Neurohypophysial Terminals Research Articles

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.

Effects of calcium and sodium on ATP-induced vasopressin release from rat isolated neurohypophysial terminals.

ATP-receptors (P2X2, P2X3, P2X4 & P2X7) are found in neurohypophysial terminals (NHT). These purinergic receptor subtypes are known to be cation selective. Here we confirm that both sodium (Na+ ) and calcium (Ca2+ ) are permeable through these NHT purinergic receptors, but to varying degrees (91% vs. 9%, respectively). Furthermore, extracellular calcium inhibits the ATP-current magnitude. Thus, the objective of this study was to determine the effects of extracellular Na+ vs. Ca2+ on ATP-induced vasopressin (AVP) release from populations of rat isolated NHT. ATP (200 μM) perfused exogenously for 2 minutes in Normal Locke's buffer caused an initial transient increase in AVP release followed by a sustained increase in AVP release which lasted for the duration of the ATP exposure. Replacing extracellular NaCl with NMDG-Cl had no apparent effect on the ATP-induced transient increase in AVP release but abolished the sustained AVP release induced by ATP. Furthermore, removal of extracellular calcium resulted in no ATP-induced transient increase in AVP release, but had no effect on the delayed, sustained increase in AVP release. The ATP-induced calcium-dependent transient increase in AVP release was >95% inhibited by 10 μM of the P2X purinergic receptor antagonist PPADS, a dose sufficient to block P2X2 and P2X3 receptors but not P2X4 or P2X7 receptors. Interestingly, the ATP-induced calcium-independent, sodium-dependent sustained increase in AVP release was not affected by 10 μM PPADS. The ATP-induced calcium-dependent transient increase in AVP release was not affected by the P2X7 receptor antagonist BBG (100 nM). However, the ATP-induced sodium-dependent sustained AVP release was inhibited by 50%. Therefore, these results show that rat isolated NHT exhibit a biphasic response to exogenous ATP that is differentially dependent on extracellular calcium and sodium. Furthermore, the initial transient release appears to be through P2X2 and/or P2X3 receptors and the sustained release is through a P2X7 receptor. This article is protected by copyright. All rights reserved.

Kisspeptin: a new peptidergic system regulating oxytocin neurons and their reproductive plasticity in the hypothalamo-neurohypophysial system.

In mammals, oxytocin (OT) release from neurohypophysial terminals during pregnancy and lactation is pulsatile, reflecting a coordinated burst firing from parent magnocellular OT neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (Poulain & Wakerley, 1982). Synchronized electrical bursting insures that OT receptors on uterine smooth muscle (for labour contractions) and the mammary gland myoepithelium (for milk ejection) avoid desensitization, maximizing OT's effects. Investigators have long demonstrated a significant plasticity in many aspects of this system that commence in late pregnancy and persist to lactation – changes suspected to play roles in developing these coordinated bursts (Theodosis et al. 2008; Armstrong, 2015). Electron microscopic investigations revealed that astrocyte processes withdraw from between OT neurons and dendrites during lactation, allowing an increase in close neuronal membrane appositions and a change in the number of excitatory and inhibitory synapses. These changes first appear in late pregnancy, and are thought to be programmed by steroid hormone changes, namely the precipitous drop in progesterone in late pregnancy. This morphological plasticity correlates with alterations in the synaptic and presynaptic actions of both glutamate and GABA on OT neurons. Other significant changes during late pregnancy and lactation that are specific to OT neurons include calcium-dependent afterhyperpolarizations that would modulate bursting patterns, and a significant upregulation in mitogen-activated protein kinase/extracellular signal-related kinase activity. Some of the GABAergic and glutamatergic inputs to magnocellular nuclei come from nearby neurons. In the SON, for example, the surrounding perinuclear zone (PZ) provides both types of inputs, and is an area thought to mediate the selective inhibition of vasopressin (VP) neurons to hypertension. This region has diverse morphological cell types, and receives inputs from regions known to affect magnocellular function, such as the diagonal band of Broca and the septum, as well as from within the hypothalamus itself. However, a specific role of this region in modulating OT neuronal activity has not previously been demonstrated. In this issue of The Journal of Physiology, Seymour et al. (2017) describe hypothalamic periventricular kisspeptin neurons that show significant plasticity during late pregnancy. Kisspeptin, a product of the KiSS-1 gene, is a relatively newly discovered peptide, previously implicated in the control of gonadotropin releasing hormone, but also with significant tumour suppressive actions (Murphy, 2005). In the brain, the kisspeptin periventricular hypothalamic neurons have widespread connections, including to and around the SON and paraventricular nuclei, areas controlling the peripheral release of OT and VP. In the present study, a striking increase in the density of axons within the PZ of the SON (but not in the SON itself) containing kisspeptin-like immunoreactivity was found during late pregnancy, and this was correlated with an upregulation of kisspeptin expression in the rostral periventricular hypothalamic neurons that project to the PZ. Furthermore, this increase appears specific to that cell group, as arcuate nucleus kisspeptin neurons are dramatically downregulated in the same period. These results were coincident with a significant ability of centrally administered kisspeptin to excite OT, but not VP neurons, in late pregnancy. Although mRNA for a kisspeptin receptor, Kiss1R, was found weakly localized to the PZ–SON area, its expression was not increased during late pregnancy, suggesting an upregulation of receptor numbers does not mediate this increased excitation. Indeed, the precise target of the centrally administered kisspeptin was not determined in this study, and as Seymour et al. (2017) suggest, it remains possible that its actions on OT neurons were not mediated by the Kiss1R, but possibly by related peptide receptors, or, by amplification of the Kiss1R signaling pathway. There are other questions that await further research for answers. Is kisspeptin critical for the activation of OT neurons during late pregnancy and lactation, and particularly, do its actions influence the synchronous bursting of these neurons and the consequent pulsatile OT release? Does stimulation of these neurons in the rostral periventricular area reproduce the state-dependent effects of centrally applied kisspeptin on OT neurons, and if so, is this mediated through the PZ? Are PZ neurons directly sensitive to kisspeptin? If so, what is their neurotransmitter phenotype, and are they among the PZ neurons that directly innervate SON neurons? Despite the above caveats, the novel results of this paper demonstrate for the first time a reproductive state-related plasticity in a defined system impacting on OT neurons, the periventricular-to-PZ kisspeptin projection. Whereas previous studies over the past four decades have demonstrated multiple types of neuronal and glial plasticity related to OT neurons during pregnancy and lactation, none have indicated this type of plasticity in a specifically located population of neurons thought to regulate OT neuron activity. Seymour et al. (2017) once again train focus on the PZ as an area potentially critical for integrating signals destined to impact VP and OT release through its locally connecting neurons, whether they be GABAergic, glutamatergic, or other cell types providing neuroactive inputs to the SON.

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.

GABA UPTAKE BY GAT1 MODULATES LONG-TERM OPTICAL CHANGES FOLLOWING ELECTRICAL STIMULATION OF THE PITUITARY GLAND NEUROINTERMEDIATE LOBE

Intrinsic optical changes that follow infundibular stalk stimulation of the neurointermediate lobe of the mouse pituitary gland exhibit three different phases that reflect three distinct physiological events. The first (E-wave) is the rapid light-scattering increase that is associated with a nerve terminal volume increase (mechanical spike), and that accompanies excitation of the neurohypophysial terminals by the invading action potential; the second (S-wave) is the slower light-scattering decrease that is tightly correlated with the secretion of the peptide hormones oxytocin and arginine vasopressin, and the third is the long-duration response (R-wave) that reflects cell volume changes in the pars intermedia. We have studied the E-wave and the S-wave in earlier publications. The R-wave, considered here, is sensitive to chloride replacement as well as to blockade of chloride channels. By blocking GABAAreceptors (which are ligand-gated chloride channels) with pharmacological agents, and by applying GABA directly into the bathing solution, or evoking its release from GABAergic inputs, we have demonstrated that this long-duration optical response is sensitive to chloride movements and reflects GABA-induced changes in the intrinsic optical properties of the pars intermedia. The full time-course of this optical response takes minutes and, therefore, has to embody some other process (or processes) related to the restoration of resting physiological chloride concentrations, following the opening and closing of GABAA-receptor channels. Here we demonstrate that the shape of the R-wave, the long-lasting light-scattering signal, is indeed affected by the activity of GAT1, one of the sodium- and chloride-dependent GABA transporters.

P2X7 Receptors Mediate Hormone Release in nerve terminals of the Neurohypophysis (NH)

P2X7R expression in neurons is controversial; however, studies suggest that it may play a role in hormone release in the hypothalamic NH system (HNS). We examined this in isolated NH terminals (NHT). Application of the P2X7R agonist, BzATP, to NHT resulted in increased levels of sustained AVP release, compared with ATP. Further experiments showed that the sustained release was inhibited by the P2X7R antagonist brilliant blue‐G (BBG). Confocal microscopy showed P2X7R‐immunoreactivity (IR) in slice sections and in isolated terminals of the NH. However, the cell bodies in the hypothalamus that project to the NH were devoid of P2X7R‐IR. Patch clamp studies revealed that subpopulations of NHT displayed differential sensitivity to ATP in evoking ion currents. One population showed a pEC50 of 5.2 ± 0.1 M and the second showed a pEC50 of 3.9 ± 0.2 M (T‐test, p< 0.0002, n=5–6), the latter being consistent with P2X7R. In low divalent cationic media the BzATP, gave a pEC50 of 5.6 ± 0.15 and pretreatment with 10 and 300 nM BBG inhibited currents by 51.8 ± 13.1 and 12.3 ± 4.2 percent of control, respectively (1 way ANOVA, P < 0.0001, n=5). The poor sensitivity to ATP and the marked inhibition of BzATP‐mediated current by BBG are hallmarks of P2X7R function. These results lead us to conclude that P2X7R is present in NHT and play a functional role in hormone release in the HNS. (Study supported by NIH grant NS29470 to JRL)

Molecular Tolerance of Voltage‐Gated Calcium Channels is Evident After Short Exposures to Alcohol in Vasopressin‐Releasing Nerve Terminals

Voltage-gated calcium channels (VGCCs) in rat neurohypophysial terminals exhibit molecular tolerance to alcohol, including desensitization to the drug and increased current density, after 3weeks of alcohol drinking. Moreover, after this time, terminals from drinking rats exhibit diminished alcohol inhibition of vasopressin (AVP) release. 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. 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 20mM EtOH for 1hour 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. These data indicate that a brief alcohol exposure affects subsequent alcohol sensitivity of VGCCs and neuropeptide release from presynaptic terminals.

Role of Intracellular Calcium in Release from Nerve Terminals

Oxytocin (OT) and vasopressin (AVP) are released from terminals in the neurohypophysis (NH). These neuropeptides (NP) are contained in large dense core vesicles (LDCV). Ryanodine receptors (RyR) in NH terminals can induce spontaneous focal Ca2+ transients (DeCrescenzo, et al., 2004 J. Neurosci.24:1226-1235), making them ideal for studying the role of intraterminal Ca2+ in NP release.Fluorescent immunolabeling and immunogold micrographs of NH terminals show that RyR are localized specifically to LDCV. Furthermore, a large conductance non-specific cation channel, previously identified in the LDCV membrane with properties similar to that of a RyR (Lee, et al., 1992 Neuron 8:335-342), is pharmacologically affected in the same characteristic manner as a RyR.We found that individual Ca2+ release events alone are not enough to drive peptide release (McNally, et al., 2009 J. Neurosci. 29:14120-14126). However, these RyR sensitive events could potentially play a role in modulating NP release. To test this hypothesis, the association of LDCV within an area 0.45 µm of the plasma membrane was assessed using immunolabeling of Neurophysins I (OT) and II (AVP) in isolated NH terminals. We found that the amount of membrane associated NP-immunoreactivity varies significantly between terminal types and that this distribution pattern can be enhanced by agonist concentrations of ryanodine, but only in OT terminals.Importantly, Ca2+-evoked NP release from permeabilized-terminals was increased by agonist concentrations of ryanodine and decreased by antagonist concentrations of this drug. Amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Agonist concentrations of ryanodine were also able to increase the asynchronous phase of low frequency electrically-stimulated capacitance increases in NH terminals. Thus, the ryanodine-sensitive mobilization of LDCV seems to have a functional role in modulating secretion of NP from NH terminals. [Support: NS29470, NS40966, P60037094900000].

GABA Uptake by GAT-1 Modulates Long-Term Optical Changes following Secretion in the Mouse Neurohypophysis

Intrinsic optical changes in the mouse pituitary gland associated with secretion exhibit three phases (waves) that reflect three distinct physiological events. The first (E-wave) is the rapid light scattering change that is associated with a nerve terminal volume increase (mechanical spike) that accompanies excitation of the neurohypophysial terminals by the invading action potential (Kim et al., Biophys. J. 92(9) 3122-3129 (2007)); the second (S-wave) is the slower, opposite change in light scattering that reflects the secretion of the peptide hormones, oxytocin and arginine vasopressin, and the third is the long duration response (recovery phase or R-wave) that reflects cell volume changes in the pars intermedia. This recovery phase is sensitive to chloride replacement and to blockade of chloride channels. Experiments that employ pharmacological blockers of GABAA receptors (chloride channels), as well as direct bath application of GABA, alter the R-wave in a manner that reflects GABA-induced changes in the intrinsic optical properties of the pars intermedia (Kosterin et al., Neuroendocrinology 92, 158-167 (2010)). Because the full time-course of this optical response takes minutes, we suggested that, in addition to chloride-channel opening / closing, some additional processes related to the restoration of resting physiological chloride concentrations may occur. Indeed, we demonstrate here that the time-course of this long term light scattering signal is also affected by the turnover of GAT-1, the sodium- and chloride-dependent GABA-transporter.Supported by RO1 040966.

Functional P2X7 Receptor Expression in the Magnocellular Neurons of the Hypothalamic Neurohypophysial System (HNS)

Our laboratory has presented evidence for P2x7 receptors (P2x7R) in isolated terminals of the neurohypophysis (NH) (Cuadra et al., 2010, Biophys. J.97: 1474). In this study we further examined P2x7R immunoreactivity (IR) and function. A stereo-specific ecto-P2x7R antibody was used for IR studies (Kim el al., 2001, JBC, 276:23262) in the rat HNS: supraoptic nucleus (SON), paraventricular nucleus (PVN) and NH. We found limited P2x7R IR in the SON and the PVN, which was absent from the somata of OT- and AVP-neurons. P2x7R IR was co-localized in some cell bodies containing the glial specific marker, GFAP. In contrast, P2x7R IR was abundantly seen on the membrane of NH terminals (NHT), with dense IR in terminals lining the capillary borders. This was confirmed in isolated NHT that showed IR puncta on the membranes of both AVP- and OT-containing terminals. Initial results using ratiometric calcium imaging with Fura-2 in isolated NHT showed differential [Ca2+]iresponses to 100 μM vs. 1 mM ATP; some NHT responded to both doses while others responded only to 1 mM ATP. A similar distribution in response was observed with ATP generated ion currents during patch clamp recordings. NHT responded with either high sensitivity (HS) or low sensitivity (LS) to ATP. The HS group corresponded to AVP-NHT, which is reported to express P2x2R, P2x3R, P2x4R and P2x7R (Knott et al., 2005, Pfluger Arch, 405:381). In contrast, the LS group corresponded to OT-NHT, which is consistent with a P2x7R response. Together these data suggest that in the HNS P2x7R is expressed chiefly in the AVP- and OT-secreting terminals of magnocellular neurons. (Supported by NIH grant NS29470 to JRL)

Exogenous oxytocin (OT) ameliorates pulmonary damage caused by Escherichia coli (E. coli) infection in chronically stressed rats

This study examines the hypothesis that exogenous OT will reduce pulmonary damage caused by E. coli infection in stressed rats. Four groups of male albino rats were used as follows: 1) rats received an intratracheal (i.t.) injection of 0.2 mL E. coli suspension containing 1·109 bacteria/mL in saline; 2) rats were subjected to footshock (FS) stress for 1–11 days; 3) rats were subjected to FS stress for 7 days and then received an i.t. injection of E. coli; 4) rats subjected to FS stress had concurrently received i.m. injections of OT (0,02 IU/mL) for 7 days and then were injected with E. coli. The lungs, hypothalamus, and neurohypophysis were analyzed by light and electron microscopy, immunohistochemistry on days 1 to 11 (groups: 1–2) and days 8 to 11 (groups: 3–4). We found that an E. coli infection in chronically stressed rats caused a greater inflammatory response, cell death and fibrosis than were seen in non‐FS animals. The detrimental changes in the former coincided with increased inhibition of OT release from neurohypophysial terminals. The injections of OT reduced the number of apoptotic cells (~40%) and production of MMP‐9 (~75%), while it stimulated the expression of Bcl‐2, Clara cell protein (CC‐16), and lactoferrin in the lungs. Thus, we concluded that exogenous OT shows a therapeutic effect against E. coli‐induced lung injury under condition of chronic stress, when the release of endogenous neuropeptide is impaired.

Voltage‐dependent κ‐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.

Differential modulation of N‐type calcium channels by µ‐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.

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.

Modulation of Neuroscretory Granule Mobilization and Neuropeptide Release by Ryanodine Receptors in Neurohypophysial Terminals

The neuropeptides oxytocin (OT) and vasopressin (AVP) are contained in large dense core vesicles (LDCV) and are released at the neurohypophysis (NH). The mobilization and translocation of vesicles to exocytotic release sites is modulated by cytosolic Ca2+. Intracellular structures and organelles able to store and release Ca2+ are significant contributors of cytosolic Ca2+. The presence of ryanodine receptors (RyR) in LDCV of NH terminals, coupled with the demonstration that pharmacological activation of these receptors can induce spontaneous focal Ca2+ transients, make them ideal modulators of cytosolic levels of Ca2+, and therefore, vesicle mobilization and subsequent neuropeptide (NP) release.To test this hypothesis, the association of LDCV in an area within 0.45 μm of the plasma membrane was assessed using immunolabeling of Neurophysin I (OT) and II (AVP) along with high stringency deconvolution techniques in isolated NH terminals. We found that the total amount of membrane associated NP-immunoreactivity varies significantly between terminal type; significantly higher in OT than in AVP terminals. This membrane associated distribution pattern can be enhanced by exposure to agonist concentrations (1-5 μM) of ryanodine in OT terminals only. RyR antagonists, 8-Br-cADPR or higher concentrations (>10 μM) of ryanodine had the opposite effect; significantly reducing the amount of OT associated with the membrane area.Additionally, Ca2+-evoked NP release from permeabilized terminals was increased by agonist concentrations of ryanodine and conversely, decreased by antagonist concentrations of this drug. Agonist concentrations of ryanodine were also able to increase the asynchronous phase of low frequency electrically stimulated capacitance increases from isolated NH terminals. Thus, the ryanodine-sensitive mobilization of secretory granules seems to have a functional role in modulating secretion of neuropeptides from NH terminals. [Supported by UMass Grant P60037094900000 (SOM) and NIH Grant NS29470 (JRL)]

Voltage-Dependent Kappa Opioid Modulation of Calcium Currents Elicited by Action Potential Waveforms in Neurohypophysial Terminals

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca2+ channels results in less calcium influx into the terminal and, presumably, a reduction in transmitter release. In the nerve terminals of the neurohypophysis, voltage-gated calcium channels (VGCC) are primarily controlled by membrane voltage and their activity can be modulated, in a voltage-dependent manner, by their interaction with G-protein subunits. Endogenous opioids also affect (inhibit) these calcium channels, upon binding to μ- and κ-receptors at the terminals.Voltage-dependent relief of G-protein inhibition of VGCC is 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 μ- and κ-receptors. The κ-opioid VGCC inhibition is relieved by such pre-pulses, suggesting that this receptor is involved in a voltage-dependent membrane-delimited G-protein pathway. In contrast, μ-opioid inhibition of VGCC is not relieved by such pre-pulses, indicating a voltage-independent diffusible second-messenger signaling pathway. Furthermore, κ-opioid inhibition is also relieved during stimulation with action potential bursts with physiological characteristics. This indicates the possibility of activity-dependent modulation in vivo.Differences in the facilitation of Ca2+ channels due to specific G-protein modulation during a burst of action potentials may contribute to the fine-tuning of Ca2+-dependent neuropeptide release in other central nervous system synapses, as well. [Supported by NIH Grant NS29470].

Potassium Accumulation Dominates Short-Term Depression of Neurohypophysial Excitability

Excitability of the axons and nerve terminals in the mammalian neurohypophysis strongly depends on the temporal pattern and intensity of stimulation. Within a given stimulus train, excitability tends to facilitate initially and begins to depress significantly during persistent stimulation. In this work we present both experimental evidence and numerical simulations indicating that depression of neurohypophysial excitability is dominated by activity-dependent potassium accumulation.

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.