bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth Hormone‐Releasing Hormone Receptor (GNRH‐R) and Growth Hormone Secretagogue Receptor (GHS‐R) mRNA Levels During Postnatal Development in Male and Female Rats
Authors: Kamegai  , Wakabayashi  , Kineman  , Frohman
Published: March 2003

Experimental evidence suggests that differential pituitary sensitivity to hypothalamic signals exerts a role in mediating both age and sex dependent patterns of growth hormone (GH) release and synthesis. One mechanism by which pituitary sensitivity to hypothalamic GH regulators could be modified is by the differential synthesis of their pituitary receptors. In the present report we therefore studied the age and sex dependency of the expression of receptors for two known stimulators of GH release, growth hormone‐releasing hormone (GHRH) and the synthetic peptidyl and non‐peptidyl GH secretagogues (GHSs). Pituitary GHRH receptor (GHRH‐R) and GHS receptor (GHS‐R) mRNA levels were measured by reverse transcriptase‐polymerase chain reaction (RT‐PCR) in male and female rats at postnatal day 1, 10, 30 and 75. We also examined the age‐ and sex‐dependent expression of the GHS‐R in whole hypothalamic extracts, since the GHS‐R is also expressed in a variety of nuclei within the hypothalamus and has been linked to central regulation of the GH‐axis. Pituitary GHRH‐R mRNA concentrations were age‐dependent; the highest levels were observed in d1 pituitaries and then declined with age, reaching a nadir by d30. These results are in concordance with the age‐related decline in pituitary GHRH sensitivity. In contrast, the ontogenic pattern of GHS‐R expression was bimodal; GHS‐R mRNA concentrations in d1 and d30 pituitaries were approximately twice those at d10 and d75. These results mirror the transient increase in GHS sensitivity observed around the onset of puberty, suggesting that gonadal steroids mediate GHS‐R expression. GHRH‐R mRNA levels were comparable in males and females within each age while GHS‐R mRNA levels were gender dependent. At d30, male GHS‐R mRNA levels were ≈30% greater than in their female counterparts. This was reversed at d75, when females had 89% more GHS‐R mRNA per pituitary and 65% more per somatotrope than did age‐matched males. These sexual differences further support a role for gonadal steroids in the modulation of pituitary GHS‐R synthesis. The ontogenic and gender‐specific pattern of hypothalamic GHS‐R expression differed from that observed for the pituitary. Hypothalamic GHS‐R mRNA levels increased with age but exhibited no significant sex difference at each age tested. Taken together, these data demonstrate that changes in the levels of pituitary GHS‐R mRNA, but not GHRH‐R mRNA, are associated with changes in the gonadal steroid environment, thereby implicating the GHS/GHS‐R signalling system as a control point in the establishment and maintenance of sexually dimorphic patterns of GH secretion.
Evidence for Oestrogen Receptor α‐Immunoreactivity in Gonadotrophin‐Releasing Hormone‐Expressing Neurones
Authors: Butler  , Sjöberg  , Coen
Published: March 2003

Having used the cingulate cortex to demonstrate the validity of our methods for detecting hitherto unrecognized oestrogen receptor alpha (ERα)‐immunoreactive neurones, we have now employed immunoprecipitation and double‐label immunohistochemistry to investigate whether the ERα protein is present in gonadotrophin‐releasing hormone (GnRH)‐containing cells. The immortalized GnRH cell line GT1–7 and GnRH neurones within the rat preoptic area were found to possess ERα‐immunoreactivity (ERα‐IR). These observations indicate that oestrogen may regulate the synthesis and release of GnRH by direct actions on GnRH neurones.
Acute and Long‐Term Treatments with the Selective Serotonin Reuptake Inhibitor Citalopram Modulate the HPA Axis Activity at Different Levels in Male Rats
Authors: Jensen  , Jessop  , Harbuz  , Mørk  , Sánchez  , Mikkelsen
Published: March 2003

It is well established that the maximal therapeutic effect of selective serotonin reuptake inhibitors (SSRI) are achieved in depressive patients after several weeks of treatment, but the adaptive processes leading to the therapeutic effects are unclear. It has been shown that hyperactivity in the hypothalamic‐pituitary‐adrenal (HPA) axis in depressive patients is affected by long‐term antidepressant treatment. These changes occur in association with the mood normalising effect, suggesting that antidepressants affect the HPA axis and this effect is associated with the therapeutic effect. Male Wistar rats were treated with the SSRI, citalopram, to investigate time‐related changes in components that may be involved in the desensitization of the HPA axis. A single injection of citalopram (10 mg/kg, s.c.), increased the plasma levels of ACTH and corticosterone in a dose‐dependent manner and increased the number of c‐Fos containing cells in the hypothalamic paraventricular nucleus. A daily treatment with the same compound (10 mg/kg, s.c.) for 14 days decreased the expression of POMC mRNA (≈40%). In addition, a blunted response to citalopram was observed in animals long‐term treated with citalopram. Also CRF‐stimulated cAMP accumulation in the pituitary was altered. In conclusion, acute citalopram activated the HPA‐axis at the hypothalamic level and long‐term citalopram treatment desensitized the HPA‐axis at the pituitary level. These results support the hypothesis that the therapeutic effects of long‐term antidepressant treatments reduce HPA axis responsiveness.
Testosterone Metabolism In Rat Brain Is Differentially Enhanced By Phenytoin‐Inducible Cytochrome P450 Isoforms
Authors: Rosenbrock  , Hagemeyer  , Singeç  , Knoth  , Volk
Published: March 2003

Many cytochrome P450 (P450) isoforms are selectively inducible by xenobiotics, e.g. pharmaceuticals like the anti‐epileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones and are of great importance, especially in early brain development. In this study, the hydroxylation of testosterone by rat brain microsomes from control and phenytoin‐induced animals was examined by use of high performance liquid chromotography (HPLC) provided with a photodiode array detector (PDA). In control rats, testosterone is converted by cytochrome(s) P450 to 6α‐hydroxytestosterone (OHT) as the main metabolite and 6β‐OHT as well as androstenedione as minor metabolites. After phenytoin treatment, brain microsomes showed a strong increase of testosterone metabolism to 2α‐, 6β‐, 16α‐, 16β‐OHT and androstenedione, whereby 16α‐OHT was the main degradation product. These metabolites indicated the action of isoforms of the P450 subfamilies CYP2B, CYP2C and CYP3A. Inhibition experiments with antibodies against CYP2B1/2 and with the CYP2B specific inhibitor orphenadrine indicated the occurrence of members of this subfamily which are known to catalyse the oxidation of testosterone to 16α‐OHT, 16β‐OHT and androstenedione. Western blots revealed the phenytoin‐inducible expression of CYP2B1 and the constitutive expression of CYP3A. The latter is involved in the 6β‐hydroxylation of testosterone which was found correspondingly in control microsomes. Distinct CYP2C isoforms involved in the hydroxylation of testosterone in phenytoin‐induced microsomes are not yet identified. The highly increased testosterone metabolism by phenytoin‐dependent induction of specific cytochrome P450 isoforms in adult rat brain illustrates the potential influence of exogenous substances on internal regulative and metabolic pathways in the brain.
Leptin Receptor‐ and STAT3‐Immunoreactivities in Hypocretin/Orexin Neurones of the Lateral Hypothalamus1
Authors: Håkansson  , De Lecea  , Sutcliffe  , Yanagisawa  , Meister
Published: March 2003

Hypocretins/orexins are recently characterized peptides that are synthesized in neurones of the lateral hypohalamus and stimulate food intake in rats. To clarify whether leptin may interact with hypocretin/orexin to reduce ingestive behaviour, the presence of leptin receptor‐immunoreactivity in hypocretin/orexin‐containing neurones was examined. Many leptin receptor‐and hypocretin/orexin‐immunoreactive neurones were demonstrated in the lateral hypothalamic area and perifornical region. Both direct double‐labelling and elution‐restaining methods showed that leptin receptor‐immunoreactivity was present in the vast majority of hypocretin/orexin‐containing neurones. Immunoreactivity for STAT3, a transcription factor activated by leptin, was also demonstrated in hypocretin/orexin‐containing neurones. Isolated hypocretin/orexin cell bodies in the dorsal part of the lateral hypothalamic area and the ventral perifornical region were shown to contain immunoreactivity for galanin, another peptide known to affect feeding. Galanin neurones were also seen to contain leptin receptor‐and STAT3‐immunoreactivity. Melanin‐concentrating hormone (MCH)‐containing neurones constituted a cell population within the lateral hypothalamus distinct from the one containing hypocretin/orexin‐immunoreactivity, as shown by elution‐restaining methodology. The presence of leptin receptor‐and STAT3‐immunoreactivities in hypocretin/orexin‐containing neurones of the lateral hypothalamus suggests that leptin may directly regulate these hypothalamic neurones, most likely via an inhibitory action on hypocretin/orexin expression and/or secretion resulting in reduced food intake.
Effects of Ageing and Dehydroepiandrosterone Administration on Pro‐Opiomelanocortin mRNA Expression in the Anterior and Intermediate Lobes of the Rat Pituitary
Authors: Givalois  , Li  , Pelletier
Published: March 2003

There is still controversy about the influence of ageing on the activity of the hypothalamo‐pituitary‐adrenocortical (HPA) axis in the rat. The first objective of the present study was to evaluate the influence of ageing on the activity of the HPA axis by measuring pituitary proopiomelanocortin (POMC, the precursor of ACTH and αH) mRNA levels in the anterior and intermediate lobes in young (50–55‐day old) and aged (18‐month‐old) rats of both sexes. The second goal of the study was to evaluate the effect of 2.5 day administration of dehydroepiandrosterone (DHEA), a steroid precursor which has been shown to improve some ageing‐associated deficits. In the young male anterior pituitary, DHEA induced a 17.5% increase in POMC mRNA levels. In aged males, anterior pituitary POMC mRNA levels were 22% lower than those detected in young animals. DHEA treatment produced a 26% increase, then completely restoring mRNA levels when compared to those found in young vehicle‐treated males. In the young female, DHEA did not induce any changes in anterior pituitary POMC mRNA. In aged females a 24% reduction in the hybridization signal was observed. This reduction was completely reversed by DHEA which induced a 45% increase over the levels observed in vehicle‐treated aged animals. In the intermediate lobe, the results were very similar to those obtained in the anterior lobe, although the observed effects induced by ageing and DHEA were less striking. These results together with previous ones indicating an age‐related decrease in corticotropin‐releasing hormone (CRH) neuronal activity suggest that ageing is associated with a decrease in HPA axis activity. They also demonstrate that a short‐term DHEA treatment can exert a beneficial influence by reversing the decrease in pituitary POMC mRNA expression which occurs as a consequence of ageing.
Evidence for Expression of Galanin Receptor Gal‐R1 mRNA in Certain Gonadotropin Releasing Hormone Neurones of the Rostral Preoptic Area
Authors: Mitchell  , Bouret  , Prévot  , Jennes  , Beauvillain
Published: March 2003

Previous studies have shown that galanin plays an important role in the regulation of gonadotropin releasing hormone (GnRH) release. At present, it is not known if this role is exerted by direct or indirect interactions between galanin producing neurones and GnRH neurones. The objective of this study was to determine whether or not GnRH neurones could express galanin receptor Gal‐R1 mRNA. Dual in‐situ hybridization experiments were carried out with digoxigenin‐labelled cRNA probes encoding GnRH in combination with 35S‐labelled riboprobe encoding the galanin receptor Gal‐R1. In order to detect possible variations in the expression of the Gal‐R1 mRNA under different physiological conditions, male rats, intact female rats throughout the phases of the oestrous cycle, ovariectomized (OVX) and steroid‐treated rats were analysed. The results show that many cells expressing Gal‐R1 mRNA were present throughout the preoptic area. Gal‐R1 mRNA‐expressing cells were observed in very close proximity with GnRH neurones. In the female rat, some GnRH neurones located in the rostral preoptic area/vascular organ of the lamina terminalis expressed Gal‐R1 mRNA. These double‐labelled cells were observed at all times of the oestrous cycle, except during diestrus at 08.00 h and pro‐oestrus at 18.00 h. Conspicuously, at oestrus 1800 h, we found that 21.6% of rostral GnRH neurones expressed the Gal‐R1 mRNA. In addition, dual‐labelled GnRH neurones were seen in OVX animals but not in oestrogen plus progesterone‐treated ones. In the male rat, colocalization of GnRH mRNA and Gal‐R1 receptor mRNA was not observed. In the medial preoptic area, no double‐labelled GnRH neurones were detected regardless of the endocrine conditions. These results suggest that, in addition to a possible indirect action of galanin on GnRH cells via neurones located at close proximity, the effects of galanin on GnRH can be mediated by direct activation of galanin receptors in rostral GnRH neurones. This study also shows that expression of Gal‐R1 mRNA in GnRH cells is influenced by the levels of circulating gonadal steroids.
N‐Terminal Splice Variants of the Type I PACAP Receptor: Isolation, Characterization and Ligand Binding/Selectivity Determinants
Authors: Dautzenberg  , Mevenkamp  , Wille  , Hauger
Published: March 2003

Three full‐length cDNAs encoding functional splice variants of the pituitary adenylate cyclase‐activating polypeptide (PACAP) type 1 receptor (PAC1) were isolated from Y‐79 retinoblastoma cells and human cerebellum. Although the third intracellular loops of the three splice variants were identical, their N‐terminal extracellular domains differed. The first full‐length PAC1 variant, PAC1normal (PAC1n), encoded the entire N‐terminus, whereas the second variant named PAC1short (PAC1s) was deleted by 21 amino acids (residues 89–109). Finally, the third variant, named PAC1very short (PAC1vs), was deleted by 57 amino acids (residues 53–109). Using semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR) analysis, it was established that all three variants were expressed in neuronal tissues. Binding‐ and cAMP studies using human embryonic kidney 293 (HEK293) cells stably transfected with PAC1n, PAC1s and PAC1vs showed significant differences in the affinities and selectivities towards PACAP38, PACAP27 and VIP. PAC1n bound PACAP38 and PACAP27 with affinities in the low nanomolar range whereas VIP was bound with up to 400‐fold lower affinity. PAC1vs preferentially bound PACAP38 (Ki=121 nM) and PACAP27 (Ki=129 nM) over VIP (Ki>1000 nM) but with 100‐fold lower affinity than PAC1n. Surprisingly, PAC1s unselectively bound all three ligands with high affinity. These data indicate that residues 53–88 within the N‐terminal domain of the PAC1 are important for high affinity ligand binding, whereas residues 89–109 determine the receptor’s ligand selectivity.
Oestrogen Receptor α is Essential for Female‐Directed Chemo‐Investigatory Behaviour but is not Required for the Pheromone‐Induced Luteinizing Hormone Surge in Male Mice
Authors: Wersinger S. R. , Rissman E. F.
Published: March 2003

The expression of normal masculine sexual behaviour requires testosterone. Testosterone can bind to androgen receptors, either in its native form, or after reduction to other androgen metabolites. In addition, testosterone can be aromatized to oestrogen, and bind to oestrogen receptor α and/or β. Male copulatory behaviour is deficient in mice lacking functional oestrogen receptor α gene (ERαKO mice). We sought to determine which aspect(s) of masculine sexual behaviour is compromised in the ERαKOs. Specifically, we asked whether ERαKO males have reduced motivation and/or an inability to recognize oestrous females. We found significant differences between mice of different genotypes in the amount of chemo‐investigatory behaviour displayed and in the target of their investigation. Wild‐type males spent more time investigating ovariectomized, oestradiol‐treated females, than either males, or ovariectomized females that had not received hormone priming. ERαKO males spent little time investigating any of the stimulus mice and showed no preferences. To test the hypothesis that this lack of chemo‐investigatory behaviour is due to the inability of ERαKO males to detect and respond to female pheromones, we exposed males to chemosensory cues (soiled bedding) from females. Males resided in clean, or female‐soiled, cage bedding for 60 min. Next, blood was collected and plasma luteinizing hormone (LH) assayed. We also assessed Fos‐like immunoreactivity (Fos‐ir) in several neural regions involved in processing chemosensory cues. Despite the fact that male ERαKOs spend little time engaged in chemo‐investigation of females, their neuroendocrine responses to female‐soiled bedding were similar to those seen in wild‐type males. Our data suggest that the normal coupling between the neuroendocrine response to females and the generation of sexual behaviour is disrupted in ERαKO mice. Responses to female pheromones do not require ERα. However, normal male sexual performance requires the ERα gene.
The Negative Feedback Action of Progesterone on Luteinizing Hormone Release is not Associated with Changes in GnRH mRNA Expression in the Ewe1
Authors: Robinson J. E. , Healey A. E. , Harris T. G. , Messent E. A. , Skinner D. C. , Taylor J. A. , Evans N. P.
Published: March 2003

Progesterone is the ovarian hormone that times events in the ovine reproductive cycle. When elevated, this ovarian hormone acts centrally to inhibit both the tonic and surge modes of gonadotrophin releasing hormone (GnRH) release. Two studies were performed to address the underlying neural mechanisms. The first tested the hypothesis that the rapid rise in GnRH release, that results from an acute fall in progesterone concentrations (such as occurs following luteolysis), is temporally associated with a rapid rise in the cellular content of GnRH mRNA. Three groups of ovariectomised (OVX) ewes were treated with exogenous progesterone for 10 days, while one remained steroid free (OVX, n=7). To determine the effects of acute progesterone (P) withdrawal, ewes were killed on day 10 while implants were still in place (OVX+P, n=6) or 4 (OVX‐P4, n=7) or 12 h (OVX‐P12, n=7) after progesterone removal. Coronal sections through the rostral portion of the medial preoptic area (rPOA) were processed for cellular in‐situ hybridization for GnRH mRNA. An increase in progesterone concentrations markedly suppressed luteinizing hormone (LH) release, while removal of the implants caused progesterone concentrations to fall (P<0.01) within 1 h and LH pulse frequency to increase (P<0.05) within 4 h. Despite these progesterone‐induced changes in LH/GnRH release there were no differences in the cellular content of GnRH mRNA among the four groups. In the second study, three groups of ovariectomised ewes were used to determined whether the inhibitory actions of early (EL; n=8) and mid‐luteal (ML; n=8) phase concentrations of progesterone on LH release are accompanied by a decrease in GnRH mRNA expression. P inhibited the secretion of LH in a dose dependant manner; pulses of LH were virtually absent in the ML group. Despite this marked inhibitory steroid action, there was no significant difference in the cellular content of GnRH mRNA among the OVX, OVX (EL) and OVX (ML) groups. Thus, both the negative feedback actions of physiological concentrations of progesterone on GnRH release and the rapid escape from progesterone‐inhibition are independent of changes in the cellular content of GnRH mRNA. These data suggest that the mechanism by which progesterone controls the timing of events in the ovine oestrous cycle is primarily by altering the secretion of GnRH rather than GnRH biosynthesis.
Effect of Interleukin‐1β on Gonadotropin‐Releasing Hormone (GnRH) and GnRH Receptor Gene Expression in Castrated Male Rats
Authors: Kang  , Kim  , Leonhardt  , Jarry  , Wuttke  , Kim
Published: March 2003

Increasing evidence suggests that interleukin‐1β (IL‐1β) regulates luteinizing hormone (LH) release primarily through modulation of the gonadotropin‐releasing hormone (GnRH) neuronal activity. This study was undertaken to elucidate the effect of IL‐1β on GnRH as well as GnRH receptor (GnRHR) gene expression in the preoptic area. IL‐1β (100 ng/rat) or saline was administered into the lateral ventricle of castrated rats. RNA samples were isolated from micropunches of the preoptic area and mediobasal hypothalamus from individual brain slices and GnRH mRNA levels in the preoptic area and GnRHR mRNA levels in the mediobasal hypothalamus were determined by competitive reverse transcription‐polymerase chain reaction (RT‐PCR) protocols. Serum LH concentrations were decreased from 1 h to 3 h after IL‐1β treatment, but rebounded at 5 h, while serum concentrations of follicle‐stimulating hormone (FSH) and prolactin were not altered. There were no significant changes in GnRH mRNA levels from the micropunched preoptic area, while GnRHR mRNA levels from the preoptic area and mediobasal hypothalamus micropunch samples, but not in the anterior pituitary, showed a pattern similar to the serum LH profile following i.c.v. administration of IL‐1β. We then examined the effect of IL‐1β on the translational efficiency of the GnRH mRNA. After the separation and fractionation of polyribosome‐associated cytoplasmic RNA from the hypothalamic fragments containing the preoptic area‐anterior hypothalamic area of control (saline‐treated) and IL‐1β‐treated group 3 h after administration, GnRH transcript levels were examined from the each fraction. IL‐1β decreased the translational efficiency of the transcribed GnRH mRNA. These results clearly demonstrate that central administration of IL‐1β suppresses the translational activity of GnRH mRNA. Moreover, GnRHR may play an important role in the modulation of GnRH neuronal activity through GnRHR‐expressing neurones (or glia) in the hypothalamus.
Temperature and Photoperiod Interact to Affect Reproduction and GnRH Synthesis in Male Prairie Voles
Authors: Kriegsfeld L. J. , Trasy A. G. , Nelson R. J.
Published: March 2003

Prairie voles (Microtus ochrogaster), like most rodent species, exhibit a phenotypic polymorphism in reproductive response to winter conditions or to short day lengths in the laboratory. Laboratory studies on seasonally breeding species have traditionally focused on the role of photoperiod in modulating reproduction and other seasonal adaptions. However, because animals use proximate environmental factors in addition to photoperiod to phase seasonal adaptions with the appropriate time of year, the present study investigated the interaction of photoperiod and temperature on reproductive function and the gonadotropin‐releasing hormone (GnRH) neuronal system. Male prairie voles were housed in either long (LD 16:8) or short (LD 8:16) photoperiods. Voles in each photoperiodic condition were also exposed to either mild (20 °C) or low (8 °C) temperatures. After 10 weeks, voles were killed and their brains were processed using in situ hybridization for mRNA for proGnRH. The results suggest that GnRH synthesis is not affected by exposure to a single inhibitory proximate factor (i.e. short days or low temperatures alone), even when reproduction is inhibited, whereas a combination of inhibitory proximate factors leads to a decrease in GnRH synthesis (i.e. fewer neurones staining for mRNA for proGnRH). These data suggest that the neuroendocrine mechanisms regulating seasonal alterations in reproductive function are likely to differ between harsh and mild winters.
Early Environmental Regulation of Glucocorticoid Feedback Sensitivity in Young Adult Monkeys
Authors: Lyons D. M. , Yang C. , Mobley B. W. , Nickerson J. T. , Schatzberg A. F.
Published: March 2003

Variations in maternal care induce in neonatal rodents life‐long changes in glucocorticoid feedback regulation of the hypothalamic‐pituitary‐adrenal axis. This aspect of plasticity in neuroendocrine development has not been established in primates. We assessed, in young adult squirrel monkeys, postnatal rearing effects on cortisol‐induced suppression of corticotropin‐releasing factor (CRF) stimulated secretion of adrenocorticotropic hormone (ACTH). Offspring of randomly bred monkeys were periodically removed from natal groups between 13 and 21 weeks of age. In two other postnatal rearing conditions, systematic differences in maternal availability were produced by manipulating the effort required of lactating mothers to successfully find food. All offspring were subsequently administered, 3–5 years later on two occasions, an intravenous ovine CRF injection preceded 60 min earlier by placebo or cortisol pretreatment. The difference between CRF‐stimulated time‐integrated secretion of ACTH following placebo vs cortisol pretreatment served as an index of glucocorticoid negative feedback. Difference scores were greatest in monkeys previously separated from natal groups. This finding was not attributable to significant rearing condition differences in plasma cortisol levels achieved following pretreatment with exogenous cortisol, nor plasma ACTH levels produced when the CRF injection was preceded by pretreatment with placebo. The results suggest that postnatal experiences altered glucocorticoid feedback in monkeys at least through early adulthood. This conclusion supports retrospective reports indicating that, for humans with major mood and anxiety disorders, systematic differences in glucocorticoid feedback may reflect neural mechanisms in development linking early life stress with psychopathology in adulthood.
Permissive Effect of Centrally Administered Oxytocin on the Excitatory Response of Oxytocin Neurones to Ventral Tegmental Stimulation in the Suckled Lactating Rat
Authors: Cosgrave A. S. , Richardson C. M. , Wakerley J. B.
Published: March 2003

The mesencephalic ventral tegmentum has been implicated in the milk‐ejection reflex and modulation of inputs from this region could provide a mechanism whereby central oxytocin facilitates synchronous bursting of oxytocin neurones during suckling. Experiments were therefore undertaken to investigate the effect of intracerebroventricular (i.c.v.) oxytocin on the response of oxytocin neurones to ventral tegmental stimulation. Oxytocin neurones were recorded in the supraoptic nucleus of urethane‐anaesthetized lactating rats during suckling, and their response to single shock stimulation of the ventral tegmentum was monitored using peri‐stimulus time‐interval histograms. Before i.c.v. oxytocin, oxytocin neurones were either unresponsive to ventral tegmental stimulation, or displayed a small inhibition. However, after administration of oxytocin (2.2 ng i.c.v.), seven out of eight neurones tested displayed a pronounced excitatory response (onset latency 78.4 ± 4.8 ms, duration 73.4 ± 8.3 ms). This permissive effect on the excitatory response was only observed in the presence of suckling, and followed the same time course as facilitation of the milk‐ejection reflex, being maximal immediately before each facilitated bursting response in the oxytocin neurones. The response to ventral tegmental stimulation remained unaltered after intraperitoneal administration of hypertonic saline to cause a generalized increase in the excitability of the oxytocin neurones. Moreover, i.c.v. oxytocin had no effect on the response of oxytocin neurones to stimulation of a descending input from the medial septum. In conclusion, administration of i.c.v. oxytocin has a selective permissive effect on the excitation of oxytocin neurones from the ventral tegmentum, and this supports previous in vitro studies suggesting that centrally released oxytocin may act as a modulator of afferent transmission to the magnocellular nuclei. This effect on the afferent excitation of oxytocin neurones may provide a mechanism whereby i.c.v. oxytocin facilitates suckling‐evoked bursting activity.
Rat Adrenal Transplants are Reinnervated: An Invalid Model of Denervated Adrenal Cortical Tissue
Authors: Ulrich‐Lai Y. M. , Engeland W. C.
Published: March 2003

Adrenal autotransplantation is a widely used approach to investigate the potential for neural modulation of adrenal cortical function. It is believed that regenerating adrenal transplants are not reinnervated, thereby providing a model to investigate adrenal function in the absence of neural modulation. However, the hypothesis that adrenal transplants become reinnervated has not been directly tested. The purpose of the present study was to characterize the time course, extent, and nature of the reinnervation of the regenerating adrenal transplant and to assess whether the recovery of steroidogenic function and enzyme expression correlates temporally with the presence of innervation. Using immunohistofluorescent detection of tyrosine hydroxylase (TH), neuropeptide Y (NPY), calcitonin gene‐related peptide (CGRP), and vasoactive intestinal peptide (VIP), the innervation of regenerating adrenals was assessed 14–30 days after transplantation of adrenal capsules beneath the kidney capsule in rats. Extensive reinnervation by TH‐, NPY‐, and VIP‐positive fibres was present by 14 days after transplantation including regions of the adrenal capsule and cortex, with only minimal reinnervation by CGRP‐positive fibres up to 30 days. TH‐ and NPY‐positive chromaffin cells were also observed in the regenerating transplants. In addition, there was marked recovery of steroidogenic function and steroidogenic enzyme expression up to 30 days. The finding that nerve fibres are present in the transplants during the re‐establishment of steroidogenic function and enzyme expression suggests that innervation may modulate the regeneration and functional recovery of adrenal transplants. In an attempt to prevent reinnervation of transplants, adrenal capsules were autotransplanted to denervated kidneys. Immunohistochemical analysis showed that, despite extensive denervation of the kidney tissue, the reinnervation and regeneration of the adrenal transplants still occurred. These data demonstrate the marked capacity of the regenerating adrenal to become reinnervated and reinforces the conclusion that adrenal transplants are an invalid model of denervated adrenal cortical tissue.
Glutamatergic Regulation of Gonadotropin Releasing Hormone mRNA Levels During Development in the Mouse
Authors: Ford H. , Ebling F. J. P.
Published: March 2003

The aims of these studies were to investigate the time course of the increase in gonadotropin releasing hormone (GnRH) mRNA levels during sexual development in the mouse, and to test the hypothesis that the neurotransmitter glutamate regulates the GnRH secretory system via actions at the level of GnRH gene expression. GnRH mRNA abundance was estimated by measuring silver grains generated by in situ hybridization of an 35S‐labelled oligonucleotide probe. There was a significant increase in GnRH mRNA abundance between the day of birth (P0) and postnatal day 2 (P2) in male mice, but no further increases at later ages when overt pubertal changes are manifest. GnRH mRNA levels also increased significantly between P0 and P2 in female mice. Treatment with the glutamate agonist NMDA caused a significant increase in GnRH mRNA levels in neonatal (P0) mice and adult male mice within 30 min of treatment, which is consistent with previous studies in the rat implicating glutamate in the regulation of GnRH mRNA stability. Treatment with the glutamate antagonist CGP40116 caused an equally rapid decrease in GnRH mRNA levels in adult mice and in mice on P5 after the neonatal increase in GnRH gene expression, but was without effect in mice on P0, prior to the developmental increase. These observations that the effect on GnRH mRNA levels of blocking endogenous glutamatergic signalling depends upon the developmental stage suggest that endogenous glutamate maintains GnRH mRNA levels in adult mouse, and is a potential regulator of the developmental increase seen in the neonatal period.
Chronic Iodine Deprivation Attenuates Stress‐Induced and Diurnal Variation in Corticosterone Secretion in Female Wistar Rats
Authors: Nolan L. A. , Windle R. J. , Wood S. A. , Kershaw Y. M. , Lunness H. R. , Lightman S. L. , Ingram C. D. , Levy A.
Published: March 2003

Many millions of people throughout the world are at risk of developing iodine deficiency‐associated disorders. The underlying effects of iodine deficiency on neuroendocrine function are poorly defined. We have studied stress‐induced and diurnal variation in corticosterone secretion in female rats rendered chronically hypothyroid by feeding them an iodine‐free diet for 6 months. Corticosterone secretory responses in iodine deficient animals were compared to those seen in animals rendered hypothyroid with propylthiouracil and untreated controls. By using a well‐validated, automated blood sampling system to collect small samples of blood over the complete daily cycle in unrestrained animals, we have demonstrated for the first time that the normal diurnal rhythm of corticosterone secretion is lost in chronic iodine deficiency and that the corticosterone secretory response to the psychological stress of 10 min exposure to white noise is attenuated. Despite restoration of circulating triiodothyronine and thyrotropin releasing hormone‐ and thyroid stimulating hormoneβ‐transcript prevalence in the hypothalamus and pituitary, respectively, 1 month after restoration of normal iodine‐containing diet both the diurnal variation in corticosterone levels and the corticosterone secretory response to the noise stress remained reduced in amplitude compared to control animals. Thus, chronic hypothyroidism induced by iodine deficiency significantly attenuates hypothalamo‐pituitary‐adrenal axis activity, an effect that persists after functional recovery of the thyroid axis.
Effects of Serotonin, GABA and Neuropeptide Y on Seabream Gonadotropin Releasing Hormone Release In Vitro from Preoptic‐Anterior Hypothalamus and Pituitary of Red Seabream, Pagrus major
Authors: Senthilkumaran B. , Okuzawa K. , Gen K. , Kagawa H.
Published: March 2003

The effects of serotonin (5‐HT), GABA and neuropeptide Y (NPY) on in vitro release of seabream (sb) gonadotropin releasing hormone (GnRH) from slices of the preoptic‐anterior hypothalamus (P‐AH) and pituitary of red seabream were studied. 5‐HT, GABA and NPY all stimulated the release of sbGnRH from the P‐AH but not from the pituitary of immature red seabream. They also stimulated sbGnRH release from the P‐AH with a similar potency during the course of gonadal development. Specific agonists and/or antagonists of 5‐HT, GABA and NPY showed that 5‐HT and GABA utilize 5‐HT2 and GABAA receptor subtypes, respectively, to mediate their action, and that NPY employs at least NPYY1 and NPYY2 receptor subtypes to stimulate sbGnRH release. Combinations of different antagonists for 5‐HT, GABA and noradrenaline/adrenaline did not block the stimulatory influence of NPY on release of sbGnRH, indicating that the action of NPY on the sbGnRH neuronal system is probably direct.
GABAergic Nature of Hypothalamic Leptin Target Neurones in the Ventromedial Arcuate Nucleus
Authors: Ovesjö M.‐L. , Gamstedt M. , Collin M. , Meister B.
Published: March 2003

Leptin is an adipose tissue‐derived cytokine hormone, which reduces body weight via interactions with hypothalamic neurones. Leptin receptors capable of activating the JAK‐STAT signal transduction pathway are expressed at high levels in the hypothalamus, particularly in the arcuate nucleus. In order to identify the chemical mediators of leptin's action in the hypothalamus, we have examined whether GABA neurones of the hypothalamic arcuate nucleus contain leptin receptors and the leptin‐activated transcription factor STAT3. GABAergic neurones, as visualized by antisera to the GABA‐synthesizing enzyme glutamic acid decarboxylase (GAD) and GABA, were demonstrated in the ventromedial and ventrolateral parts of the arcuate nucleus. GABA neurones in the ventromedial arcuate nucleus were shown to contain leptin receptor immunoreactivity, as revealed using an antiserum generated to a sequence common to all isoforms of the leptin receptor (Ob‐R), as well as an antiserum generated to the carboxy‐terminal end of the long leptin receptor (Ob‐Rb), and immunoreactivity for the leptin‐induced signal transduction molecule STAT3. Ventromedial GABA neurones were also shown to contain neuropeptide Y, whereas ventrolateral proopiomelanocortin‐containing neurones lacked GAD and GABA immunoreactivity. Levels of mRNA for GAD65, GAD67 and the vesicular GABA transporter (VGAT) were analysed in the arcuate nucleus of leptin‐deficient ob/ob mice and lean control mice by in situ hybridization. No significant differences in GAD65, GAD67 or VGAT mRNA were detected in the arcuate nucleus of ob/ob mice as compared to lean control mice. The presence of leptin receptor and STAT3 in GABAergic arcuate neurones, but absence of changes in gene transcription for GAD and VGAT mRNA suggests, that leptin does not transcriptionally regulate the expression of proteins involved in GABAergic transmission in arcuate neurones. However, mechanisms other than transcriptional regulation for leptin to influence arcuate GABA neurones may exist.
Ablation of Pituitary Pro‐Opiomelanocortin (POMC) Cells Produces Alterations in Hypothalamic POMC mRNA Levels and Midbrain µ Opioid Receptor Binding in a Conditional Transgenic Mouse Model
Authors: Zhou Y. , Unterwald E. M. , Ho A. , LaForge K. S. , Yuferov V. P. , Kreuter J. , Sirianni M. J. , Allen R. G. , Kreek M. J.
Published: March 2003

The hypothalamic‐pituitary‐adrenal (HPA) axis is regulated by stress‐related excitatory inputs, and various inhibitory and negative‐feedback controls by glucocorticoids and opioids, including pro‐opiomelanocortin (POMC)‐derived peptides. The role of POMC‐derived peptides of pituitary origin in the modulation of brain POMC mRNA expression and opioid receptor binding was investigated using a line of transgenic mice that express a fusion gene composed of the pituitary expression‐specific promoter region of the POMC gene driving the herpes simplex viral‐1 thymidine kinase (TK). Male adult mice were treated with the antiherpes agent ganciclovir that selectively ablates cells expressing TK. Following treatment, POMC mRNA levels, measured by quantitative solution hybridization/RNase protection assays, were decreased by 48% in the pituitary of the TK+/+ mice, reflecting an expected loss of the pituitary corticotrope POMC cells. This treatment also significantly lowered pituitary β‐endorphin immunoreactivity content and plasma concentrations of corticosterone. In contrast, POMC mRNA levels were increased by 79% in the hypothalamus of the TK+/+ mice with pituitary POMC cell ablation. Binding of [3H]DAMGO to µ opioid receptors, as measured by quantitative autoradiography, was significantly reduced in several brain regions including the central grey, median raphe and superficial grey layer of the superior colliculus. These regions are innervated by hypothalamic POMC neurones. No significant differences in binding to either κ or δ opioid receptors were found in the brain regions studied. These results suggest that POMC‐derived peptides of pituitary origin may exert a tonic negative‐feedback effect on hypothalamic POMC neurones. In turn, the downregulation of central µ opioid receptors in this model may be mediated through a mechanism related to hypothalamic POMC overexpression.
Photorefractoriness of Immune Function in Male Siberian Hamsters (Phodopus sungorus)
Authors: Prendergast B. J. , Wynne‐Edwards K. E. , Yellon S. M. , Nelson R. J.
Published: March 2003

Short days induce multiple changes in reproductive and immune function in Siberian hamsters. Short‐day reproductive inhibition in this species is regulated by an endogenous timing mechanism; after approximately 20 weeks in short days, neuroendocrine refractoriness to short‐day patterns of melatonin develops, triggering spontaneous recrudescence of the reproductive system. It is unknown whether analogous mechanisms control immune function, or if photoperiodic changes in immune function are masked by prevailing photoperiod. In Experiment 1, 3 weeks of exposure to long days was not sufficient to induce long‐day‐like enhancement of in vitro lymphocyte proliferation in short‐day adapted male Siberian hamsters. Experiment 2 tested the hypothesis that immunological photorefractoriness is induced by prolonged exposure to short days. Adult male hamsters were gonadectomized or sham‐gonadectomized and housed in long (14 h light/day) or short (10 h light/day) photoperiods for 12, 32 or 40 weeks. Somatic and reproductive regression occurred after 12 weeks in short days, and spontaneous recrudescence was complete after 32–40 weeks in short days, indicative of somatic and reproductive photorefractoriness. In gonad‐intact hamsters, 12 weeks of exposure to short days decreased the number of circulating granulocytes and increased the number of B‐like lymphocytes. After 32 weeks in short days, these measures were restored to long‐day values, indicative of photorefractoriness; castration eliminated these effects of photoperiod. In both intact and castrated hamsters, in vitro proliferation of splenic lymphocytes was inhibited by 12 weeks of exposure to short days. After 40 weeks in short days lymphocyte proliferation was restored to long‐day values in intact hamsters, but remained suppressed in castrated hamsters. These results suggest that short‐day‐induced inhibition of lymphocyte function does not depend on gonadal regression, but that spontaneous recrudescence of this measure is dependent on gonadal recrudescence. In Experiment 3, in vitro treatment with melatonin enhanced basal proliferation of lymphocytes from male hamsters exposed to short days for 12 weeks, but had no effect on lymphocytes of photorefractory hamsters or long‐day control hamsters. Lymphocytes of castrated hamsters were unresponsive to in vitro melatonin, suggesting that photoperiodic changes in gonadal hormone secretion may be required to activate mechanisms which permit differential responsiveness to melatonin depending on phase in the annual reproductive cycle. Together, these data indicate that, similar to the reproductive system, the immune system of male Siberian hamsters exhibits refractoriness to short days.
Leptin Uptake by Serotonergic Neurones of the Dorsal Raphe
Authors: Fernández‐Galaz M. C. , Diano S. , Horvath T. L. , Garcia‐Segura L. M.
Published: March 2003

The effects of leptin on food intake, metabolism, sleep patterns and reproduction may be mediated, in part, by the midbrain serotonergic systems. Here, we report on the distribution of neurones that accumulate leptin in the raphe nuclei of male and female rats after intracerebroventricular administration of mouse recombinant leptin labelled with digoxigenin. Direct leptin‐targeted cells were present in the periventricular grey, pontine and raphe nuclei. Confocal microscopy revealed that raphe neurones which accumulated leptin were predominantly serotonergic. The temporal pattern of leptin accumulation by raphe neurones showed a marked gender difference: 6 h after leptin administration, all male and female rats showed massive leptin binding in the dorsal raphe, while 30 min after leptin treatment, only 10% of male rats exhibited leptin‐labelled cells in contrast to 50% of females. The present observations reveal that leptin can be selectively accumulated by serotonergic neurones in the raphe nuclei and that this mechanism is gender specific. These findings support the idea that the midbrain serotonergic system is an important mediator of the effects of leptin on brain function and may provide an explanation for gender differences in metabolism regulation and its coordination with higher functions of the brain.
Sexual Differentiation of Astrocyte Morphology in the Developing Rat Preoptic Area
Authors: Amateau S. K. , McCarthy M. M.
Published: March 2003

The preoptic area is an important brain region controlling sex‐typic behaviour and physiology, and astrocytes of this region are responsive to steroids perinatally. Utilizing glial fibrillary acidic protein immunocytochemistry, the morphology of astrocytes in the preoptic area of male and female rat pups was examined on the day of birth and on postnatal day 3. As early as the day of birth, astrocytes of the male preoptic area exhibit both significantly greater primary process length and number of primary processes, and these differences remain at postnatal day 3. Application of exogenous steroid to females suggested that gonadal steroids, in particular oestradiol, mediate the sex difference. Pups received 100 µg of steroid on the day of birth and again on postnatal day 1, and astrocyte morphology was assessed on postnatal day 3. Both oestradiol and testosterone induced significant changes in process length and number compared to vehicle‐treated controls. Astrocytes of oestradiol‐treated females did not differ on PN3 from those of PN3‐untreated males. Exposure to the nonaromatizable steroid, dihydrotestosterone, had no effect on any attribute of astrocyte morphology. This suggests the effects induced by testosterone are mediated by oestradiol following local aromatization of the steroid, and not through direct activation of the androgen receptor. Astrocytes are important in synapse formation and efficacy, and we hypothesize a role for astrocyte complexity and differentiation in the establishment of synaptic patterning.
Cerebral Insulin Increases Brain Response to Glucose
Authors: Alquier T. , Leloup C. , Atef N. , Fioramonti X. , Lorsignol A. , Pénicaud L.
Published: March 2003

The hypothalamus participates in the regulation of carbohydrate metabolism involving a feedback loop between the brain and the periphery in which glucose‐sensitive hypothalamic areas appear to be involved. We have previously shown that a glucose injection (9 mg/kg) in the carotid artery toward the brain, in an amount that did not modify glycaemia, caused a rapid and transient increase in plasma insulin concentrations. To determine whether central insulin could influence this response, we investigated the change in central glucose‐induced insulin secretion in intracerebroventricular (i.c.v) insulin‐injected rats and in hyperinsulinaemic obese Zucker rats. Central glucose‐induced insulin secretion was increased by 50% in i.c.v. insulin‐injected rats compared to control rats. When a similar test was performed at a lower dose of glucose (3 mg/kg), a significant insulin secretion was observed only in rats submitted to a prior central insulin injection. These data indicate an increase in the brain response to glucose after insulin treatment. Using an identical lower glucose dose, we also demonstrated an enhanced brain glucose sensitivity in hyperinsulinaemic and insulin‐resistant obese Zucker rats. Together, these results indicate that acute i.c.v. insulin or pathological hyperinsulinaemic state (i.e. obese Zucker rat) modulates the nervous control of insulin secretion by increasing the brain response to glucose.