Techniques for studying the role of electrical activity in control of secretion by normal anterior pituitary cells.
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Animals↗
Biomedical subjects
W T Mason
Publications and source records attributed to W T Mason.
Central release of oxytocin, vasopressin and neurophysin by magnocellular neurone depolarization: evidence in slices of guinea pig and rat hypothalamus.
Using slices of rat hypothalamus maintained in vitro, we have examined release of oxytocin and vasopressin under conditions of increased neuronal activity. We report here that when the supraoptic or paraventricular nucleus is depolarized with high K+ solutions, hormone is released into areas close to the nucleus. Similar experiments with guinea pig hypothalamus suggest that neurophysin may also be co-released with oxytocin and vasopressin. Use of acetylcholine to selectively stimulate vasopressin neurones appears to evoke a rise in local release of vasopressin but not oxytocin. These results suggest that under conditions of increased neuronal activity, hormones normally secreted from the neurohypophysis are secreted locally into the hypothalamus.
Acetylcholine↗
Patch clamp recordings of single ion channel activation by gonadotrophin-releasing hormone in ovine pituitary gonadotrophs.
Gonadotrophs of the ovine pars tuberalis have been studied using the patch clamp technique for recording of single ion channel currents. We report that gonadotrophin-releasing hormone (GnRH) acts on these cells to open an inward-current cation channel which is permeable to Ca2+. When measured in cell-attached patches with 5 mM extracellular Ca2+, the GnRH-activated channel has a unit slope conductance of 8.0 +/- 2.6 pS (range 4-14 pS). The channel conductance is increased to 13.6 +/- 2.4 pS when the external medium contains 95 mM Ba2+ as the charge carrier. GnRH action appears to be mediated through an internal messenger system, since GnRH does not need to be in direct contact with these channels in order to cause their opening. This internal messenger system is unlikely to be Ca2+ itself. In addition, two other voltage-dependent outward-current channels have also been detected, both of which are permeable to K+, but differentiated in the cell-attached recording mode by widely different conductance values of 20-30 and 100-120 pS, respectively, and a reversal potential of -90 to -100 mV. The higher conductance channel is sensitive to internal Ca2+, and its probability of opening is increased in the presence of GnRH.
Animals↗
Intracellular recordings from bovine anterior pituitary cells: modulation of spontaneous activity by regulators of prolactin secretion.
The electrophysiological properties of cultured bovine anterior pituitary cells were examined using intracellular recordings. Although all cells had high input resistances (mean 332 M omega), membrane potentials (Vm) showed a wide distribution (-32 to -80 mV). In 9 cells with low Vm (mean -38 mV) spontaneous Ca2+-dependent action potentials were observed (mean frequency 3.4 hertz). In the majority of silent cells (mean Vm -58 mV), application of TRH caused a reduction in membrane resistance and a transient hyperpolarization of Vm. This initial response was followed by a small, sustained depolarization. In 4/18 cells this depolarization led to the appearance of action potentials in previously silent cells. In 15/18 cells application of dopamine caused an increase in the membrane conductance and led to a sustained hyperpolarization of 30-35 mV. The response reversibly blocked the propagation of action potentials in spontaneously active cells. This effect could also be reversed by the dopamine receptor antagonists, sulpiride and haloperidol. In all cells, elevating the extracellular potassium concentration caused a concentration-dependent depolarization and decrease in the membrane input resistance. In some cells this effect was associated with an increased frequency of action potentials. Electrophysiological responses to TRH, dopamine, and elevated potassium were correlated with changes in the release of PRL. It is concluded that by their effects on Vm these factors modulate spontaneous electrical properties and may regulate the entry of calcium necessary for hormone secretion from lactotrophs.
Action Potentials↗
Single channel recordings obtained from basolateral membranes of isolated rabbit enterocytes.
Epithelial cells isolated by hyaluronidase incubation from rabbit small intestine were used to explore the presence of ionic channels by the patch-clamp method. Recordings were made from cell-attached or excised patches of basolateral membrane. Evidence was obtained for the presence of at least two kinds of channels conducting potassium currents. One of these can be shown to be activated by Ca2+.
Animals↗
Staining of the magnocellular nuclei of the rat hypothalamus by a monoclonal antibody directed against the alpha-subunit of the nicotinic cholinergic receptor.
Previous work has shown the vasopressin-secreting cells of the supraoptic nucleus receive a cholinergic input from a region dorsolateral to the nucleus, and that acetylcholine (ACh) activation of these cells is blocked by nicotinic antagonists. This work reports that a monoclonal antibody, directed against the alpha-subunit of the nicotinic ACh receptor isolated from Electrophorus electricus, stains certain hypothalamic cells and processes, particularly in regions associated with vasopressin-containing neurones.
Acetylcholinesterase↗
Electrophysiological recordings from gonadotrophs. Evidence for Ca2+ channels mediated by gonadotrophin-releasing hormone.
The electrophysiological properties of gonadotrophs have been studied in vitro, using the ovine adenohypophyseal pars tuberalis as a naturally enriched source of this cell type. Trypsin-dispersed pars tuberalis cells maintained in primary tissue culture had a membrane potential of -72 +/- 4 mV (mean +/- SEM) and an input resistance of 314 +/- 38 M omega. Spontaneous action potentials were not observed; however, a single spike could be induced by depolarizing current injection. The hypophysiotrophic peptide gonadotrophin-releasing hormone (GnRH) increased membrane voltage fluctuations, but these fluctuations (+/- 5-10 mV) did not induce action potentials or changes in membrane potential or resistance. Power spectra obtained from analysis of this noise indicated that the fundamental event underlying GnRH action has a mean life-time of 38.4 +/- 4.5 ms. The observations that cells incubated in recording medium secreted luteinizing hormone in response to GnRH and that the GnRH-induced increase in voltage noise was inhibited by Ca2+ channel antagonists support the hypotheses (1) that gonadotrophin secretion is initiated by GnRH-induced Ca2+ channel activation and (2) that action potentials are not a prerequisite for gonadotrophin release.
Animals↗
Complex action potential waveform recorded from supraoptic and paraventricular neurones of the rat: evidence for sodium and calcium spike components at different membrane sites.
Magnocellular neurones of the supraoptic and paraventricular nuclei display a complex waveform when recorded extracellularly. The present work has examined the possible reasons for the complexity of this waveform by making extracellular recordings from antidromically identified neurones in the anaesthetized rat and extra- and intracellular recordings from similar neurones in the hypothalamic slice preparation, where external solutions can be changed. In extracellularly recorded units, action potentials had two positive going components. The first of these was abolished by tetrodotoxin. The second, slower component was abolished when external Ca++ concentration was lowered, but enhanced in magnitude and duration when Ba++ was placed in the external medium. The second component was sensitive to electrode movement and was not observed to the same degree when intracellular recordings were made. In light of these observations and the known morphology of these neurones, it is suggested that the magnocellular neurone action potential is comprised of a fast Na+-dependent component and a slower component dependent on Ca++ entry which may originate from a part of the cell other than the soma. The most likely site for this Ca++ component to occur is at the cell dendrite.
Action Potentials↗
Axon collaterals of supraoptic neurones: anatomical and electrophysiological evidence for their existence in the lateral hypothalamus.
The magnocellular neurones of the supraoptic nucleus which synthesize and secrete vasopressin and oxytocin have been commonly regarded as simple "output" neurones in that they receive an input, generate an action potential and in turn release hormone from their terminals in the posterior pituitary. Three lines of evidence are presented which suggest that rat supraoptic nucleus neurons also have axon collaterals which terminate in the hypothalamus close to the nucleus. Small injections of horseradish peroxidase were made directly into the nucleus in hypothalamic slices, allowing visualization of the axons of supraoptic neurones. Collaterals of these axons could be observed in regions both dorsal and dorsolateral to the supraoptic nucleus. In a separate series of experiments, sections of perfusion-fixed hypothalamus were stained for vasopressin and oxytocin using specific antisera. Peptide-containing collaterals of both types were observed near the supraoptic nucleus, in a region similar to that seen after horseradish peroxidase injections. Finally, electrophysiological studies were carried out on hypothalamic slices containing the supraoptic nucleus. A small concentric bipolar stimulating electrode was placed directly into the nucleus and activity of lateral hypothalamic neurones within 0.1-1 mm of the nucleus was recorded. Of 68 neurones studied, 52 were excited by supraoptic stimulation via a synaptic pathway that could be blocked by Ca2+ -free solutions containing 18 mM Mg2+. These studies suggest that supraoptic neurones communicate via axon collaterals with other neurones in the lateral hypothalamus, in addition to their previously well characterised functional role in neurosecretion.
Animals↗
Excitation by dopamine of putative oxytocinergic neurones in the rat supraoptic nucleus in vitro: evidence for two classes of continuously firing neurones.
Neurones from the supraoptic nucleus in the rat hypothalamic slice preparation in vitro have been recorded from using conventional extracellular techniques. Neurones which fired in a slow continuous manner (putative oxytocinergic cells) increased their firing pattern in response to dopamine applied in the perfusate or pressure ejected onto the slice. Of these neurones, 85% were inhibited by spiperone, haloperidol or cis-flupenthixol applied in the bath. A small proportion (approximately 15%) of neurones excited by dopamine (DA) were not affected by these DA antagonists but were inhibited strongly by (+/-)-sulpiride. Similar results were also obtained on a small number of cells recorded from an 'island-slice' preparation, utilized to isolate the observed responses to an area immediately adjacent to the supraoptic nucleus. These results may be relevant to earlier work on in vivo anaesthetized preparations using intracerebroventricular injections of DA and antagonists, and indicate that such substances may act directly on a synapse(s) within the magnocellular nucleus in addition to other possible actions within the neurohypophyseal tract. The differential response to the dopamine antagonists indicate that the majority of continuously firing neurones in the supraoptic nucleus are of the DI receptor class, but the presence of possible D2 receptors on a small proportion of these neurones suggests that a second type of continuously firing neurone may exist in this nucleus.
Animals↗
Electrical properties of neurons recorded from the rat supraoptic nucleus in vitro.
The electrical properties of neurons in the supraoptic nucleus (so.n.) have been studied in the hypothalamic slice preparation by intracellular and extracellular recording techniques, with Lucifer Yellow CH dye injection to mark the recording site as being the so.n. Intracellular recordings from so.n. neurons revealed them to have an average membrane potential of -67 +/- 0.8 mV (mean +/- s.e.m.), membrane resistance of 145 +/- 9 M omega with linear current-voltage relations from 40 mV in the hyperpolarizing direction to the level of spike threshold in the depolarizing direction. Average cell time constant was 14 +/- 2.2 ms. So.n. action potentials ranged in amplitude from 55 to 95 mV, with a mean of 76 +/- 2 mV, and a spike width of 2.6 +/- 0.5 ms at 30% of maximal spike height. Both single spikes and trains of spikes were followed by a strong, long-lasting hyperpolarization with a decay fitted by a single exponential having a time constant of 8.6 +/- 1.8 ms. Action potentials could be blocked by 10(-6) M tetrodotoxin. Spontaneously active so.n. neurons were characterized by synaptic input in the form of excitatory and inhibitory postsynaptic potentials, the latter being apparently blocked when 4 M KCl electrodes were used. Both forms of synaptic activity were blocked by application of divalent cations such as Mg2+, Mn2+ or Co2+. 74% of so.n. neurons fired spontaneously at rates exceeding 0.1 spikes per second, with a mean for all cells of 2.9 +/- 0.2 s-1. Of these cells, 21% fired slowly and continuously at 0.1 - 1.0 s-1, 45% fired continuously at greater than 1 Hz, and the remaining 34% fired phasically in bursts of activity followed by silence or low frequency firing. Spontaneously firing phasic cells showed a mean burst length of 16.7 +/- 4.5 s and a silent period of 28.2 +/- 4.2 s. Intracellular recordings revealed the presence of slow variations in membrane potential which modified the neuron's proximity to spike threshold, and controlled phasic firing. Variations in synaptic input were not observed to influence firing in phasic cells.
Action Potentials↗
Mapping of cholinergic neurons associated with rat supraoptic nucleus: combined immunocytochemical and histochemical identification.
Recent electrophysiological experiments have suggested that electrical stimulation of an area dorsolateral to the rat supraoptic nucleus (SON) activates a cholinergic pathway to the vasopressin neurons of the SON. As no detailed information is available concerning the distribution and projections of the cholinergic neurons in this area, we have sought to provide this using a combination of choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry. In some cases, these techniques were applied to the same neurons. Almost all neurons just outside of the SON that showed ChAT-like immunoreactivity also stained densely for AChE. These cells were distributed in a region dorsolateral to the SON. Light, punctate AChE staining around SON neurons was observed predominantly in the more ventral and posterior parts of the nucleus and were suggestive of synaptic terminals. Cholinergic fibres were found to enter the SON mainly from a lateral direction, turning in an anterior or posterior direction inside the nucleus. These results support the conclusion of earlier studies that the major cholinergic input to the SON arises in its immediate vicinity. We hypothesize that these ChAT/AChE-positive neurons are those responsible for cholinergically mediated, osmotically-stimulated release of vasopressin.
Acetylcholinesterase↗
Control of neurosecretory cell activity in the hypothalamic slice preparation.
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Angiotensin II↗
Synaptic activation of phasic bursting in rat supraoptic nucleus neurones recorded in hypothalamic slices.
Using slices of rat hypothalamus (400-500 micron thick), intracellular and extracellular recordings were made of activity from eight-eight neurones in the supraoptic nucleus (s.o.n.). Electrical stimulation with single stimuli dorsolateral to s.o.n. was excitatory to fifty-nine phasically firing cells (67% of total, 95% of phasic cells). In intracellularly recorded cells, such stimulation reliably evoked excitatory post-synaptic potentials which often gave rise to action potentials. Trains of stimuli reliably triggered bursts of action potentials which continued after stimulation had ceased. Stimulation more dorsal or more lateral to the critical region or in the optic tract adjacent to the s.o.n. did not evoke responses. Stimulation dorsomedial to the nucleus produced only direct, probably antidromic, activation of s.o.n. neurones. Application of acetylcholine (ACh) by microperifusion in the s.o.n. region mimicked the effect of electrical stimulation by evoking prolonged discharge in eight of eight tested phasically firing s.o.n. neurones. Non-phasic, continuously firing neurones were either inhibited or unaffected by electrical stimulation in the critical region. The discharge pattern of unaffected cells (six cells) was not modified by locally applied ACh, although they were excited by local application of sodium glutamate. The excitatory, synaptically mediated, responses to stimulation in the dorso-lateral region were blocked reversibly by the nicotinic blockers, d-tubocurarine chloride and hexamethonium bromide (in seven of seven cells tested), but were unaffected by the muscarinic blocker, atropine, even at high concentrations (two of two cells tested). Thus, this activation appears to be mediated by nicotinic receptors. In separate experiments with the position of stimulating and recording electrodes reversed, s.o.n. stimulation was effective in antidromically activating one cell of sixty-eight recorded extracellularly in the dorsolateral region. Some slowly firing s.o.n. neurones (less than 4 Hz) were inhibited by electrical stimulation in the same area in which phasically active cells were excited. In these cases, stimulation produced large summating i.p.s.p.s. and/or inhibition of ongoing activity for the duration of the stimulus train. These results support evidence from earlier studies that the cholinergic input to s.o.n. neurones originates from cells in its close proximity, and suggests this input to be via a monosynaptic pathway.
Acetylcholine↗
Cultured neurones from the mature bovine mediobasal hypothalamus contain LHRH but not catecholamine.
The in vitro culture of mature neurones from bovine mediobasal hypothalamus (MBH) is reported, providing a model for studies of mammalian neurosecretion at the cellular level. Explant tissue cultures of mature bovine MBH containing the arcuate nucleus were examined for LHRH, ACTH and catecholamines with a view to investigating the control of prolactin and gonadotropin secretion. LHRH immunoreactivity was found in both neuronal and non-neuronal cells in the outgrowth monolayer region of the explant. Neurones in this region appeared able to attach to a substrate and regenerate, in monolayer culture, well developed neurites characterized by beaded swellings as observed in vivo. Neither ACTH immunoreactivity nor catecholamine fluorescence was detected. Cultured neurones and astrocytes were labelled by tetanus toxin and anti-GFAP, respectively. Double labelling of cultures with tetanus toxin and anti-LHRH demonstrated the neuronal nature of many LHRH-immunoreactive cells. Radio-immunoassay data confirmed the presence of LHRH in the cultures but application of 60 mM KC1 failed to evoke hormone release. These studies have confirmed the view of previous workers that hypothalamic control of prolactin secretion in the bovine may be very different from that thought to occur in non-ruminants such as the rat and guinea pig. Finally, this work demonstrates that a cultured system from the mature bovine may prove a good model for study of neuronal regulation of gonadotropin secretion by the bovine mediobasal hypothalamus.
Adrenocorticotropic Hormone↗
Influence of vasopressin upon firing patterns of supraoptic neurons: a comparison of normal and Brattleboro rats.
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Action Potentials↗
The supraoptic nucleus as an osmoreceptor.
The "Progress' review is written to challenge the prevalent belief that neurones of the supraoptic nucleus are not involved in the reception of osmotic stimuli. Evidence is presented that these neurones are depolarised by physiological increases in the osmotic pressure of the extracellular fluid. However, generation of action potentials in these depolarised cells still requires the presence of synaptic input from currently unidentified neurones. We suggest that the osmoreceptor regulating vasopressin secretion is probably composed of more than one type of neurone and discuss this suggestion in the context of selected experiments previously cited as evidence that the supraoptic nucleus is not involved in osmoreception.
Animals↗
Supraoptic neurones of rat hypothalamus are osmosensitive.
It has been found that cells in the region of the supraoptic nucleus (SON) of the hypothalamus are sensitive to osmotic stimuli in a physiological range, and have studied the mechanism by which the osmotic sensitivity arises. An in vitro hypothalamic brain slice preparation has been used to make intracellular recordings from the SON. Cells lying in the SON respond to small increases (9--40 mosmol l-1) in the osmolarity of their external environment with a marked increase in firing rate. They respond to NaCl and mannitol although not to glucose. The osmotic sensitivity of SON neurones has a complex origin at both a pre- and a postsynaptic level, being composed first of depolarization of the primary SON neurone by an increase of extracellular osmolarity and second by an increased rate of occurrence of excitatory synaptic events which markedly augment firing rate. These results are consistent with Jewell and Verney's suggestion that osmoreception in the mammalian brain occurs in the region of the anterior hypothalamus, and extends this localization by indicating that the SON neurones are themselves both directly osmosensitive and part of an osmoreceptive complex.
Action Potentials↗