Q.Z. Yang

Electrophysiology of excitatory and inhibitory afferents to rat histaminergic tuberomammillary nucleus neurons from hypothalamic and forebrain sites

Anatomical evidence exists for projections to the tuberomammillary nucleus (TM) from the nucleus of the diagonal band of Broca (DBB) and the lateral preoptic area (LPO). The physiological effects of activating these inputs were studied by recording postsynaptic responses intracellularly from TM cells during both electrical stimulation and local nanodrop application of glutamate in horizontally cut brain slices. Electrical stimulation of the DBB, LPO and anterior lateral hypothalamic area (LH) usually evoked fast IPSPs (approximately 75% of responses) which were blocked by bicuculline or picrotoxin, suggesting GABA(A) mediation. The remaining excitatory responses evoked by stimulation of the LPO and LH were blocked by non-NMDA receptor antagonists (CNQX or NBQX) and the NMDA receptor antagonist, AP-5. Glutamate applied to the above areas induced postsynaptic responses in TM cells similar to those seen with electrical stimulation. Spontaneous firing in TM cells was suppressed by glutamate applied in the DBB. Glutamate applied in the LPO or LH evoked both inhibitory and excitatory responses. Changes in PSPs and firing rates were interpreted to result from glutamate activation of the neurons in the DBB, LPO and LH areas with inhibitory or excitatory connections to recorded TM neurons. These results support previous anatomical findings and suggest that inhibitory and excitatory synaptic control of TM activity is exerted by the DBB, LPO and LH areas.

Histamine mediates fast synaptic inhibition of rat supraoptic oxytocin neurons via chloride conductance activation

Axons from the histaminergic neurons of the tuberomammillary nucleus project to both the anterior and tuberal portions of the supraoptic nucleus. Histamine is known to activate vasopressin neurons via a histamine receptor subtype 1 and to increase release of vasopressin, but effects on oxytocin neurons have been previously unexplored. Here we investigated the effects of tuberomammillary nucleus electrical stimulation as well as of histamine antagonists on supraoptic nucleus oxytocin and vasopressin neurons in slices of rat hypothalamus. Electrical stimulation evoked short constant latency (approximately 5 ms), fast (4-6 ms onset to peak) inhibitory postsynaptic potentials in oxytocin neurons and, as shown previously, fast excitatory postsynaptic potentials in vasopressin neurons. These synaptic responses followed paired-pulse stimulus frequencies up to 100 Hz and were, thus, probably reflecting monosynaptic connections. Inhibitory postsynaptic potentials were selectively blocked by histamine receptor subtype 2 antagonists (either cimetidine or famotidine) and by picrotoxin but not by histamine receptor subtype 1 antagonists or bicuculline. Similar synaptic responses to tuberomammillary nucleus stimulation were found in 16 of 16 neurons immunocytochemically identified as oxytocinergic and in seven putative oxytocin neurons. Perifusion of the slice with low chloride medium (4.8 mM) reversed stimulus-evoked inhibitory postsynaptic potentials. We conclude that histaminergic neurons monosynaptically contact both oxytocin and vasopressin cells of the supraoptic nucleus and inhibit the former via activation of chloride channels which can be blocked by the histamine receptor subtype 2 antagonists, famotidine and cimetidine.

Direct evidence for electrical coupling among rat supraoptic nucleus neurons

Transfer of the fluorescent dye, Lucifer yellow (LY), from an intracellularly injected neuron to one or more other neurons is accepted as indirect evidence of electrotonic interactions among such dye coupled cells. Direct evidence requires that at least two coupled cells be recorded from simultaneously and such evidence in the CNS has been gained only for hippocampal pyramidal neurons. Since interpretations of the functional significance of dye coupling among magnocellular neuroendocrine cells depend upon its relation to electrical coupling, we sought to obtain direct evidence for electrotonic interactions in such neurons. Over 150 pairs of supraoptic nucleus (SON) neurons in hypothalamic slices were recorded from intracellularly using one LY and one potassium acetate electrode in each instance. Of these, 9 pairs were studied in sufficient detail to determine that they were electrically coupled. Most of the remaining pairs were determined not to be coupled. In each coupled pair of cells, membrane voltage changes due to spontaneously occurring or current evoked action potentials, as well as current evoked hyperpolarizations, in one cell were reflected in similar, though attenuated changes in the other cell. All of these changes occurred simultaneously in the two neurons. Spontaneously arising postsynaptic potentials in the two cells were temporally uncorrelated. In each case that electrical coupling was observed, dye coupling resulted from LY injection. Coupling ratios ranged from 0.05 to 0.2. Capacitative coupling between the recording electrodes as an artifact was ruled out since cells in the same tissue penetration as the coupled cell showed no responses to membrane voltage changes in the primary cell; no responses were seen with the second electrode placed extracellularly or in the medium; and similar coupling potentials were also seen when one cell was recorded without a second electrode present. We conclude that electrical coupling exists among magnocellular neurons of the SON and that the incidence of dye coupling is a reasonable estimate of the incidence of electrical coupling. These electrotonic interactions probably play important roles in the coordination of firing among magnocellular neurosecretory neurons.

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.

Magnocellular tuberomammillary nucleus input to the supraoptic nucleus in the rat: anatomical and in vitro electrophysiological investigations.

Anatomical and electrophysiological methods were used to investigate the existence and role of inputs from the magnocellular tuberomammillary nucleus to the supraoptic nucleus. After injecting either Fluoro-Gold or rhodamine-labeled latex microspheres into the supraoptic nucleus, consistent patterns of retrogradely labeled neurons within the tuberomammillary nucleus were observed. The results indicate that both subdivisions of the supraoptic nucleus, the tuberal and the anterior, receive input from the tuberomammillary nucleus. Injections into the tuberal supraoptic nucleus tended to label more cells in the contralateral tuberomammillary nucleus, while injections into the anterior supraoptic nucleus may label more cells on the ipsilateral side. The in vitro intracellular electrophysiological results support the anatomical findings and extend them in several ways. Some tuberomammillary neurons were found to project to the supraoptic nuclei on both sides of the brain. Intracellular Lucifer Yellow injections into tuberomammillary cells after electrophysiological recording revealed labeled axons that were traceable into the supraoptic nucleus, where apparent varicosities (possible en passant terminals) were seen. Magnocellular tuberomammillary nucleus neurons had characteristic passive and active membrane properties and morphology, similar to histaminergic neurons in this area studied by other workers. Finally, in two of the 21 cases, Lucifer Yellow injection into one neuron revealed dye-coupled pairs of tuberomammillary neurons. Previous work by others has shown that histamine excited cells in the tuberal subdivision of the supraoptic nucleus, stimulating vasopressin release, and that the tuberomammillary nucleus provides histaminergic input to the anterior portion of the supraoptic. The present findings show that the tuberomammillary nucleus supplies input to both subdivisions of the supraoptic nucleus and that this input is provided bilaterally. Taken together with previous work, these data suggest that the tuberomammillary nucleus provides histaminergic input to the supraoptic nucleus and may be involved specifically with vasopressin release.

Activation of excitatory amino acid inputs to supraoptic neurons. I, Induced increases in dye-coupling in lactating, but not virgin or male rats

Mitral cells of the main and accessory olfactory bulbs have been shown to project monosynaptically to the supraoptic nucleus (SON) via the lateral olfactory tract (LOT) which uses excitatory amino acid transmitters. Data collected during characterization of these projections suggested that synaptic activation of SON neurons via LOT stimulation in slices influenced the incidence of dye-coupling. The present study pursued this suggestion using horizontally cut slices from male, virgin female and lactating rats. Neurons were confirmed to be excited by electrical stimulation of the tract, injected with Lucifer yellow, and synaptically activated for 10 min at 10 Hz (n = 92). Another 94 neurons were similarly confirmed and injected, but received no further stimulation. In an additional 8 slices, injected neurons were antidromically activated for 10 min at 10 Hz. Analyses done on 194 injected neurons from the 3 groups showed that synaptic activation resulted in a significant (P less than 0.01) increase in the incidence of coupling only in tissue from lactating rats. This increase was entirely due to larger numbers of cells being coupled dendrodendritically to the injected cells in the stimulated slices. Antidromic activation did not influence coupling. Increased coupling occurred among both oxytocin and vasopressin cell types. This is the first report of increased coupling resulting from synaptic activation in mammalian CNS. Changes seen only in lactating rats may be related to their altered SON ultrastructural morphology (i.e. dendritic bundling). Strong olfactory and vomeronasal input associated with some maternal behaviors may increase neuronal coupling and enhance hormone release in response to other incoming stimuli (e.g. suckling, dehydration).

NMDA and non-NMDA receptors on rat supraoptic nucleus neurons activated monosynaptically by olfactory afferents.

The recently discovered efferent projections from the main and accessory olfactory bulbs to the supraoptic nucleus (SON) were further investigated. Intracellular electrophysiological methods were used to determine (a) if these projections are monosynaptic, (b) which excitatory amino acid (EAA) receptor subtypes mediate responses to activation of these pathways and (c) whether the same receptor subtypes mediate responses of phasically firing (vasopressin) and continuously firing (putative oxytocin) neurons. Recordings were made from SON neurons in large explants and 500 microns thick horizontal slices, containing 2-6 mm of the piriform cortex and lateral olfactory tract (LOT). This allowed recording of synaptic responses to selective stimulation of the LOT. EPSPs in SON neurons faithfully followed stimulus frequencies of 50-100 Hz, indicating that these inputs were monosynaptic. Stimulus-evoked EPSPs were blocked by the non-specific EAA antagonist, kynurenate. Perifusion of the slice with Mg(2+)-free medium revealed the presence of NMDA receptors in addition to the non-NMDA receptors on both phasically and continuously firing cells, indeed, on all cells tested. Partial blockade of these EPSPs in Mg(2+)-free medium could be achieved with either the NMDA antagonist, AP5, or the non-NMDA antagonist, CNQX or NBQX. Full blockade of the stimulus-evoked EPSPs was effected by adding both types of antagonists to the medium, although spontaneous EPSPs were still observed in several cells. These results are consistent with prior studies showing both receptor subtypes in the SON. This is the first demonstration that afferent stimulation activates both subtypes in the same SON neuron regardless of its peptide content.

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.

Supraoptic nucleus afferents from the main olfactory bulb--II. Intracellularly recorded responses to lateral olfactory tract stimulation in rat brain slices.

To establish the functional nature of the anatomically demonstrated main olfactory bulb inputs to the supraoptic nucleus, electrophysiological responses of intracellularly recorded supraoptic neurons to lateral olfactory tract stimulation were recorded in horizontal slices of basal forebrain and hypothalamus. A total of 71 synaptically influenced neurons were studied in slices from adult rats of both sexes. Of these, 60 cells (84%) were monosynaptically activated by olfactory tract stimulation; seven cells (10%) were activated via polysynaptic pathways; and four cells (6%) were characterized by long latency inhibitory responses. Lucifer Yellow was injected into 64 cells and subsequent immunocytochemical identification of 44 of these neurons showed that both oxytocin and vasopressin cells, in approximately equal numbers, were excited by olfactory stimulation. Polysynaptically mediated excitation, however, was only associated with oxytocin cells (six of the six identified cells). These results corroborate anatomical tract tracing data showing main olfactory bulb efferents to both supraotic neurons and to neurons of the perinuclear zone. Also supported are earlier speculations of olfactory participation in release of oxytocin and vasopressin during various physiological states.

Dye coupling among supraoptic nucleus neurons without dendritic damage: differential incidence in nursing mother and virgin rats

To assess the possibility that dye coupling among neurons in hypothalamic slices might require dendrotomy, as has been suggested for neocortical neurons, dye coupling was studied in horizontally cut slices containing the supraoptic nucleus (SON). Since the dendrites of SON neurons project toward the pial surface, dendritic damage due to slicing can be avoided in the horizontal plane. Intracellular injections of Lucifer Yellow into individual SON neurons in slices from male, virgin female and lactating, mother rats yielded the following results. When dendrotomy occurred there was a significantly lower incidence of dye coupling than was observed when dendrites were intact. Higher order coupling (3 or more cells dye coupled after a single injection) was only seen among neurons without dendrotomy. Independently of dendritic damage, incidence of dye coupling in nursing mothers was reliably greater than for virgins, confirming previous results from coronal slices. The results of this study indicate that dendrotomy is not an inducer of dye coupling in SON neurons. Taken together with other recent findings, these data suggest that a reinterpretation of the effects of dendrotomy on cortical cell dye coupling may be in order.