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.     

Distribution of somatostatinlike immunoreactivity in the brain of the little brown bat (Myotis lucifugus)

The distribution of somatostatinlike immunoreactive (SLI) perikarya, axons, and terminals was mapped in subcortical areas of the brain of the little brown bat, Myotis lucifugus, using light microscopic immunocytochemistry. A preponderance of immunoreactivity was localized in reticular, limbic, and hypothalamic areas including: 1) in the forebrain: the bed nucleus of the stria terminalis; lateral preoptic, dorsal, anterior, lateral and posterior hypothalamic areas; amygdaloid, periventricular, arcuate, supraoptic, suprachiasmatic, ventromedial, dorsomedial, paraventricular, lateral and medial mammillary, and lateral septal nuclei; the nucleus of the diagonal band of Broca and nucleus accumbens septi; 2) in the midbrain: the periaqueductal gray, interpeduncular, dorsal and ventral tegmental, pretectal, and Edinger-Westphal nuclei; and 3) in the hindbrain: the superior central and parabrachial nuclei, nucleus incertus, locus coeruleus, and nucleus reticularis gigantocellularis. Other areas containing SLI included the striatum (caudate nucleus and putamen), zona incerta, infundibulum, supramammillary and premammillary nuclei, medial and dorsal lateral geniculate nuclei, entopeduncular nucleus, lateral habenular nucleus, central medial thalamic nucleus, central tegmental field, linear and dorsal raphe nuclei, nucleus of Darkschewitsch, superior and inferior colliculi, nucleus ruber, substantia nigra, mesencephalic nucleus of V, inferior olivary nucleus, inferior central nucleus, nucleus prepositus, and deep cerebellar nuclei. While these results were similar in some respects to those previously reported in rodents, they also provided interesting contrasts.     

Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.     

Long‐term effects of a single exposure to immobilization: A C‐fos mRNA study of the response to the homotypic stressor in the rat brain

A single exposure to a severe emotional stressor such as immobilization in wooden boards (IMO) causes long‐term (days to weeks) peripheral and central desensitization of the hypothalamic‐pituitary‐adrenal (HPA) response to the same (homotypic) stressor. However, the brain areas putatively involved in long‐term desensitization are unknown. In the present experiment, adult male rats were subjected to 2 h of IMO and, 1 or 4 weeks later, exposed again to 1 h IMO together with stress‐naive rats. C‐fos mRNA activation just after IMO and 1 h after the termination of IMO (post‐IMO) were evaluated by in situ hybridization. Whereas in most brain areas c‐fos mRNA induction caused by the last IMO session was similar in stress‐naive (controls) and previously immobilized rats, a few brain areas showed a reduced c‐fos mRNA response: ventral lateral septum (LSv), medial amygdala (MeA), parvocellular region of the paraventricular hypothalamic nucleus (pPVN), and locus coeruleus (LC). In contrast, an enhanced expression was observed in the medial division of the bed nucleus stria terminalis (BSTMv). The present work demonstrates that a previous experience with a stressor can induce changes in c‐fos mRNA expression in different brain areas in response to the homotypic stressor and suggests that LSv, MeA, and BSTMv may be important for providing signals to lower diencephalic (pPVN) and brainstem (LC) nuclei, which results in a lower physiological response to the homotypic stressor. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Autoradiographic localization of 3Hdihyrotestosterone in the preoptic area,hypothalamus, and amygdala of a male rhesus monkey

In a preliminary study, autoradiography was used to localize target cells for ³Hdihydrotestosterone (DHT), a non-aromatizable androgen, in the brain of the rhesus monkey. One castrated male was injected intravenously with 2 mCi of ³HDHT (0.42 μg/kg), and was killed one hour later. Neurons that concentrated radioactivity in their nuclei were located in widespread areas of the brain, which included the medial and suprachiasmatic preoptic nuclei, bed nucleus of the stria terminalis, lateral septal nucleus, anterior hypothalamic area, ventromedial, arcuate, or dorsemedial, and paraventricular hypothalamic nuclei, ventral premammillary nucleus, and medial, cortical, basal accessory, and lateral amygdaloid nuclei. These results indicate that the topographic distribution of androgen target neurons is considerably wider than that observed in a study using ³Htestosterone (T) in the male rhesus monkey (1). However, further work is needed to elucidate these differences before attempting correlations between behavioral activity and androgen receptors in the brain.     

Expression pattern of Zac1 mouse gene, a new zinc-finger protein that regulates apoptosis and cellular cycle arrest, in both adult brain and along development

Using in situ hybridization, we analyzed the expression pattern of the Zac1 gene in mouse brain during the embryonic and postnatal development. Zac1 is a new gene that regulates extensive apoptosis and cell cycle arrest through unrelated pathways. At embryonic stages, strong expression was observed in brain areas with active proliferation (ventricular zone and numerous neuroepithelius) and in nervous system (neural retina and neural tube). In addition, some areas with differentiation activity were noticeably labeled such as arcuate nucleus and amygdaloid region of the brain together with other embryonic sites (hindlimb, forelimb and somites). From P0 onwards, the expression appeared in some proliferative areas, such as subventricular zone and cerebellum (external granular layer and Purkinje cells) and in some synaptic plasticity areas, such as the dorso and ventromedial hypothalamic nuclei, arcuate nucleus, ventral thalamic nucleus.     

Taurine Levels in Discrete Brain Nuclei of Rats

Concentrations of taurine have been measured in 44 microdissected rat brain nuclei or areas. Taurine is ubiquitously present and distributed unevenly in the rat brain: the ratio of the highest (pyriform cortex) to lowest (midbrain reticular formation) concentrations is 4.7:1. High taurine levels were found in cerebral cortical areas, caudate-putamen, cerebellum, median eminence, and supraoptic nucleus. Acute pain stress reduced taurine levels in the hypothalamus and the lower brainstem nuclei but not in cortical areas. Increased locomotor and behavioral activities following a high dose of amphetamine elevated taurine concentrations significantly in the substantia nigra and locus ceruleus.     

Long-term effects of a single exposure to immobilization: a c-fos mRNA study of the response to the homotypic stressor in the rat brain

A single exposure to a severe emotional stressor such as immobilization in wooden boards (IMO) causes long-term (days to weeks) peripheral and central desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the same (homotypic) stressor. However, the brain areas putatively involved in long-term desensitization are unknown. In the present experiment, adult male rats were subjected to 2 h of IMO and, 1 or 4 weeks later, exposed again to 1 h IMO together with stress-naive rats. C-fos mRNA activation just after IMO and 1 h after the termination of IMO (post-IMO) were evaluated by in situ hybridization. Whereas in most brain areas c-fos mRNA induction caused by the last IMO session was similar in stress-naive (controls) and previously immobilized rats, a few brain areas showed a reduced c-fos mRNA response: ventral lateral septum (LSv), medial amygdala (MeA), parvocellular region of the paraventricular hypothalamic nucleus (pPVN), and locus coeruleus (LC). In contrast, an enhanced expression was observed in the medial division of the bed nucleus stria terminalis (BSTMv). The present work demonstrates that a previous experience with a stressor can induce changes in c-fos mRNA expression in different brain areas in response to the homotypic stressor and suggests that LSv, MeA, and BSTMv may be important for providing signals to lower diencephalic (pPVN) and brainstem (LC) nuclei, which results in a lower physiological response to the homotypic stressor.     

Plasma hyperosmolality increases G protein and 3',5'-cyclic adenosine monophosphate synthesis in the paraventricular and supraoptic nuclei

Hyperosmotic stimuli produce profound changes in cellular morphology and biosynthetic activities within the hypothalamic paraventricular and supraoptic nuclei (SON) of the rat. The mechanisms by which osmoreceptive signals are transduced within these nuclei are poorly understood. We examined several components of the cAMP-associated second messenger system after giving rats 2% saline to drink for one week, a strong hyperosmotic stimulus. We found that mRNA levels for both the stimulatory and inhibitory guanine-nucleotide binding protein alpha-subunits were increased in the paraventricular nucleus and SON. In the SON, these changes were accompanied by increased basal cAMP levels, cholera toxin-stimulated adenylate cyclase, and Gs alpha. Our results suggest that Gs alpha levels are not saturated with respect to adenylate cyclase coupling and that osmoreception activates the cAMP second messenger system.     

The Differential Response of Protein Kinase A to Cyclic AMP in Discrete Brain Areas Correlates with the Abundance of Regulatory Subunit II

Abstract: We analyzed the expression and relative distribution of mRNA for the regulatory subunits (RIα, RIIα, and RIIβ) and of 150-kDa RIIβ-anchor proteins for cyclic AMP (cAMP)-dependent protein kinase (PKA) into discrete brain regions. The subcellular distribution of both holoenzyme and free catalytic subunit was evaluated in the same CNS areas. In the neocortex and corpus striatum high levels of RIIβ paralleled the presence of specific RII-anchoring proteins, high levels of membrane-bound PKA holoenzyme, and low levels of cytosolic free catalytic activity (C-PKA). Conversely, in brain areas showing low RIIβ levels (cerebellum, hypothalamus, and brainstem) we found an absence of RII-anchoring proteins, low levels of membrane-bound holoenzyme PKA, and high levels of cytosolic dissociated C-PKA. Response to cAMP stimuli was specifically evaluated in the neocortex and cerebellum, prototypic areas of the two different patterns of PKA distribution. We found that cerebellar holoenzyme PKA was highly sensitive to cAMP-induced dissociation, without, however, a consistent translocation of C-PKA into the nucleus. In contrast, in the neocortex holoenzyme PKA was mainly in the undissociated state and poorly sensitive to cAMP. In nuclei of cortical cells cAMP stimulated the import of C-PKA and phosphorylation of cAMP-responsive element binding protein. Taken together, these data suggest that RIIβ (whose distribution is graded throughout the CNS, reaching maximal expression in the neocortex) may represent the molecular cue of the differential nuclear response to cAMP in different brain areas, by controlling cAMP-induced holoenzyme PKA dissociation and nuclear accumulation of catalytic subunits.     

Biochemical Mapping of Peptidyl-Glycine α-Amidation Activity in the Rat CNS

Peptidyl-glycine alpha-amidation enzyme activity has been measured in 36 nuclei or areas in the rat CNS and pituitary using D-Tyr-Phe-Gly as the substrate. The distribution of this enzyme is highly uneven, with highest activity levels (greater than 30 pmol/mg of protein/h) in hypothalamic nuclei, substantia grisea centralis, and nucleus ruber; moderate activity levels (10-30 pmol/mg of protein/h) in globus pallidus, septum, midbrain, pons, medulla oblongata, and cervical spinal cord; and low activity levels (1-10 pmol/mg of protein/h) in other telencephalic and thalamic structures. Almost no alpha-amidation activity (less than 0.5 pmol/mg of protein/h) was detected in cerebellar cortex. The Km values in several brain regions are of the same order.     

Maternal obesity alters adiposity and monoamine function in genetically predisposed offspring

The impact of maternal obesity on brain monoamine function in adult offspring of dams selectively bred to express diet-induced obesity (DIO) or diet resistance (DR) was assessed by making dams obese or lean during gestation and lactation. After 12 wk on chow and 4 wk on a 31% fat diet, offspring hypothalamic nucleus size and [(3)H]nisoxetine binding to norepinephrine transporters (NET) and [(3)H]paroxetine binding to serotonin transporters (SET) were measured. Offspring of obese DIO dams became more obese than all other groups, but maternal obesity did not alter weight gain in DR offspring (25). Maternal obesity was associated with 10-17% enlargement of ventromedial nuclei (VMN) and dorsomedial nuclei in both DIO and DR offspring. Offspring of obese DIO dams had 25-88% lower NET binding in the paraventricular nuclei (PVN), arcuate nuclei, VMN, and the central amygdalar nuclei, while offspring of obese DR dams had 43-67% higher PVN and 90% lower VMN NET binding and a generalized increase in SET binding across all hypothalamic areas compared with other groups. Thus maternal obesity was associated with alterations in offspring brain monoamine metabolism, which varied as a function of genotype and the development of offspring obesity.     

Hypothalamic paraventricular injections of ghrelin: effect on feeding and c-Fos immunoreactivity

The paraventricular hypothalamic nucleus (PVN) appears to integrate orexigenic properties of a novel peptide, ghrelin. Thus, we examined central mechanisms underlying feeding generated by intra-PVN ghrelin. We established that 0.03 nmol of PVN-injected ghrelin was the lowest dose increasing food consumption and it induced c-Fos immunoreactivity (a marker of neuronal activation) in the PVN itself, as well as in other feeding-related brain areas, including the hypothalamic arcuate and dorsomedial nuclei, central nucleus of the amygdala, and nucleus of the solitary tract. We conclude that the PVN, as part of larger central circuitry, mediates orexigenic properties of ghrelin.     

Distribution of gastrin-releasing peptide immunoreactivity in the brain of the collared dove (Streptopelia decaocto)

The distribution of immunoreactivity after applying an antibody against gastrin-releasing peptide (GRP) was studied in the brain of the collared dove (Streptopelia decaocto). In the forebrain GRP-immunoreactive (GRP-ir) cells were found in the hyperstriatum accessorium, medial and lateral parts of the neostriatum, corticoidea dorsolateralis and temporoparieto-occipitalis areas, hippocampus, pre- and parahippocampal areas and prepiriform cortex. In the brainstem, GRP-ir cells were restricted mainly to the substantia nigra and ventral tegmental nucleus. Areas with densely packed GRP-ir clusters of varicosities were the medial intermediate hyperstriatum ventrale and lateral septal nucleus; dense GRP-ir neuropil was found in the parolfactory lobe, and in the dorsal half of the intermediate and caudal archistriatum. The ventral lamina medullaris contained many GRP-ir fibers. Forebrain areas devoid of immunoreactivity were the basal nucleus, ectostriatum, rostral archistriatum, most of the paleostriatum augmentatum and the lateral bed nucleus of the stria terminalis. Moderate densities of GRP-ir elements were found in the other telencephalic areas and further in, among others, the preoptic and hypothalamic region, ventral area of Tsai, cerulean nuclei, parabrachial complex, dorsal glossopharyngeal and vagus motor nuclei and medial nuclei of the solitary complex. The observations are compared with data from the literature and the implications for the definition of specific centers within the avian brain are discussed, with emphasis on systems with a role in visceral and motivational functions and in learning.     

刺激大鼠穹窿下器官诱发饮水和脑内c—fos表达的胆碱能机制

实验以饮水行为脑内c-fos表达为指标,,观察刺激大鼠穹窿下器官（SFO）的效应,结果显示,刺激SFO能诱发明显的饮水行为,与此同时,前脑8个部位（终板血管器官,正中视前核,室旁核,视上核,下丘脑外侧区,穹窿周核背侧区,丘脑联合核和无名质）和后脑3个部位（最后区,孤束核和壁旁外侧核）的Fos蛋白表达明显增强,免疫组化双重染色结果显示,刺激SFO能诱导视上核和室旁核中部分神经元呈Fos蛋白和加压素共同表达。脑室注射阿托品能部分阻断刺激SFO诱发的饮水行为,脑内上述各部位所诱导的Fos蛋白表达也明显减弱,以上结果提示,M胆碱能机制参与 刺激SFO诱发的饮水行为和脑内Fos蛋白的表达。     

Distribution of Preproatrial Natriuretic Peptide mRNA in Rat Brain Detected by In Situ Hybridization of DNA Oligonucleotides: Enrichment in Hypothalamic and Limbic Regions

The expression and distribution of mRNA encoding preproatrial natriuretic peptide (ppANP) in rat brain has been investigated by in situ hybridization of two 35S-labeled synthetic DNA oligonucleotides, based on a cDNA clone sequence that encodes rat ppANP. The highest relative concentrations of ppANP mRNA were detected in the medial preoptic hypothalamic nucleus ("anteroventral/third ventricle region") and the medial habenula. Moderate concentrations of ppANP mRNA were observed in the CA1 pyramidal cells of the hippocampus, the endopiriform nucleus, the arcuate nucleus, the zona incerta, and cells of the pontine tegmental and peduculopontine nuclei. Several of these regions, including the habenula and the hypothalamic areas, have previously been reported to contain atrial natriuretic peptide (ANP)-like immunoreactivity, but the expression of ppANP mRNA in CA1 pyramidal cells suggests the occurrence of differential translation of ppANP mRNA into protein product in different brain regions, or the existence of different immunological forms of the peptide. The abundance of ppANP mRNA in brain was relatively low in comparison with that previously reported for many other mRNA species encoding other brain neuropeptides. These results demonstrate that ANP gene expression occurs in discrete neuronal populations of the CNS and that studies of the regulation of this expression should now be possible using quantitative in situ hybridization.     

The simultaneous quantification of dopamine,norepinephrine and epinephrine in micropunched rat brain nuclei by on-line trace enrichment HPLC with electrochemical detection: Distribution of catecholamines in the limbic system

A method is described for the simultaneous quantification of dopamine, norepinephrine and epinephrine in microdissected rat brain nuclei using on-line trace enrichment high performance liquid chromatography (HPLC) and electrochemical detection (EC). The method is specific, sensitive and rapid with lower limits of detection comparable to those of radioenzymatic and mass fragmentographic techniques. The ideal application of the method to determinations in discrete microdissected brain nuclei is illustrated by mapping the topographic distribution of these three catecholamines in the limbic system of the rat. Dopamine is well represented and remarkably heterogeneously distributed in the amygdaloid nuclei. Norepinephrine concentrations are high in the interstitial (bed) nucleus of the stria terminalis and the paraventricular, periventricular, dorsomedial and medial preoptic hypothalamic nuclei, though generally more evenly distributed among the limbic nuclei than dopamine. Epinephrine was reliably quantified in a minority of the limbic nuclei examined with the highest concentration found in the paraventricular hypothalamic nuclei. The method described is ideally suited for examination of the pharmacological and functional heterogeneity of dopamine-, norepinephrine- and epinephrine-containing neuronal systems at the level of discrete brain nuclei.     

Vasopressin-like peptides in the rat brain: immunologic and chromatographic behavior and their response to water deprivation

The immunologic and chromatographic behavior of vasopressin-like immunoreactivity (VP-LI) extracted from rat brain tissue has been studied. VP-LI present in acid extracts of hypothalamic, hippocampal, and septal tissue was found to be immunologically identical to synthetic AVP. When extracts of hypothalamic tissue were fractionated using high-performance liquid chromatography, Arg⁸-vasopressin (AVP) was shown to be the predominant immunoreactive species. In contrast, in addition to AVP, extrahypothalamic brain tissue extracts also contained a small second vasopressin-immunoreactive peak.The effect of water deprivation on brain vasopressin content and chromatographic profile was also studied. This treatment depleted VP-LI content in the posterior pituitary but did not greatly alter that of hypothalamic or extrahypothalamic brain. Microdissection studies showed that VP-LI content was reduced, but only in a restricted number of extrahypothalamic brain nuclei, and that water deprivation failed to alter or increased content in other areas. The results suggest that VP-ergic neurons in the rat brain may be differentially activated.