neuropeptide release physiologically.

µ-Opioid inhibition of Ca2+ currents and secretion in isolated terminals of the neurohypophysis occurs via ryanodine-sensitive Ca2+ stores.

µ-Opioid agonists have no effect on calcium currents (I(Ca)) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, µ-opioid receptor (MOR)-mediated inhibition of I(Ca) is reliably demonstrated using the perforated-patch configuration. This suggests that the MOR-signaling pathway is sensitive to intraterminal dialysis and is therefore mediated by a readily diffusible second messenger. Using the perforated patch-clamp technique and ratio-calcium-imaging methods, we describe a diffusible second messenger pathway stimulated by the MOR that inhibits voltage-gated calcium channels in isolated terminals from the rat neurohypophysis (NH). Our results show a rise in basal intracellular calcium ([Ca(2+)]i) in response to application of [D-Ala(2)-N-Me-Phe(4),Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a MOR antagonist. Buffering DAMGO-induced changes in [Ca(2+)]i with BAPTA-AM completely blocked the inhibition of both I(Ca) and high-K(+)-induced rises in [Ca(2+)]i due to MOR activation, but had no effect on κ-opioid receptor (KOR)-mediated inhibition. Given the presence of ryanodine-sensitive stores in isolated terminals, we tested 8-bromo-cyclic adenosine diphosphate ribose (8Br-cADPr), a competitive inhibitor of cyclic ADP-ribose (cADPr) signaling that partially relieves DAMGO inhibition of I(Ca) and completely relieves MOR-mediated inhibition of high-K(+)-induced and DAMGO-induced rises in [Ca(2+)]i. Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca(2+)]i and inhibition of I(Ca) and high-K(+)-induced rises in [Ca(2+)]i while not affecting KOR-mediated inhibition. Antagonist concentrations of ryanodine also blocked MOR-mediated inhibition of electrically-evoked increases in capacitance. These results strongly suggest that a key diffusible second messenger mediating the MOR-signaling pathway in NH terminals is [Ca(2+)]i released by cADPr from ryanodine-sensitive stores.

P2X7 receptors in neurohypophysial terminals: evidence for their role in arginine-vasopressin secretion.

Arginine-vasopressin (AVP) plays a major role in maintaining cardiovascular function and related pathologies. The mechanism involved in its release into the circulation is complex and highly regulated. Recent work has implicated the purinergic receptor, P2X7R, in a role for catecholamine-enhanced AVP release in the rat hypothalamic-neurohypophysial (NH) system. However, the site of P2X7R action in this endocrine system, and whether or not it directly mediates release in secretory neurons have not been determined. We hypothesized that the P2X7R is expressed and mediates AVP release in NH terminals. P2X7R function was first examined by patch-clamp recordings in isolated NH terminals. Results revealed that subpopulations of isolated terminals displayed either high ATP-sensitivity or low ATP-sensitivity, the latter of which was characteristic of the rat P2X7R. Additional recordings showed that terminals showing sensitivity to the P2X7R-selective agonist, BzATP, were further inhibited by P2X7R selective antagonists, AZ10606120 and brilliant blue-G. In confocal micrographs from tissue sections and isolated terminals of the NH P2X7R-immunoreactivity was found to be localized in plasma membranes. Lastly, the role of P2X7R on AVP release was tested. Our results showed that BzATP evoked sustained AVP release in NH terminals, which was inhibited by AZ10606120. Taken together, our data lead us to conclude that the P2X7R is expressed in NH terminals and corroborates its role in AVP secretion.

Molecular tolerance of voltage-gated calcium channels is evident after short exposures to alcohol in vasopressin-releasing nerve terminals.

BACKGROUND: Voltage-gated calcium channels (VGCCs) in rat neurohypophysial terminals exhibit molecular tolerance to alcohol, including desensitization to the drug and increased current density, after 3 weeks of alcohol drinking. Moreover, after this time, terminals from drinking rats exhibit diminished alcohol inhibition of vasopressin (AVP) release. METHODS: We took advantage of organotypic cultures (explants) of the hypothalamo-neurohypophysial system (HNS) to extend our analysis of molecular tolerance to 2 classes of the VGCC. The isolated HNS explant allows much finer temporal resolution of molecular tolerance than do voluntary drinking paradigms. After exposure of the HNS explant to alcohol, terminals are isolated by mechanical treatment and plated in a dish. Patch clamp recording techniques are used to obtain VGCC currents, and immunohistochemistry is used to determine VGCC distribution. A release assay is used to provide functional readout of AVP release. RESULTS: We show that even a brief, 1-hour exposure to a clinically relevant concentration of alcohol is sufficient to evoke similar changes to those observed after several weeks of exposure. Acute ethanol (EtOH) exposure inhibits high K(+) -induced AVP release from naïve terminals. However, terminals pre-exposed to 20 mM EtOH for 1 hour become tolerant to EtOH, and subsequent exposure has significantly less effect on high K(+) -induced AVP release. Electrophysiological recordings indicate that among different types of VGCCs present in the neuronal terminal, the L-type is the most affected by alcohol. The current density of L-type current is significantly increased (approximately 50%), while its responsiveness to alcohol is significantly diminished (approximately 50%), after brief alcohol exposure. Fluorescent imaging results were consistent with the electrophysiology and suggest that the increased current density of VGCCs after brief exposure is attributable to combined synthesis of 1.2 and 1.3 subtypes of the L-type VGCC and redistribution of channel protein into terminal plasma membrane. CONCLUSIONS: These data indicate that a brief alcohol exposure affects subsequent alcohol sensitivity of VGCCs and neuropeptide release from presynaptic terminals.

The spatial and temporal regulation of the hormonal signal. Role of mitochondria in the formation of a protein complex required for the activation of cholesterol transport and steroids synthesis.

The mitochondria are critical for steroidogenesis since the ability of cholesterol to move into mitochondria to be available for cytochrome P450, CYP11A1, determines the efficacy of steroid production. Several proteins kinases, such as PKA, MEK and ERK which are essential to complete steroidogenesis, form a mitochondria-associated complex. The protein-protein interactions between kinases and key factors during the transport of cholesterol takes place in the contact sites between the two mitochondrial membranes; however, no mitochondrial targeting sequence has been described for these kinases. Here we discuss the possibility that mitochondrial reorganization may be mediating a compartmentalized cellular response. This reorganization could allow the physical interaction between the hormone-receptor complex and the enzymatic and lipidic machinery necessary for the complete steroid synthesis and release. The movement of organelles in specialized cells could impact on biological processes that include, but are not limited to, steroid synthesis.

Modulation/physiology of calcium channel sub-types in neurosecretory terminals.

The hypothalamic-neurohypophysial system (HNS) controls diuresis and parturition through the release of arginine-vasopressin (AVP) and oxytocin (OT). These neuropeptides are chiefly synthesized in hypothalamic magnocellular somata in the supraoptic and paraventricular nuclei and are released into the blood stream from terminals in the neurohypophysis. These HNS neurons develop specific electrical activity (bursts) in response to various physiological stimuli. The release of AVP and OT at the level of neurohypophysis is directly linked not only to their different burst patterns, but is also regulated by the activity of a number of voltage-dependent channels present in the HNS nerve terminals and by feedback modulators. We found that there is a different complement of voltage-gated Ca(2+) channels (VGCC) in the two types of HNS terminals: L, N, and Q in vasopressinergic terminals vs. L, N, and R in oxytocinergic terminals. These channels, however, do not have sufficiently distinct properties to explain the differences in release efficacy of the specific burst patterns. However, feedback by both opioids and ATP specifically modulate different types of VGCC and hence the amount of AVP and/or OT being released. Opioid receptors have been identified in both AVP and OT terminals. In OT terminals, µ-receptor agonists inhibit all VGCC (particularly R-type), whereas, they induce a limited block of L-, and P/Q-type channels, coupled to an unusual potentiation of the N-type Ca(2+) current in the AVP terminals. In contrast, the N-type Ca(2+) current can be inhibited by adenosine via A(1) receptors leading to the decreased release of both AVP and OT. Furthermore, ATP evokes an inactivating Ca(2+)/Na(+)-current in HNS terminals able to potentiate AVP release through the activation of P2X2, P2X3, P2X4 and P2X7 receptors. In OT terminals, however, only the latter receptor type is probably present. We conclude by proposing a model that can explain how purinergic and/or opioid feedback modulation during bursts can mediate differences in the control of neurohypophysial AVP vs. OT release.

Differential modulation of N-type calcium channels by micro-opioid receptors in oxytocinergic versus vasopressinergic neurohypophysial terminals.

Opioids modulate the electrical activity of magnocellular neurons (MCN) and inhibit neuropeptide release at their terminals in the neurohypophysis. We have previously shown that micro-opioid receptor (MOR) activation induces a stronger inhibition of oxytocin (OT) than vasopressin (AVP) release from isolated MCN terminals. This higher sensitivity of OT release is due, at least in part, to the selective targeting of R-type calcium channels. We now describe the underlying basis for AVP's weaker inhibition by MOR activation and provide a more complete explanation of the complicated effects on neuropeptide release. We found that N-type calcium channels in AVP terminals are differentially modulated by MOR; enhanced at lower concentrations but increasingly inhibited at higher concentrations of agonists. On the other hand, N-type calcium channels in OT terminals were always inhibited. The response pattern in co-labeled terminals was analogous to that observed in AVP-containing terminals. Changes in intracellular calcium concentration and neuropeptide release corroborated these results as they showed a similar pattern of enhancement and inhibition in AVP terminals contrasting with solely inhibitory responses in OT terminals to MOR agonists. We established that fast translocation of Ca(2+) channels to the plasma membrane was not mediating current increments and thus, changes in channel kinetic properties are most likely involved. Finally, we reveal a distinct Ca-channel beta-subunit expression between each type of nerve endings that could explain some of the differences in responses to MOR activation. These results help advance our understanding of the complex modulatory mechanisms utilized by MORs in regulating presynaptic neuropeptide release.

Voltage-dependent kappa-opioid modulation of action potential waveform-elicited calcium currents in neurohypophysial terminals.

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca(2+) channels results in less calcium influx into the terminals and presumably a reduction in transmitter release. In the neurohypophysis (NH), Ca(2+) channel kinetics, and the associated Ca(2+) influx, is primarily controlled by membrane voltage and can be modulated, in a voltage-dependent manner, by G-protein subunits interacting with voltage-gated calcium channels (VGCCs). In this series of experiments we test whether the kappa- and micro-opioid inhibition of Ca(2+) currents in NH terminals is voltage-dependent. Voltage-dependent relief of G-protein inhibition of VGCC can be achieved with either a depolarizing square pre-pulse or by action potential waveforms. Both protocols were tested in the presence and absence of opioid agonists targeting the kappa- and micro-receptors in neurohypophysial terminals. The kappa-opioid VGCC inhibition is relieved by such pre-pulses, suggesting that this receptor is involved in a voltage-dependent membrane delimited pathway. In contrast, micro-opioid inhibition of VGCC is not relieved by such pre-pulses, indicating a voltage-independent diffusible second-messenger signaling pathway. Furthermore, relief of kappa-opioid inhibition during a physiologic action potential (AP) burst stimulation indicates the possibility of activity-dependent modulation in vivo. Differences in the facilitation of Ca(2+) channels due to specific G-protein modulation during a burst of APs may contribute to the fine-tuning of Ca(2+)-dependent neuropeptide release in other CNS terminals, as well.

Ionic conditions modulate stimulus-induced capacitance changes in isolated neurohypophysial terminals of the rat.

Peptidergic nerve terminals of the neurohypophysis (NH) secrete both oxytocin and vasopressin upon stimulation with peptide-specific bursts of action potentials from magnocellular neurons. These bursts vary in both frequency and action potential duration and also induce in situ ionic changes both inside and outside the terminals in the NH. These temporary effects include the increase of external potassium and decrease of external calcium, as well as the increase in internal sodium and chloride concentrations. In order to determine any mechanism of action that these ionic changes might have on secretion, stimulus-induced capacitance recordings were performed on isolated terminals of the NH using action potential burst patterns of varying frequency and action potential width. The results indicate that in NH terminals: (1) increased internal chloride concentration improves the efficiency of action potential-induced capacitance changes, (2) increasing external potassium increases stimulus-induced capacitance changes, (3) decreasing external calcium decreases the capacitance induced by low frequency broadened action potentials, while no capacitance change is observed with high frequency un-broadened action potentials, and (4) increasing internal sodium increases the capacitance change induced by low frequency bursts of broadened action potentials, more than for high frequency bursts of narrow action potentials. These results are consistent with previous models of stimulus-induced secretion, where optimal secretory efficacy is determined by particular characteristics of action potentials within a burst. Our results suggest that positive effects of increased internal sodium and external potassium during a burst may serve as a compensatory mechanism for secretion, counterbalancing the negative effects of reduced external calcium. In this view, high frequency un-broadened action potentials (initial burst phase) would condition the terminals by increasing internal sodium for optimal secretion by the physiological later phase of broadened action potentials. Thus, ionic changes occurring during a burst may help to make such stimulation more efficient at inducing secretion. Furthermore, these effects are thought to occur within the initial few seconds of incoming burst activity at both oxytocin and vasopressin types of NH nerve terminals.

Individual calcium syntillas do not trigger spontaneous exocytosis from nerve terminals of the neurohypophysis.

Recently, highly localized Ca(2+) release events, similar to Ca(2+) sparks in muscle, have been observed in neuronal preparations. Specifically, in murine neurohypophysial terminals (NHT), these events, termed Ca(2+) syntillas, emanate from a ryanodine-sensitive intracellular Ca(2+) pool and increase in frequency with depolarization in the absence of Ca(2+) influx. Despite such knowledge of the nature of these Ca(2+) release events, their physiological role in this system has yet to be defined. Such localized Ca(2+) release events, if they occur in the precise location of the final exocytotic event(s), may directly trigger exocytosis. However, directly addressing this hypothesis has not been possible, since no method capable of visualizing individual release events in these CNS terminals has been available. Here, we have adapted an amperometric method for studying vesicle fusion to this system which relies on loading the secretory granules with the false transmitter dopamine, thus allowing, for the first time, the recording of individual exocytotic events from peptidergic NHT. Simultaneous use of this technique along with high-speed Ca(2+) imaging has enabled us to establish that spontaneous neuropeptide release and Ca(2+) syntillas do not display any observable temporal or spatial correlation, confirming similar findings in chromaffin cells. Although these results indicate that syntillas do not play a direct role in eliciting spontaneous release, they do not rule out indirect modulatory effects of syntillas on secretion.

Endogenous ATP potentiates only vasopressin secretion from neurohypophysial terminals.

Exogenous ATP induces inward currents and causes the release of arginine-vasopressin (AVP) from isolated neurohypophysial terminals (NHT); both effects are inhibited by the P2X2 and P2X3 antagonists, suramin and PPADS. Here we examined the role of endogenous ATP in the neurohypophysis. Stimulation of NHT caused the release of both AVP and ATP. ATP induced a potentiation in the stimulated release of AVP, but not of oxytocin (OT), which was blocked by the presence of suramin. In loose-patch clamp recordings, from intact neurohypophyses, suramin or PPADS produces an inhibition of action potential currents in a static bath, that can be mimicked by a hyperpolarization of the resting membrane potential (RMP). Correspondingly, in a static versus perfused bath there is a depolarization of the RMP of NHT, which was reduced by either suramin or PPADS. We measured an accumulation of ATP (3.7 +/- 0.7 microM) released from NHT in a static bath. Applications of either suramin or PPADS to a static bath decreased burst-stimulated capacitance increases in NHT. Finally, only vasopressin release from electrically stimulated intact neurohypophyses was reduced in the presence of Suramin or PPADS. These data suggest that there was sufficient accumulation of ATP released from the neurohypophysis during stimulations to depolarize its nerve terminals. This would occur via the opening of P2X2 and P2X3 receptors, inducing an influx of Ca2+. The subsequent elevation in [Ca2+](i) would further increase the stimulated release of only vasopressin from NHT terminals. Such purinergic feedback mechanisms could be physiologically important at most CNS synapses.

Identification of the neuropeptide content of individual rat neurohypophysial terminals.

The objective of this study was to develop a method that could reliably determine the arginine vasopressin (AVP) and/or oxytocin (OT) content of individual rat neurohypophysial terminals (NHT) >or=5 microm in diameter, the size used for electrophysiological recordings. We used a commercially available, highly sensitive enzyme-linked immunoassay (ELISA) kit with a sensitivity of 0.25 pg to AVP and of 1.0pg to OT. The NHT content of AVP (2.21+/-0.10 pg) was greater than OT (1.77+/-0.08 pg) and increased with terminal size. AVP-positive terminals (10.2+/-0.21 microm) were larger in diameter than OT-positive terminals (9.1+/-0.24 microm). Immunocytochemical techniques indicated that a higher percentage (58%) of smaller terminals contained OT, and that a higher percentage (42%) of larger NHTs were colabeled. Similar percentages of AVP-positive terminals were obtained between immunocytochemical (73%) and ELISA (72%) methods when NHTs were assayed for AVP alone, but there was a higher percentage of OT terminals when using immunocytochemistry (43%) compared to ELISA (26%). The percent of AVP-positive (60%) and OT-positive (18%) terminals decreased when NHT were assayed for both AVP and OT. Therefore, the best method to reliably identify AVP-positive NHTs is to assay only for AVP, since this allows the conclusion that AVP-negative terminals contain only OT.

Endogenous adenosine inhibits CNS terminal Ca(2+) currents and exocytosis.

Bursts of action potentials (APs) are crucial for the release of neurotransmitters from dense core granules. This has been most definitively shown for neuropeptide release in the hypothalamic neurohypophysial system (HNS). Why such bursts are necessary, however, is not well understood. Thus far, biophysical characterization of channels involved in depolarization-secretion coupling cannot completely explain this phenomenon at HNS terminals, so purinergic feedback mechanisms have been proposed. We have previously shown that ATP, acting via P2X receptors, potentiates release from HNS terminals, but that its metabolite adenosine, via A(1) receptors acting on transient Ca(2+) currents, inhibit neuropeptide secretion. We now show that endogenous adenosine levels are sufficient to cause tonic inhibition of transient Ca(2+) currents and of stimulated exocytosis in HNS terminals. Initial non-detectable adenosine levels in the static bath increased to 2.9 microM after 40 min. These terminals exhibit an inhibition (39%) of their transient inward Ca(2+) current in a static bath when compared to a constant perfusion stream. CPT, an A(1) adenosine receptor antagonist, greatly reduced this tonic inhibition. An ecto-ATPase antagonist, ARL-67156, similarly reduced tonic inhibition, but CPT had no further effect, suggesting that endogenous adenosine is due to breakdown of released ATP. Finally, stimulated capacitance changes were greatly enhanced (600%) by adding CPT to the static bath. Thus, endogenous adenosine functions at terminals in a negative-feedback mechanism and, therefore, could help terminate peptide release by bursts of APs initiated in HNS cell bodies. This could be a general mechanism for controlling transmitter release in these and other CNS terminals.

Dihydropyridine receptors and type 1 ryanodine receptors constitute the molecular machinery for voltage-induced Ca2+ release in nerve terminals.

Ca2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of -80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of -80 to -60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+.