Monosodium glutamate lesions in rat hypothalamus studied by immunohistochemistry for gonadotropin releasing hormone, neurotensin, tyrosine hydroxylase, and glutamic acid decar☐ylase and by autoradiography for [H]estradiol

Adult male and female rats treated neonatally with monosodium glutamate (MSG) exhibit lesions in the arcuate nucleus of the hypothalamus. Immunohistochemical analysis of the distribution of tyrosine hydroxylase (TH), glutamic acid decar☐ylase (GAD), neurotensin (NT) and gonadotropin-releasing hormone (GnRH) reveals substantial destructions of tuberoinfundibular dopamine and NT systems accompanied by a marked reduction of immunoreactivity in the median eminence. GAD immunoreactivity in the arcuate nucleus and median eminence is greatly reduced, while GnRH containing structures in the mediobasal hypothalamus are not noticeably affected. Evaluation of autoradiograms after intravenously administered [³H]estradiol in the ventral hypothalamus indicate an almost complete loss of target neurons in the arcuate nucleus but not in the nearby ventromedial nucleus. The results suggest that: (a) NT- and dopamine-containing neurons of the arcuate nucleus project to the median eminence via tuberoinfundibular NT and dopaminergic pathways; (b) GABA in the median eminence originates to a major extent from neurons of the arcuate nucleus through a tuberoinfundibular GABAergic system; (c) GnRH is produced in the rat outside the arcuate nucleus; (d) the MSG-induced lesion in the basal tuberal region abolishes or strongly diminishes estradiol target neurons in the arcuate nucleus.     

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.     

Neurotensin in the rat median eminence: the possible sources of neurotensin-like fibers and varicosities in the external layer

The possible sources of neurotensin-like immunoreactive axons in the median eminence were studied after several experimental surgical approaches including unilateral lateral retrochiasmatic area transection, midsagittal knife cut through the median eminence, complete surgical isolation of the medial basal hypothalamus and bilateral paraventricular nucleus lesions. Both immunohistochemical and radioimmunoassay data demonstrate that neurotensin-containing neuronal located in the hypothalamic arcuate nuclei represent the main source of neurotensin occurring in the external zone of the median eminence of the rat: (1) neither the complete isolation of the medial basal hypothalamus nor the transection of the major neuronal input channel to the median eminence in the lateral retrochiasmatic area altered neurotensin-like immunoreactivity in the median eminence; (2) bilateral lesioning of the paraventricular nucleus resulted in insignificant changes of neurotensin level in the median eminence; and (3) two days after lesioning the median eminence an increased amount of retrogradely accumulated neurotensin-like immunoreactivity was found in several perikarya of the arcuate nuclei due to the blockage of axonal transport in the transected fibers. Retrograde accumulation of neurotensin-like material in other cells scattered in the anterior hypothalamus (in the paraventricular, paraventricular and anterior hypothalamic nuclei) indicates that in addition to the arcuate neurons these neurons may also participate in the neurotensin innervation of the median eminence.     

Relationship of Neuronal Nitric Oxide Synthase Immunoreactivity to GnRH Neurons in the Ovariectomized and Intact Female Rat

The present study has used a rat neuronal nitric oxide synthase (nNOS) antibody to examine the relationship of nNOS immunoreactivity to GnRH neurons in the ovariectomized and intact diestrous and proestrous rat. A striking band of nNOS-immunoreactive cells was identified in the rostral preoptic area which began in the median preoptic nucleus and organum vasculosum of the lamina terminalis and formed an inverted Y-type distribution above the rostral third ventricle at the level of the anteroventral periventricular nucleus. Another band of nNOS-immunoreactivity was found extending through the internal zone of the median eminence into the arcuate nucleus. Although nNOS immunoreactivity was not detected within GnRH neuronal cell bodies in any of the experimental groups, GnRH perikarya located in the rostral preoptic area, but not elsewhere, were found to be surrounded by nNOS-containing cells. In the median eminence, nNOS and GnRH immunoreactivities were distributed separately in the internal and external zones, respectively.
These results provide evidence that, regardless of their pattern of activity, GnRH neurons in the female rat do not express nNOS. Instead, a close anatomical relationship between nNOS-immunoreactive cells and GnRH perikarya and fibers has been identified within specific sub-regions of the rostral preoptic area and in the median eminence. Such findings are compatible with a role for NO at both sites in regulating the release of GnRH throughout the estrous cycle.     

Somatostatin-containing neuron systems in the rat hypothalamus: retrograde tracing and immunohistochemical studies

By employing a combination of the immunohistochemistry for somatostatin (SRIF) and retrograde tracing with biotinylated wheat germ agglutinin (b-WGA) injected into the posterior pituitary (group 1) or into the median eminence (group 2), functional topography of hypothalamic SRIF neurons was determined in the rat hypothalamus. In group 1, large numbers of WGA-labeled neurons appeared in the rostral periventricular region and in the magnocellular division of the paraventricular and supraoptic nuclei; none of them were SRIF immunoreactive. In group 2, WGA-labeled neurons were numerous in the rostral periventricular region, the parvicellular division of the paraventricular nucleus, and the arcuate nucleus; most of the WGA-labeled neurons in the rostral periventricular region and some in the paraventricular nucleus were SRIF immunoreactive, but none in the arcuate nucleus showed immunoreactivity for SRIF. It is concluded that, in the rat hypothalamus, the locations of neurons containing hypophysiotrophic SRIF are confined within the rostral periventricular region and the parvicellular paraventricular nucleus. Our results do not support previous suggestions that SRIF immunoreactive axons innervate the posterior lobe of the pituitary.     

The distribution of growth-hormone-releasing factor (GRF) immunoreactivity in the central nervous system of the rat: an immunohistochemical study using antisera directed against rat hypothalamic GRF

Immunohistochemical methods have been used to chart the distribution of rat hypothalamic growth-hormone-releasing factor (rhGRF) immunoreactivity in the brains of normal and colchicine-treated adult albino rats. The results suggest the existence of at least two distinct rhGRF-containing systems: one responsible for delivery of the peptide to portal vessels in the median eminence, and one whose relationship, if any, to hypophysiotropic function is less direct. A dense plexus of rhGRF-stained fibers was found throughout the external lamina of the median eminence that is the route by which the peptide is delivered to the anterior pituitary. This projection appears to arise primarily from a group of rhGRF-immunoreactive neurons centered in the arcuate nucleus. Some 1,000-1,500 rhGRF-positive neurons were counted on each side of the brain in rats pretreated with colchicine. Colocalization studies, using a sequential double staining technique, indicated that a subset of rhGRF-immunoreactive neurons in the arcuate region contain neurotensin immunoreactivity. No evidence was obtained for colocalization of rhGRF with either of two pro-opiomelanocortin-derived peptides (alpha-melanocyte-stimulating hormone, adrenocorticotropic hormone (1-24)) in individual neurons in the arcuate nucleus. Much smaller groups of neurons were localized in the parvicellular division of the paraventricular nucleus of the hypothalamus and in the dorsomedial nucleus, and it is unclear whether they contribute to the plexus of rhGRF-stained fibers in the median eminence. The only other region in the rat brain in which rhGRF-stained cells were found reliably was in the area that roughly encapsulates the caudal aspect of the ventromedial nucleus of the hypothalamus. Because cells in this region are not known to project to the median eminence, they may be assumed to contribute to the extrahypophysiotropic rhGRF-stained projections outlined below. From the level of the arcuate and ventromedial nuclei, rhGRF-immunoreactive fibers could be traced along the base of the brain and through the periventricular system to discrete terminal fields limited almost exclusively to the hypothalamus and adjoining parts of the basal telencephalon. All parts of the periventricular region of the hypothalamus receive an input, including the preoptic and anterior parts in which somatostatin-containing neurons that project to the median eminence are clustered. Other prominent terminal fields were localized in discrete parts of the dorsomedial, paraventricular, suprachiasmatic, and premammillary nuclei, and in the medial preoptic and lateral hypothalamic areas.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphologic evidence that neurokinin B modulates gonadotropin-releasing hormone secretion via neurokinin 3 receptors in the rat median eminence

Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual-label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin-releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB-immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH-ir somata exhibited NK3R staining. ProNKB and NK3R-ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB-ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis.     

Tuberoinfundibular neurons in the basomedial hypothalamus of the rat: electrophysiological evidence for axon collaterals to hypothalamic and extrahypothalamic areas.

In pentobarbital or urethane anesthetized rats, the activity of 889 mediobasal hypothalamic neurons was studied for evidence of a response to median eminence stimulation. Evidence of antidromic invasion, which indicated a projection to the median eminence, identified 134 cells (15%) as 'tuberoinfundibular' neurons. Antidromic spike latencies ranged from 0.5 to 14.0 msec (4.3 +/- 2.9 S.D.); conduction velocities were under 1.0 m/sec and were generally slower for tuberoinfundibular neurons located closest to the ventral surface of the hypothalamus. Certain tuberoinfundibular neurons followed paired median eminence shocks at frequencies up to 500 Hz; an increase in both the threshold and the latency for the second antidromic spike was observed with interstimulus intervals under 4 msec. Only 38% of tuberoinfundibular neurons were spontaneously active; 24 of 29 spontaneously active neurons displayed evidence of recurrent inhibition with durations up to 150 msec and at latencies which approximated that of the antidromic spike but which did not depend upon antidromic invasion. Similar responses were observed from 33 spontaneously active non-tuberoinfundibular neurons. Evidence of orthodromic excitation in response to median eminence shocks was observed from 22 other medial hypothalamic neurons. Latencies for excitation ranged from 1.5 to 9.0 msec (mean 4.5 +/- 2.1 S.D.). Simultaneous antidromic invasion from other hypothalamic and extrahypothalamic sites was observed from 8 tuberoinfundibular neurons. These sites included the anterior hypothalamic area (2 cells), the preoptic area (3 cells) and the thalamic nucleus medialis dorsalis (3 cells). These results indicate the presence of axon collaterals within the tuberoinfundibular system; some appear to terminate locally within the hypothalamus, while others extend rostrally and dorsally into extrahypothalamic areas. These connections may provide pathways for extrahypothalamic distribution of peptides which regulate adenohypophyseal secretion, and suggest that these peptides may subserve alternate regulatory roles within the central nervous system.     

Immunocytochemical localization of GnRH in the hypothalamus of the bat Miniopterus schreibersii schreibersii.

The distribution of gonadotropin-releasing hormone (GnRH) has been examined in the hypothalamus of adult female bats, Miniopterus schreibersii schreibersii, along annual reproductive cycle by means of light-microscopic immunocytochemistry. GnRH-immunoreactive cells are localized throughout the medial basal hypothalamus, being specially numerous in the arcuate nucleus. They are generally bipolar or monopolar neurons with smooth contour, although spiny cells were also found exclusively in the arcuate nucleus from periovulatory bats. Depletion in both number and immunocytochemical labelling of GnRH perikarya is detected in pregnant and lactating bats. GnRH-immunoreactive fibers are distributed in the internal layer of the median eminence and the infundibular stalk. Pregnant and lactating animals show lower GnRH fibers content and a less intense labelling than in periovulatory and hibernating conditions. The results suggest that changes in the secretory activity of GnRH neurons may be associated with hibernation and delayed implantation that suffer these mammalian species.     

Electrophysiologic evidence for connections between the supraoptic and the arcuate/ventromedial hypothalamic nuclei in the rat

Extracellular action potentials were recorded from 48 single units located in the hypothalamic arcuate and ventromedial nuclei. Fifteen percent of the cells were identified as projecting to the median eminence and some of these cells may have belonged to the tuberoinfundibular dopaminergic systems. Responses of all cells to stimulation of the ipsilateral supraoptic nucleus were recorded; 17% of ventromedial nucleus neurons were antidromically identified as projecting to the supraoptic nucleus. None of the latter cells was also identified as projecting to the median eminence. Three of six identified tuberoinfundibular and eight unidentified ventromedial nucleus cells were found to be excited by stimulation of the supraoptic nucleus. One arcuate cell identified as projecting to the median eminence was nonresponsive to supraoptic stimulation. Orthodromic inhibitory responses were recorded from 17% of all cells recorded but no inhibitory responses were recorded from cells identified as projecting to the median eminence. We suggest that these results may provide some neurophysiologic explanations for the observed interrelationships between oxytocin and prolactin secretion, and between vasopressin and growth hormone secretion.     

Type II glucocorticoid receptors in the ovine hypothalamus: distribution,influence of estrogen and absence of co-localization with GnRH

There is a strong association between the stress-induced increase in cortisol secretion and perturbation of the neuroendocrine reproductive axis. Previous studies implicate a neural target for glucocorticoids and it is possible that cortisol may act directly on gonadotropin releasing hormone (GnRH) neurons and, thus, luteinizing hormone release, through type II glucocorticoid receptors (GRs). In this study we investigated the effect of estradiol on GR immunoreactivity and determined whether GnRH neurons contain GRs. GRs were dispersed throughout most diencephalic structures but were most concentrated within the medial preoptic area and arcuate nucleus. GR cell numbers were significantly higher in these two areas in ewes pre-treated only with progesterone compared to ewes pre-treated with estradiol plus progesterone; there was no variation in the paraventricular nucleus between groups. No colocalization between GnRH and GRs was observed at any level of the brain. These results suggest that estrogen may down-regulate GRs and glucocorticoids do not act directly on GnRH neurons in the ewe.     

Neuronal morphology and neuropil structure in the stomatogastric ganglion of the lobster, Homarus americanus

The hypothalamus is a major site of somatostatin (SST) production and action. SST is synthesized in several hypothalamic nuclei and involved in a variety of functions. Using SST receptor (SSTR)-specific antibodies, we localized SSTR subtypes in the rat hypothalamus. In addition, we also demonstrated SSTRs colocalization with SST, NADPH-diaphorase (NADPH-d), and tyrosine hydroxylase (TH). SSTR1 is strongly localized in neurons in all major hypothalamic nuclei as well as in nerve fibers in the zona externa of the median eminence and the ependyma of the third ventricle. SSTR2 is also well expressed in most regions but with a relatively lower abundance in comparison to SSTR1. In contrast, SSTR3 is localized primarily in the paraventricular nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, and median eminence. SSTR4-like immunoreactivity is mainly confined to the arcuate nucleus, ventromedial hypothalamic nucleus, median eminence, and ependymal cells of third ventricle, with the rare SSTR4-positive neuron in the paraventricular nucleus. SSTR5 is the least expressed subtype occurring only in few cells in the inner layer of the median eminence. Overall, SSTR1 is the predominant subtype, followed by SSTR2, 4, 3, and 5. Combined immunofluorescence, immunocytochemistry, and histochemistry were used to demonstrate SSTRs colocalization with SST, TH, and NADPH-d. SSTRs colocalization with SST, TH, and NADPH-d displays in a region and receptor specificity. Colocalization of SST and NADPH-d with SSTRs in hypothalamic regions was similar, suggesting that SST and NADPH-d producing cells are same. In contrast, TH was selectively coexpressed with SSTRs in the hypothalamus in a receptor-specific manner. Taken together, these data suggest that SSTRs may interact with NADPH-d and TH to exert a physiological role in concert within the hypothalamus.     

Location of the Neuroendocrine Gonadotropin-Releasing Hormone Neurons in the Monkey Hypothalamus by Retrograde Tracing and Immunostaining*,**

In order to localize neuroendocrine gonadotropin-releasing hormone (GnRH) neurons in the monkey hypothalamus, four juvenile cynomolgus macaques (one female, three males) were each given two or three microinjections (0.2 to 0.3 μl per site) of the retrograde tracer wheat germ agglutinin-apoHorseradish peroxidase-10 nm colloidal gold into the superficial, median eminence region of the infundibular stalk. Five to 15 days following surgery, the brains were fixed by perfusion and vibratomed at 40 μm in the frontal plane. Every 12th section was immunostained with rabbit anti-GnRH using the peroxidase anti-peroxidase technique with diaminobenzidine as the chromogen. Neuroendocrine GnRH neurons were easily identified in tissue sections as brown, immunostained cell bodies containing more than three distinct, dark blue, tracer-filled lysosomes. Neuronal counts from each complete series of sections were compiled by anatomical region, and the percentages of GnRH and neuroendocrine GnRH neurons determined. The highest proportion of neuroendocrine GnRH neurons (with projections to the median eminence) occurred in the ventral hypothalamic tract, especially in its medial third (71%), and in the supraoptic decussation just anterior to it. Proportions decreased moving laterally into the middle third (58%) and lateral third (25%) of the ventral hypothalamic tract. Further anterior and lateral, progressively smaller but significant neuroendocrine GnRH contributions were found in the supraoptic nucleus (57%) and lateral hypothalamus (33%), and in the medial preoptic area (26%). Although the medial preoptic area contained a greater percentage of the total GnRH-immunoreactive cell bodies (36%) than the ventral hypothalamic tract (27%), as a whole, the ventral hypothalamic tract contained 60% of the neuroendocrine GnRH neurons compared to only 25% from the medial preoptic area. Large numbers of GnRH cell bodies found in the diagonal band of Broca near the organum vasculosum of the lamina terminalis were not retrogradely labeled. GnRH neurons were not observed in the arcuate nucleus, the few in the paraventricular nucleus were not neuroendocrine, and the contribution from the periventricular zone was negligible. Our results here are the first to identify the neurons giving rise to the neuroendocrine GnRH system in juvenile monkeys. The data indicate that more GnRH neurons close to the infundibulum serve a neuroendocrine (perhaps hypophysiotropic) role than do those in more anterior areas. Furthermore, they suggest that the ventral hypothalamic tract is the most important, and perhaps most influential, neuroendocrine GnRH cell group in primates. The data substantiate the observed autonomy of the medial basal hypothalamus in controlling gonadotropin secretion and menstrual cyclicity in these animals. However, they also infer that perhaps 60% of the GnRH neurons do not project to the primate median eminence, and thus may serve other non-neuroendocrine functions.     

Effect of electrical stimulation of the arcuate nucleus on neurochemical estimates of tuberoinfundibular and tuberohypophysial dopaminergic neuronal activities

The activity of nigrostriatal dopaminergic neurons has been estimated biochemically by measuring the rates of dopamine (DA) synthesis (accumulation of dihydroxyphenylalanine (DOPA) after NSD 1015) and turnover (decline of DA concentrations after alpha-methyltyrosine) in the striatum. It has been assumed that the activities of tuberoinfundibular dopaminergic (TIDA) and tuberohypophysial dopaminergic (THDA) neurons can also be estimated by making the same measurements in the terminals of these neurons in the median eminence and the posterior pituitary, respectively. In the present study, this assumption was tested directly by measuring the rates of DA synthesis and turnover in the median eminence and posterior pituitary following electrical stimulation of TIDS and THDA cell bodies in the arcuate nucleus. Electrical stimulation of the arcuate nucleus increased the rate of DOPA accumulation and the alpha-methyltyrosine-induced decline of DA concentrations in the median eminence and in the neural and intermediate lobes of the posterior pituitary. gamma-Butyrolactone (GBL), an anesthetic that selectively inhibits DA impulse flow, reduced the rates of DA synthesis and turnover in the median eminence. GBL also increased prolactin secretion which is tonically inhibited by DA released from TIDA neurons. Serum prolactin levels were significantly decreased by arcuate nucleus stimulation in GBL-anesthetized rats. These results indicate that the rates of DA synthesis and turnover within the median eminence and posterior pituitary reflect the activities of TIDA and THDA neurons, respectively.     

Dopamine transporter immunohistochemistry in median eminence,amygdala, and other areas of the rat brain

In an extension of our previous work, an antibody directed against a peptide from the N-terminal region of DAT was used to localize specific dopamine transporter immunoreactivity (DAT-IR) in several regions of rat brain. Apparent axons and varicosities were found in the zona incerta, external layer of the median eminence, various nuclei of the amygdala, the cortex-amygdala transition zone, and in periglomerular regions in the olfactory bulb. Apparent stained neuronal perikarya and dendrites were observed in the arcuate nucleus and olfactory bulb. These regions are known to have dopaminergic neurons and innervations, although there was not a perfect correspondence between DAT-IR and the known distribution of dopaminergic neurons. A possible explanation is that different dopamine containing cell groups express different levels of DAT mRNA and protein, as we have previously shown. Also in the tuberoinfundibular neurons, for example, DAT-IR was preferentially localized to distal axons in the median eminence, suggesting intracellular compartmentation. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Catecholamine neurons in the squirrel monkey hypothalamus

Summary Catecholamine neurons were found in the periventricular nucleus, arcuate nucleus, and a region around the mammillothalamic tract in the hypothalamus of the squirrel monkey. These neurons did not fluoresce in control or monoamine-oxidase-inhibited animals, and exhibited fluorescence only after intraventricular injections of alpha-methyl-norepinephrine. Dense accumulations of Catecholamine varicosities appeared in the periventricular nucleus, external contact zone of the median eminence, and perivascular region of the median eminence; the arcuate nucleus contained no more than a moderate density of Catecholamine varicosities. The median eminence fluorescence had a regional character, with greatest intensity in the caudal portion.     

An Immunohistochemical Study on the Expressional Dynamics of Kisspeptin Neurons Relevant to GnRH Neurons Using a Newly Developed Anti-kisspeptin Antibody

To investigate the reported discrepancy regarding the immunohistochemical expression of kisspeptin neurons, we produced a new antibody against synthetic peptide containing the same amino acid residual sequence as rat kisspeptin10. Although the antibody showed cross-reactivities against neurons other than kisspeptin neurons, these cross-reactivities were excluded by preabsorption with neuropeptide FF (NPFF). Immunohistochemistry using the antibody preabsorbed with NPFF showed specific kisspeptin immunoreactivities (IRs) in the arcuate nucleus (Arc), the inner layer of the median eminence, and the infundibulum in the rat hypothalamus. IRs were more intense in the adult female rats than in the males. This sexual dimorphism became observable at the 7th day after birth. These IRs intensified with age. Ovariectomy enhanced the IRs in the Arc in the female rats. In contrast, regarding IRs in the anteroventral periventricular nucleus (AVPV), only a few immunoreactive fibers were detected in the adult rats. We applied this antibody for the investigation of the interaction between kisspeptin fibers and gonadotropin-releasing hormone (GnRH) neurons. No direct morphological interaction between the cell bodies of GnRH neurons and kisspeptin fibers was observed in the medial preoptic area. Many projections of kisspeptin fibers were found close to the GnRH fibers in the lateral part of the median eminence. However, we did not observe any direct contact between kisspeptin fibers and the GnRH fibers. These results suggest that kisspeptin neurons regulate GnRH neurons not via the synaptic contact but via another information transmission process that is not synapse-dependent, such as volume transmission.     

β-endorphin alters dopamine uptake by the dopamine neurons of the hypothalamus and striatum

The study of hypothalamic dopamine (DA) neurons is complicated by the difficulty in distinguishing DA neurons from norepinephrine (NE) neurons and by the fact that they comprise only a small proportion of the catecholamine neuron population of the hypothalamus. We have studied DA uptake into nerve terminals of hypothalamic DA neurons using a synaptosomal preparation. Desmethylimipramine (DMI) was used to prevent uptake into synaptosomes from NE neurons, thus pharmacologically isolating dopaminergic from noradrenergic nerve terminals. This DMI-insensitive DA uptake in hypothalamus had all the properties of a high affinity uptake process; it was saturable, dependent on incubation time and incubation temperature, increased linearly with increasing amounts of tissue and was completely abolished by excess unlabeled DA. Also, it was completely abolished by benztropine, an inhibitor of amine uptake into DA neurons. We believe that DMI-insensitive DA uptake into hypothalamic synaptosomes represents uptake into DA nerve terminals.The DMI-insensitive DA accumulation in discrete areas of hypothalamus correlated well with the known prevalence of DA neurons relative to NE neurons in these areas: median eminence > median eminence-arcuate nucleus > mediobasal hypothalamus > whole hypothalamus. Comparison of the affinity constants for DA uptake into synaptosomes incubated without DMI revealed a 2-fold higher affinity constant for DA uptake in median eminence compared with striatum, but affinity constants in all the other hypothalamic regions examined (mediobasal hypothalamus, hypothalamus minus median eminence, whole hypothalamus) were identical to that of striatum. In contrast, comparison of the affinity constants for DA uptake in the presence of DMI revealed a 2–3-fold higher affinity constant for DA uptake in all these hypothalamic regions compared with striatum. It appears the tuberoinfundibular DA neurons and the other DA neurons of the hypothalamus have a high affinity uptake system for DA, although affinity for DA in all of these hypothalamic DA neurons appears to be 2–3-fold lower than that in striatal DA neurons. The data also suggest that the much larger mediobasal hypothalamus may serve as a model for studies of DA uptake into tuberoinfundibular DA neurons of the median eminence.     

GRF Neurons in the rat hypothalamus

The growth hormone-releasing factor (GRF)-containing neuronal system was immunohistochemically studied in the rat hypothalamus. The immunolabeled cell bodies were determined by intraventricular administration of colchicine 24 h before killing. In intact animals, the neurons appeared in the ventral portion of the arcuate nucleus (group 1) and in the area surrounding the ventromedial nucleus (group 2). Most of the cell bodies also indicated immunoreactivity for tyrosine hydroxylase (TH). The immunoreactive fibers accumulated showing a palisade arrangement in the external layer of the median eminence. The rats treated neonatally with monosodium glutamate revealed group 2 neurons and a few immunoreactive fibers in the median eminence. Half-anterolateral deafferentation of the medial basal hypothalamus, which was performed to isolate group 1 neurons or both group 1 and 2 neurons from the other brain parts, did not remarkably affect the appearance of the fibers in the median eminence. However, the perikarya were hypertrophic and strongly immunolabeled for GRF and TH. It is concluded that the fibers containing GRF in the median eminence derive mostly from group 1 neurons, and that the neurons may be regulated by an inhibitory mechanism by other neurons on the outside of the deafferented hypothalamic islands. GRF synthesized in group 2 neurons may act on other neurons as a neurotransmitter-like substance.     

Coexpression of dynorphin and neurokinin B immunoreactivity in the rat hypothalamus: Morphologic evidence of interrelated function within the arcuate nucleus

Considerable evidence suggests that dynorphin and neurokinin B (NKB) neurons in the hypothalamic arcuate nucleus participate in the sex-steroid regulation of reproduction. In the present study, we used dual-label immunofluorescence to explore the distribution of prodynorphin and proNKB immunoreactivity in the rat hypothalamus. Additionally, we investigated whether arcuate prodynorphin-ir (immunoreactive) neurons expressed the neurokinin 3 receptor (NK3R) or nuclear estrogen receptor-alpha (ERalpha). We found that the majority of prodynorphin-ir neurons in the rat arcuate nucleus expressed proNKB, whereas nearly all (99%) of the proNKB neurons were immunoreactive for prodynorphin. The arcuate nucleus was the only site in the hypothalamus where neuronal somata coexpressing prodynorphin and proNKB-immunoreactivity were identified. A dense plexus of double-labeled prodynorphin/proNKB-ir fibers was found within the arcuate nucleus extending to the median eminence and throughout the periventricular zone of the hypothalamus. Prodynorphin/proNKB fibers were also identified in the paraventricular nucleus, anterior hypothalamic area, medial preoptic area, median preoptic nucleus, anteroventral periventricular nucleus, and bed nucleus of the stria terminalis in a distribution consistent with previously described arcuate nucleus projections. Interestingly, the majority of prodynorphin-ir neurons in the arcuate nucleus expressed NK3R, and nearly 100% of the prodynorphin-ir neurons contained nuclear ERalpha. Our results suggest that there is a close functional relationship between dynorphin and NKB peptides within the arcuate nucleus of the rat, which may include an autofeedback loop mediated through NK3R. The diverse hypothalamic projections of fibers expressing both prodynorphin and proNKB provide evidence that these neurons may participate in a variety of homeostatic and neuroendocrine processes.