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To understand the contribution of intrinsic membrane properties to the different in vivo firing patterns of oxytocin (OT) and vasopressin (VP) neurones, in vitro studies are needed, where stable intracellular recordings can be made. Combining immunochemistry for OT and VP and intracellular dye injections allows characterisation of identified OT and VP neurones, and several differences between the two cell types have emerged. These include a greater transient K + current that delays spiking to stimulus onset, and a higher Na + current density leading to greater spike amplitude and a more stable spike threshold, in VP neurones. VP neurones also show a greater incidence of both fast and slow Ca 2+ -dependent depolarising afterpotentials, the latter of which summate to plateau potentials and contribute to phasic bursting. By contrast, OT neurones exhibit a sustained outwardly rectifying potential (SOR), as well as a consequent depolarising rebound potential, not found in VP neurones. The SOR makes OT neurones more susceptible to spontaneous inhibitory synaptic inputs and correlates with a longer period of spike frequency adaptation in these neurones. Although both types exhibit prominent Ca 2+ -dependent afterhyperpolarising potentials (AHPs) that limit firing rate and contribute to bursting patterns, Ca 2+ -dependent AHPs in OT neurones selectively show significant increases during pregnancy and lactation. In OT neurones, but not VP neurones, AHPs are highly dependent on the constitutive presence of the second messenger, phosphatidylinositol 4,5-bisphosphate, which permissively gates N-type channels that contribute the Ca 2+ during spike trains that activates the AHP. By contrast to the intrinsic properties supporting phasic bursting in VP neurones, the synchronous bursting of OT neurones has only been demonstrated in vitro in cultured hypothalamic explants and is completely dependent on synaptic transmission. Additional differences in Ca 2+ channel expression between the two neurosecretory terminal types suggests these channels are also critical players in the differential release of OT and VP during repetitive spiking, in addition to their importance to the potentials controlling firing patterns., (© 2018 British Society for Neuroendocrinology.)

Oxytocin (OT) and vasopressin (VP) hormone release from neurohypophysial terminals is controlled by the firing pattern of neurosecretory cells located in the hypothalamic supraoptic (SON) and paraventricular nuclei. Although glutamate is a key modulator of the electrical activity of both OT and VP neurons, a differential contribution of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) has been proposed to mediate glutamatergic influences on these neurons. In the present study we examined the distribution and functional properties of synaptic currents mediated by AMPARs and NMDARs in immunoidentified SON neurons. Our results suggest that the properties of AMPA-mediated currents in SON neurons are controlled in a cell type-specific manner. OT neurons displayed AMPA-mediated miniature EPSCs (mEPSCs) with larger amplitude and faster decay kinetics than VP neurons. Furthermore, a peak-scaled nonstationary noise analysis of mEPSCs revealed a larger estimated single-channel conductance of AMPARs expressed in OT neurons. High-frequency summation of AMPA-mediated excitatory postsynaptic potentials was smaller in OT neurons. In both cell types, AMPA-mediated synaptic currents showed inward rectification, which was more pronounced in OT neurons, and displayed Ca2+ permeability. On the other hand, NMDA-mediated mEPSCs of both cell types had similar amplitude and kinetic properties. The cell type-specific expression of functionally different AMPARs can contribute to the adoption of different firing patterns by these neuroendocrine neurons in response to physiological stimuli.

Parenchymal injections of puromycin were known to induce Golgi-like silver impregnations upon treatment with the cupric-silver method. We have found lateral ventricular injections (5 microliter, 0.1 M) also effective. Such injections consistently led to detailed Golgi-like profiles of cells in a number of hypothalamic regions including one, the paraventricular nucleus, which traditionally is quite recalcitrant to Golgi methods. It is concluded that this technique is a reliable alternative to Golgi methods for the study of many hypothalamic regions.

Single unit activity was recorded from 400-500 mu m thick slices of rat hypothalamus, using either NaCl- or horseradish peroxidase-filled glass micropipettes. Spontaneous activity was present in the following hypothalamic loci: anterior hypothalamic-preoptic area, nucleus circularis, nucleus of the diagonal band of Broca, paraventricular accessory nucleus, paraventricular nucleus (all portions), periventricular regions of the anterior hypothalamus, and the suprachiasmatic nucleus. The supraoptic nucleus was the only major cell group studied to exhibit no spontaneous activity. Cells of the paraventricular and circularis nuclei were spontaneously active, displayed firing rates and patterns of activity similar to those recorded in vivo for magnocellular elements of the hypothalamus, and in some cases responded to increases in the osmolality of the bathing medium with altered firing rates and/or patterns of activity. Many cells in these preparations were characterized by phasic, bursting patterns of activity. Slow, irregular and regular, continuous activity was also frequently observed, as is typical in vivo. Median firing rates were in the range of 4-6 spikes/sec, somewhat faster than the rates usually reported for anesthetized in vivo preparations. These rates are more similar to those observed in unanesthetized monkeys or rats with diencephalic islands. Extracellular HRP marking provided a high degree of localization for many of the recorded cells. These results indicate that the hypothalamic slice preparation is useful for studies in which it is desirable to eliminate extrahypothalamic connections and in which it is necessary to exercise a fine degree of control over the extracellular environment of the cells.

gamma-Aminobutyric acid (GABA) has been identified in axon terminals innervating neurons of the supraoptic nucleus and has been shown to inhibit the electrical activity of supraoptic neurons when applied iontophoretically. This study examines the effects of GABA and GABA antagonists on vasopressin (VP) release from organ-cultured explants of the hypothalamo-neurohypophyseal system (HNS). The GABA antagonists bicuculline and picrotoxin stimulated VP release in a concentration-dependent manner. These observations suggest that VP release by HNS explants is tonically inhibited by GABA. Exposure of HNS explants to GABA (10(-8)-10(-3) M) did not consistently alter basal VP release. This was true even when penicillin, which can block GABA-activated chloride channels, was omitted from the medium. Similarly, nipecotic acid, an agent that potentiates GABA activity by inhibiting GABA uptake, did not alter basal VP release; stimulation of VP release by acetylcholine and increases in osmolality was not diminished by the addition of 10(-5) M GABA. The failure of exogenous GABA to modify basal and stimulated VP release suggests that GABAergic inhibition of VP release is maximally activated by endogenously released GABA in cultured HNS explants. This is consistent with evidence for a local source of GABA in the supraoptic nucleus and suggests that one role of GABA in the regulation of VP release is that of a potent local inhibitory neurotransmitter.

Subcutaneous injections of isotonic saline induced nucleolar proliferation in supraoptic neurons in animals sacrificed approximately 5 min postinjection. The magnitude of this proliferation was sustained 4 and 8 hr postinjection. Polyethylene glycol (PG) injections depleted blood volume 4 and 8 hr after the injection, but the percentage of SON cells with multiple nucleoli in these animals was not different from saline-injected controls. The anterior (SOa) portion of the SON in rats given 2% NaCl to drink instead of water for three days contained more cells with multiple nucleoli than controls. This effect was enhanced after five days ingestion, and accompanied by a similar response in the tuberal portion of SON (SOt). Rehydration for ten days after three days of 2% NaCl intake brought the percentage of cells with multiple nucleoli down to control levels. Cell area in SON cells paralleled nucleolar responses during dehydration and rehydration. The results demonstrate the sensitivity of nucleolar proliferation in SON to environmental changes ranging from osmotic to neurogenic stress.

Cell area, percentage of cells with more than one nucleolus, and percentage of cells with the nucleolus apposed to the nuclear membrane (marginated) were compared in the medial and lateral magnocellular portions of the paraventricular nucleus (PVN) during water deprivation and voluntary rehydration. Lateral cells were larger and had a greater percentage of cells with multiple nucleoli in all conditions of deprivation and rehydration. In addition laterals were found to have more cells with marginated nucleoli in these cells with only one nucleoli. Medial and lateral cells showed similar responses to deprivation and rehydration.

Single unit, extracellular electrical activity of 97 hypothalamic supraoptic neurons was recorded with micropipettes from rat hypothalamo-neurohypophysial explants maintained acutely in vitro. Spontaneously firing as well as quiescent neurons were encountered in roughly in roughly equal numbers. Of the spontaneously active neurons, 36.3% fired in a phasic mode quantitatively similar to that of "vasopressin-secreting' neurons recorded in vivo under conditions of enhanced hormone release. A small portion (approximately 14%) of the recorded supraoptic neurons had spikes which showed constant latency and high frequency following responses to stimulation of the neural stalk, responses characteristic of antidromically stimulated neurons. The mean latency and the range of latencies to these responses were similar to that recorded in vivo. Hypothalamo-neurohypophysial explants maintained in vitro thus provide a useful tool for investigating the electrical activity of supraoptic neurosecretory cells. Since the explant contains both the supraoptic neurons and their neurohypophysial projection path, this preparation offers an opportunity for characterizing further the relationship between electrical activity and hormone release.

Projection neurons in the rat hypothalamic paraventricular nucleus (pv) were identified using the retrograde transport of horseradish peroxidase (HRP) following injections of this tracer into the neurohypophysis or jugular vein. The objective was to determine if parvocellular pv neurons might be labelled by either injection in addition to the well known magnocellular contingent. Injections confined to the neurohypophysis resulted in the labelling of only magnocellular pv neurons, while intrajugular injections which produced spread of the HRP to the arcuate nucleus-median eminence region resulted in the labelling of both parvocellular and magnocellular pv elements. Thus parvocellular pv neurons may project to the arcuate nucleus and/or the external layer of the median eminance but not to the neurohypophysis.

Processes of magnocellular neurosecretory cells in the rat supraoptic nucleus which project along the pial surface in the ventral glial lamina were investigated using immunocytochemistry, Golgi stains and electron microscopy. Immunocytochemical studies revealed that although both oxytocin- and vasopressin-containing processes were evident in the ventral glial lamina, vasopressin-containing processes predominated. Ventral processes were thicker and of a different morphology than dorsal axon-like processes which joined the hypothalamo-neurohypophysial tract and exhibited large varicosities along their length or at their apparent termination. Golgi stains revealed that classically defined dendrites of supraoptic neurons projected primarily ventrally and often invaded the ventral glial lamina. No axons were traced to the lamina. Ultrastructurally, processes within the ventral glial lamina characterized as dendrites could be stained immunocytochemically for neurophysin and were post-synaptic to a variety of presynaptic elements. The results suggest that many dendrites from magnocellular neurosecretory cells in the supraoptic nucleus project to the ventral glial lamina and form a restricted, receptive plexus. The previously demonstrated coexistence of catecholamine-containing varicosities and other axon types with these processes in the lamina indicates an important role for supraoptic dendrites in integrating a wide variety of information relevant to neurohypophysial hormone release.

Evidence is presented indicating that norepinephrine (NE) inhibits vasopressin (VP) release from the rat hypothalamo-neurohypophyseal explant under some, but not all, conditions in vitro. NE at 10(-5) M inhibited basal VP release and inhibited acetylcholine-induced release in a concentration-dependent fashion. However, the induction of VP release caused by the addition of NaCl (sufficient to yield a 10 mosmol/kg H2O increase in culture medium osmolality) was not reduced by NE in concentrations as high as 10(-5) M. An alpha-adrenergic receptor mediation of the inhibition of VP release by NE was suggested by the ability of phentolamine and phenoxybenzamine, but not propranolol, to block this effect. In addition, phentolamine at 10(-4) M, but not equimolar amounts of propranolol, increased VP release when added alone on day 2 of culture, but not on days 3 or 4. Histofluorescence examination of additional explants revealed that endogenous catecholamine was still present on day 2 within varicosities in the supraoptic nucleus, but diminished by days 3 and 4, suggesting that endogenous NE could influence basal VP release. The results indicate that NE can inhibit spontaneous and cholinergically stimulated VP release from the hypothalamo-neurohypophyseal explant, but osmotic stimulation renders the explants insensitive to attenuation of VP release by NE.