bluuebunny/crossref_metadata_2025 huggingface dataset:
Arginine Vasopressin‐lnduced Sensitization in Brain: Facilitated Inositol Phosphate Production Without Changes in Receptor Number
Authors: Poulin Paule , Pittman Quentin J.
Published: September 2006

Arginine vasopressin (AVP) has been shown to have a unique sensitization effect whereby repeated injection of AVP into a lateral cerebral ventricle or a mediobasal region of the rat forebrain below the lateral septum and including the anterior hypothalamus referred to as the ventral septal area, causes enhanced motor responses to the ligand. To elucidate possible neuronal mechanisms responsible for AVP sensitization, 1) we determined the dose and the time required for the development and expression of AVP sensitization, and 2) we tested the hypotheses that AVP sensitization may result in a) alteration of septal AVP V1 receptor affinity or number, and/or b) alteration of septal AVP V1 receptor signal transduction (phosphatidylinositol hydrolysis) mechanisms. Our behavioral data show that the magnitude of AVP sensitization varies with dose and time, and the effect is dependent on the time interval between injections, in that an initial intracerebroventricular AVP injection enhances the sensitivity of the animals to the motor effects of similar AVP injections given 6 h to 6 days later but not to injections given hourly or weekly. No changes in septal AVP binding site density and affinity, as measured by [3H]AVP binding to septal synaptic plasma membrane, were found in sensitized animals; [3H]inositol monophosphate stimulation in response to AVP in septal slices, however, was found to be significantly enhanced. This enhanced [3>H]inositol monophosphate stimulation appears specific to a V1‐type receptor because it was significantly reduced in the presence of the V1 receptor antagonist, d(CH2)5Tyr(Me)AVP, and was not found using oxytocin or the V2 receptor agonist, DDAVP. Our results therefore indicate that receptor binding, while critical to peptide neurotransmitter action, is not the sole factor for determining responsiveness. Rather, an appropriate schedule of AVP administration, which may cause changes in postreceptor effector system(s) such as inositol phosphate hydrolysis, appears most important.
Effects of Lipocortin 1 and Dexamethasone on the Secretion of Corticotrophin‐Releasing Factors in the Rat: in vitro and in vivo Studies
Authors: Loxley H. D. , Cowell A‐M. , Flower R. J. , Buckingham J. C.
Published: September 2006

Lipocortin 1 (LC1: also called annexin 1) was first described as a putative second messenger protein for the anti‐inflammatory steroids in peripheral tissues. In the present study, in vitro and in vivo methods were used to examine its potential role within the hypothalamus as a mediator of the regulatory actions of the glucocorticoids on the hypothalamo‐pituitary‐adrenocortical axis of the rat.

In the in vitro studies, the effects of human recombinant LC1 (hu‐r‐LC1) on the concomitant release of the two major corticotrophin‐releasing factors (CRF‐41 and arginine vasopressin, AVP) from isolated hypothalami removed from chronically adrenalectomized rats were compared with those of dexamethasone in the presence and absence of appropriate secretagogues, namely phospholipase A2 (PLA2), interleukin‐6 (IL‐6) and a non‐specific depolarizing agent, K+ (56 mM). The spontaneous release of CRF‐41 in vitro was unaffected by either hu‐r‐LC1 (5 to 100 ng/ml) or dexamethasone (1 μM). Both compounds however reduced the release of the neuropeptide evoked by IL‐6 (5 ng/ml) but failed to modify the secretory responses to PLA2 (25 U/ml) or K+ (56 mM). Dexamethasone (1 μM) had no effect on the basal release of AVP but effectively blocked the secretion of the peptide induced by either IL‐6 (10 ng/ml) or PLA2 (25 U/ml). In complete contrast, hu‐r‐LC1 (5 to 100 ng/ml) stimulated the release of AVP and potentiated the secretory responses to IL‐6 (10 ng/ml) and PLA2 (25 U/ml) but not to K+ (56 mM). The hypothalamic responses to PLA2 stimulation (25 U/ml) were associated with significant (P<0.01) increases in prostaglandin E2 release which, in some instances, were potentiated by hu‐r‐LC1 (5 to 20 ng/ml).

In vivo, administration of histamine (0.6 mg/100 g body wt, ip) produced significant (P<0.01) increases in the serum corticosterone concentration and in the hypothalamic LC1 content. Neither hu‐r‐LC1 (0.6 to 1.2 μg) nor a polyclonal anti‐LC1 antibody (3 μl, diluted 1:200), injected intracerebroventricularly (icv), influenced either the resting serum corticosterone concentration or the hypersecretion of the steroid evoked by histamine stress. A lower dose of the recombinant protein (0.3 μg icv) also failed to alter basal corticosterone release but, in contrast to the higher doses, potentiated the pituitary‐adrenocortical responses to histamine.

The results suggest that LC1 may contribute to some aspects of peptide release in the hypothalamus but that its actions are not necessarily related to those of the glucocorticoids.
Hormonal Control of Sex Differences in the Brain, Behavior and Accessory Sex Structures of Whiptail Lizards (Cnemidophorus Species)
Authors: Wade Juli , Huang Junn‐Ming , Crewst David
Published: September 2006

The effects of steroid hormones on sexual dimorphisms in the brain, behavior and accessory sex structures were investigated in two species of whiptail lizards. The studies were conducted both in adults and hatchlings of a sexually reproducing species (Cnemidophorus inornatus) and an all‐female species (C. uniparens) which displays ‘sexual’ behaviors typical of males and females. Adults were gonadectomized and approximately 3 months later given either a Silastic capsule filled with sex steroid or an empty capsule. Young animals of both species were left intact and given a capsule on the day of hatching. An additional group of C. uniparens was ovariectomized on the day of hatching. Following treatment, measures of oviduct (estrogen‐dependent), renal sex segment (androgen‐dependent) and wolffian duct (androgen‐dependent) hypertrophy were taken in some experiments. Animals were also tested for sexual behavior in some of the studies. The volumes of the anterior hypothalamus‐preoptic area and ventromedial hypothalamus were measured in each individual. Estrogen, testosterone and dihydrotestosterone stimulated peripheral structures at both time periods in both sexes and species. The hormones also stimulated courtship and copulatory behaviors in many of the adult animals. However, testosterone in the anterior hypothalamus‐preoptic area of male C. inornatus was the only treatment which produced parallel effects on the volume of a brain area and the behaviors which it controls. These data add whiptail lizards to the list of species in which steroid hormones affect the volume of brain regions in adulthood, but suggest that such changes in morphology are not necessarily predictive of functional differences.
Is Vasopressin Preferentially Released from Corticotropin‐Releasing Factor and Vasopressin Containing Nerve Terminals in the Median Eminence of Adrenalectomized Rats?
Authors: de Goeij Dimphena C. E. , Berkenbosch Frank , Tilders Fred J. H.
Published: September 2006

The effects of adrenalectomy (ADX) on the amounts of immunoreactive corticotropin‐releasing factor (CRFj) and arginine vasopressin (AVPi) that are stored in the zona externa of the median eminence (ZEME) were investigated by means of quantitative immunocytochemistry. Although ADX of male Wistar rats for 1 week or 4 weeks did not affect CRFi in the ZEME as compared to sham‐operated or intact controls, AVPi showed a progressive accumulation. The ratio of AVPi over CRFi in the ZEME had already increased 1 day after ADX. However, it should be noted that the exact changes in CRFi and AVPj as measured by radioimmunoassay and/or quantitative immunocytochemistry were dependent on the substrain of rats used. The secretion rate of CRFi and AVPj was estimated in 1 week and 4 week ADX rats, by measuring the disappearance rate of CRFi and AVPi from the ZEME after blockade of fast axonal transport, by a low non‐toxic dose of colchicine (5 μg per rat). In contrast to intact rats, where this dose of colchicine did not affect CRFi or AVPi in the ZEME, ADX rats showed a progressive depletion of the CRFi and AVPi stores as measured 2.5 and 5 h later. In 1 week ADX rats, CRFi and AVPi both disappeared at a rate of 7% to 8% of their stores per hour. In contrast, after 4 weeks of ADX the fractional disappearance rates of CRFi and AVPi were different and were 3% and 8% of the content per hour, respectively. This indicates that in long‐term ADX rats the chance of AVP being released from the ZEME is more than twice that of CRF. Since most, if not all, of the CRF; containing nerve terminals in the ZEME of ADX rats costore AVP, we hypothesize that in long‐term ADX rats AVP may be preferentially secreted from AVP and CRF costoring nerve terminals.
Altered Ratios of Beta‐Endorphin:Beta‐Lipotropin Released from Anterior Lobe Corticotropes with Increased Secretory Drive.
Authors: Young Elizabeth A. , Day Robert , Schafer Martin , Watson Stanley J. , Akil Huda
Published: September 2006

A number of stimuli including acute footshock and electrically‐induced seizures lead to release of β‐endorphin immunoreactivity from the anterior pituitary corticotropes. Gel filtration of this β‐endorphin immunoreactivity indicates that approximately 3‐fold more β‐endorphin than β‐lipotropin is released into plasma following these acute stressors. A similar preponderance of β‐endorphin over β‐lipotropin is seen in the media of short‐term anterior lobe cell suspensions stimulated with ovine corticotropin‐releasing hormone. Previous studies indicated that footshock stress, when administered repeatedly, can increase the biosynthesis of anterior lobe proopiomelanocortin (POMC) as indicated by increased steady state adrenocorticotropin/β‐endorphin content as well as increased POMC mRNA levels and increased POMC biosynthesis and rate of processing as measured by pulse‐labeling and pulse‐chase studies. The goal of the present studies was to determine whether this increased biosynthetic drive results in an alteration in the end products secreted with repeated stress. Acute footshock in a rat which has received 14 days of chronic footshock releases proportionately more β‐lipotropin than is released in a naive rat. Chronic electrically‐induced seizures, which also increase anterior lobe POMC derived peptide stores, lead to a similar shift in the ratio of β‐lipotropin:β‐endorphin released following stress. These data suggest that chronic drive and the subsequent changes in POMC peptide stores may lead to a decrease in the proportion of β‐endorphin size immunoreactivity in the releasable pool of the anterior lobe corticotrope, thus altering the hormonal signal from the anterior lobe corticotrope.
Glucocorticoid Receptor Function in Rat Pituitary Intermediate Lobe is Inhibited by an Endogenous Protein
Authors: Sheppard Karen E. , Autelitano Dominic J. , Roberts James R. , Blum Mariann
Published: September 2006

Though glucocorticoids inhibit proopiomelanocortin (POMC) gene expression and POMC‐derived peptide release from corticotroph cells of the anterior pituitary, the regulation of this gene by glucocorticoids is less clear in the melanotroph cell of the pituitary intermediate lobe. To examine the difference between glucocorticoid sensitivity of the anterior lobe (AL) and intermediate lobe (IL) of the pituitary we have examined glucocorticoid receptor (GR) status of these two tissues and the role glucocorticoids play in regulating IL POMC gene expression. The rate of in vivo GR gene transcription, measured by nuclear run‐on assay was consistently higher in the pituitary neurointermediate lobe (NIL) compared with the AL of the same animals. On a concentration basis, cytoplasmic GR mRNA in the NIL was similar to that found in the AL, and GR binding using [3H]dexamethasone (DEX) as ligand demonstrated similar concentrations of specific [3H]DEX binding in acutely isolated AL and NIL tissues. The specific Type II corticosteroid receptor ligand RU28362 displaced [3H]DEX binding to levels equivalent to non‐specific binding, thus indicating that DEX was binding only to Type II corticosteroid receptors. To assess the direct action of glucocorticoids on POMC gene expression, NIL cells were cultured for 7 days and then treated with DEX. One hour DEX treatment of NIL primary cultures had no effect on levels of POMC heteronuclear RNA levels; in contrast, DEX induced a rapid and potent inhibition of POMC heteronuclear RNA levels in cells treated with the protein synthesis inhibitor puromycin. These data suggest that GR is present in the normal IL of the rat pituitary, but that its ability to regulate POMC gene expression is inhibited by the presence of one or more rapidly turned over proteins.
Estradiol Modulates Insulin‐Like Growth Factor I Receptors and Binding Proteins in Neurons from the Hypothalamus
Authors: Pons S. , Torres‐Aleman I.
Published: September 2006

Trophic effects of 17β‐estradiol (βE2) on in vitro developing hypothalamic cells have been reported. Insulin‐like growth factor I (IGF‐I) is also a potent trophic factor for cultured hypothalamic cells. An interaction between sexual steroids and insulin‐like growth factors (IGFs) in modulating growth of hypothalamic cells has been suggested. Thus, we tested whether βE2 modulates the levels of IGF‐I, its membrane receptor and its binding proteins in rat hypothalamic culturs. Using both neuron‐ and glial‐enriched cultures obtained from fetal rat hypothalami we found that addition of βE2 elicited a significant increase in IGF‐I receptor levels in neurons, without affecting its affinity. On the other hand, the three different IGF‐binding proteins (IGFBPs) found in the conditioned medium of the cultures were differentially modulated by βE2 in the two types of cells studied. Overall, neuronal cultures produced greater amounts of IGFBPs after treatment with βE2, with IGFBP2 reaching significantly higher levels. On the contrary, treatment with βE2 did not significantly alter the amounts of IGFBPs produced by glial cells. Finally, the levels of immunoreactive IGF‐I found either in the medium or in cellular extracts in both neuronal and glial cultures were not modified by treatment with βE2. These results strongly support previous observations of a trophic synergistic interaction between IGFs and βE2 on hypothalamic cells. Thus, an increase in IGF‐I receptors and/or IGFBPs after exposure to βE2 may result in an enhanced response of hypothalamic neurons to IGF‐I. Further, the present findings strengthen our recent observation that the effects of βE2 on hypothalamic glial cells are neuronally mediated, since IGF‐I receptors and IGFBPs are modulated by this sex hormone in neurons, but not in glial cells.
Arginine Vasotocin Gene Expression and Hormone Synthesis During Ontogeny of the Chicken Embryo and the Newborn Chick
Authors: Mühlbauer E. , Hamannt D. , Xu B. , Ivell R. , Udovic B. , Ellendorff F. , Grossmann R.
Published: September 2006

Chicken embryos at different developmental stages (embryonal day (E) 6 to 21) and chicks at posthatch day 1 (D1) were monitored for the development of their hypothalamo‐neurohypophysial system as indicated by the kinetics of arginine vasotocin (AVT) gene expression via mRNA concentration and brain AVT content. Our data concerning the onset of gene expression support previous results from our laboratory and others about an early activation of the AVT gene transcriptional and translational activity around E6. We could detect measurable amounts of AVT in chicken embryo brains at E6 and an exponential increase during further development until D1. Dot blots of hypothalamic RNA extracts indicated that AVT gene transcript concentrations rose between E12 and E17 and slightly dropped thereafter. Northern hybridization showed that this drop was caused by a decrease of full length message and an increase of smaller transcripts during late embryonal and D1 stages, probably an AVT mRNA specific degradation phenomenon. The dissociation between the increase of AVT concentration and AVT mRNA concentration visible at the D1 stage might be due to accumulation and storage of AVT in the magnocellular neurons, preferentially in their axon terminals in the neurohypophysis. Blood samples taken from E14 onwards revealed a constant increase in plasma osmolality and plasma AVT concentration. Our data suggest that, in the chicken, AVT seems to be required early during embryonal development, either for osmoregulatory or further unknown functions.
Fos Expression in the Rat Brain Following Vaginal‐Cervical Stimulation by Mating and Manual Probing
Authors: Tetel Marc J. , Getzinger Michael J. , Blaustein Jeffrey D.
Published: September 2006

Vaginal‐cervical stimulation (VCS), provided by mating or manual probing, induces many reproductive behavioral and endocrine changes in female rats. These changes include an increase in lordosis duration, heat termination and pseudopregnancy. Electro‐physiological and [14C]2‐deoxy‐D‐glucose studies collectively show that neurons in the medial preoptic area, ventromedial hypothalamus and midbrain central gray respond to manual VCS. In the present study we immunocytochemically labeled brain sections for Fos, the protein product of the immediate early gene c‐fos, to detect VCS‐responsive neurons in hormone‐primed animals receiving VCS by mating or manual probing. In Experiment 1, females receiving mounts and intromissions were compared to: 1) vaginally‐masked females receiving mounts but no VCS, 2) females exposed to an intact anesthetized male or 3) females not exposed to males or the testing arena. Those animals receiving VCS showed a dramatic increase in the number of Fos‐immunoreactive cells in the medial preoptic area, posterodorsal portion of the medial amygdala and bed nucleus of the stria terminalis, as well as the dorsomedial hypothalamus, ventromedial hypothalamus and midbrain central gray. These effects of VCS were confirmed in Experiment 2 in animals receiving manual vaginal‐cervical probing. These findings extend previous electrophysiological and [14C]2‐deoxy‐D‐glucose studies by providing evidence that additional brain areas respond to VCS by mating, as well as manual probing.
Chronic but not Acute Exposure to Stress is Associated with Hypothalamic Vasoactive Intestinal Polypeptide (VIP) Release into Median Eminence
Authors: Gavalda Amadeu , Benyassi Ahmed , Arancibia Sandor , Armario Antonio
Published: September 2006

The influence of stress on hypothalamic VIP release into the pituitary portal blood has not been assessed at present despite the fact that this peptide has been implicated in the control of several pituitary hormones and especially in the release of prolactin (PRL) caused by stress. In the present work the effect of stress on the in vivo release of VIP into the pituitary portal blood of male rats was evaluated by means of push‐pull perfusion (PPP) of median eminence (ME). VIP content in the PPP liquid was successfully measured and their levels agree well with pituitary portal blood levels measured by other authors. Whereas plasma PRL levels strongly increased during acute immobilization (IMO), no changes in VIP secretion into the ME were observed. VIP release into the ME was also unaffected by exposure to ether. In contrast, in chronically immobilized rats a significant increase in VIP release into the ME was obtained in response to acute IMO. The present data argue against a major role of hypothalamic VIP in PRL release caused by acute stress and show that chronic stress qualitatively alters the signal of hypothalamic VIP to the pituitary.
Inhibitory Effects of Natriuretic Peptides on Vasopressin Neurons Mediated through cGMP and cGMP‐Dependent Protein Kinase in vitro
Authors: Akamatsu Naoki , Inenaga Kiyotoshi , Yamashita Hiroshi
Published: September 2006

The effects of natriuretic peptides on electrical activity and cellular cGMP levels were studied in neurons of the supraoptic nucleus (SON) of rat hypothalamic slice preparations. Intracellular and extracellular recordings showed that bath application of A type natriuretic peptide (ANP) at 100 nM or B type natriuretic peptide (BNP) at 100 to 300 nM decreased the firing rate and hyperpolarized the membrane potential in phasically firing (putative vasopressin) neurons. Non‐phasically firing (putative oxytocin) neurons did not respond to these natriuretic peptides in firing rate or membrane potential. The membrane‐permeable cGMP analogue 8‐bromo cGMP at 0.5 mM and the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX) at 50 μM mimicked the inhibitory effects of ANP and BNP. The specific inhibitor of cGMP phosphodiesterase 1‐(3‐chloroanilino)‐4‐phenylphthalazine (MY5445) at 30 μM also decreased the firing rate of SON neurons. The cGMP‐dependent protein kinase inhibitor N‐(2‐(methylamino)ethyl)‐5‐isoquinoline‐sulfonamide dihydrochloride (H8) at 1 μM abolished the inhibition by natriuretic peptides. We measured cGMP and cAMP contents in discrete SON regions and compared the change of the contents before and after application of ANP and BNP. The increases in cellular cGMP accumulation were 430% for ANP and 120% for BNP, although they did not cause significant change of cAMP accumulation. The results suggest that the inhibitory effects of natriuretic peptides on putative vasopressin neurons are mediated through cGMP and cGMP‐dependent protein kinase.
Effects of Morphine and Naloxone on Prolactin and Growth Hormone Gene Expression in the Male Rat Pituitary Gland
Authors: Dobado‐Berrios Pablo‐Manuel , Li Songuyn , de Yebenes Ester Garcia , Pelletier Georges
Published: September 2006

It is generally admitted that opioids can stimulate the release of both prolactin (PRL) and growth hormone (GH). In order to investigate the role of opioids in the regulation of PRL and GH gene expression in the rat pituitary, we studied the effects of chronic administration of the opioid drug morphine and an opiate receptor antagonist naloxone on both PRL and GH gene expression as measured by in situ hybridization. Four‐day treatment with morphine (40 mg/kg/day) produced a 12% increase in PRL mRNA levels. Conversely, naloxone (4 mg/kg/day) decreased the autoradiographic reaction by 10%. The concomitant administration of morphine and naloxone induced no significant changes in PRL gene expression. On the other hand, treatment with morphine produced a 22% decrease in GH mRNA levels, an effect which was prevented by the concomitant administration of naloxone. When injected alone, naloxone did not modify the hybridization signal. These results clearly indicate that opioids are involved not only in the regulation of GH and PRL release but also in the gene expression of the two hormones. The discordance observed between the acute effects of morphine on GH release and the effect of the opioid drug on mRNA levels remains to be clarified.
Plasticity in Expression of Immunoreactivity for Neuropeptide Y, Enkephalins and Neurotensin in the Hypothalamic Tubero‐lnfundibular Dopaminergic System during Lactation in Mice
Authors: Ciofi Philippe , Crowley William R. , Pillez André , Schmued Lawrence L. , Tramu Gérard , Mazzuca Marc
Published: September 2006

In lactating nursing vs lactating pup‐deprived mice, single or multiple immunolabeling was performed to compare immunoreactivities (ir) for neuropeptide Y (NPY), enkephalins (ENK) and neurotensin (NT) in the tyrosine hydroxylase (TH)‐ir hypothalamic tubero‐infundibular dopaminergic (TIDA) system. NPY‐, ENK‐ and NT‐irs were intensely expressed and coexisted in virtually all TH‐ir endings in the median eminence (ME) of nursing mice. Removal of the pups induced a marked depletion of the peptide‐irs from the ME TH‐ir endings. In the arcuate nucleus (ARC) of colchicine‐treated nursing mice which received peripheral injections of Fluoro‐Gold (FG) to retrogradely label neuroendocrine cells, virtually all dorsal A12 TH‐ir perikarya simultaneously expressed, with individual variations, NPY‐, ENK‐ and NT‐irs, and all contained FG. These results suggest that the synthesis of NPY, ENK and NT is enhanced in TIDA neurons during lactation and that these neuromessengers may be co‐released together with DA from the ME to regulate the suckling‐induced prolactin secretion at the hypothalamic and/or pituitary levels.
Antiovulatory Doses of Antagonists of LH‐RH Inhibit LH and Progesterone but not FSH and Estradiol Release
Authors: Kovács Magdolna , Koppán Miklós , Mezö Imre , Teplán István , Flerkó Béla
Published: September 2006

The differential regulation of immunoactive FSH and LH secretion by endogenous LH‐RH was studied using LH‐RH antagonists (Ac‐D‐Trp1,2, D‐Cpa2, D‐Lys6, D‐Ala10LH‐RH (MI‐1544) and (Ac‐D‐Nal1, D‐Phe(pCI2), D‐Trp3, D‐Cit6, D‐Ala10LH‐RH (SB‐030) in ovariecto‐mized (OVX) and regularly cycling rats. Single injections of 10 μg and 100 μg doses and long‐term treatment with 10 μg doses of MI‐1544 were used in OVX animals. Serum and pituitary LH and FSH, as well as serum estradiol and progesterone was determined by RIA during and/or after the treatment. Single injections of MI‐1544 in OVX animals caused prompt (in 2 h) and long‐lasting (for more than 24 h) suppression of the serum LH, while no or late decrease (after more than 6 h) of the serum FSH. Long‐term treatment with the same analog decreased the serum LH (by 50%) and moderately increased the pituitary LH (by 21%) but did not change the serum and the pituitary FSH concentrations. In normal rats, long‐term treatment with both of our analogs also resulted in divergent alterations in the LH and FSH concentrations. Serum LH dropped to undetectable levels, while serum FSH did not change significantly. Pituitary LH increased (by 31 to 41%), while FSH decreased (by 27 to 38%). Marked depression was found in the serum progesterone (by 64%) but no significant change in the serum estradiol levels, after the long‐term treatment for 21 days. The ovarian cycles were interrupted, and no ovulation appeared during the treatment. Significant decrease was detectable in the weight of the ovaries (by 46%), whereas the weight of the uteri did not change or slightly elevated (by 22%), after the treatment with SB‐030 or MI‐1544, respectively. The clear antagonistic effect of the analogs on the LH‐RH‐stimulated LH and FSH release was tested in the in vitro superfused rat pituitary cell system. In this system our analogs provented both the LH‐and the FSH‐releasing effect of the LH‐RH. The inhibitory effect was longer‐lasting on the FSH than on the LH release. Our studies give evidences that the long‐term treatment with antiovulatory doses of LH‐RH antagonists suppress the LH and progesterone but not the FSH and estradiol secretion and indirectly support the earlier publications suggesting the presence of FSH‐releasing factor(s) in the CNS.
Gonadal Steroids have Paradoxical Effects on Brain Oxytocin Receptors
Authors: Insel Thomas R. , Young Larry , Witt Diane M. , Crews David
Published: September 2006

Specific brain receptors for oxytocin have been described in several mammalian species. The distribution of these receptors differs greatly across species and in the rat, receptor binding in specific brain regions appears to depend upon gonadal steroids. This study used in vitro receptor autoradiography to examine the effects of testosterone on oxytocin receptor binding in the mouse forebrain. Three groups of male mice were compared: castrates treated with blank capsules, castrates treated with testosterone filled capsules, and intact males. Irrespective of steroid treatment, the distribution of oxytocin receptors in mouse forebrain differed markedly from patterns previously described in the rat. In addition to these species differences in receptor distribution, testosterone had effects in the mouse which differed from the induction of receptors previously reported in the rat. In the mouse ventromedial nucleus of the hypothalamus, binding in the untreated castrate males was approximately double that observed in either the intact or the testosterone‐treated castrates. In other regions of the mouse brain, such as the intermediate zone of the lateral septum, binding to oxytocin receptors was increased with testosterone treatment. These results suggest that the brain oxytocin receptor varies across species not only in its distribution but also in its regional regulation by gonadal steroids. These apparently paradoxical changes in oxytocin receptor binding may result from either direct or indirect effects of gonadal steroids in mouse brain.
Differential Effects of Estrogen and Progesterone on Levels of POMC mRNA Levels in the Arcuate Nucleus: Relationship to the Timing of LH Surge Release
Authors: Petersen S. L. , Keller M. L. , Carder S. A. , McCrone S.
Published: September 2006

Beta‐endorphin is thought be an important inhibitor of LHRH neuronal activity and also to play a role in conveying information about changes in steroid levels to LHRH neurons. We have previously shown that the mRNA encoding the precursor of ß‐endorphin, proopiomelanocortin (POMC), fluctuates during the estrous cycle with the most dramatic changes occurring on proestrus. POMC mRNA levels decline before the onset of LH surge release but then dramatically rise and remain elevated during the surge. In the present studies we tested the hypothesis that the decline in POMC mRNA levels immediately before the proestrous LH surge is mediated by estrogen and the rise during the surge by progesterone. To test this hypothesis, we compared changes in POMC mRNA levels between ovariectomized (OVX) and OVX estrogen (E2)‐treated rats and between OVX E2‐treated rats with and without progesterone. Animals were examined at hourly intervals after the administration of progesterone, then at every 4h during the LH surge. Using in situ hybridization histochemistry, we found that E2 decreased POMC mRNA levels in OVX rats before the onset of the LH surge and further suppressed levels during the surge. Compared to animals treated with E2 alone, progesterone advanced the time at which both the LH surge began and the time at which POMC mRNA levels declined. After a transient decline, POMC mRNA levels rose in these progesterone‐treated animals and remained elevated throughout the period of the LH surge. These results support the hypothesis that progesterone times the LH surge and limits its appearance to one day by exerting a biphasic effect on the activity of ß‐endorphinergic neurons of the arcuate nucleus.
Autonomie Control of Penile Erection: Modulation by Testosterone in the Rat
Authors: Giuliano François , Rampint Olivier , Schirar Alain , Jardin Alain , Rousseau Jean‐Paul
Published: September 2006

The role of testosterone on peripheral autonomie control of penile erection was studied in rats. Erectile response to cavernous nerve stimulation was measured by intracavernous pressure associated with arterial blood pressure monitoring in anesthetized adult males. Comparison was performed between control (Co), castrated (Ox) and castrated, testosterone‐replaced (OxT) rats. Ox rats exhibited smaller erectile responses. Testosterone replacement restored these responses in OxT rats. To identify the peripheral target of testosterone, postganglionic neurons of the major pelvic ganglion, innervating the corpora cavernosa through the cavernous nerves, were separated from the spinal cord by preganglionic axotomy of the pelvic nerves in three other groups of rats (PNx). Erectile response was unchanged in PNx rats, decreased in OxPNx more than in Ox rats, and restored by testosterone replacement (OxPNxT rats). We ruled out the participation of a somatic component in the erectile response in this model as there was no difference between curarized and Co rats. We infer that testosterone enhances the erectile response of cavernous nerve stimulation, acting peripherally to the spinal cord. Arguments are provided that the sites of action for testosterone or its metabolites are situated on neurons rather than on penile erectile tissue. Proerectile postganglionic parasympathetic neurons seem to be the exact target for gonadal steroids.
Expression of Growth Hormone Receptor Gene in Rat Hypothalamus
Authors: Minami Shiro , Kamegai Jun , Hasegawa Osamu , Sugihara Hitoshi , Okada Kenmei , Wakabayashi Ichiji
Published: September 2006

Growth hormone receptor (GHR) mRNA‐expressing cells in the hypothalamus were observed using hybridization histochemistry in adult male rats. Digoxigenin‐labeled cRNA corresponding to the extracellular part of rat GHR was used as a probe. Northern blotting analysis of hypothalamic total RNA from adult male rats revealed that the 4.5 kilobase (kb) transcript of the GHR gene corresponding to the GHR messenger RNA (mRNA) predominated over the 1.2 kb transcript corresponding to GH‐binding protein mRNA. GHR mRNA‐containing cells were observed in the arcuate nucleus (ARC), the periventricular nucleus (PeV), ventrolateral region of the ventromedial nucleus, the paraventricular nucleus and the supraoptic nucleus. To further understand the significance of the GHR gene expression in the hypothalamus, the effect of in vivo manipulation of GH on the somatostatin (SS) gene expression in the ARC and PeV, and the GRF gene expression in the ARC was observed among adult male rats using in situ hybridization histochemistry. Ten days after hypophysectomy, the SS mRNA level in the ARC as well as PeV was significantly lower than that in the respective nuclei of sham‐operated control rats, while the GRF mRNA level in the ARC was significantly higher than that in the ARC of control animals. Subcutaneous injection of recombinant human GH (0.33 mg) to hypophysectomized rats every 12 h for 5 days restored the SS mRNA level in the ARC and PeV, and reduced the GRF mRNA level in the ARC to that of control animals. The data suggest that GH directly acts on the hypothalamic PeV and ARC, and alters the gene expression of SS and GRF.
Re‐Evaluation of Gonadotropin‐Releasing Hormone (GnRH) Action on Pituitary Cell Differentiation with Special Regard to its Effect on LH and TSH Cell Types
Authors: Héritier Anne G. , Dubois Paul M.
Published: September 2006

We have previously demonstrated that TRH induces several hypophyseal cell type differentiation. In the present study, we re‐evaluated the effect of GnRH on gonadotroph differentiation, and we determined whether gonadotropin is implicated in the appearance of other pituitary cell types. These effects were compared to the influence of TRH on LH and TSH cell differentiation. Pituitary primordia of 11 day‐old rat embryos were cultured in a synthetic medium until the equivalent of term, and the LH and TSH cell differentiation was visualized using an immunohistochemical technique. The effect of GnRH on pituitary hormone cells was evaluated by measuring the percentage of immunoreactive area per section.

GnRH, when added on the first day of culture, induced LH and TSH cell differentiation in a dose‐dependent manner. GnRH was more effective than TRH in inducting gonadotropic phenotype expression. This suggests that GnRH, which evokes LH release, is the most effective factor inducing gonadotroph differentiation in early fetal life. However, GnRH was as effective as TRH in thyrotroph differentiation. Lactotrophs were detected in primordia cultured with GnRH. As lactotroph phenotype differentiation is controlled by the a‐subunit of glycoproteic hormone, further studies are required to determine whether or not GnRH effect on lactotroph is direct. GnRH did not affect the corticotroph and somatotroph differentiation. These results confirm that hypothalamic factors such as TRH or GnRH are capable of inducing more than one pituitary cell phenotypes.
Colocalization of Natriuretic Peptide and Estrogen Receptor Immunoreactivities in Preoptic Nuclei in the Female Rat
Authors: Watson Robert E. , Hutchinson Robert K. , Langub M. Chris , Landis John W. , Seksaria Shikha , Rainey David M. , Keil Lanny C.
Published: September 2006

Estrogen is known to play an important role in regulating reproductive function in female rats through actions exerted at the preoptic area, a part of the brain that is markedly sexually dimorphic and which contains abundant estrogen receptors. A critical question to our understanding of estrogen's action on the brain is to identify the types of neurons that contain estrogen receptors (ER). Previous studies have shown that atrial natriuretic peptide (ANP) is in abundance in the preoptic area, and that ANP and other natriuretic peptides are capable of regulating gonadotropin secretion. In an effort to determine whether ERs are present in natriuretic peptide‐immunoreactive (NP‐ir) neurons in the preoptic area of the rat, double label immunocytochemistry was performed. Since ER‐ir, as demonstrated with antibody H222 is known to be localized predominantly in cell nuclei, while NP‐ir is present in the cytoplasm, single cells can be double labeled. Diaminobenzidine tetrahydrochloride was used for localization of NP‐ir neurons, while nickel‐enhanced diaminobenzidine tetrahydrochloride was used for localization of ER‐ir. The results revealed that many nuclei throughout the preoptic area contained neurons that were ER‐ir or NP‐ir and that a substantial number were double labeled. Cell counts in selected preoptic nuclei and components, including the anteroventral periventricular nucleus, periventricular preoptic nucleus, medial part of the medial preoptic nucleus, and central part of the medial preoptic nucleus revealed that 13.6%, 11.1%, 13.5%, and 24.4%, respectively, of the NP‐ir neurons in these nuclei also contained ER‐ir. Collectively, a total of 14.9% of the NP‐ir neurons in these nuclei also contained ER‐ir. These results support the hypothesis that estrogen may regulate the activity of NP‐ir cells in the preoptic area and thereby may be capable of altering functions associated with NPs in this region of the brain, including not only regulation of gonadotropin secretion, but also control of blood pressure and fluid balance.
Projections from Ventrolateral Hypothalamic Neurons Containing Progestin Receptor‐ and Substance P‐Immunoreactivity to Specific Forebrain and Midbrain Areas in Female Guinea Pigs
Authors: Ricciardi Kirsten H. Nielsen , Blaustein Jeffrey D.
Published: September 2006

Many neurons within the ventrolateral hypothalamus in guinea pigs contain estrogen‐induced progestin receptors as well as substance P. Retrograde tracing combined with immunocytochemistry was used to determine the specific projections of this subset of steroid‐ sensitive cells. Unilateral Fluoro‐Gold injections into the dorsal midbrain, including the central gray, labeled a large proportion of the ventrolateral hypothalamic neurons immunoreactive for both progestin receptors and substance P (approximately 30%); substantially fewer of these neurons were labeled by unilateral Fluoro‐Gold injections into the preoptic area (approximately 6%), medial amygdala (approximately 10%), or the bed nucleus of the stria terminalis (approximately 11 %). The projections of progestin receptor‐immunoreactive neurons in the ventrolateral hypothalamus were similar to those of progestin receptor/substance P double‐labeled neurons, while a slightly lower percentage of the ventrolateral hypothalamic, substance P‐immunoreactive neurons tended to project to each of these areas. These pathways may prove to be components of the neural circuitry underlying a variety of functions influenced by gonadal steroid hormones and substance P, such as female sexual behavior, salt intake, nociception and aggression.
Interleukin‐1β and lnterleukin‐6 Stimulate Neurohypophysial Hormone Release in vitro
Authors: Yasin Sharifah A. , Costa Alfredo , Forsling Mary L. , Grossman Ashley
Published: September 2006

Interleukin‐1 (IL‐1) and interleukin‐6 (IL‐6) have been reported to stimulate the release of corticotrophin‐releasing hormone (CRN) in vitro, the response being antagonized by the cyclo‐oxygenase inhibitor, indomethacin. The effects of cytokines on the other major ACTH‐releasing hormone, vasopressin (AVP), and the other neurohypophysial hormone, oxytocin, have been little studied, and the published data are conflicting. We have therefore used a previously validated rat hypothalamic expiant model to evaluate whether IL‐1β and IL‐6 can directly activate the AVP and oxytocin neurosecretory system. In addition, we have also investigated the effects of inhibition of cyclo‐oxygenase (CO) and lipoxygenase (LO) activities on the stimulated release of AVP and oxytocin by means of a series of antagonists, including a specific LO pathway inhibitor. The static rat hypothalamic incubation system used involves fresh hypothalamic expiants with consecutive 20‐min incubations, and estimation of AVP and oxytocin concentrations in the medium by specific and sensitive radioimmuno‐assays. It was found that IL‐1β produced a dose‐dependent increase in the release of AVP and oxytocin at doses of 10 and 100 U/ml (P<0.005). Only at the higher dose of 100 U/ml was IL‐6 able to increase significantly AVP and oxytocin release (P<0.05). These stimulatory effects of IL‐1β and IL‐6 were blocked by cyclo‐oxygenase inhibitors, indomethacin (28 μM) and ibuprofen (100 nM), but not by the lipooxygenase inhibitor, BW A4C (10 μg/ml), suggesting that prostaglandins are involved in this process. Thus, cytokines are clearly able to modulate the neurohypophysial system in vitro, the effects probably being mediated by cyclo‐oxygenase products.
Photoperiodically‐lnduced Cycles in the Secretion of Prolactin in Hypothalamo‐Pituitary Disconnected Rams: Evidence for Translation of the Melatonin Signal in the Pituitary Gland
Authors: Lincoln G. A. , Clarke I. J.
Published: September 2006

Long term changes in the secretion of prolactin were monitored in groups of hypothalamo‐pituitary disconnected rams (HPD rams, n = 8) and control rams (HPD sham‐operated and unoperated, n = 8) while exposed to an artificial lighting regimen of alternating 16‐weekly periods of long days (16L : 8D) and short days (8L : 16D) for 72 weeks, and during a treatment with subcutaneous constant‐release implants of melatonin under long days. The HPD rams showed all the clinical characteristics of complete pituitary disconnection (diabetes insipidus, gonadal regression and slight obesity), and were unresponsive to a range of provocation tests (exposure to a barking sheep dog, cannulation of the jugular vein, injection of serotonin and NMDA) which caused acute changes in the blood plasma concentrations of prolactin in the controls. Nevertheless, there was a clearly defined cycle in the blood concentrations of prolactin in the HPD rams related to the imposed lighting regimen with values 10‐fold higher under long days compared to short days (HPD mean ± SEM: 90.1 ± 24.7 vs 9.4 ± 2.0 μl, long vs short day respectively, P < 0.001). The temporal pattern was very similar to that observed in the controls, although the concentrations of prolactin were higher in the HPD rams and more variable (control mean ± SEM: 55.6 ± 3.6 vs 3.0±0.5 μl, long vs short day, P < 0.001). There was a corresponding cycle in the growth and moulting of the wool in the HPD rams consistent with a biological response to the photoperiodically‐induced changes in the secretion of prolactin. The diurnal rhythm in the blood concentrations of prolactin was absent in the HPD rams, but there was a normal rhythm in the secretion of melatonin. The treatment of the animals with constant‐release implants of melatonin under long days caused a marked decrease in the blood concentrations of prolactin in both the HPD and control rams. The overall conclusion is that the endogenously generated daily melatonin signal which encodes daylength acts directly in the pituitary gland to mediate the effects of photo‐period on the secretion of prolactin. The photo‐period transduction pathway thus by‐passes the hypothalamus.
Selective Enhancement of an A Type Potassium Current by Dexamethasone in a Corticotroph Cell Line
Authors: Pennington Anne J. , Kelly John S. , Antoni Ferenc A.
Published: September 2006

Perforated patch recording was used to examine the effect of the synthetic steroid dexamethasone on the whole cell potassium (K+) current, in the mouse corticotroph tumour cell line AtT20/D16‐16. In 15 out of 52 control cells (29%) there was a rapidly‐activating, rapidly‐ inactivating K+ current of the A type, the amplitude of which was strongly dependent on the holding potential in use prior to its activation by depolarising voltage pulses, and which was blocked by 1 mM 4‐aminopyridine (4‐AP, n = 5). The effect of dexamethasone (100nM, 2h, 37°C) was that the A current increased in prevelance (24 out of 31 cells, 77%), lost its dependence on holding potential (over the range studied), and as a result became significantly larger than in controls, for certain voltage steps (peak A current density was 18.5 +2.4 pA/pF (n = 12) for control cells and 26.3 ± 3.9 pA/pF (n = 18) for dexamethasone treated cells, for a step to +30mV from ‐60mV, values are mean ± SEM). All cells exhibited a slowly‐activating, sustained K+ current, which was unaffected by changes in the holding potential, unaffected by 4‐AP and consisted of at least 3 components: one blocked by 30 mM tetraethylammonium(TEA) or 100 nM charybdotoxin (CTX); a second blocked by 100 nM apamin; and a third not blocked by TEA, CTX, apamin, clofilium (100 nM) or niflumic acid (0.1 mM). Dexamethasone produced no change in the slowly‐activating, sustained current nor in any of its individual components. The effect of dexamethasone on the A current was completely blocked by 0.1 mM puromycin, a protein synthesis blocker, while puromycin alone did not affect the size or frequency of the A current, nor alter the slowly‐activating, sustained current. Secretion studies using 4‐AP confirmed that the A current has a role in stimulated adrenocorticotrophic hormone (ACTH) secretion. In summary, AtT20 cells contain at least four types of K+ current: an A current and 3 currents contributing to the slowly‐activating current. Selective enhancement of the A current by dexamethasone, shown here to require synthesis of new protein, is one of the mechanisms whereby glucocorticoids exert inhibitory control on ACTH secretion.
Localization of Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) in the Hypothalamus‐Pituitary System in Rats: Light and Electron Microscopic Immunocytochemical Studies
Authors: Kimura Shigeharu , Ohshige Yoshimi , Lin Liping , Okumura Tadashi , Yanaihara Chizuko , Yanaihara Noboru , Shiotani Yahe
Published: September 2006

The localization of pituitary adenylate cyclase‐activating polypeptide (PACAP) in the hypothalamus‐pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1–38 (R0831). In light microscopic study, intensely PACAP‐immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP‐immunoreactivity was examined by avidin‐biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB‐reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough‐surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.