Gamma-aminobutyric acidB receptors and the development of the ventromedial nucleus of the hypothalamus

Gamma-aminobutyric acid (GABA) is a highly abundant neurotransmitter in the brain and the ligand for GABA(A), GABA(B), and GABA(C) receptors. Unlike GABA(A) and GABA(C) receptors, which are chloride channels, GABA(B) receptors are G-protein linked and alter cell-signaling pathways. Electrophysiological studies have found GABA(B) receptors in cultured embryonic hypothalamus, but the distribution of these receptors remains unknown. In the present study, we examined the expression of GABA(B) receptors in the ventromedial nucleus of the hypothalamus (VMH) during embryonic mouse development. GABA(B) receptors were present in the VMH at all ages examined, from embryonic day 13 to postnatal day 6. Using a brain slice preparation, we examined the effect of GABA(B) receptor activation on cell movement in the embryonic VMH as the nucleus forms in vitro. The GABA(B) receptor agonist baclofen decreased the rate of cell movement in a dose-dependent manner. Baclofen reduced cell movement by up to 56% compared with vehicle-treated controls. The percentage of cells moving per field and the angles of cell movement were not affected. With our previous findings of GABA(A) receptor activation, it is likely that GABA influences VMH development via multiple mechanisms.     

Amygdaloid and pontine projections to the ventromedial nucleus of the hypothalamus

Amygdaloid and pontine projections to the feline ventromedial nucleus of the hypothalamus (HVM) were studied with retrograde transport of horseradish peroxidase (HRP) and anterograde transport of tritiated amino acids. Following injections of HRP into HVM, amygdaloid neurons were labeled in the ipsilateral cortical and medial nuclei and the ventral portion of the parvocellular part of the basal nucleus. In experiments in which HRP was injected into the tuberal hypothalamus following stria terminalis lesions, it was determined that amygdaloid neurons projecting to HVM by way of the stria terminalis were located in the cortical and medial nuclei while those projecting through another route, presumably the ventral amygdalofugal pathway, were found in the rostral part of the medial nucleus and the parvocellular basal nucleus. Following HRP injection into lateral hypothalamus at the level of HVM, labeled neurons were seen in the magnocellular basal nucleus. After preoptic injections, neurons containing the HRP reaction product were in cortical and medial nuclei and magnocellular and parvocellular parts of the basal nucleus. In addition to cells in the amygdala, rostral pontine neurons were labeled after HRP injections into HVM. The cells were located ipsilateral to the injection, mostly in the dorsal nucleus of the lateral lemniscus, lateral and dorsolateral to the brachium conjunctivum. The pontine cells labeled following HVM injections of HRP were different from those labeled following lateral hypothalamic and preoptic region injections. The pontine projection to HVM was confirmed using axoplasmic transport autoradiography. A mixture of tritiated leucine and tritiated proline was injected into the lateral pontine region labeled after HRP injections into HVM. Labeled axons ascending in the medial forebrain bundle terminated throughout the rostro-caudal extent of HVM.     

Responses to cholecystokinin in the ventromedial nucleus of the rat hypothalamus in vivo

The peptide cholecystokinin (CCK) is a short‐term satiety signal released from the gastrointestinal tract during food intake. From the periphery, CCK signalling travels via the vagus nerve to reach the brainstem from which it is relayed higher into the brain. The hypothalamus is a key integrator of appetite‐related stimuli and the ventromedial nucleus of the hypothalamus (VMN) is thought to have an important role in the regulation of satiety. We investigated the effect of intravenous injections of CCK on the spontaneous firing activity of single VMN neurons in urethane‐anaesthetised rats in vivo. We found that the predominant effect of CCK on the electrical activity in the VMN is inhibitory. We analysed the responses to CCK according to electrophysiologically distinct subpopulations of VMN neurons and found that four of these VMN subpopulations were inhibited by CCK, while five were not significantly affected. Finally, CCK‐induced inhibitory response in VMN neurons was not altered by pre‐administration of intravenous leptin.     

Ibotenic acid lesions reduce noradrenergic activation in ventromedial hypothalamus during hypoglycemia

Noradrenergic and GABAergic systems in the ventromedial hypothalamus (VMH) are activated during hypoglycemia and initiate part of the compensatory counterregulatory response. Norepinephrine (NE) terminals innervating the VMH originate in glucosensing hindbrain areas, but whether NE activity in the VMH is under local control or in the hindbrain is unclear. To elucidate the role of neurons intrinsic to the VMH on NE release in the VMH during hypoglycemia, ibotenic acid (IBO), an NMDA receptor agonist that selectively destroys cell bodies, was used. In a 2 x 2 factorial study, IBO (3-5 microg/0.5 microL) or vehicle was stereotaxically administered into the VMH of male Sprague-Dawley rats. One week later, NE concentration in the VMH was measured by microdialysis during insulin-induced hypoglycemia (2.0 U/kg) or euglycemia (saline control). Baseline levels of NE were not statistically different (p=0.10) in IBO-treated compared with vehicle-treated rats (13.3+/-2.8 nM vs. 7.9+/-1.1 nM). The initial increase in interstitial NE concentration during hypoglycemia in control rats was absent in IBO-treated rats (p<0.01). In IBO-treated hypoglycemic rats, NE concentrations increased after 45 min to a similar level observed in control rats during the first 20 min of hypoglycemia. These results are consistent with the suggestion that local neurons in the VMH respond to hypoglycemia and modify NE activation in the VMH during hypoglycemia.     

Age-dependent expression of microtubule-associated protein 2 in the ventromedial nucleus of the hypothalamus

A distinctive developmental pattern of microtubule-associated protein 2 (MAP2) was detected in the ventromedial hypothalamus of rats. A region of more intense MAP2 immunoreactivity in this nucleus was present at birth, became prominent in a ringed appearance by postnatal day 4 and disappeared in the third postnatal week. This period of selective MAP2 expression in particular subcortical regions may signify important functions of MAP2 in the stabilization of developing dendritic structure.     

Gonadal steroids regulate immunoreactive tachykinin in the ventromedial nucleus of the rat hypothalamus

The ventromedial nucleus of the hypothalamus (VMH) is an important site in the mediation of female receptive behavior (lordosis); however, the sequence of events that eventually facilitate this estrogen-dependent behavior is only partially understood. Here, evidence is presented, suggesting that an interaction between gonadal steroids and tachykinin peptides is involved. Tachykinin-immunoreactive (TACir) neurons were distributed throughout the anterior (VMHa), dorsomedial (VMHdm), central (VMHc), and ventrolateral (VMHvl) subdivisions in the adult male and female rat. Numbers of cells in the VMH of intact males and males that had been castrated for three weeks were similar. Among females, numbers of TACir cells in the VMHvl were significantly increased over numbers seen in ovariectomized females by a two day (1.5X) and by a two week (1.6X) exposure to estrogen. In the VMHa, numbers of TACir cells were also significantly higher in females that received replacement estrogen compared to ovariectomized females. Since estrogen concentration by TACir neurons is common in the VMHvl and rare in the VMHa, estrogen may have both direct and indirect effects on tachykinin synthesis. In the caudal pole of the VMH, staining intensity of TACir fibers varied with endocrine state. In intact males, castrated males, and ovariectomized females, fibers were lightly stained. In females exposed to estrogen for two days, fibers were moderately intense, and in females exposed to estrogen for two weeks, they formed a darkly stained plexus. These responses to estrogen suggest that production of tachykinin peptides in the VMH is involved in facilitation of female typical sexual behavior.     

Identification of urocortin 3 afferent projection to the ventromedial nucleus of the hypothalamus in rat brain

Urocortin 3 (Ucn 3) is a corticotrophin-releasing factor related neuropeptide highly expressed in the brain. Ucn 3 nerve fibers heavily innervate the hypothalamic ventromedial nucleus (VMH), and Ucn 3 injection into the VMH suppresses feeding. Currently, the origin of the Ucn 3 afferent input into the VMH is unknown. In the present study, anatomical tracing shows that the major Ucn 3 afferent input to the VMH resides in the anterior parvicellular part of the paraventricular nucleus of the hypothalamus (PVHap) and the adjacent posterior part of the bed nucleus of stria terminalis (pBNST). VMH also receives moderate Ucn 3 input from the medial amygdala. Ucn 3 neurons located immediately caudal to the PVHap/pBNST in the rostral perifornical hypothalamic area (rPFH) provide only minimal input. The paucity of rPFH-VMH Ucn 3 projection is consistent with the finding that only Ucn 3 neurons in the rPFH co-expressed enkephalin (Enk), and Ucn 3/Enk double-labeled nerve fibers and terminals were observed predominately in the lateral septum (LS), whereas only a few double-labeled fibers were found in other brain areas including the VMH. Furthermore, retrograde tracing demonstrates that Ucn 3 neurons in the rPFH project to the LS. In conclusion, the present study determines that the major Ucn 3 afferent into the VMH originates from the PVHap/pBNST. Moreover, anatomical heterogeneity is observed in the hypothalamic Ucn 3 neuron population as the rostral part (PVHap/pBNST) of the population projects to the VMH and the caudal part (rPFH) co-localizes with Enk and provides major afferent input to the LS.     

Intrinsic synapses in the ventromedial nucleus of the hypothalamus: an ultrastructural study

To test the ideas that neurons intrinsic to a hypothalamic region might participate in steroid hormone effects and that intrinsic synapses might have unusual morphological features, we deafferented the ventromedial nucleus of the hypothalamus (VMH) in ovariectomized female rats with a Halasz knife. Some rats were given estrogen; others were endocrine controls. The main result of this study was that a large number of synapses in VMH survived the circumscribing cut. In fact, they comprised between one-half and two-thirds the number counted in unoperated controls. These were apparently from neuron cell bodies intrinsic to the medial basal hypothalamic island. Their morphology was not clearly different from that expected from unoperated controls or from other brain regions. A significant estrogen effect, an increase in the number of axodendritic synapses per unit area as seen in animals without surgical transections, was not present in the deafferented groups. This suggests that the hormone effect is due to synapses on VMH neurons from cell groups outside the medial basal hypothalamic island. If functional effects of estrogen mediated through VMH do not rely on intrinsic neurons, they must use estrogen-concentrating neurons that project to other brain regions.     

An autoradiographic study of the efferent connections of the ventromedial nucleus of the hypothalamus

Efferent projections from the ventromedial nucleus of the hypothalamus (VMN) were traced using tritiated amino acid autoradiography in albino rats. Ascending fibers passed through the anterior hypothalamus. Labelled fibers and terminal fields were seen in the preoptic area, bed nucleus of the stria terminalis, substantia innominata, the anterior amygdaloid area, diagonal bands of Broca and lateral septum. Fibers also projected laterally from VMN and entered the supraoptic commissures and zona incerta. These lateral projections were responsible for the fibers observed in the cerebral peduncle, the amygdala, the thalamus and the reticular formation. Fibers descending in a medial position projected through the posterior hypothalamus and then swept dorsally to terminate in the mesencephalic and pontine central grey. A projection from VMN into the median eminence was noted. The overall patterns of projection from different parts of VMN were similar; differences that existed were primarily in the relative strengths of the different projections. The efferent projections from VMN are extensive, well organized, and would appear capable of supporting significant physiological actions on extra-hypothalamic structures.     

The efferent connections of the ventromedial nucleus of the hypothalamus of the rat.

The efferent connections of the ventromedial nucleus of the hypothalamus (VMH) of the rat have been examined using the autoradiographic method. Following injections of small amounts (0.4-2.0 muCi) of tritium labeled amino acids, fibers from the VMH can be traced forward through the periventricular region, the medial hypothalamus and the medial forebrain bundle to the preoptic and thalamic periventricular nuclei, to the medial and lateral preoptic areas, to the bed nucleus of the stria terminalis and to the ventral part of the lateral septum. Some labeled axons continue through the bed nucleus of the stria terminalis into the stria itself, and hence to the amygdala, where they join other fibers which follow a ventral amygdalopetal route from the lateral hypothalamic area and ventral supraoptic commissure. These fibers terminate in the dorsal part of the medial amygdaloid nucleus and in the capsule of the central nucleus. A lesser number of rostrally directed fibers from the VMH crosses the midline in the ventral supraoptic commissure and contributes a sparse projection to the contralateral amygdala. Descending fibers from the VMH take three routes: (i) through the medial hypothalamus and medial forebrain bundle; (ii) through the periventricular region; and (iii) bilaterally through the ventral supraoptic commissure. These three pathways are interconnected by labeled fibers so that it is not possible to precisely identify their respective terminations. However, the periventricular fibers seem to project primarily to the posterior hypothalamic area and central gray, as far caudally as the anterior pole of the locus coeruleus, while the medial hypothalamic and medial forebrain bundle fibers apparently terminate mainly in the capsule of the mammillary complex, in the supramammillary nucleus and in the ventral tegmental area. The ventral supraoptic commissure fibers leave the hypothalamus closely applied to the medial edges of the two optic tracts. After giving off their contributions to the amygdala, they continue caudally until they cross the dorsal edge of the cerebral peduncle to enter the zona incerta. Some fibers probably terminate here, but others continue caudally to end in the dentral tegmental fields, and particularly in the peripeduncular nucleus. Within the hypothalamus, the VMH appears to project extensively to the surrounding nuclei. However, we have not been able to find evidence for a projection from the VMH to the median eminence. Isotope injections which differentially label the dorsomedial or the ventrolateral parts of the VMH have shown that most of the long connections (to the septum, amygdala, central tegmental fields and locus coeruleus) originate in the ventrolateral VMH, and there is also some evidence for a topographic organization within the projections of this subdivision of the nucleus.     

Limbic projections to the ventromedial hypothalamus of the opossum

Limbic projection systems which influence neuronal activity of the ventromedial nucleus of the hypothalamus of the opossum were studied by an evoked potential method. Bipolar concentric recording electrodes were placed in the ventromedial nucleus to monitor activity while exploring the forebrain with a stimulating electrode delivering single rectangular pulses (0.5 msec, 0.4 ma, 0.5–1/sec). Responses in the ventromedial nucleus were consistently evoked by stimulating three forebrain systems. From the dorsal septum, responses with one monophasic component were evoked (latency 3–9 msec). Stimulation more ventrally in the septum evoked, in addition, a second slower component. Stimulation of the corticomedial amygdala produced biphasic responses in the ventromedial nucleus at latencies of 4–9 msec; stimulation of the stria terminalis, the efferent pathway from this division of the amygdaloid complex, yielded almost identical responses at 4- to 6-msec latencies. Responses in the ventromedial nucleus were also recorded to stimulation of the dorsal hippocampus and adjacent fimbria (latencies, 3–7 msec). These responses demonstrate three possibly direct limbic influences on the ventromedial nucleus of a metatherian mammal, and provide new data on the anatomic relationships of this behaviorally significant hypothalamic area.     

Concomitant regulation of running activity and metabolic change by the ventromedial nucleus of the hypothalamus

The aim of this study was to elucidate the involvement of kainate (KA) type glutaminergic, GABAergic and adrenergic receptors in the ventromedial nucleus of the hypothalamus (VMH) in inducing running activity and metabolic adaptations. Injection of either KA or bicuculline methiodide (BM), a GABA_A receptor antagonist, into the VMH of conscious rats resulted in an increase in plasma glucose, norepinephrine, epinephrine and corticosterone, as well as running activity. KA or BM increased plasma glucose and catecholamines even under urethane anesthesia. Co-injection of either α- or β-adrenergic receptor antagonist, i.e. phentolamine or timolol, respectively, with KA into the VMH of conscious rats elicited only a slight increase in plasma glucose and catecholamines, though it successfully induced hyper-running. However, plasma corticosterone was higher in the animals injected with adrenergic blockers, suggesting that an insufficient supply of energy substrates would enhance the activity of the hypothalamo-pituitary-adrenal system. We conclude that: (1) KA type glutaminergic and GABAergic receptors in the VMH are involved in regulating running activity and the sympathetic nervous system; (2) the brain noradrenergic system may mediate the KA action on the sympathetic nervous system.     

Spontaneous discharge characteristic of neurons in the ventromedial nucleus of the rat hypothalamus in vivo

The ventromedial nucleus of the hypothalamus (VMN) is one of the main central regulators of two vital behaviours in rat: feeding behaviour and sexual behaviour. To better understand how these behaviours are regulated in the brain requires assessing how physiological stimuli are encoded by the electrical activity of populations of neurons, but there is still little known about the electrical activity of neurons in the VMN, in particular how it is regulated in vivo . Here, we recorded spontaneous firing activity from single VMN neurons in urethane-anaesthetized rats in vivo , and characterized their electrophysiological identities. For each of 271 cells, we constructed hazard functions from interspike interval histograms to show how the excitability of the cell changes with time after a spike. We completed the statistical characterization of each cell by analysis of its mean firing rate and coefficient of variation, and features of its interspike interval distribution, including kurtosis and skew (around the mean and around the mode). We thereby identified nine subpopulations of neurons in the VMN, which we named according to the main features of their firing pattern. One of the subpopulations fires very regularly, another almost randomly and another in intermittent clusters of two–three spikes, but perhaps the most interesting subpopulation are 'oscillatory cells' whose activity seems to be governed by an extrinsic 3-Hz rhythm. Whether these electrophysiologically distinct populations are also functionally and neurochemically distinct has now to be tested.     

Local gamma-aminobutyric acid and glutamate circuit control of hypophyseotrophic corticotropin-releasing factor neuron activity in the paraventricular nucleus of the hypothalamus

Paraventricular corticotropin-releasing factor (CRF) neurons play a pivotal role in regulating neuroendocrine responses to stress. The mechanisms by which synaptic inputs control the activity of these neurons are not well understood. The present study was undertaken to determine the role of the intrinsic gamma-aminobutyric acid (GABA)- and glutamatergic neural circuits of the hypothalamic paraventricular nucleus (PVN) in the control of CRF neural activity. We show that in organotypic cultures of the PVN, blockade of the intrinsic GABAergic neurotransmission by the GABAA receptor antagonist bicuculline resulted in a significant increase in CRF secretion. The bicuculline-induced CRF secretory activity was abolished by the coadministration of the selective alpha-amino-3-hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Electrical stimulation of the CRF cell division elicited glutamatergic extracellular field potentials that were dramatically enhanced by bicuculline and were suppressed by CNQX. These results show that the functional activity of CRF neurons in organotypic cultures of the PVN is under a tonic inhibitory influence of an intrinsic GABAergic circuit. Suppression of GABAergic transmission appears to have a permissive role for inducing an increased secretory activity of CRF neurons that is driven by an excitatory glutamatergic network via AMPA/kainate receptors.     

Hyperprolactinemia: its electrophysiologic and pharmacologic effect on neurons of the ventromedial nucleus of the hypothalamus

Extracellular action potentials were recorded from 691 neurons in the ventromedial hypothalamus (VMH) of urethane anesthetized female rats under acute and chronic sham-operated and hyperprolactinemic conditions. Hyperprolactinemia was produced by transplanting pituitaries under the kidney capsules. Neuronal excitability was recorded and analyzed during spontaneous, baseline activity and following the iontophoretic application of prolactin, dopamine (DA) and luteinizing hormone-releasing hormone (LH-RH). No statistically significant changes were observed in the spontaneous electrical activity of VMH neurons under the conditions tested. Of the responsive neurons, approximately 90% of the neurons recorded and tested with prolactin displayed an increase in firing activity while DA produced a decrease, independent of the endogenous plasma prolactin levels (basal or elevated). However, the response to LH-RH was modified in the chronic hyperprolactinemic animal. The predominant response of VMH neurons to LH-RH in acute sham-operated and hyperprolactinemic as well as in chronic sham-operated animals was one of inhibition, while in the chronic hyperprolactinemic animal, the application of LH-RH initiated excitation rather than inhibition. These results provide evidence that chronic (long-term) exposure to elevated prolactin levels is a sufficient stimulus to modify the neuronal response pattern of VMH nerve cells to iontophoretically applied LH-RH but not to prolactin nor to DA.