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1. Using ion-sensitive microelectrodes, a transient reduction in the local volume of neural lobe extracellular space was found to accompany the elevation in extracellular potassium induced by stimulation of the neural stalk. The volume decrease and potassium increase had similar stimulus-response curves when stimulus frequency was varied from 1 to 40 Hz, with maximal response at 20 Hz. The curves for stimulus duration diverged, as a near maximal potassium response was reached in 4-16 s with a 20-Hz stimulus, while the extracellular volume decrease was maximal at 64 s. 2. The volume decrease, but not the potassium increase, was strongly inhibited by lowering bath temperature and moderately inhibited by furosemide and by lowering extracellular chloride concentration. Both the volume and the potassium response were enhanced by ouabain. 3. In conclusion, shrinkage of the local extracellular space in neural lobe during nerve activity is mediated by a metabolically active process which is only partially dependent upon extracellular chloride concentration and anion-cation co-transport, but is relatively independent of Na(+)-K+ pump activity. A transient shrinkage in extracellular space during increased neurohypophysial nerve activity would be expected to play a role in hormone diffusion, ion buffering, and extracellular current flow.

The distribution of the neurohypophysial prohormone products neurophysin and C-terminal glycopeptide within dense core vesicles were investigated at the ultrastructural level using quantitative immunogold labeling of rapidly frozen rat neural lobes. In vasopressin vesicles, gold particles after labeling for the glycopeptide were found significantly closer to membrane (chi = 16 nm) than were particles after labeling for neurophysin (chi = 32 nm). This distribution supports a previous histochemical localization of glycopeptide to the rim of vasopressin vesicles and suggests there is morphological segregation of prohormone products within the vesicle.

Increased neural activity of neurosecretory cells is accompanied by large increases in extracellular K+. The possibility that elevations of this ion might involve fluid redistribution and thus affect the size of the extracellular space and the relationship between pituicytes and axons in the rat neural lobe was explored using rapid freezing and freeze-substitution. Neural lobes were incubated for 15 min before freezing either in a normal medium or one containing a 10 mM increase in KCl (high KCl), a 10 mM increase in KCl balanced by an equimolar reduction in NaCl (high KCl-low NaCl), or only a 10 mM reduction in NaCl (low NaCl). A quantitative assessment of the region of good fixation was made to determine the relative fractions occupied by axons, pituicytes and the extracellular space near the neurohaemal contact zone. In addition, the percentage of basal lamina contacted by pituicytes and axons was calculated, as was the degree of enclosure of axons by pituicytes. In neural lobes incubated in normal medium, the extracellular space accounted for approximately 30% of the cross-sectional area of the neuropil and could be divided into two domains: an extended perivascular space (28-29% of total area); and a narrow (approximately 24 nm; approximately 1% of total) space between closely apposed neurosecretory processes or between these processes and pituicytes. Pituicytes occupied almost 60% of the basal lamina at the neurohaemal contact zone, while axons occupied approximately 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid freezing of freshly dissected or incubated neural lobes was explored as a means of obtaining ultrastructural preservation of the more natural state of this tissue. A quantitative assessment of the region of good fixation was made in order to determine the relative fractions occupied by axons, pituicytes and the extracellular space. The immunocytochemical distributions of neurophysins and the glycopeptide portion of the vasopressin precursor were evaluated using the immunogold technique in order to determine the relative numbers of oxytocin and vasopressin fibre types in the fixed region, and the subcellular localization of these antigens. The uncut surface of rat neural lobes was rapidly frozen against a highly polished copper plug and freeze-substituted in osmium-acetone either immediately after dissection (approximately 2 min), or after a 15 min incubation period in vitro in an oxygenated, balanced salt solution. Substituted neural lobes were prepared for either conventional electron microscopy, or for immunogold labelling of neurophysins and the glycopeptide precursor to vasopressin. Membranes, subcellular organelles and extracellular matrix were well preserved 10 microns deep to the contacted surface. The extracellular space accounted for approximately 30% of the cross-sectional area of the neuropil and could be divided into two domains: an extended perivascular space (28-29% of total area); and a narrow (approximately 24 nm; approximately 1% of total) space between closely apposed neurosecretory processes or between these processes and pituicytes. Pituicytes accounted for about 30% of the area and axons 20-25%. Pituicytes occupied close to 60% of the basal lamina at the neurohaemal contact zone, while axons occupied approximately 20%. There were no differences between neural lobes frozen immediately after dissection and those incubated for 15 min in any of these measures, suggesting minimal fluid redistribution. Gold particles were specifically localized over large (100-200 nm) dense core vesicles, and less frequently over multivesicular bodies and lysosomes. No etching of the plastic or reduction of osmium was necessary to achieve labelling. Specific labelling of one set of terminals and axons (about 80%) was observed with the monoclonal antibody previously shown to be specific for oxytocin-neurophysin, while in neighbouring sections the remaining 20% of the processes were labelled with the antiserum to the vasopressin precursor, or with non-specific antibodies to neurophysins. In conclusion, ultrarapid freezing preserves a large extracellular space in the neural lobe and provides for high resolution morphological and immunocytochemical studies of neurohypophysial structure.

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.

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.