Arcuate Nucleus-median Eminence Complex Research Articles

Synchronized Network Oscillations in Rat Tuberoinfundibular Dopamine Neurons: Switch to Tonic Discharge by Thyrotropin-Releasing Hormone

The pituitary hormone, prolactin, triggers lactation in nursing mothers. Under nonlactating conditions, prolactin secretion is suppressed by powerful inhibition from hypothalamic tuberoinfundibular dopamine (TIDA) neurons. Although firing pattern has been suggested as integral to neuroendocrine control, the electrical behavior of TIDA cells remains unknown. We demonstrate that rat TIDA neurons discharge rhythmically in a robust 0.05 Hz oscillation. The oscillation is phase locked between neurons, and while it persists during chemical synaptic transmission blockade, it is abolished by gap junction antagonists. Thyrotropin-releasing hormone (TRH) potently stimulates prolactin release, an effect assumed to take place in the pituitary. In TIDA cells, TRH caused a transition from phasic to tonic firing through combined pre- and postsynaptic effects. These findings suggest a model for prolactin regulation where a TIDA network switch from oscillations to sustained discharge converts dopamine from an antagonist at high concentrations to a functional agonist as dopamine output from the network decreases.

An androgen-dependent sexual dimorphism visible at puberty in the rat hypothalamus

Morphological studies in rodents have well documented the masculinization of the perinatal brain by estradiol derived from aromatized testosterone, and the resulting irreversible quantitative sex-differences generated in cell numbers or expression of chemical phenotypes. Here, using immunohistochemistry, we explored how this applies to the postnatal development and masculinization of the neurokinin B (NKB)-containing system of the arcuate nucleus/median eminence complex (ARC/ME). In adult rats, NKB-immunoreactive neurons exhibit an unusual, qualitative sexual dimorphism of their ventral axonal projections: to the neuropil in females, to capillary vessels in males. In adults, there was no sex-difference in the numbers of NKB-immunoreactive perikarya or capillary vessels in the ARC/ME, suggesting that this sexual dimorphism cannot be explained by the existence of supernumerary structures. At birth (day 0) the NKB system was immature in both sexes, and while its adult features emerged progressively until puberty in females, they did not develop before puberty (day 40) in males, revealing a sexual dimorphism only late postnatally. When males were orchidectomized at day 30, the masculine distribution of NKB-immunoreactive axons expected at day 40 was not seen, while it was apparent after chronic treatment with testosterone or dihydrotestosterone, suggesting a testicular masculinizing action via androgen receptors at puberty. Moreover in these prepubertal-orchidectomized males, the distribution of NKB-immunoreactive axons was surprisingly feminized by chronic estradiol alone, suggesting that NKB neurons are not irreversibly programmed before puberty. Last, in adult females, the distribution of NKB-immunoreactive axons was feminine 30 days after ovariectomy, and it was masculinized after concurrent chronic dihydrotestosterone, suggesting that NKB neurons remain responsive to androgens late in reproductive life.Thus, the sexual differentiation of the hypothalamus proceeds well beyond the perinatal period and includes the epigenetic action of non-aromatizable androgens upon subsets of neurons that have retained bipotent features.

Capsaicin increases GFAP and glutamine synthetase immunoreactivity in rat arcuate nucleus and median eminence.

Antibodies to glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) were used to determine the effect of s.c. capsaicin (after 75 min) on astroglial cells in the rat arcuate nucleus-median eminence (ARC-ME). Compared to vehicle, capsaicin significantly increased GFAP and GS immunoreactivity in the ARC-ME. Co-localization of GFAP and GS was observed in the ARC-ME complex. Since GS is primarily responsible for glutamate-glutamine metabolism, the increase in total immunostaining for GFAP-and GS- staining suggests a functional adjustment to cope with some of the capsaicin-induced effects. Together with the involvement of nitric oxide synthase in the ARC-ME response to capsaicin, these observations indicate activity-dependent plasticity of the neuron-glia network in response to this stressful/noxious stimulus.

Exogenous Glutamate Enhances Glutamate Receptor Subunit Expression during Selective Neuronal Injury in the Ventral Arcuate Nucleus of Postnatal Mice

Administration of high doses of glutamate (Glu) leads to selective neurodegeneration in discrete brain regions near circumventriclular organs of the early postnatal mouse. The arcuate nucleus-median eminence complex (ARC-ME) appears to be the most Glu-sensitive of these brain regions, perhaps because of the intimate relationships between its neurons and specialized astroglial tanycytes. To investigate the mechanism of Glu-induced neuronal loss, we administered graded doses of the sodium salt of glutamate (MSG) to postnatal mice, measured their plasma Glu concentrations, and performed microscopic analyses of the ARC-ME region 5 h after treatment. Nursing, 7-day-old mouse pups (CD1, Charles River, Hollister, Calif.) were injected subcutaneously with single doses of 0.1–0.5 or 1.0–4.0 mg of MSG per g BW, or with water vehicle alone. Mice were decapitated 5 h later and the brains immediately fixed by immersion in buffered aldehydes. Frontal vibratome tissue sections at comparable levels of the ARC-ME were examined by light microscopy. A dose of 4.0 mg MSG/g BW caused neurodegeneration throughout the ARC region, while 1.0 mg/g MSG resulted in less extensive damage. Injection of 0.2 mg MSG/g BW, which raised plasma Glu concentrations 17-fold after 15 min, was the minimum dose tested at which nuclear and cytoplasmic changes were observed in a small group of subependymal neurons near the lateral recesses of the third ventricle. Higher doses of 0.3–0.5 mg MSG caused injury to additional neurons situated farther laterally, but damage remained confined to the ventral region of the ARC nucleus. Ultrastructural examination showed some subependymal neurons with pyknotic nuclei, reduced cytoplasmic volume, and swollen subcellular organelles, while others had fragmented and condensed nuclear material. Immunostaining for tyrosine hydroxylase indicated that dopamine neurons were spared at the threshold dose, but suffered damage after higher doses of MSG. Immunostaining for Glu receptor subtypes revealed that 0.2 mg MSG/g BW enhanced neuronal expression of NMDAR1 and of GluR2/4, and that higher doses of MSG preferentially increased NMDAR1 expression in injured neurons. These results extend previous reports of Glu sensitivity in the ARC-ME region of 7-day postnatal mice. A dose of 0.2 mg MSG/g BW s.c. causes clear but discrete injury to specific subependymal neurons of undetermined phenotype near the base of the third ventricle. Slightly higher doses of MSG evoke damage of additional neurons confined to the ventral region of the ARC traversed by tanycytes. These same greater amounts of MSG promote dose-related increase in the expression of NMDAR1 more than of GluR2/4 in injured ARC neurons, suggesting that elevated Glu receptor levels may contribute to or be related to neuronal cell death. Taken together with previous findings, the data suggest that Glu responsitivity in the ARC-ME of the postnatal mouse may result from transient developmental conditions involving the numerical ratios and juxtaposition between tanycytes and neurons, expression of Glu receptors, and perhaps other ontogenetic factors which may not persist in the mature adult.

Increased neuropeptide Y concentrations in specific hypothalamic regions of lactating rats: Possible relationship ot hyperphagia and adaptive changes in energy balance

Lactation is accompanied by hyperphagia and a reduction in brown adipose tissue (BAT) thermogenesis, which are unexplained. Neuropeptide Y (NPY) powerfully stimulates feeding and inhibits BAT thermogenesis when injected into the paraventricular nucleus and other specific regions of the rat hypothalamus. We have tested the hypothesis that hypothalamic NPY activity is increased in lactating rats. Lactating rats consumed over four times as much food as nonlactating controls ( n = 10; p < 0.001). Final plasma insulin concentrations in lactating rats were lower than in controls ( 6.8 ± 0.8 vs. 11.7 ± 2.1 pmol/l ; p < 0.05) although plasma glucose and corticosterone concentrations were comparable ( p > 0.05). Lactating rats showed significantly higher NPY levels than controls in specific hypothalamic regions, namely the arcuate nucleus-median eminence complex (a 41% rise; p < 0.001), paraventricular nucleus (35%; p < 0.001), ventromedial nucleus (66%; p = 0.003), and dorsomedial nucleus (78%; p < 0.001). Other hypothalamic regions showed no significant differences between groups. Increased NPY concentrations in specific hypothalamic regions, particularly the arcuate nucleus where NPY is synthesized, suggest increased activity of the hypothalamic NPYergic system in lactation. Neuropeptide Y may mediate hyperphagia and reduced BAT thermogenesis in lactation. Hypoinsulinemia may be a stimulus to hypothalamic NPY in lactation, as has been postulated in other conditions of negative energy balance.

Peptide- and transmitter-containing neurons in the mediobasal hypothalamus and their relation to GABAergic systems: possible roles in control of prolactin and growth hormone secretion.

Indirect immunofluorescence histochemistry was used to study the relation among GABAergic, catecholaminergic, cholinergic, and peptidergic neurons in the rat mediobasal hypothalamus. By employing a direct double-labelling procedure using sheep antiserum against glutamic acid decarboxylase (GAD), mouse monoclonal and rabbit antibodies to neurotensin (NT) and rabbit antisera to tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), galanin (GAL), growth hormone-releasing factor (GRF), or somatostatin (SOM), it was demonstrated that GAD-positive fibers and terminals in the external part of the median eminence co-contained immunoreactivity for TH, NT, GAL or GRF, but not for SOM. In the internal part of the median eminence-infundibular stalk, GAD-positive/NT-, GAL-, and GRF-negative and GAD-positive/TH-positive fiber plexa were shown. When a recently developed direct triple-labelling procedure with biotin-conjugated mouse secondary antibodies in conjunction with diethylaminocoumarin (DAMC)-conjugated avidin was employed, presence of GAD/GAL/NT- as well as GAD/GRF/NT-containing varicosities could be demonstrated close to hypophysial portal vessels. In colchicine-pretreated animals, GAD was shown to coexist with TH, NT, or GAL in cell bodies in both the dorsomedial and ventrolateral domains of the arcuate nucleus, but with GRF only in the ventrolateral division. ChAT-positive neurons in the ventrolateral region were also TH-positive. In the ventrolateral arcuate nucleus, triple-labelling followed by elution-restaining showed GAD/NT/GAL/TH-immunoreactivities in the same cells. Similarly, double-labelling with two following elution-restaining steps showed several NT/GAL/GRF/TH-containing cell bodies in this part of the arcuate nucleus. GAD-positive cells in the anterior hypothalamic periventricular area and fibers in the pituitary neurointermediate lobe were also TH-positive. The results demonstrate complex patterns of storage of chemical messengers in neurons of the arcuate nucleus-median eminence complex. Possible neuroendocrine interactions of these systems in the control of prolactin and growth hormone secretion are discussed.

Incorporation of amino acids into proteins of the hypothalamus of prepuberal female rats after estradiol treatment.

The arcuate nucleus-median eminence complex (AM) undergoes major structural and functional changes during normal puberty or if exposed to a pulse of estradiol in the prepuberal period. Those changes are expressed by increased synaptogenesis and by a drastic alteration in the feedback control of anterior pituitary gland hormone release. In this study we investigated the effects of estradiol benzoate (EB) on specific proteins in this hypothalamic area. Prepuberal, 25-day-old female rats were administered 10 micrograms of EB s.c. in oil or sesame oil vehicle. The animals were decapitated either 17 or 42 h after treatment. The brains were removed, blocked and serially sections at 300 micron using a Vibratome. The AM was dissected out and incubated for 6 h in a medium containing 35S-methionine and 35S-cysteine. Proteins from the AM were separated by two-dimensional gel electrophoresis, and the gels were exposed to X-ray film. The resulting autofluorographs were analyzed by scanning densitometry. The results show that the incorporation of labeled amino acids was increased in 10 proteins and decreased in 2 proteins in rats killed 17 h after EB. At 42 h after EB, 6 proteins showed an increased incorporation of amino acids and two proteins showed a decrease. Our results suggest that one or several of these proteins might be involved in the neuroendocrine and structural changes observed in the AM during puberty.

The hypothalamic arcuate nucleus-median eminence complex: immunohistochemistry of transmitters, peptides and DARPP-32 with special reference to coexistence in dopamine neurons.

In this paper, we describe the results of a series of experiments which have examined the distribution within the arcuate nucleus of the hypothalamus of neurons containing the following immunoreactivities: TH-LI, GAD-LI, NT-LI, GAL-LI, GRF-LI, Met-ENK-LI, Leu-ENK-LI, Met-ENK-7-LI, Met-ENK-8-LI, metorphamide-LI, DYN-LI, NPY-LI, SOM-LI, FMRFamide-LI, and CLIP-LI and ependymal tanycytes containing DARPP-32-LI. Using elution-restaining and double antibody staining techniques we have established numerous patterns of coexistence of these various neurotransmitters and neuropeptides. Thus, neurons containing TH-LI were, in some instances, also found to contain GAD-LI, NT-LI, GAL-LI, GRF-LI, Met-ENK-8-LI, Leu-ENK-LI, or DYN-LI or combinations of these compounds. For example, some TH-IR neurons also contained GAL-LI and GRF-LI, while other TH-IR. neurons were also seen to contain GRF- and NT-LI. These neurons may, in fact, contain even more compounds. NPY-IR neurons and those containing SOM-LI and CLIP-LI were distinct and separate from those containing TH-LI. The distribution of these different neurochemical types of neurons and their patterns of coexistence are summarized in Fig. 34, while the relative distribution patterns of immunoreactive fibres in the median eminence are summarized in Fig. 35.

Dual effect of verapamil on K +-evoked release of endogenous dopamine from arcuate nucleus-median eminence complex

The effect of verapamil, a calcium-entrance blocker, on K +-evoked release of endogenous dopamine from tuberoinfundibular neurons incubated in vitro was studied. This compound, added to the incubation medium, at the dose of 10 −6 M, significantly reinforced K +-induced dopamine release, whereas, at higher doses (10 −5, 5 × 10 −5 and 10 −4 M), it completely prevented the stimulated dopamine release. The results obtained with the higher doses showed the calcium dependence of K +-evoked release of endogenous dopamine from central neurons. The opposite effect, seen with the lower dose of verapamil, could be due to different pharmacological properties of the drug.

EFFECTS OF ANTERIOR HYPOTHALAMIC IMPLANTATION OF GONADAL STEROIDS ON SERUM AND PITUITARY GONADOTROPHINS (FOLLICLE-STIMULATING HORMONE AND LUTEINIZING HORMONE) IN THE CASTRATED MALE RAT

Castrated rats were stereotaxically implanted with 1 microng oestradiol benzoate, 5 microng testosterone isobutyrate or, as a control, 10 microng cholesterol, in the hypothalamus. The effects of the steroids on plasma and pituitary gonadotrophins (FSH and LH) were assessed by radioimmunoassay. Our results indicate that, in the male rat, in addition to the arcuate nucleus-median eminence complex, the preoptic suprachiasmatic area is able to control synthesis and secretion of both gonadotrophins, and that it is sensitive to oestradiol and testosterone.