Testosterone 5 alpha-reductase in discrete hypothalamic nuclear areas in the rat: effect of castration

The conversion of testosterone into 5 alpha-dihydrotestosterone (DHT) has been studied in different hypothalamic nuclear areas and in the superficial layers of the cerebral cortex of normal and castrated male rats. The tissue fragments utilized in each incubation have been punched from frozen brain sections utilizing calibrated needles. Castration has been performed 12 (short term) and 180 (long term) days before sacrifice. The nuclear areas studied include: the medial preoptic nucleus (MPN), the lateral preoptic nucleus (LPN), the anterior hypothalamic nucleus (AHN), the lateral hypothalamic nucleus (LHN), the posterior hypothalamic nucleus (PHN), the nucleus ventromedialis (HVM), the arcuate nucleus (AR), the median eminence (ME), the nucleus paraventricularis (HPV), the supraoptic nucleus (SO) and the suprachiasmatic nucleus (SC). The possible effect of castration on the 5 alpha-reductase, were assessed in the MPN,LPN,AHN,LHN,PHN and in the cerebral cortex. The results indicate that, in the male rat: 1) the lateral preoptic(LPN) and the lateral hypothalamic nuclei(LHN) possess a 5 alpha-reductase activity higher than that present in the cerebral cortex and in the other hypothalamic nuclei considered; 2)the suprachiasmatic nucleus (SC) apparently possesses a testosterone metabolizing activity lower than that found in any other nervous structures studied so far; 3) castration does not seem to influence the 5 alpha-reductase activity either in the hypothalamic nuclear structures considered or in the cerebral cortex.     

Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus

Summary The rat hypothalamus was studied at the light microscopic level with the use of single and double immunocytochemical staining methods. It was shown that the rat supraoptic and paraventricular hypothalamic nuclei, and their accessory neurosecretory nuclei, do not contain magnocellular somatostatin neurons. The distribution of the hypothalamic parvocellular somatostatin cells is described. The parvocellular component of the rat hypothalamic paraventricular nucleus is, at least partly, composed of somatostatin cells: they form a fairly well circumscribed periventricular cell mass. The rat suprachiasmatic nuclei contain separate somatostatin neurons and vasopressin neurons. Scattered somatostatin cells are present in the entire arcuate nucleus. In addition to the periventricular somatostatin cells located in the preopticanterior hypothalamic area and in the arcuate nucleus, the rat hypothalamus also contains numerous scattered somatostatin cells located distant from the third ventricle.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

A histochemical study of the primary catecholamines in the hypothalamic neurons of the rat in relation to the ontogenetic and sexual differentiation

Summary The alterations in the content of the primary catecholamines in the hypothalamus have been studied with the histochemical technique of para-formaldehyde induced fluorescence.In the adult normal rats, independent of the sex, the fluorescence is located in the cell bodies of a few arcuate neurons, around the perikarya of the arcuate, para-ventricular and supra-optic neurons, and in the nerve endings of the arcuate neurons in the median eminence.The appearance of the primary catecholamines takes place at the 20th day of gestation in the para-ventricular and arcuate-peri-ventricular regions. In the supra-optic nucleus the fluorescent nerve terminals are not seen before birth. In the outer layer of the median eminence the fluorescence develops around the 5th post-natal day. No sexual differences were observed in the maturation of the primary catecholamines during the ontogenic development of the rat.More fluorescent cell bodies and nerve endings are seen in the arcuate neurons during the late diestrus than during estrus. The number and intensity of the catecholamine fluorescent neurons in the arcuate nucleus increases during the pregnancy. Castration increases slightly the number and intensity of the fluorescent cell bodies in the arcuate nucleus, but it diminishes the fluorescence in the median eminence. The changes were compensated by a treatment with testosterone propionate. Hypophysectomy alone has no effect on the fluorescence of the hypothalamic neurons.Supported by a grant from The Finnish Medical Society Duodecim.     

The neurons containing dense core vesicles in the mouse hypothalamic arcuate nucleus: distribution and influence of ovarian activity

The hypothalamic arcuate nucleus (ARC) was electron microscopically studied in diestrous (D) and estrous (E) mice, and mice ovariectomized (OX) 3 weeks before. In the middle region of the ARC (mARC), the number of dense core vesicles (DCVs) appearing in a section of each neuronal perikaryon was examined, and the location of the neurons containing DCVs was mapped. About 10% of the neurons in the mARC contained many (5 or more) DVCs in the perikarya in a section. These neurons mainly localized in the ventromedial portion of the mARC, which shows that the cytoarchitecture of the mouse ARC is heterogeneous. The localization and the frequency of these neurons were not influenced by the variance of ovarian activity. A DCV was composed of an electron-dense core and a membrane halo, and was likely to be formed in the cisternae of the Golgi apparatus. The size of DCVs was estimated at 100 nm as the median diameter and was stable independent of the ovarian activity. However, the formation of DCVs was likely influenced by the ovarian activity, because DCVs in D mice were more intimately associated with the Golgi apparatus than in E or OX mice. In the neuronal perikarya of the ventromedial portion of the mARC, Nissl bodies developed in OX mice and nucleolus-like bodies developed in E mice. These results suggest that the neurons of the ventromedial portion of the mARC change their function according to the variance of ovarian activity and, therefore, are involved in the estrogen feedback mechanism.     

大鼠下丘脑内一氧化氮合酶阳性神经元的分布

用ＮＡＤＰＨ－ｄ组织化学方法观察了大白鼠下丘脑内一氧化氮合酶（ＮＤＳ）阳性神经元的分布及形态特征。结果显示：在视上核、室旁核的大细胞部、环状核、穹窿周核、下丘脑外侧区、下丘脑腹内侧核、下丘脑背内侧核、乳头体区大部分核团均可见一氧化氮合酶阳性神经元聚集成团。在视前内侧区、视前外侧区、下丘脑前区、下丘脑背侧区、下丘脑后区、室周核、室旁核小细胞部及穹窿内可见散在的一氧化氮合酶阳性神经元。室周核内可见呈阳性反应的接触脑脊液神经元的胞体及突起。一氧化氮合酶阳性神经元大多可见突起,有的突起上可见１～２级分支,并可见膨体。下丘脑大部分区域内可见阳性神经纤维。弓状核内可见许多弧形纤维连于第三脑室室管膜和正中隆起。     

Electron microscopic observations on extraneuronal lipofuscin in the monkey brain

Protein synthesizing activity of the rat hypothalamic arcuate nucleus following partial or total deafferentation of the medial basal hypothalamus was studied by light and electron microscopic autoradiography when administering tritiated leucine into the lateral ventricle. There were significantly more grains over the arcuate nucleus 21 days after disconnection of this hypothalamic region than over the intact nucleus. Isolation of a temporal cortical region induced similar changes in the isolated area, although this effect was not so pronounced as in the arcuate region. Data suggest that the protein synthesizing activity of arcuate neurons increases significantly after interruption of neural connections of the medial basal hypothalamus. It is assumed that the effect is primarily due to transneuronal alteration and/or interruption of inhibitory afferents.     

Target neurons of endogenous androgens immunocytochemically localized in male rat hypothalamus

A polyclonal antiserum against androgens, i.e., testosterone, 5alpha-dihydrotestosterone and androstenedione, was tested to reveal target neurons of endogenous androgens in the hypothalamus of both intact and castrated male rats. Paraffin sections of hypothalamus and testis were immunostained by using Avidin-Biotin Complex method and 3-3' diaminobenzidine to visualize the immunoperoxidase complex. Conventional control experiments for method and antiserum specificity were performed. The antiserum proved to be specific for androgens, i.e., testosterone, 5alpha-dihydrotestosterone and androstenedione. The nuclear labeling observed in tissues stained by this procedure is consistent with the hypothesis that the labeled neurons contained DHT, which is the main testosterone metabolite active in the cell nucleus. The antiserum was effective in staining not only the hypothalamic neurons of intact males with normal serum levels of testosterone but also the hypothalamic neuron of castrated males with very low serum levels of testosterone. Evidence is presented indicating that the immunostaining technique represents a more specific and sensitive method to identify target neurons of endogenous androgens than autoradiography.     

Morphological interaction between galanin-like peptide- and dopamine-containing neurons in the rat arcuate nucleus

Galanin-like peptide (GALP) is a 60-amino acid neuropeptide that plays an important role in the neuronal regulation of feeding, energy balance and reproduction. GALP is produced in the hypothalamic arcuate nucleus, an area containing, amongst other neuron types, two populations of neurons in which we were interested: a population of GALP-containing neurons which regulate energy balance and reproduction, and a second population consisting of tuberoinfundibular dopaminergic neurons which suppress prolactin secretion from the adenohypophysis. To characterize morphologically the relationship between GALP and dopamine-containing neurons in the arcuate nucleus, a double immunofluorescence study was performed on cryosections from rat brain. Immunohistochemical double labeling studies revealed that GALP-immunoreactive nerve fibers made direct contact on tyrosine hydroxylase (TH)-containing neuronal cell bodies in the arcuate nucleus. These results suggest that GALP-containing neurons innervate tuberoinfundibular dopaminergic neurons.     

Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus.

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.     

Reserpine-induced immunocytochemical change of neuropeptide Y in the hypothalamic arcuate nucleus

The effect of reserpine on neuropeptide Y immunoreactive (NPY-IR) neurons in the rat hypothalamic arcuate nucleus was examined by immunocytochemical techniques. Although only NPY-IR fibers and terminals were distributed in this nucleus in untreated and saline treated rats, single treatment of reserpine (10 mg/kg, i.p.) visualized abundant NPY-IR neuronal cell bodies: the increase began at 12 h of postinjection, reached its maximal level at 48 h, and returned to its normal level at 96 h. Pretreatment of nialamide, a monoamine oxidase inhibitor, prevented these acute reserpine-induced changes, suggesting reserpine acts on NPY neurons through monoaminergic mechanism. Chronic treatment of haloperidol (5 mg/kg, once daily for 5 days) a dopamine receptor antagonist, could induce the similar increase of NPY immunoreactivity. However, interruption of adrenergic and serotonergic neurotransmissions by chronic treatment of propranorol and methysergide, or chemical lesions of ascending noradrenergic and serotononergic pathways by 6-hydroxydopamine and 5,6-dihydroxytryptamine, could not induce any immunoreactive increase of NPY in arcuate neurons. These findings strongly suggest that reserpine-induced NPY increase occurs through dopaminergic afferents in hypothalamic arcuate neurons. Special issue dedicated to Dr. Kinya Kuriyama.     

Different distributions of nitric oxide synthase-containing neurons in the mouse and rat hypothalamus.

The distribution of nitric oxide synthase-containing neurons was studied in the rat and mouse hypothalamus by immunohistochemistry and NADPH-diaphorase histochemistry. Immunostaining and NADPH-diaphorase staining of hypothalamic neurons were comparable in all hypothalamic nuclei of either species except in the arcuate nucleus that stained positive for nitric oxide synthase immunoreactivity but negative for NADPH-diaphorase reactivity. The presence of nitric oxide synthase-immunopositive neurons in the arcuate nucleus was confirmed by nitric oxide synthase immunofluorescence viewed under the confocal microscope at 1 microm thickness. Cross-species comparison showed that, in general, the number and intensity of nitric oxide synthase-containing neurons were much higher in the rat than in the mouse hypothalamus. Differences in the distribution of nitric oxide synthase-containing neurons between these two rodents were found in most hypothalamic nuclei. In particular, two dense clusters of nitric oxide synthase-containing neurons were found in the paraventricular and supraoptic nuclei of the rat hypothalamus in contrast to their scarcity in the same nuclei of the mouse hypothalamus.     

Expression of neuropeptide Y and agouti-related peptide in the hypothalamic arcuate nucleus of newborn neurogenin3 null mutant mice

Mice deficient in neurogenin 3 (Ngn3) fail to generate pancreatic endocrine cells and intestinal endocrine cells. Hypothalamic neuropeptides implicated in the control of energy homeostasis might also be affected in Ngn3 homozygous null mutant mice. We investigated the expression of two prominent orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), in the hypothalamic arcuate nucleus of newborn wild-type and Ngn3 null mutant mice. Immunohistochemical analysis demonstrated that, in Ngn3 null mutants, the number of NPY-immunoreactive neurons and nerve fibers was markedly increased in the arcuate nucleus, and the nerve fibers were widely distributed in the hypothalamic area, including the paraventricular and dorsomedial nuclei. Little increase of AgRP immunoreactivity was detected in the arcuate nucleus of mutant mice. In situ hybridization analysis confirmed the increased population of the NPY neurons in the arcuate nucleus of the mutants. The NPY mRNA level, as estimated by laser capture microdissection and real-time quantitative polymerase chain reaction, was 371% higher in Ngn3 null mutants than in wild-type mice. AgRP mRNA levels did not differ significantly between the null mutants and wild-type mice. Thus, up-regulation of the hypothalamic NPY system is probably a feature characteristic of Ngn3 null mice.     

Neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity in the cat hypothalamus

Immunohistochemical studies were conducted on sections of cat hypothalamus in order to determine the distribution of neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity. A large number of neurons in the arcuate nucleus were stained after incubation of sections with antisera to either substance. Analysis of serial sections suggested that each neuron revealed with one antiserum was also revealed with the other antiserum, indicating the co-existence of alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity within these arcuate neurons. In contrast, a more diffuse group of lateral hypothalamic neurons which extended from the retrochiasmatic level to the posterior hypothalamus were stained only with the antiserum directed against alpha-melanocyte stimulating hormone. The present results largely confirm findings in the rat hypothalamus, although the lateral hypothalamic group of alpha-melanocyte stimulating hormone immunoreactive neurons appears to be more extensive in the cat.     

Morphological evidence for neural interactions between leptin and orexin in the hypothalamus

Both leptin and orexin have been recently discovered as peptides involved in feeding regulation. The morphological evidence of neural interaction between leptin and orexin, one considered to inhibit food intake and the other to stimulate it in the central nervous system (CNS), was studied by use of double immunostaining method. The leptin receptor-like immunoreactive (LR-LI) neurons in the hypothalamic arcuate nucleus and ventromedial nucleus were innervated by orexin-like immunoreactive (OX-LI) neurons. The distribution of LR-LI neurons in the hypothalamus was very similar to that of OX-LI neurons. These results may suggest that leptin and orexin are intimately correlated with each other and that they reciprocally regulate feeding at the hypothalamic level.     

Steroid control of gonadotropin-releasing hormone secretion: associated changes in pro-opiomelanocortin and preproenkephalin messenger RNA expression in the ovine hypothalamus

The endogenous opioid peptides have been implicated in mediating the actions of estrogen and progesterone on GnRH release. We used in situ hybridization histochemistry to determine whether steroid-induced changes in GnRH/LH release in the female sheep are associated with changes in the cellular mRNA content of the precursors for beta-endorphin (pro-opiomelanocortin; POMC) and met-enkephalin (pre-proenkephalin; PENK). Two specific hypotheses were tested. First, that the inhibitory actions of progesterone are associated with an increase in opioid gene expression in specific hypothalamic nuclei. Our data support this hypothesis. Thus, an increase in progesterone was associated with increased POMC gene expression in the arcuate nucleus and PENK in the paraventricular nucleus. Further, the increase in POMC was restricted to regions of the arcuate nucleus that contain steroid sensitive beta-endorphin neurons. Our second hypothesis, that gene expression for the two opioid precursors would decrease prior to the start of the estradiol-stimulated GnRH surge, was not supported. Rather, POMC (but not PENK) gene expression in the arcuate nucleus was significantly higher in estradiol-treated animals than controls at the peak of the GnRH surge. These data suggest that beta-endorphin neurons in subdivisions of the arcuate nucleus and enkephalin neurons in the paraventricular nucleus are part of the neural network by which progesterone inhibits LH release. While enkephalin neurons may not play a role in estrogen positive feedback, increases in POMC mRNA in the arcuate nucleus at the time of the GnRH peak may be important for replenishing beta-endorphin stores and terminating estrous behavior.     

Relative number and distribution of murine hypothalamic proopiomelanocortin neurons innervating distinct target sites

Proopiomelanocortin (POMC) neurons send projections widely throughout the brain consistent with their role in regulating numerous homeostatic processes and mediating analgesia and reward. Recent data suggest that POMC neurons located in the rostral and caudal extents of the arcuate nucleus of the hypothalamus may mediate selective actions, however it is not clear if POMC neurons in these regions of the arcuate nucleus innervate specific target sites. In the present study, fluorescent microspheres and cholera toxin B were used to retrogradely label POMC neurons in POMC-DsRed transgenic mice. The number and location of POMC cells projecting to the supraoptic nucleus, periaqueductal gray, ventral tegmental area, paraventricular nucleus, lateral hypothalamic nucleus, amygdala and the dosal vagal complex was determined. Tracer injected unilaterally labeled POMC neurons in both sides of the arcuate nucleus. While the total number of retrogradely labeled cells in the arcuate nucleus varied by injection site, less than 10% of POMC neurons were labeled with tracer injected into any target area. Limited target sites appear to be preferentially innervated by POMC neurons that reside in the rostral or caudal extremes of the arcuate nucleus, whereas the majority of target sites are innervated by diffusely distributed POMC neurons. The modest number of cells projecting to each target site indicates that relatively few POMC neurons may mediate potent and specific physiologic responses and therefore disturbed signaling in a very few POMC neurons may have significant consequences.     

Role of melanocortin signaling in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis

The melanocortin signaling system is orchestrated by two, independent groups of neurons in the hypothalamic arcuate nucleus with opposing functions that synthesize either alpha-melanocyte stimulating hormone (alpha-MSH) or agouti-related protein (AGRP). These neurons exert regulatory control over hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus (PVN) at least in part through direct, overlapping, monosynaptic projections to the PVN. Alpha-MSH has an activating effect on hypophysiotropic TRH neurons via the phosphorylation of CREB, and when administered exogenously, can completely reverse fasting-induced suppression of TRH mRNA in the PVN. AGRP has a potent inhibitory effect on the hypothalamic-pituitary-thyroid axis in normally fed animals, mediated through actions at melanocortin 4 receptors. Inhibition of the HPT axis by fasting may be explained by inhibition of melanocortin signaling as a result of a reduction in alpha-MSH and increase in AGRP. Neuropeptide Y may also modulate the effects of the melanocortin signaling system during fasting by potentiating the inhibitory actions of AGRP on hypophysiotropic TRH neurons to prevent the phosphorylation of CREB and through direct inhibitory effects on alpha-MSH-producing neurons in the arcuate nucleus.     

雌激素受体在老年和青年猕猴下丘脑中表达的比较

为了观察老年猕猴下丘脑中雌激素受体的表达及变化情况,运用免疫组织化学SABC 法,对青年和老年猕
猴下丘脑雌激素受体的表达特点进行了研究。结果发现,雌激素受体免疫阳性细胞主要分布于下丘脑的室周灰
质、室旁核、腹外侧核、腹内侧核、弓状核等,雌激素受体阳性产物主要定位于细胞核和细胞质中。与青年猕
猴比较,老年猕猴下丘脑各个核团的雌激素受体在表达强度及阳性细胞数量上均显著或极显著低于青年猕猴
(P<0.05 或 P<0.01)。表明,老年猕猴下丘脑雌激素受体的表达显著低于青年猕猴,可能为老年猕猴雌激素缺
乏所致。     

Proliferation of lamellar whorls in arcuate neurons of the hypothalamus of castrated and morphine-treated male rats

Summary A comprehensive ultrastructural examination of one cross-sectional level (middle 1/3) of the arcuate nucleus of the hypothalamus (AH) of male rats several weeks after castration or after two weeks of morphine treatment confirmed a marked increase in lamellar whorls of endoplasmic reticulum in AH neurons in each group. A comparable incidence of AH whorls was not detected in rats treated with lactose, those treated for only 1–3 days with morphine, or in those given testosterone plus morphine for 2 weeks. It is postulated that the testosterone deficiency following either castration or chronic morphine treatment stimulated the observed increase in AH whorls. A close correspondence was noted between the distribution within the AH of neurons containing whorls and those reported by others to contain luteinizing hormone releasing hormone (LH-RH). The possibility that whorls may be a marker for hypothalamic neurons which play a role in the LH-RH regulatory system warrants further consideration.Supported by Grants DA-00259 and NS-09156 from the United States Public Health ServiceResearch Scientist Development Award MH-38894Research Scientist Development Award MH-70180     

Glucose-sensing neurons of the hypothalamus

Specialized subgroups of hypothalamic neurons exhibit specific excitatory or inhibitory electrical responses to changes in extracellular levels of glucose. Glucose-excited neurons were traditionally assumed to employ a 'beta-cell' glucose-sensing strategy, where glucose elevates cytosolic ATP, which closes KATP channels containing Kir6.2 subunits, causing depolarization and increased excitability. Recent findings indicate that although elements of this canonical model are functional in some hypothalamic cells, this pathway is not universally essential for excitation of glucose-sensing neurons by glucose. Thus glucose-induced excitation of arcuate nucleus neurons was recently reported in mice lacking Kir6.2, and no significant increases in cytosolic ATP levels could be detected in hypothalamic neurons after changes in extracellular glucose. Possible alternative glucose-sensing strategies include electrogenic glucose entry, glucose-induced release of glial lactate, and extracellular glucose receptors. Glucose-induced electrical inhibition is much less understood than excitation, and has been proposed to involve reduction in the depolarizing activity of the Na+/K+ pump, or activation of a hyperpolarizing Cl- current. Investigations of neurotransmitter identities of glucose-sensing neurons are beginning to provide detailed information about their physiological roles. In the mouse lateral hypothalamus, orexin/hypocretin neurons (which promote wakefulness, locomotor activity and foraging) are glucose-inhibited, whereas melanin-concentrating hormone neurons (which promote sleep and energy conservation) are glucose-excited. In the hypothalamic arcuate nucleus, excitatory actions of glucose on anorexigenic POMC neurons in mice have been reported, while the appetite-promoting NPY neurons may be directly inhibited by glucose. These results stress the fundamental importance of hypothalamic glucose-sensing neurons in orchestrating sleep-wake cycles, energy expenditure and feeding behaviour.