Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase enzymes. The maximum concentrations of these enzymes are found within the hypothalamus and amygdala, where they appear to play an important role in the process by which androgens affect both behavior and neuroendocrine function. In the present study, we measured the levels of aromatase activity (AA) in 20 nuclei and brain regions of the adult rat brain. Individual nuclei were microdissected from 600-micron frozen sections. Tissues from 3 animals were pooled, and AA was measured by an in vitro radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. We report that AA is heterogeneously distributed within the rat brain. The greatest amounts of activity were found in the bed nucleus (n.) of the stria terminalis (700 protein fmol/h . mg) and in the medial (MA) and cortical amygdala (400-600 fmol/h . mg protein) of the male. There was an evident rostral-caudal and medial-lateral gradient in AA throughout the diencephalon. Activity was high in the periventricular preoptic n. and medial preoptic n.; intermediate in the suprachiasmatic preoptic n., anterior hypothalamus, periventricular anterior hypothalamus, and ventromedial n.; and low in the arcuate n.-median eminence, lateral preoptic n., supraoptic n., dorsomedial n., and lateral hypothalamus. Regions devoid of measurable AA included the medial and lateral septum, caudate-putamen, hippocampus, and parietal cortex. In the female, AA was greatest in the MA and cortical amygdala. We found that AA in the MA, stria terminalis n., suprachiasmatic preoptic n., periventricular preoptic in., medial preoptic n., anterior hypothalamus, and ventromedial n. was significantly greater (P less than 0.05) in males than in females. Orchidectomy reduced AA to levels seen in females, and administration of testosterone to castrated males restored AA in these areas. No significant sex differences were observed in any other hypothalamic or amygdaloid nuclei, although AA was increased by testosterone treatment in the periventricular anterior hypothalamus, arcuate n.-median eminence, and lateral hypothalamus. Our results provide a quantitative profile of AA in specific hypothalamic and limbic nuclei of the rat brain as well as information on the control of AA within these discrete regions.     

Glutamatergic and GABAergic innervation of human gonadotropin-releasing hormone-I neurons

Amino acid (aa) neurotransmitters in synaptic afferents to hypothalamic GnRH-I neurons are critically involved in the neuroendocrine control of reproduction. Although in rodents the major aa neurotransmitter in these afferents is γ-aminobutyric acid (GABA), glutamatergic axons also innervate GnRH neurons directly. Our aim with the present study was to address the relative contribution of GABAergic and glutamatergic axons to the afferent control of human GnRH neurons. Formalin-fixed hypothalamic samples were obtained from adult male individuals (n = 8) at autopsies, and their coronal sections processed for dual-label immunohistochemical studies. GABAergic axons were labeled with vesicular inhibitory aa transporter antibodies, whereas glutamatergic axons were detected with antisera against the major vesicular glutamate transporter (VGLUT) isoforms, VGLUT1 and VGLUT2. The relative incidences of GABAergic and glutamatergic axonal appositions to GnRH-immunoreactive neurons were compared quantitatively in two regions, the infundibular and paraventricular nuclei. Results showed that GABAergic axons established the most frequently encountered type of axo-somatic apposition. Glutamatergic contacts occurred in significantly lower numbers, with similar contributions by their VGLUT1 and VGLUT2 subclasses. The innervation pattern was different on GnRH dendrites where the combined incidence of glutamatergic (VGLUT1 + VGLUT2) contacts slightly exceeded that of the GABAergic appositions. We conclude that GABA represents the major aa neurotransmitter in axo-somatic afferents to human GnRH neurons, whereas glutamatergic inputs occur somewhat more frequently than GABAergic inputs on GnRH dendrites. Unlike in rats, the GnRH system of the human receives innervation from the VGLUT1, in addition to the VGLUT2, subclass of glutamatergic neurons.     

Expression of thyroid hormone receptor beta 2 in rat hypothalamus.

A polymerase chain reaction based assay was used to evaluate expression of thyroid hormone receptor beta 2 mRNA in rat hypothalamus. Expression was detected in the arcuate, ventromedial and paraventricular nuclei, as well as the median eminence. Trace expression was found in the dorsomedial nucleus, but no expression of thyroid hormone receptor beta 2 was detected in the lateral hypothalamus or the preoptic region. The results indicate that, contrary to previous belief, expression of thyroid hormone receptor beta 2 is not confined to the anterior pituitary.     

Quantitative histochemical studies of the hypothalamus: dehydrogenase enzymes following androgen sterilization.

Four enzymes, selected as representative of major metabolic pathways (malic dehydrogenase, of the citric acid cycle, lactic dehydrogenase, of glycolysis, glucose-6-phosphate dehydrogenase, of the pentose pathway and glutamic dehydrogenase, of glutamate metabolism), were measured by quantitative histochemical methods in individual hypothalamic nuclei of adult neonatally androgenized female rats. Malic dehydrogenase (MDH) was significantly reduced in nuclei of the anterior hypothalamus: the suprachiasmatic, supraoptic and anterior. Lactic dehydrogenase (LDH) increased significantly in the lateral preoptic area. Glucose-6-phosphate dehydrogenase G-6-PDH was also significantly elevated in anterior hypothalamic nuclei: medial preoptic, lateral preoptic, supraoptic and paraventricular. Glutamic dehydrogenase (GDH) was generally elevated throughout the hypothalamus with significant increases of activity occurring in the paraventricular, lateral ventromedial, arcuate, medial mamillary and posterior nuclei.     

Hypophysiotropic thyrotropin-releasing hormone and corticotropin-releasing hormone neurons of the rat contain vesicular glutamate transporter-2

TRH and CRH are secreted into the hypophysial portal circulation by hypophysiotropic neurons located in parvicellular subdivisions of the hypothalamic paraventricular nucleus (PVH). Recently these anatomical compartments of the PVH have been shown to contain large numbers of glutamatergic neurons expressing type 2 vesicular glutamate transporter (VGLUT2). In this report we presented dual-label in situ hybridization evidence that the majority (>90%) of TRH and CRH neurons in the PVH of the adult male rat express the mRNA encoding VGLUT2. Dual-label immunofluorescent studies followed by confocal laser microscopic analysis of the median eminence also demonstrated the occurrence of VGLUT2 immunoreactivity within TRH and CRH axon varicosities, suggesting terminal glutamate release from these neuroendocrine systems. These data together indicate that the hypophysiotropic TRH and CRH neurons possess glutamatergic characteristics. Future studies will need to address the physiological significance of the endogenous glutamate content in these neurosecretory systems in the neuroendocrine regulation of thyroid and adrenal functions.     

On the origin of Leu-enkephalin and Met-enkephalin in the rat neurohypophysis

The posterior lobe of the pituitary contains large amounts of Leu- and Met-enkephalin (LE and ME, respectively). A marked depletion of ME (81.9%) and LE (94.5%) in the posterior pituitary occurred after transection of the pituitary stalk. This indicates that most, if not all, of the enkephalins are in processes of central neurons. In the present study, I attempted to determine the source(s) of the LE- and ME-containing fibers in the posterior pituitary by examining the effects of hypothalamic lesions or fiber transections on the LE and ME levels. Lesions of the hypothalamic paraventricular nuclei caused ME and LE levels in the posterior pituitary to decrease significantly (55.6% and 27.6%, respectively). Deafferentation of the medial basal hypothalamus (creating islands of tissue containing the ventromedial and arcuate nuclei) resulted in a marked reduction in LE (94.1%) and ME (54.7%). Treating neonatal rats with monosodium glutamate resulted in a selective destruction of arcuate nucleus neurons, but did not affect LE and ME concentrations in the posterior pituitary. Thus, about half of the ME in the posterior pituitary seems to be provided by neurons in the vicinity of the paraventricular and ventromedial nuclei, whereas only about one quarter of the LE in the posterior pituitary is in processes of the paraventricular nucleus neurons. The remainder of the LE is contributed to the posterior pituitary by neurons outside the medial basal hypothalamus, probably by the supraoptic nucleus neurons. These findings are consistent with the hypothesis that LE and ME may be localized in separate populations of nerve endings in the neurohypophysis and may have different roles.     

Agouti-related protein containing nerve terminals innervate thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Gene expression for agouti-related protein (AGRP), an endogenous antagonist of melanocortin receptors, has been localized to the hypothalamic arcuate nucleus, where it colocalizes with neuropeptide Y (NPY). Having reported that the NPY innervation of hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus (PVN) originates primarily from NPY-producing neurons in the arcuate nucleus, here we examined the possibility that TRH neurons in the PVN are similarly innervated by AGRP nerve terminals. Using immunohistochemistry, AGRP-containing cell bodies were found almost exclusively in the arcuate nucleus, but their projections were distributed widely in the hypothalamus, most conspicuously in the paraventricular (PVN), arcuate and dorsomedial nuclei, and the posterior hypothalamic area. Ablation of the arcuate nucleus by the neonatal administration of monosodium glutamate obliterated nearly all AGRP-immunoreactivity in the hypothalamus. In the PVN, double-labeling light and electron microscopic immunohistochemistry revealed that TRH neurons receive dense innervation by AGRP nerve terminals, with the frequent occurrence of axosomatic and axodendritic synapses (mainly of the symmetrical type). These findings provide morphological basis to hypothesize a role for AGRP in the arcuato-paraventricular pathway, in the down-regulation of the hypothalamic-pituitary-thyroid axis, which occurs as an adaptive response to starvation.     

Fasting, leptin treatment, and glucose administration differentially regulate Y(1) receptor gene expression in the hypothalamus of transgenic mice

NPY is a potent orexigenic signal and represents a key component of targets through which leptin exerts a regulatory restraint on body adiposity. Part of the orexigenic effects of NPY are mediated by hypothalamic NPY-Y(1) receptors. Here we studied the effect of fasting, leptin, and glucose administration on Y(1) receptor gene expression using a transgenic mouse model carrying a mouse Y(1) receptor/LacZ fusion gene. Transgene expression was determined by quantitative analysis of beta-galactosidase histochemical staining in the paraventricular, arcuate, ventromedial, and dorsomedial hypothalamic nuclei and in the medial amygdala, as a control region. Food deprivation for 72 h decreased transgene expression in the paraventricular nucleus but not in the arcuate nucleus. Leptin treatment, that was per se ineffective, counteracted the decrease of transgene expression induced in the paraventricular nucleus by 72 h fasting. Supplementing the drinking water with 10% glucose increased beta-galactosidase expression both in the paraventricular nucleus and arcuate nucleus of control mice. Finally, none of the treatments altered transgene expression in the dorsomedial hyphothalamic, ventromedial, and amygdaloid nuclei. Results suggest that changes in energetic balance affect Y(1) receptor expression in the paraventricular and arcuate nuclei and that leptin regulates the NPY-Y(1) system in the paraventricular nucleus. Different regulatory signals might modulate the NPY-Y(1) transmission in the dorsomedial hyphothalamic and ventromedial hyphothalamic nuclei.     

Immunohistochemical localization of substance P-like immunoreactivity in the hypothalamus of the lizard, Podarcis sicula, R

The distribution of substance P (SP) immunoreactivity was investigated in the hypothalamus (preoptic area included) of the lizard by single and double immunocytochemical procedures, SP-immunopositive cell bodies were seen in the paraventricular nucleus and periventricular hypothalamic gray (including the paraventricular organ) together with some more lateral elements. Extensive nerve fibers were seen in the white matter and surrounding the paraventricular and supraoptic neurons, and more caudally reaching the hypothalamic periventricular gray, suggesting a massive involvement of SP-like substance in the control of hypothalamic neuroendocrine areas.     

Expression of vesicular glutamate transporter-2 in gonadotropin-releasing hormone neurons of the adult male rat

Isoforms of the recently cloned vesicular glutamate transporters (VGLUT1-3) selectively accumulate glutamic acid into synaptic vesicles in excitatory axon terminals and are viewed as reliable markers for glutamatergic neurons. Our present studies provided dual-label in situ hybridization evidence that virtually all (99.5%) GnRH neurons express VGLUT2 mRNA in the preoptic region of the adult male rat. Dual-label immunofluorescent experiments were carried out to examine the presence of VGLUT2 protein in GnRH axon terminals. Confocal laser microscopic analysis of the organum vasculosum of the lamina terminalis and the external zone of the median eminence, the major termination fields for GnRH-secreting axons, demonstrated the frequent occurrence of VGLUT2 immunoreactivity in GnRH axon terminals. Together these mRNA hybridization and immunocytochemical data indicate that GnRH neurons of the adult male rat possess marked glutamatergic characteristics. The physiological significance of endogenous glutamate in the regulation of gonadotropin secretion requires clarification.     

The distribution of cells containing estrogen receptor-alpha (ERalpha) and ERbeta messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females

We have used in situ hybridization to compare the distributions of estrogen receptor alpha (ERalpha) and ERbeta messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusion-fixed brain tissue was collected from luteal phase ewes and intact rams (n = 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides. A number of sex differences were observed, with females having a greater density of labeled cells than males (P < 0.001) and a greater number of silver grains per cell (P < 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P < 0.05) cell density for ERalpha mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P < 0.05) of ERalpha mRNA-containing cells than males. There was a trend (P = 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ERalpha mRNA. In both sexes, there was considerable overlap in the distributions of ERalpha and ERbeta mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that contained a higher (P < 0.001) density of ERalpha than ERbeta mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P < 0.001), paraventricular nucleus (males only, P < 0.05), and retrochiasmatic nucleus (females only, P < 0.05) had a greater density of ERalpha than ERbeta mRNA-containing cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ERalpha, whereas only ERbeta mRNA-containing cells were seen in the zona incerta. The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ERalpha and ERbeta mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ERbeta mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ERbeta may not be involved in these functions.     

Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons

The neuropeptide kisspeptin has recently been implicated as having a critical role in the activation of the GnRH neurons to bring about puberty. We examined here the postnatal development of kisspeptin neuronal populations and their projections to GnRH neurons in the mouse. Three populations of kisspeptin neurons located in the 1) anteroventral periventricular nucleus (AVPV) and the preoptic periventricular nucleus (PeN), 2) dorsomedial hypothalamus, and 3) arcuate nucleus were identified using an antisera raised against mouse kisspeptin-10. A marked 10-fold (P<0.01), female-dominant sex difference in the numbers of kisspeptin neurons existed in the AVPV/PeN but not elsewhere. Kisspeptin neurons in the AVPV/PeN of both sexes displayed a similar pattern of postnatal development with no cells detected at postnatal day (P) 10, followed by increases from P25 to reach adult levels by puberty onset (P<0.01; P31 females and P45 males). This pattern was not found in the dorsomedial hypothalamus or arcuate nucleus. Dual immunofluorescence experiments demonstrated close appositions between kisspeptin fibers and GnRH neuron cell bodies that were first apparent at P25 and increased across postnatal development in both sexes. These studies demonstrate kisspeptin peptide expression in the mouse hypothalamus and reveal the postnatal development of a sexually dimorphic continuum of kisspeptin neurons within the AVPV and PeN. This periventricular population of kisspeptin neurons reaches adult-like proportions at the time of puberty onset and is the likely source of the kisspeptin inputs to GnRH neurons.     

Anorexigenic effects of central neuropeptide K are associated with hypothalamic changes in juvenile Gallus gallus

The central mechanisms that mediate neuropeptide K (NPK) associated anorexia are poorly understood in any species, and information in this area of avian biology is totally lacking. Thus, the effects of intracerebroventricular NPK treatment were studied in Cobb-500 chicks (Gallus gallus). In Experiment 1, NPK caused decreased feed intake, but did not affect water intake or whole blood glucose concentration. In Experiment 2, NPK-treated chicks had increased c-Fos immunoreactivity in the parvicellular division of the paraventricular nucleus and arcuate nucleus. The lateral hypothalamus, ventromedial hypothalamus, dorsomedial hypothalamus, periventricular nucleus, magnocellular division of the paraventricular nucleus, and the superchiasmatic nucleus were not affected by NPK treatment. In Experiment 3, the number of feed pecks, exploratory pecks, jumps, escape attempts, and distance moved were decreased, while time spent standing was increased. None of the NPK-treated chicks sat or entered deep rest. In Experiment 4, blockage of corticotrophin releasing factor receptors did not affect NPK-induced anorexia. Thus, we conclude that NPK is a regulator of chick appetite and the effects may be mediated directly in the arcuate nucleus and parvicellular division of the paraventricular nucleus.     

Embryonic birthdate of hypothalamic leptin-activated neurons in mice

The hypothalamus plays a critical role in the regulation of energy balance. Neuroanatomical and mouse genetic data have defined a core circuitry in the hypothalamus that mediates many of the effects of leptin on feeding and energy balance regulation. The present study used 5-bromo-2'-deoxyuridine (a marker of dividing cells) and a neuronal marker to systematically examine neurogenesis in the mouse embryonic hypothalamus, particularly the birth of neurons that relay leptin signaling. The vast majority of neurons in hypothalamic nuclei known to control energy balance is generated between embryonic days (E) 12 and E16, with a sharp peak of neurogenesis occurring on E12. Neurons in the dorsomedial and paraventricular nuclei and the lateral hypothalamic area are born between E12 and E14. The arcuate and ventromedial nuclei exhibit a relatively longer neurogenic period. Many neurons in these nuclei are born on E12, but some neurons are generated as late as E16. We also examined the birth of leptin-activated cells by coupling the 5-bromo-2'-deoxyuridine staining with cFos immunohistochemistry. Remarkably, the majority of leptin-activated cells in the adult hypothalamus were also born during a discrete developmental window on E12. These results provide new insight into the development of hypothalamic neurons that control feeding and identify important developmental periods when alterations in the intrauterine environment may affect hypothalamic neurogenesis and produce long-term consequences on hypothalamic cell numbers.     

Acute effects of estradiol on circulating luteinizing hormone and prolactin concentrations and on serotonin turnover in individual brain nuclei

Two experiments tested whether serotonergic neurons participate in the acute feedback effects of estradiol on LH and PRL secretion. In the first study, ovariectomized female rats received oil vehicle or estradiol benzoate and were killed 3 h later. The turnover of serotonin [5-hydroxytryptamine (5-HT)] in microdissected brain nuclei was assessed from its accumulation after monoamine oxidase inhibition. Estradiol treatment produced a gradual rise in PRL and a decline in LH, and potentiated the accumulation of 5-HT in the medial preoptic, ventromedial, and cortical amygdaloid nuclei, suggesting increased turnover. Concentrations of the metabolite, 5-hydroxyindole acetic acid, were also elevated in these areas. 5-HT turnover was unaffected by estradiol in the interstitial nucleus of the stria terminalis, periventricular, dorsomedial, or lateral amygdaloid nuclei. The second experiment tested whether localized 5-HT depletions in these estradiol-activated nuclei blocked the acute effects of the steroid on LH or PRL. Stereotaxic microinjections of the 5-HT neurotoxin, 5,7, dihydroxytryptamine were made into the preoptic area, cortical amygdala, or medial basal hypothalamus in ovariectomized rats, pretreated with desmethylimipramine. Such treatment reduced 5-HT, without affecting catecholamines, in the targeted structures. Localized 5-HT depletion in the preoptic area and amygdala blunted the elevation of PRL, but not the decline of LH, after estradiol. 5-HT depletion in the medial basal hypothalamus elevated, whereas 5-HT depletion in the medial preoptic area lowered, resting levels of LH. These results suggest that: 1) estradiol may acutely increase PRL secretion by activating serotonergic projections to the medial preoptic and cortical amygdaloid nuclei, and 2) serotonergic neurons innervating the preoptic area stimulate LH release, whereas serotonergic systems in the medial basal hypothalamus inhibit LH release.     

Regional distribution of rat growth hormone releasing factor-like immunoreactivity in rat hypothalamus

A heterologous RIA for rat GH-releasing factor (rGRF) was established using synthetic rGRF-(1-43)OH for both standard reference and radioiodination with the antiserum produced against human GRF-(1-44) NH2. The regional distribution of rGRF-like immunoreactivity (LI) in rat hypothalamus was examined according to the Palkovits microdissection method and compared with that of somatostatin (SRIF)-LI. Both rGRF- and SRIF-LI contents (mean +/- SE; nanograms per mg protein) were highest in the median eminence (rGRF, 32.28 +/- 11.42; SRIF, 109.9 +/- 19.2) and next most abundant in the arcuate nucleus (rGRF, 3.50 +/- 0.47; SRIF, 12.88 +/- 0.60). Only a small amount of rGRF-LI was found in the ventromedial (1.41 +/- 0.51) and dorsomedial (1.16 +/- 0.15) nuclei and the anterior hypothalamic area (1.29 +/- 0.42), whereas rGRF-LI was not detected in the other nuclei of the hypothalamus. A considerable amount of SRIF-LI was contained in the periventricular, ventromedial, and paraventricular nuclei and the anterior hypothalamic area, in accordance with other reports. Gel filtration chromatography revealed that hypothalamic extracts contained a major peak of rGRF-LI corresponding to rGRF-(1-43)OH and two peaks of SRIF-LI equivalent to SRIF-(1-28) and SRIF-(1-14), respectively. These findings indicate that rGRF-LI is localized in the median eminence and arcuate nucleus in the rat and that rGRF-, SRIF-(1-28)-, and SRIF-(1-14)-LI are present in a 1:2.10:6.29 ratio on a molar basis.     

Differential patterns of Fos induction in the hypothalamus of the rat following central injections of galanin-like peptide and galanin

Galanin and its newly discovered relative galanin-like peptide (GALP) are neuropeptides that are implicated in the neuroendocrine regulation of body weight and reproduction. GALP has been shown to bind in vitro to galanin receptor subtypes 1 and 2, but whether it has its own specific receptor(s) is unknown. We reasoned that if GALP acts through a receptor that is distinct from galanin receptors, then GALP should activate central pathways that are different from those activated by galanin. The purpose of this study was to determine whether galanin and GALP produce different patterns of neuronal activation within the hypothalamus. Quantitative analysis of Fos immunoreactivity showed that galanin induced a significantly greater number of Fos-positive nuclei in the paraventricular nucleus compared with GALP (P < 0.001); however, compared with galanin, GALP induced significantly more Fos-positive cells in the horizontal limb of the diagonal band of Broca, caudal preoptic area, arcuate nucleus, and median eminence (P < 0.05). These observations suggest that GALP and galanin act through different receptor-mediated pathways to exert their effects on the regulation of body weight and reproduction and identify target cells for GALP's specific actions in the hypothalamus, including the preoptic area, paraventricular and arcuate nuclei, and the median eminence.