Peter Cobbett

Immunoreactivity to vasopressin- but not oxytocin-associated neurophysin antiserum in phasic neurons of rat hypothalamic paraventricular nucleus

Bursts of action potentials were recorded intracellularly from 11 phasically firing magnocellular neurons in the paraventricular nucleus in slices of rat hypothalamus. The bursts of overshooting, often broadening action potentials (63-87 mV peak-to-peak) were superimposed on depolarizing plateau potentials. Phasic activity was recorded before and/or after the neurons were injected with the fluorescent dye Lucifer Yellow CH. Injected neurons were first examined in whole slices, and subsequently, in sectioned material, characterized immunocytochemically using antisera to vasopressin- and oxytocin-associated neurophysins (VP-NP and OT-NP respectively). The 11 injections produced 8 single dye filled neurons and 3 pairs of dye-coupled neurons, 14 dye-filled cells in all. Six of the single cells and all the dye coupled pairs were immunoreactive with VP-NP antiserum and not reactive with OT-NP antiserum. Most of these neurons were in areas of the nucleus in which VP-NP reactive cells predominated, but two were surrounded by OT-NP reactive cells. Two single, dye-filled, phasically active, magnocellular neurons failed to show immunoreactivity to either antiserum.

Dye coupling among immunocytochemically identified neurons in the supraoptic nucleus: Increased incidence in lactating rats

The hypothesis that electrotonic spread among oxytocinergic neurons contributes to synchronized bursting in the lactating rat leads to the prediction that coupling among oxytocinergic neurons would be stronger and more abundant in lactating than in non-lactating animals. We tested this prediction using, as an index of electrical coupling, transfer among neurons of the fluorescent dye Lucifer Yellow CH, which crosses gap junctions. Intracellular injections (total of 159) of the dye were made in supraoptic nucleus neurons in hypothalamic slices from virgin female and lactating rats. In virgins, 86 injections resulted in 76 single, 8 coupled pairs and 2 triplets of dye-filled neurons. In contrast, 73 injections in lactators yielded 51 single, 16 coupled pairs and 6 triplets, (greater than 100% increase) a difference significant at P less than 0.001. Immunocytochemical identification of the dye-filled cells revealed that there was an increase over virgins in coupling among both oxytocinergic and vasopressinergic neurons. These results are consistent with the hypothesis that electrical coupling is involved in synchronizing oxytocin cell bursting in lactators. They are also consistent with published data indicating that vasopressin neurons are metabolically activated (show increased glucose uptake) during suckling and may show correlated activity.

Characterization of three types of potassium current in cultured neurones of rat supraoptic nucleus area.

1. Whole‐cell, voltage‐clamp recordings were obtained from neurones of the supraoptic area of neonatal rats in dissociated cell culture. Recordings were made from neurones having the same morphology as those which were vasopressin or oxytocin immunoreactive. 2. Three types of voltage‐activated K+ current were identified on the basis of their kinetics, voltage sensitivities, Ca2+ dependence and pharmacology. The currents corresponded to the delayed rectifier current (IK), the A‐current (IA), and the Ca2+‐dependent current (IK(Ca] described in other neurones. 3. IK had a threshold of ‐40 mV, a sigmoidal time course of activation, and was sustained during voltage steps lasting less than 300 ms. The underlying conductance was voltage dependent reaching a maximum at +30 mV (mean maximum conductance 4.09 nS). The activation time constant was also voltage dependent declining exponentially from 4.5 ms at ‐30 mV to 1.8 ms at +50 mV. 4. IA was transient, and was activated from holding potentials negative to ‐70 mV; the maximum conductance (mean 5.9 nS) underlying the current was obtained at +10 mV. The activation and inactivation time constants were voltage dependent: the activation time constant declined exponentially between ‐40 mV (2.2 ms) and +40 mV (0.65 ms). 5. IK and IA were attenuated by the K+ channel blockers tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP). TEA blocked the conductance underlying IK but appeared to alter the kinetics of IA. In contrast, 4‐AP blocked the conductance underlying IA and, to a lesser extent, IK. 6. IK and IA were activated independently of external Ca2+ and the voltage activation of Ca2+ channels since these currents were recorded in the presence of Co2+, a Ca2+ channel blocker. 7. IK(Ca) was recorded only when Ca2+ (2 mM) was present in the external medium. From a holding potential of ‐30 mV, IK(Ca) had a threshold of ‐20 mV, was maximal at about +20 mV and declined at more positive potentials. This current was sustained during voltage steps lasting 100 ms and was abolished by addition of Co2+ (2 mM) to the medium. 8. The possible roles of the three K+ currents in regulating the characteristic firing behaviour of supraoptic neurones previously recorded in vivo and in vitro are discussed.

Dye-coupled magnocellular peptidergic neurons of the rat para ventricular nucleus show homotypic immunoreactivity

Magnocellular neurons in rat hypothalamic slices are known to exhibit dye coupling: the transfer of the fluorescent dye, Lucifer Yellow, from an intracellularly-injected neuron to one or more nearby neurons. The question of the hormonal identity of coupled cells and the possibility of dye coupling as an artefact led us to determine the immunoreactivity of dye-coupled magnocellular neurons in the paraventricular nucleus of the rat hypothalamus using antisera to oxytocin- and vasopressin-associated neurophysins. In 23 pairs, one triplet, and one quadruplet, immunoreactivity to one or the other antiserum was always exclusive, and dye coupling was always homotypic, that is, coupled neurons in each instance were reactive to the same antiserum. The quadruplet, triplet and 17 pairs were immunoreactive to vasopressin-associated neurophysin, and oxytoxin-associated neurophysin immunoreactivity was observed in the remaining pairs. Immunoreactivity to each antiserum was found for somasomatic and non somasomatic modes of coupling and for coupled neurons in the three magnocellular areas of the nucleus. A relationship between mode of coupling and hormone content was not detected. The data support the hypothesis that coupling is a real, functionally significant mechanism for coordinating neuronal activity in this nucleus, particularly under conditions of high hormone demand. They do not support the idea that coupling is artefact. The possibility of a relationship between hormone content and mode of coupling, and the projection pathway(s) of the coupled neurons of each type require further study.

Extranuclear axon collaterals of paraventricular neurons in the rat hypothalamus: intracellular staining, immunocytochemistry and electrophysiology.

Recent studies have suggested that some paraventricular nucleus (PVN) neurons projected to more than one target and, thereby, perhaps coordinate some aspects of seemingly diverse functions. We have systematically investigated the existence, location, hormonal contents and functional integrity of some axon collaterals arising from PVN neurons. This was done using intracellular injections of the fluorescent dye, Lucifer Yellow, extracellular ejections of horseradish peroxidase (HRP), immunocytochemistry with antisera directed against vasopressin (VP) and oxytocin (OX) and electrophysiological analysis of synaptic activation of perifornical neurons in response to electrical stimulation of the PVN in hypothalamic slices. Each of the three morphological techniques revealed clear axon collaterals, arising in the lateral hypothalamus and generally ventrolateral to the PVN. Most branching axons appeared to have a small number of branch points, and many collaterals appeared to terminate near their parent axon. Electrical stimulation of the PVN was found to activate synaptically perifornical neurons located in the areas where the other methods revealed collaterals. Stimulation outside of the nucleus was ineffective unless current intensities were increased 10-30-fold over those applied to the PVN. We conclude that many PVN neurons, at least some of these containing OX and other VP, give rise to axons that branch in the perifornical and more ventral lateral hypothalamus, and that some of their collaterals probably terminate on neurons close to the PVN.

Incidence of dye coupling among magnocellular paraventricular nucleus neurons in male rats is testosterone dependent

Recently published work in the rat has shown that: the incidence of electrical coupling, as measured by dye coupling, is decreased from control levels by 8 days of drinking hypertonic saline; an index of circulating testosterone, seminal vesicle weight, is also decreased by 8 days of saline drinking; and both plasma and urinary vasopressin levels are reduced in castrated males, but can be returned to normal with testosterone replacement. These findings have led to the hypothesis that dye coupling, particularly that involving vasopressinergic cells, may be affected by gonadal steroids. We have investigated the effects of castration and testosterone replacement on the incidence of dye coupling among the neurons of the predominantly vasopressinergic magnocellular lateral paraventricular nucleus in slices of male rat hypothalamus. Incidence of dye coupling in this nucleus of castrated rats was found to be decreased by 67% from sham castrated control levels. Testosterone-filled Silastic capsules (but not empty capsules) implanted subcutaneously at the time of castration abolished the effect of castration on dye coupling. We conclude that testosterone has a powerful influence upon coupling among PVN vasopressinergic neurons and may participate in the control of vasopressin release in intact animals.

A reliable method for immunocytochemical identification of Lucifer Yellow injected, peptide-containing mammalian central neurons

An immunocytochemical procedure is described for reliably determining the hormone content of magnocellular neuroendocrine neurons that have been injected with Lucifer Yellow in slices of rat hypothalamus. The chief advantages of this procedure over others currently available are: (a) it permits whole mount observation of the tissue, and thus, of the morphology of filled cell(s) as well as of such phenomena as dye-coupling; (b) the reliability of tissue preparation and peptide determination has been optimized so that about 85% of injected cells are identified immunocytochemically; and (c) the final immunostained product is permanent, permitting bright-field examination of the injected cell. Relative advantages and limitations of this and other recently published methods are discussed.

Outward potassium currents of supraoptic magnocellular neurosecretory cells isolated from the adult guinea‐pig

1 Several types of whole‐cell outward K+ current recorded from magnocellular neurosecretory cells (MNCs) dissociated from the supraoptic nucleus of the adult guinea‐pig were identified on the basis of their voltage dependence, kinetics, pharmacology and Ca2+ dependence. 2 The predominant K+ current evoked from a holding potential of −40 mV was slowly activating, long‐lasting, tetraethylammonium (TEA) sensitive and showed little steady‐state inactivation. Also, this current was reduced by extracellular Cd2+. These data suggest that in supraoptic MNCs classical Ca2+‐insensitive, delayed rectifier channels (Kv) and Ca2+‐sensitive, non‐inactivating channels (KCa) both contribute to the sustained current. 3 A transient, low‐threshold K+ current, which was 4‐aminopyridine (4‐AP) sensitive and showed significant steady‐state inactivation, was evoked along with the sustained current from a holding potential of −90 mV. Based on these characteristics, this current corresponds to the A‐current (IK(A)) described in other neurons. 4 I K(A) was activated when Ca2+ influx was blocked or when Ca2+ was absent from the extracellular medium, suggesting that Ca2+ influx is not necessary for activation of the current. 5 In many recordings, a transient 4‐AP‐insensitive outward current was evoked from a holding potential of −40 mV. This high‐threshold transient K+ current was abolished by extracellular Cd2+ or TEA and was absent when extracellular Ca2+ was replaced by Sr2+, suggesting that it is a transient Ca2+‐dependent K+ current. 6 We conclude that the presence of multiple types of K+ current may, in part, underlie the complex firing patterns of oxytocinergic and vasopressinergic MNCs.

Sodium and potassium currents involved in action potential propagation in normal bovine lactotrophs.

1. The properties of whole‐cell and single‐channel Na+ and K+ currents in immunocytochemically identified bovine lactotrophs were studied using the patch‐clamp technique. 2. In the whole‐cell, current‐clamp mode, cells had membrane potentials of ‐94.7 +/‐ 6.7 mV and input resistances of 2‐17 G omega. Current‐induced action potentials were recorded with a threshold around ‐35 mV and amplitude of 40‐65 mV. Repetitive firing was not sustained at frequencies greater than 1‐2 Hz without total inactivation. 3. Under voltage clamp, action potentials were shown to be composed of an inward TTX‐sensitive Na+ current and an outward K+ current that was abolished by internal Cs+. 4. The isolated Na+ current had a threshold for activation around ‐35 mV and rapidly inactivated to a steady state during a test voltage pulse. Inactivation was strongly voltage‐dependent, with the Na+ current being half‐inactivated at ‐20 mV. 5. Recovery from inactivation was voltage dependent and at a holding potential of ‐60 mV, 50% reactivation was achieved after 420 ms. The implications of this long reactivation time on sustained action potential frequency are discussed. 6. Single Na+ channel activity was examined with the outside‐out patch configuration and yielded single‐channel conductances of 22.5 pS. Reconstruction of the voltage and time dependence of single‐channel currents provided an accurate picture of the whole‐cell Na+ current. 7. Whole‐cell outward current carried by K+ in the absence of Na+ and Ca2+ had a large conductance, was slowly activated and demonstrated no inactivation. A second, more rapidly activating Ca2+‐dependent K+ current could also be demonstrated. 8. Ensemble analysis of whole‐cell K+ currents in the absence of Ca2+ showed underlying single‐channel amplitudes of 1.2 pA at +10 mV, with the lactotroph having about 350 active channels at this potential. 9. Recordings of single K+ channels also demonstrated two classes of channel: a small (50 pS) voltage‐activated channel and a higher‐conductance (100 pS) Ca2+‐ sensitive channel. 10. Prolactin secretion was shown to be TTX‐insensitive but sensitive to membrane potential, demonstrated as increased release following increased external K+ but not Na+ concentration.(ABSTRACT TRUNCATED AT 400 WORDS)

γ-Aminobutyric acid as an inhibitory neurotransmitter in the rat supraoptic nucleus: Intracellular recordings in the hypothalamic slice

Intracellular recordings have been made from rat supraoptic neurones in the hypothalamic slice preparation. Application of gamma-aminobutyric acid (GABA) caused all neurones to hyperpolarise and this was accompanied by an increase in membrane conductance. GABA application examined on a variety of cells was found to have a potent influence on patterning of electrical activity, always consistent with an inhibitory action.