bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Cl– Substitution on Electrophysiological Properties, Ca2+ Influx and Prolactin Secretion of Rat Lactotropes in vitro
Authors: Garcia Liliana , Couderc Badia , Odessa Marie Françoise , Dufy-Barbe Luce , Sartor Pierre
Published: April 2003

In this study, we compared the effects of different chloride (Cl–) substitutes – methane sulfonate (CH3SO–3), bromide (Br–), nitrate (NO–3), thiocyanate (SCN–) and perchlorate (ClO–4) – on the secretory activity and calcium current activation of rat lactotropes in primary culture. We observed that CH3SO–3 decreased basal prolactin (PRL) secretion. Br– had no effect, whereas the more lyotropic anions, such as NO–3, SCN– and ClO–4, increased basal PRL secretion. The latter three substitutes induced a significant shift in the voltage dependence of T-type calcium channel activation towards hyperpolarized values. However, this shift alone cannot explain the increase in secretion. Anion permeability studies also demonstrated that the organic anion CH3SO–3 was less permeant than Cl–, whereas monovalent inorganic anions were more permeant, with the following anion permeability sequence: SCN– > ClO–4 > NO–3 > Br–. In conclusion, deprivation of Cl– ions has converse consequences on basal and induced secretion; permeating anions result in a transient increase in intracellular Ca2+ ions. This process involves voltage-dependent Ca2+ channels. We propose that an alteration in intracellular anion concentrations may influence the activation of internal effectors such as G proteins or channel proteins and, therefore, interfere with exocytosis. These effects are correlated with an external action of lyotropic anions, particularly NO–3, ClO–4 and SCN–, on the gating properties of T-type calcium channels, probably through changes in cell surface charges. The results demonstrate the modulatory effect of anions on the secretory activity of rat lactotropes and underline the specific role played by chloride in stimulus-secretion coupling.
Evidence that Neuronal Nitric Oxide Synthase but Not Heme Oxygenase Increases in the Hypothalamus on Proestrus Afternoon
Authors: Lamar Charisee A. , Bhat Ganapathy K. , Mahesh Virendra B. , Brann Darrell W.
Published: April 2003

Nitric oxide (NO) has been implicated in the control of the proestrus luteinizing hormone (LH) surge in the rat but to date no studies have attempted to measure neuronal nitric oxide synthase (nNOS) or NO production on proestrus in the hypothalamus in order to determine if endogenous NO is increased on proestrus afternoon to activate gonadotropin-releasing hormone (GnRH) neurons. To address this deficit in our knowledge, we measured nNOS mRNA and protein levels as well as NOS activity levels in rat preoptic area (POA) and medial basal hypothalamus (MBH) fragments at 12.00, 14.00, 16.00, and 18.00 h of proestrus. Serum LH levels were also assessed to determine whether NOS changes correlate to the LH surge. To determine the specificity of observed changes we also measured mRNA levels for the enzyme heme oxygenase-2, which is responsible for production of another putative gaseous transmitter, carbon monoxide. In all studies a metestrus 12.00 h control group was included since steroid and LH levels would be basal at this time as compared to proestrus. The results revealed that nNOS mRNA and protein levels, as well as NOS activity did not change significantly in the MBH on proestrus. In contrast, nNOS mRNA levels were significantly elevated in the POA at proestrus 12.00 and 14.00 h, as compared to metestrus 12.00 h. Likewise, at the protein and activity level, nNOS protein levels in the POA were significantly elevated on proestrus at 14.00 and 16.00 h, with NOS activity significantly increased at 16.00 h on proestrus. The elevation of nNOS protein and activity levels in the POA occurred at the time of initiation of the LH surge. The elevation of nNOS was specific as mRNA levels for the CO-synthetic enzyme heme oxygenase-2 did not change significantly on proestrus in the POA or MBH. As a whole, the current studies provide new evidence that nNOS is elevated in the POA on proestrus, and thus could play a role in the activation of GnRH neurons to produce the preovulatory LH surge.
Stimulation of the Gonadotropic Axis by the Neuropeptide Y Receptor Y1 Antagonist/ Y4 Agonist 1229U91 in the Male Rat
Authors: Raposinho Paula D. , Broqua Pierre , Hayward Amanda , Akinsanya Karen , Galyean Robert , Schteingart Claudio , Junien Jean-Louis , Aubert Michel L.
Published: April 2003

Neuropeptide Y (NPY) is a highly potent orexigenic substance that is also known to modulate gonadotropin secretion. Five receptor subtypes for NPY have been identified, and a potent antagonist for the receptor subtype 1 (Y1), 1229U91, also known as GW1229 or GR231118, has been described. Subsequently, 1229U91 was also shown to represent a highly potent agonist for the Y4 receptor subtype. Very unexpectedly, intracerebroventricular administration of 1229U91 elicited an intense, dose-dependent surge of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in intact male rats that lasted for 6 h. Such stimulation was absent when a potent gonadotropin-releasing hormone antagonist was administered systemically, suggesting that 1229U91 acts centrally to stimulate gonadotropin-releasing hormone release. 1229U91 administration had no effect on growth hormone, thyroid-stimulating hormone, and corticosterone secretions. In addition to 1229U91, four other parent dimer molecules described earlier produced a marked and sustained stimulation of LH when injected intracerebroventricularly that was proportional to their binding affinity for the Y4 receptor. Central administration of the specific Y1 antagonist BIBO3304 (20 µg) had no effect on LH secretion, making it unlikely for 1229U91 to stimulate LH secretion by an antagonistic action on the Y1 receptor subtype, thus suggesting a Y4 receptor mediation. In conclusion, the 1229U91 molecule displays an interesting conformational epitope that is able to generate large LH surges, possibly by activating Y4 or Y4-like receptor subtypes or by acting on a NPY receptor unrelated target.
CRH Deficiency Impairs but Does Not Block Pituitary-Adrenal Responses to Diverse Stressors
Authors: Jacobson Lauren , Muglia Louis J. , Weninger Stacie C. , Pacák Karel , Majzoub Joseph A.
Published: April 2003

We have previously observed significant, albeit decreased, corticosterone responses to restraint stress in corticotropin releasing hormone (CRH)-deficient (knockout, CRH KO) mice. Because different stressors have been shown to engage different populations of hypophysiotropic neurons, we have used hypoglycemia and hypovolemia to test whether CRH-independent pituitary-adrenal activation is evoked by stimuli other than restraint. Insulin injection in fasted CRH KO mice elicited increases in corticosterone that were markedly lower than those in wild type but marginally significant relative to corresponding KO controls. Consistent with impaired adrenocortical function, hypoglycemia-induced epinephrine secretion was reduced in female CRH KO mice. Hypovolemia produced by retro-orbital bleeding also significantly elevated corticosterone in CRH KO mice. In contrast to significant stress-induced increases in corticotropin (ACTH) in wild-type mice, those in CRH KO mice were slight, transient and difficult to detect without frequent sampling. Restraint-induced interleukin-6 (IL-6) levels were similar between wild-type and CRH KO mice, arguing against compensatory changes in IL-6 responses to restraint due to CRH deficiency. CRH infusion enhanced adrenocortical responses to restraint independently of effects on basal corticosterone levels, suggesting that pituitary-adrenal activity is augmented by factors besides CRH during stress. We conclude that although stress-induced pituitary-adrenal activity does not require acute increases in CRH, CRH is required to support the normal amplitude of adrenocortical axis responsiveness to other endocrine or neural factors during stress.
Pulsatile Gonadotropin-Releasing Hormone (GnRH) Secretion Is an Inherent Function of GnRH Neurons, as Revealed by the Culture of Medial Olfactory Placode Obtained from Embryonic Rats
Authors: Funabashi Toshiya , Daikoku Shigeo , Shinohara Kazuyuki , Kimura Fukuko
Published: April 2003

To determine whether gonadotropin-releasing hormone (GnRH) neurons in culture without the hypothalamus secrete GnRH in a pulsatile fashion, the nasal placode (NAP) was obtained at day 13.5 of gestation and cultured by a roller tube method. If the GnRH release occurs in a pulsatile fashion, it can be said that the pulse generator of GnRH exists inherently in each cell or community of cells in the culture. The concentration of GnRH in the NAP culture medium collected at 8-min intervals for 160 min after 2- to 4-week cultures showed that GnRH release occurred in a pulsatile fashion with a mean interpulse interval of 29.8 ± 2.3 min (n = 9). When the NAP was cultured with tissues of the forebrain vesicle (n = 3) or the hypothalamus (n = 4), GnRH was also released in a pulsatile fashion with similar intervals (27.3 ± 1.0 min for the NAP+forebrain vesicle culture and 36.0 ± 6.3 min for the NAP+hypothalamus culture) as those in cultures without brain tissues. It is concluded that pulsatile GnRH release is an inherent function of GnRH neurons.
The Long Form of the Leptin Receptor (OB-Rb) Is Widely Expressed in the Human Brain
Authors: Burguera Bartolome , Couce Marta E. , Long Jin , Lamsam Jesse , Laakso Karen , Jensen Michael D. , Parisi Joseph E. , Lloyd Ricardo V.
Published: April 2003

Leptin exerts important effects on the regulation of food intake and energy expenditure by acting in the brain. Leptin action is mediated by the interaction with a receptor that is alternatively spliced, resulting in at least five different isoforms. The long form (OB-Rb) has a long intracellular domain that is essential for intracellular signal transduction. The specific aim of this study was to further investigate the role that the brain may play in the pathogenesis of obesity in humans. We studied the expression of OB-R mRNA (both short or common and long isoforms) in the brains of obese, lean and diabetic subjects, by in situ hybridization, semiquantitative RT-PCR and Northern blots analysis. We used two alternative probes: one that recognizes all known splice variants (OB-Ra) and a second that recognizes only the long form, OB-Rb. Several brain regions, including hypothalamus, cerebellum, neocortex, entorrhinal cortex, amygdala, and rostral medulla, were evaluated. In situ hybridization studies revealed that both OB-Ra and OB-Rb mRNAs are widely distributed in the human brain. The specific hybridization signal with both probes was detected exclusively in the cytoplasm of the cell body, dendrites and proximal axons of neurons. Hypothalamic nuclei, Purkinje cells and dentate nuclei of the cerebellum, inferior olivary and cranial nerves nuclei in the medulla, amygdala and neurons from both neocortex and entorrhinal cortex demonstrated positive signals. The hybridization signal obtained in ependyma was lower than that in neurons and no specific hybridization was detected in glial cells. No significant differences were identified among the regions or among the three groups studied. These results match those previously obtained by us [Couce et al.: Neuroendocrinology 1997;66:145] in which the distribution of the OB-R protein in the human brain was first described. RT-PCR indicated that the OB-Rb was highly expressed in the hypothalamus and cerebellum. No significant differences of OB-Ra or OB-Rb mRNA expression were identified in lean or obese individuals in these two cerebral regions. The levels of OB-Rb were significantly higher in cerebellum compared to hypothalamus in lean and obese individuals. The original hypothesis that OB-Rb is present only in the hypothalamus needs to be reconsidered. This OB-Rb isoform seems to be widely expressed in the human brain with highest levels in the cerebellum. Obesity and hyperleptinemia appears not to be associated with down-regulation of the OB-Rb in the human brain.
Oxytocin/Alpha2-Adrenoceptor Interactions in Feeding Responses
Authors: Díaz-Cabiale Zaida , Narváez José A. , Petersson Maria , Uvnäs-Moberg Kerstin , Fuxe Kjell
Published: April 2003

The modulation of α2-adrenoceptor-induced food intake by oxytocin has been evaluated in studies on food intake and by quantitative receptor autoradiography in the hypothalamus and the amygdala of the rat. The effects of lateral intracerebroventricular administration of clonidine and oxytocin were evaluated on food intake in satiated animals. Food consumption was measured at 30, 90, 240 min and 22 h (1,320 min) after injection. The coinjection of oxytocin and clonidine was found to counteract the increase in food intake produced by clonidine (p < 0.001) in satiated rats. Receptor autoradiographic experiments showed that oxytocin significantly increased the Kd values of [3H]p-aminoclonidine α2-agonist-binding sites in the hypothalamus. Effective oxytocin concentrations ranged between 0.3 and 1 nM (p < 0.05) with a maximal action of 250% at 1 nM. The Bmax value was significantly increased (p < 0.05) for all concentrations of oxytocin. In the amygdala, oxytocin also increased both the Kd of [3H]p-aminoclonidine-binding sites by about 190% at 1 nM and the Bmax values at 1 and 3 nM (p < 0.05). Oxytocin (1 nM) also significantly and substantially (p < 0.01) increased the Kd and Bmax values of the [3H]UK 14.304 α2-agonist-binding sites in the hypothalamus and amygdala in agreement with the results obtained with the other agonist of the α2-adrenoceptor [3H]p-aminoclonidine. This effect was partially blocked by the presence of the specific oxytocin receptor antagonist, CAP. These findings suggest the existence of an antagonistic oxytocin/α2-receptor interaction in the hypothalamus and amygdala that may be of relevance for the demonstrated modulation of α2-adrenoceptor-induced feeding responses by oxytocin.
Ovine Fetal Estrogen Sulfotransferase in Brain Regions Important for Hypothalamus-Pituitary-Adrenal Axis Control
Authors: Purinton Scott C. , Wood Charles E.
Published: April 2003

Ovine parturition is initiated by increases in fetal hypothalamus-pituitary-adrenal (HPA) axis activity which, in turn, increase placental estrogen biosynthesis and ultimately increase uterine contractility. In addition to their action in the uterus, estrogens in fetal plasma augment fetal corticotropin (ACTH) secretion. In late gestation, estrone sulfate is more abundant in fetal plasma than unconjugated estrone and it is possible that there is interconversion of sulfoconjugated and unconjugated steroids within the fetal brain. We studied hypothalamus and brainstem tissue from fetal, neonatal, and adult sheep to test the hypothesis that the ovine brain contains estrogen sulfotransferase. Although no significant ontogenic pattern was revealed, the presence of estrogen sulfotransferase within the hypothalamus and brainstem was detectable. Immunohistochemistry revealed the presence of estrogen sulfotransferase in the paraventricular nucleus of the hypothalamus, the nucleus of the tractus solatarius, and the rostral ventral lateral medulla. We conclude that ovine fetal hypothalamus and brainstem contain estrogen sulfotransferase in brain regions important for HPA axis control.
Immunoneutralization of Endogenous Opioid Peptides Prevents the Suckling-Induced Prolactin Increase and the Inhibition of Tuberoinfundibular Dopaminergic Neurons
Authors: Callahan Phyllis , Klosterman Shannon , Prunty Danielle , Tompkins Janine , Janik James
Published: April 2003

Previous studies have shown that the endogenous opioid peptides, acting at specific opiate receptor subtypes, are involved in the suckling-induced prolactin secretory response. The prolactin increase elicited by suckling is due, at least in part, to an inhibition of tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus. We investigated the effects of immunoneutralization of dynorphin, leu-enkephalin and met-enkephalin on the suckling-induced prolactin increase and on the activity of the TIDA neurons in lactating female rats between days 7 and 12 postpartum. Rats were injected into the right lateral ventricle with antiserum specific for one of these three peptides. Control rats were administered equal amounts of immunoglobulin proteins. Suckling produced a profound and significant increase in prolactin levels, as well as a decrease in DOPA accumulation in the median eminence of lactating rats. Administration of immunoglobulin concentrations of up to 3.6 µg did not inhibit the prolactin secretory response to the suckling stimulus and did not prevent the suckling-induced inhibition of TIDA neurons. Antisera to all three endogenous opioid peptides abolished the suckling-induced prolactin increase and prevented the inhibition in DOPA accumulation in the median eminence. Thus, the endogenous opioid peptides, dynorphin, leu-enkephalin and met-enkephalin, are essential for the prolactin secretory response to suckling and inhibition of TIDA neuronal activity is at least one of the mechanisms of action utilized by these peptides.
Ontogeny of Immunoreactive Prostaglandin Endoperoxide Synthase Isoforms in Ovine Fetal Pituitary, Hypothalamus and Brainstem
Authors: Deauseault Deanna , Giroux Damian , Wood Charles E.
Published: April 2003

Parturition is initiated in sheep by an increase in the activity of the fetal hypothalamus-pituitary-adrenal (HPA) axis. Prostaglandins, known to augment the activity of this endocrine axis, have long been proposed as involved in the initiation of paturition. We have previously demonstrated that endogenously produced prostanoids augment the activity of the HPA axis, and we have proposed that the increased production of prostanoids within the fetal brain or pituitary at the end of gestation might be involved in the initiation of parturition. An important regulatory step in the biosynthesis of prostanoids is the activity of prostaglandin endoperoxide synthase (PGHS). The present study was designed to test the hypothesis that the abundance of one or both isoforms of PGHS (PGHS-1 and PGHS-2) increase in brain and/or pituitary at the end of gestation. We used immunoblot analysis to measure the abundance of immunoreactive PGHS-1 and PGHS-2 in pituitary, hypothalamus and brainstem collected from fetuses of known gestational ages. We found that the abundance of PGHS-1 was weakly but significantly increased at the end of gestation in the pituitary and brainstem. The abundance of PGHS-2, on the other hand, increased exponentially in the pituitary and hypothalamus with highest concentrations found in term fetuses. We conclude that these enzymes are developmentally regulated in pituitary and in brain regions important for HPA axis control. We speculate that the increased enzyme’s abundance results in increased prostanoid biosynthesis near term, and is a link in the chain of events which initiates parturition.
Regulation of C-fos Protein in Gonadotrope Cells by Oxytocin and Gonadotropin-Releasing Hormone
Authors: Abbas Maysoon M. , Evans John J.
Published: April 2003

The integrated regulation of luteinizing hormone (LH) from the anterior pituitary gland is vital to the functioning of the ovulatory cycle in the female and consists of several components acting at different time points. The best-studied is the rapid release of LH elicited by gonadotropin-releasing hormone (GnRH). The so-called primary (immediate early) response genes (PRGs), including c-fos, regulate relatively long-term activities, such as mitosis, protein synthesis, protein release and cell differentiation. Regular ovulatory cycles occur as a result of interaction of several peptide factors including the primary factor, GnRH and oxytocin, although GnRH and oxytocin do not have identical activities. We wished to determine whether oxytocin could mediate changes in expression of c-fos protein and compare its effects with those of GnRH. Anterior pituitary glands were collected from female rats at proestrus and a single-cell suspension prepared. Cells were incubated with oxytocin or GnRH at selected concentrations for various times. C-fos protein was extracted and submitted to Western blot analysis. Other cells were stained immunohistochemically for c-fos and LH following incubation with the peptides and fixation. There was an increase in c-fos protein from 15 to 60 min in Western blots of cells from all incubations. After immunohistochemistry, it was observed that both oxytocin (100 nM) and GnRH (100 nM) increased the percentage of cells that expressed c-fos protein (p < 0.001) and of cells that expressed LH (p < 0.001). The responses to the peptides were concentration dependent. We found that neither all LH-containing cells expressed c-fos, nor all c-fos-containing cells immunostained for LH. The effects of the peptides were not the same. High concentrations of GnRH (1 µM) induced the appearance of a higher percent of LH-containing cells having c-fos than did 10 nM GnRH (p < 0.01), whereas a lower percent of LH-containing cells with c-fos were observed when the oxytocin concentration was raised from 10 nM to 1 µM (p < 0.02). It appears, therefore, that the two peptides have different regulatory effects on LH-containing cells, indicating the possibility of specialized function. The results emphasize the suggestion that stimulation of LH secretion is not the sole index of gonadotrope-directed activity by a peptide. Collectively, these results indicate that the peptides oxytocin and GnRH are able to modulate processes that are associated with longer-term activities of gonadotropes and also demonstrate that specific subpopulations of LH-containing gonadotropes are stimulated to express c-fos.
Blockage of N-Methyl-D-Aspartate Receptors Decreases Testosterone Levels and Enhances Postnatal Neuronal Apoptosis in the Preoptic Area of Male Rats
Authors: Hsu Chin , Hsieh Ya-Lun , Yang Rei-Cheng , Hsu Hseng-Kuang
Published: April 2003

Sexual dimorphism has been found in the preoptic area of the hypothalamus (POA), a major site of glutamate actions via N-methyl-D-aspartate (NMDA) receptors. The sexually dimorphic nucleus of the preoptic area (SDN-POA) of male rats exhibits about seven-fold greater nuclear volume than that of females. A naturally occurring neonatal neuronal apoptosis, that can be prevented by testosterone, may contribute to this sexual difference in SDN-POA nuclear volume. Since activation of NMDA receptors in the POA induces GnRH secretion, it may be involved in both elevation of serum testosterone and prevention of neuronal death in the SDN-POA. In the present study, protein expression of NMDA receptors in the POA of male and female fetuses was quantified on the day preceding the fetal testosterone peak (embryonic day 16; ED 16). Rats were then distributed in four groups: (1) untreated males, (2) untreated females, (3) males pretreated with MK-801 (a noncompetitive NMDA receptor antagonist), and (4) females pretreated with MK-801. Serum levels of testosterone were estimated on the afternoon of ED 18. Expression of Bcl-2 and Bax, as well as neuronal apoptosis in SDN-POA, were observed on postnatal day 8. The results showed that (1) expression of NMDA receptors in the POA of male fetuses was higher than that of females on ED 16; (2) levels of testosterone were lower in MK-801 pretreated male fetuses than in intact males on ED 18; (3) expression of Bcl-2 in the POA of MK-801 pretreated male rats was significantly less than that of control males; (4) the apoptotic incidence in the SDN-POA of MK-801 pretreated male rats was significantly greater than in control males, while there was no significant difference in apoptotic incidence in the SDN-POA between MK-801 pretreated and intact females. These results suggest that the NMDA receptor is highly expressed in prenatal male fetuses, and that it might play an important role in the elevation of testosterone levels. Moreover, activation of NMDA receptors may protect SDN-POA neurons from naturally occurring neuronal death, by modulating testosterone and/or Bcl-2 expression.
Calcitonin Inhibition of Prolactin Secretion in Lactating Rats: Mechanism of Action
Authors: Tohei Atsushi , VandeGarde Blake , Arbogast Lydia A. , Voogt James L.
Published: April 2003

The effects of intracerebroventricular (10 ng/rat) or intravenous (10 or 40 µg/15 min/rat) administration of salmon calcitonin (sCT) on the prolactin (PRL) response to suckling and the activity of tyrosine hydroxylase (TH) were examined in lactating rats. Plasma concentration of PRL increased dramatically in control rats after the onset of the suckling stimulus, while administration of sCT resulted in inhibition of PRL response to suckling. The action of sCT was much more effective with intracerebroventricular administration, which totally blocked PRL release, compared to intravenous administration. The intracerebroventricular administration of sCT increased TH activity of tuberoinfundibular dopamine neuron (TIDA) in the stalk-median eminence, as measured by DOPA accumulation, while completely suppressing the PRL response to suckling. Injection of α-methyl-p-tyrosine (α-MT; 50 mg/kg), an inhibitor of TH and thus dopamine synthesis, increased PRL levels, and suckling caused a further increase in plasma concentrations of PRL. Injection of sCT (intracerebroventricularly) did not inhibit the PRL response to suckling in the presence of a depletion of dopamine. These results suggest that sCT inhibition of PRL secretion in lactating rats is mediated mainly by TIDA neurons without involvement of other neuroendocrine mechanisms.
Centrally Administered Murine-Leptin Stimulates the Hypothalamus-Pituitary- Adrenal Axis through Arginine-Vasopressin
Authors: Morimoto Isao , Yamamoto Shigeki , Kai Keiko , Fujihira Takashi , Morita Emiko , Eto Sumiya
Published: April 2003

Starvation induces a decrease in circulating leptin levels and activation of the hypothalamus-pituitary-adrenal (HPA) axis. Leptin inhibits the HPA axis in unfed rodents or genetically leptin-deficient ob/ob mice, whereas it stimulates corticotropin-releasing hormone (CRH) gene expression in the paraventricular nucleus (PVN). However, the interactions between leptin, CRH and the HPA axis are poorly understood and are likely to be complex. We recently demonstrated that central leptin administration caused increases in plasma arginine-vasopressin (AVP) and AVP gene expression of the PVN in nonstressful rats. AVP stimulates the release of adrenocorticotropic hormone (ACTH), but it also potentiates the action of CRH on ACTH release. In this study, we investigated the effects of leptin on plasma ACTH and corticosterone levels, CRH mRNA of the PVN and proopiomelanocortin (POMC) mRNA of the pituitary in nonstrained rats. Intracerebroventricularly administered leptin caused increases in plasma ACTH and corticosterone levels in dose-dependent manners. In Northern blot analyses, the leptin injection induced significant increases in the expression of CRH mRNA in the PVN and POMC mRNA in the pituitary. The increased plasma ACTH and corticosterone levels by leptin were attenuated with intracerebroventricular pretreatment of a V1a receptor antagonist (OPC-21268) or a V1a/V1b receptor antagonist (dP[Tyr(Me)2]AVP), but not with that of a V2 receptor antagonist (OPC-31260). The leptin-induced CRH mRNA expression in the PVN and POMC mRNA expression in the pituitary were also reduced by the pretreatment with OPC-21268 and dP[Tyr(Me)2]AVP. These results suggest that intracerebroventricular leptin administration activates the HPA axis by AVP receptor activation through V1a receptors in the PVN which in turn activates CRH neurons to drive ACTH and corticosterone secretion in concert with AVP in nonstrained rats.
Immunolocalization of Androgen Receptors and Aromatase Enzyme in the Adult Musk Shrew Brain
Authors: Veney Sean L. , Rissman Emilie F.
Published: April 2003

In the brain and other tissues, estrogens are produced by aromatization of androgens. Biochemical data suggest that aromatase enzyme is regulated by the androgen receptor (AR). Neurons that contain either AR or aromatase (AROM) enzyme reside in many of the same brain regions. In this report, we examined the codistribution of AR- and AROM-enzyme-immunoreactive (-ir) neurons in several regions of the adult male and female musk shrew brain. Data were collected from the intermediate nucleus of the lateral septum (LS), medial anterior (BNSTMA) and medial posterointerior (BNSTMP) divisions of the bed nucleus of the stria terminalis, medial preoptic area (mPOA), ventromedial nucleus of the hypothalamus (VMN), medial (MeA), cortical and central nuclei of the amygdala. Males had significantly more AR-ir neurons in the BNSTMP, mPOA, VMN and LS as compared to females. With the exception of the BNSTMA and LS, males had more AROM-ir neurons in each region than females. Furthermore, males had significantly more double-labeled neurons than females in the BNSTMP, mPOA, VMN, LS and MeA. The percentage of AROM-ir neurons that also contained AR immunoreactivity ranged from 13 to 82% depending on sex and region. The highest percentage of dual-labeled neurons (79% in females and 82% in males) was found in the VMN. Taken together, these data show that there is extensive cellular colocalization of AR and AROM enzyme in specific regions of the musk shrew brain. We propose that in both sexes, androgen receptors may act as transcription factors to regulate AROM enzyme.
The Effects of Testicular Denervation on Spermatogenesis in the Sprague- Dawley Rat
Authors: Chow Shih-Han , Giglio William , Anesetti Robert , Ottenweller John E. , Pogach Leonard M. , Huang Hosea F.S.
Published: April 2003

In the rat, regression of spermatogenesis during the chronic stages of spinal cord injury (SCI) occurs in the presence of normal function of the pituitary-testis hormone axis, thus suggesting that nonendocrine mechanisms might be involved. The current study examined whether disruption of neural input to the testis contributes to the cascade that leads to the regression of spermatogenesis. Four weeks after denervation of the superior spermatic nerve (SSN), testis weight was 25% lower (p < 0.01) than that of the contralateral sham-operated testis. Defects in spermatogenesis including phagocytosis of mature spermatids, vacuolization of spermatid nuclei, delayed spermiation and incomplete cellular associations were observed in >60% of the tubules. In the remaining 30–40% of tubules, the seminiferous epithelium was severely regressed. While cutting the inferior spermatic nerve (ISN) alone did not affect spermatogenesis significantly, it enhanced the effect of SSN denervation on both spermatogenesis and testis weight (p < 0.01). Spermatogenesis was totally regressed in the SSN/ISN-denervated testes. At this time, quantitatively normal spermatogonial proliferation was maintained in SSN- or ISN-denervated testes. Twelve weeks after surgery, regression of the seminiferous epithelium characterized by absence of proliferating spermatogonia, while undifferentiating spermatogonia were present, was observed in all SSN-denervated testes. At this time, regression of the seminiferous epithelia also occurred in >30% of the tubules in ISN-denervated testes. At both times, serum follicle-stimulating hormone, luteinizing hormone and testosterone levels were normal and >60% of normal testicular testosterone concentrations were maintained in the denervated testes. These results indicate that disruption of neural input to the testis is not a cause for the decrease in spermatogonial proliferation during the acute phase of SCI, but may contribute to the chronic effects of SCI on spermatogenesis.
Brain-Derived Neurotrophic Factor But Not Neurotrophin-3 Enhances Differentiation of Somatostatin Neurons in Hypothalamic Cultures
Authors: Loudes Catherine , Petit Florence , Kordon Claude , Faivre-Bauman Annie
Published: April 2003

The present work investigated whether neurotrophins could differentially affect in vitro growth and maturation of two related subsets of hypothalamic neurons, hypophysiotropic somatostatin (SRIH) neurons projecting from the periventricular area and arcuate SRIH interneurons. For this purpose, the hypothalamus of 17-day-old rat fetuses was sampled and separated into a ventral and a dorsal fragment containing respectively periventricular and arcuate regions. Each fragment was dissociated and seeded separately in defined medium. Brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), two important members of the neurotrophin family involved in neuronal differentiation and plasticity, were added to the cultures at seeding time. After 6 or 11 days in vitro, neurons were labeled with an anti-SRIH antiserum and submitted to morphometric analysis. In parallel, SRIH mRNA was estimated by semiquantitative reverse-transcriptase-polymerase chain reaction, and neuronal SRIH content, basal and depolarisation-stimulated releases measured by radioimmunoassay. The response of control, non-labeled neurons was estimated by neuronal counts and by assaying glutamic acid decarboxylase, a marker of a large majority of hypothalamic neurons. BDNF markedly increased the size and the branching number of SRIH periventricular cell bodies. Expression of SRIH mRNA, as well as SRIH content and release into the culture medium, were also stimulated by the neurotrophin. Non-SRIH neurons were not affected by the treatment. Under the same conditions, arcuate neurons exhibited a weak, mostly transient response to BDNF. NT-3 was ineffective on either neuronal subset. Immunoneutralization of Trk receptors provided further evidence for BDNF effect specificity. The results indicate that BDNF is a selective activator of the differentiation of hypophysiotropic SRIH neurons in the periventricular area of the hypothalamus.
Ontogenesis of Proopiomelanocortin and Its Processing to β-Endorphin by the Fetal and Neonatal Rat Brain
Authors: Angelogianni Panagoula , Li Hong-Ling , Gianoulakis Christina
Published: April 2003

A number of reports suggest that β-endorphin (β-END) may play an important role in the regulation of cell proliferation and neuronal differentiation. Proopiomelanocortin (POMC), the common precursor ofadrenocorticotropic hormone and β-END, is detected very early in embryonic life in hypothalamic neurons of the developing rat. However, very little is known about the degree to which POMC is processed to β-END during fetal and early postnatal life. Thus, it was the objective of the present study to estimate the hypothalamic content of POMC mRNA, as well as the biosynthesis and posttranslational processing of POMC by hypothalamic neurons on fetal day 20 and on days 1, 8 and 22 of postnatal life. Hypothalamic POMC mRNA, as determined by Northern blot analysis, was higher on fetal day 20 than on postnatal days 1, 8 and 22. A higher rate of incorporation of [3H]phenylalanine into β-END immunoreactive peptides was observed on fetal day 20 than on postnatal day 1. However, the rate of incorporation was significantly increased by day 8 of postnatal life and was similar to that on day 22. POMC was processed to β-lipotropin (β-LPH) and β-END at all ages examined, but the relative proportions of POMC:β-LPH:β-END changed during development. Thus, β-END accounted only for 34.89 ± 6.14% of the total [3H]phenylalanine-labeled β-END immunoreactive peptides on fetal day 20, while it accounted for 57.37 ± 5.20, 62.81 ± 1.38 and 79.25 ± 6.57% on days 1, 8 and 22 of postnatal life, respectively. Thus, POMC is processed to a considerable extent into β-END-sized peptides by the fetal hypothalamus and may influence brain development. Furthermore, the rate of processing of hypothalamic POMC into β-END increases with development, probably due to the increased activity of the enzymes specific for POMC processing.
Recruitment of Calcium from Intracellular Stores Does Not Occur during the Expression of Large Spontaneous Calcium Oscillations in GH3 Cells and Lactotropic Cells in Primary Culture
Authors: Zimber Michael P. , Simasko Steven M.
Published: April 2003

We used simultaneous electrophysiological and intracellular calcium microfluorometry recordings to directly test for the presence of a calcium-induced calcium release mechanism in individual GH3 cells and cells of a lactotrope-enriched primary culture. In voltage-pulse experiments, extending the duration of a depolarizing voltage-pulse increased intracellular calcium concentration ([Ca2+]i), but we did not observe any evidence for recruitment of intracellular calcium stores. Furthermore, depletion of intracellular calcium stores with thapsigargin or caffeine did not change the calculated calcium buffer capacity of the cells. In current-clamp experiments, we observed elevations in [Ca2+]i in response to spontaneous action potentials. These [Ca2+]i responses were not inhibited by thapsigargin or caffeine. We did find a significant correlation between the magnitude of spontaneous [Ca2+]i increases and action potential duration. We conclude that intracellular calcium stores are not released during the spontaneous [Ca2+]i oscillations observed in these cells, and that the magnitude of [Ca2+]i oscillations is a direct result of extracellular calcium influx that is determined in part by action potential duration.
Multinucleated Arginine-Vasopressin Neurons in the Human Supraoptic Nucleus: A Hallmark of Pulmonary Pathology
Authors: Ishunina Tatjana A. , Unmehopa Unga A. , Dolzhikov Alexandr A. , Swaab Dick F.
Published: April 2003

The human supraoptic nucleus (SON) is the main production site of plasma arginine-vasopressin (AVP). The present study aimed to define the subpopulation of patients in which multinucleated SON neurons (MNN) are present. We determined the proportion of MNN in immunocytochemically defined SON AVP population and compared AVP mRNA levels in the SON of 29 patients with and without MNN. Interestingly, MNN appeared to be present in a high proportion in patients with pneumonia and other pulmonary pathologies (Pul P). The percentage of MNN in the SON of Pul P patients turned out to be age- and sex-dependent. In young women with Pul P their proportion was 10 times higher than in women without such a pathology and in young men with Pul P their frequency was 22 times higher than in other men. In those patients with the highest proportion of MNN, i.e. young females and males with Pul P, AVP mRNA expression in the SON was the lowest. In addition, young women (≤50 years old) had lower AVP mRNA levels than young men and than elderly women (>50 years old). In conclusion, our study suggests that multinucleated neurons are a hallmark of Pul P in the human SON and that this phenomenon may be accompanied by lower AVP production in young subjects.
Deficits in Progesterone-Facilitated Sexual Behaviors and Forebrain Estrogen and Progestin Receptors in Obese Female Zucker Rats
Authors: Olster Deborah H. , Auerbach Ilene D.
Published: April 2003

Obese female Zucker rats (fa/fa) are sterile. Among their reproductive abnormalities is hyporesponsiveness to the stimulatory effects of ovarian steroid hormones on sexual behaviors. This study was designed to test the hypothesis that obese Zucker females are deficient in hypothalamic/preoptic area estrogen receptors (ERs) and/or estradiol-induced progestin receptors (PRs). Ovariectomized (OVX) lean and obese Zucker rats were tested for the display of sexual behaviors following injection of estradiol benzoate (EB, 15 or 100 µg/kg) plus progesterone (P, 2 mg/kg). As expected, obese females showed significantly lower lordosis quotients and lordosis ratings than lean animals after injection of the lower, physiological dose of EB followed by P. In contrast, obese and lean females receiving the higher EB dose, prior to P, showed similar levels of sexual receptivity. Two weeks later, these OVX lean and obese females received injections of vehicle, 15 or 100 µg/kg EB, prior to perfusion and tissue processing for PR immunocytochemistry (ICC). Additional groups of OVX virgin females of both genotypes were perfused and tissue from the preoptic area and hypothalamus was processed for ERα ICC. No genotypic differences in the number of cells containing ERα-immunoreactivity (-IR) in the medial preoptic area (MPOA), ventromedial hypothalamus (VMH) or arcuate nucleus (ARC) were noted, but obese females had significantly fewer ERα-IR cells in the anteroventral periventricular nucleus (AVPV) than lean rats. In both genotypes, the number of PR-IR cells in the AVPV, MPOA and VMH was significantly higher following injection of EB (either dose) as compared to vehicle, demonstrating estradiol induction of PRs. Only in the MPOA was there a significant difference between fat and lean females in estradiol-induced PR-IR. Obese females receiving 15 µg/kg EB had fewer PR-IR cells in the MPOA than comparably-treated lean animals. No such genotypic difference was observed following injection of the vehicle or higher dose (100 µg/kg) of EB. These data are consistent with the hypothesis that deficiencies in ERα in the AVPV and/or PRs in the MPOA may contribute to obese Zucker females’ poor responsiveness to ovarian steroid hormones.
Cross-Talk between Excitatory and Inhibitory Amino Acids in the Regulation of Growth Hormone Secretion in Neonatal Rats
Authors: Pinilla Leonor , Gonzalez Lucas C. , Tena-Sempere Manuel , Aguilar Enrique
Published: April 2003

Experiments were carried out in 5-day-old male and female rats in order to assess the respective roles of distinct γ-aminobutyric acid (GABA) receptor subtypes in the control of growth hormone (GH) secretion in the newborn rat and to identify the potential sexual dimorphism of GABA actions on GH release. The interplay of GABA and excitatory acids (EAAs) to control GH secretion was also analysed. Effects of specific GABAA, GABAB and GABAC agonists upon GH secretion as well as of co-administration of GABA and agonists and antagonists of EAA receptors were monitored in 5-day-old male rats. The data confirm that GABA stimulates GH secretion in 5-day-old rats, but indicate that the effect is sexually dimorphic, being greater in females. Stimulation of GH secretion by GABA is mainly mediated by GABAA receptors, while GABAC appears to be ineffective. Stimulatory effects of GH by GABA and muscimol, a GABAA receptor agonist, are blocked by pretreatment with (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclophepten-5-10-imine hydrogen maleate (MK-801), an antagonist of N-methyl-D-aspartate (NMDA) receptors, but not by 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]-quinoxaline-7-sulfonamide (NBQX), an antagonist of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Simultaneous administration of GABA and NMDA was additive on GH release while basal GH secretion was not affected by either bicuculline or the respective blockers of GABAA and GABAB receptors. In conclusion, our results suggest that cross-talk between inhibitory (GABA) and EAAs is involved in the control of GH secretion in neonates.
Prenatal Glucocorticoid Modifies Hypothalamo-Pituitary-Adrenal Regulation in Prepubertal Guinea Pigs
Authors: Dean Farrah , Yu Catherine , Lingas Rania I. , Matthews Stephen G.
Published: April 2003

We hypothesized that exposure to synthetic glucocorticoid during rapid brain growth (d50–52, birth = 68 days) in fetal guinea pigs modifies hypothalamo-pituitary-adrenal (HPA) function after birth, and that this involves changes in central corticosteroid receptor regulation. On the basis of our previous studies, we proposed that this effect is sex-specific. Pregnant guinea pigs were treated with dexamethasone (1 mg/kg) or vehicle on d50–51 of gestation, and juvenile offspring were euthanized at rest or following isolation stress on postnatal day 18. Dexamethasone increased the length of gestation (1.5 days) and altered body and organ (brain, heart, adrenal) growth. Resting plasma cortisol concentrations were significantly elevated in young male, but not female guinea pigs exposed to dexamethasone as fetuses. In female offspring born to dexamethasone-treated mothers, cortisol responses to isolation stress were attenuated. In males, elevated basal cortisol levels were not increased further by isolation. In the brain, hippocampal glucocorticoid receptor (GR) mRNA levels were significantly lower (10–25%) in females exposed to dexamethasone in utero. In contrast, GR mRNA levels were elevated (10–20%) in males from this prenatal treatment group. Mineralocorticoid receptor mRNA in the limbic system and GR mRNA levels in the pars distalis were unaffected. Pro-opiomelanocortin mRNA was significantly lower (30%) in the male pars intermedia following dexamethasone exposure. In conclusion, prenatal glucocorticoid exposure affects growth and HPA function as well as limbic and hypothalamic GR expression in juvenile offspring, and these effects are highly sex-specific.
Oral Testosterone Self-Administration in Male Hamsters
Authors: Johnson Luke R. , Wood Ruth I.
Published: April 2003

The addiction potential of anabolic steroids remains largely unexplored. Here, we demonstrate voluntary oral testosterone intake in hamsters. Using a 2-bottle choice test, males preferred an aqueous solution of 200 µg/ml testosterone over vehicle. However, the taste of testosterone is not highly preferred. Addition of testosterone at 400 µg/ml increased fluid consumption from the nonpreferred bottle in a 2-bottle choice test, but cholesterol at the same concentration reduced drinking, suggesting that testosterone reward is not common to all sterols. With food-induced drinking, testosterone maintained fluid intake when food was withdrawn. These data demonstrate that oral self-administration of testosterone is reinforcing in hamsters, suggesting the potential for dependence in human users.
Hypothalamo-Pituitary-Adrenal Axis Activity Is Related to the Level of Central Arousal: Effect of Sleep Deprivation on the Association of High-Frequency Waking Electroencephalogram with Cortisol Release
Authors: Chapotot Florian , Buguet Alain , Gronfier Claude , Brandenberger Gabrielle
Published: April 2003

The temporal and quantitative interrelationships between the hypothalamo-pituitary-adrenal (HPA) axis activity and the level of central arousal were studied in 10 healthy young men during daytime wakefulness. Two experimental sessions were conducted randomly between 09.00 and 18.00 h, once after nocturnal sleep and once after a night of total sleep deprivation. Spectral analysis of serial waking electroencephalography (EEG) from a short target fixation task repeated every 10 min was undertaken, along with an estimation of cortisol secretory profiles by deconvolution of plasma radioimmunoassay measures obtained from continuous blood withdrawal with regular sampling at a 10-min interval. Following nocturnal sleep, a temporal association between the HPA axis activity and the waking EEG activity was found, cortisol secretory rate following changes in frontal gamma (20–45 Hz) band power by 10 min (average R = 0.458, p < 0.001). Although it remained significant (average R = 0.276, p < 0.05), the association strength decreased significantly following total sleep deprivation (p < 0.05, Wilcoxon test). Cortisol plasma level, secretory rate and pulse amplitude were increased as well as waking EEG power in the delta (0.5–5.5 Hz), theta (5.5–8.5 Hz) and gamma frequency bands (all p values <0.05, Student t tests). The sleep deprivation-related increases in cortisol secretory rate and waking EEG gamma activity were quantitatively associated (R = 0.504, p < 0.05). These results support the existence of a common ultradian regulatory mechanism, co-ordinating HPA axis activity to the level of central arousal in man, which seems involved in the sleep deprivation-induced hyperarousal.