bluuebunny/crossref_metadata_2025 huggingface dataset:
Pineal Microdialysis of the Melatonin in Conscious Sheep: Methodology, Application to a Diurnal Rhythm and Effect of Isoproterenol
Authors: Ravault Jean Paul , Chesneau Didier , Ouvray Matthieu , Locatelli Alain
Published: November 2003

This paper describes the development of a new technique to measure melatonin contents in the pineal gland of moving sheep: the microdialysis. A dialysis probe was used to collect extracellular fluid in the sheep pineal gland, but also to inject directly into it different drugs such as isoproterenol at a very low concentration. The probe was implanted the day before the beginning of the experiment in order to obtain low levels of melatonin. This technique makes it possible to measure melatonin in the dialysate and plasma of rams submitted to 8L:16D. No melatonin either in the dialysate or in the plasma was found during the light phase. Shortly after lighting off, the melatonin concentration increased in the dialysate and plasma and remained stable during the dark phase. Melatonin concentrations began to decrease before lighting on and no detectable levels were found during the following light phase. The secretion of melatonin is, at least, under adrenergic regulation. Local infusion of isoproterenol (90 μl at 10−6 M), an agonist of β adrenergic receptor, through the probe, increased melatonin levels during 2 h, even when infusions were repeated 3 times. This demonstrates the presence of β adrenergic receptors. The technique presented in this paper could be of considerable interest for studying sheep pineal gland and its main secretion, melatonin, for example during diurnal rhythms or for studying its regulation.
Suckling‐Induced Fos‐Immunoreactivity in Subgroups of Hypothalamic POMC Neurons of the Lactating Rat: Investigation of a Role for Prolactin
Authors: Pape Jean‐Rémi , Ciofi Philippe , Tramu Gérard
Published: November 2003

Attention has recently been focused on lactation‐induced modifications of activity of neuronal populations in the arcuate nucleus (ARC) of the mediobasal hypothalamus.The ARC hosts the tubero‐infundibular dopaminergic (TIDA system) responsible for the neuroendocrine control of prolactin (PRL), and other non‐neuroendocrine neuronal populations, such as neuropeptide Y (NPY)‐ and proopiomelanocortin (POMC)‐containing systems that are important modulators of hypothalamic gonadoliberin (GnRH) secretion. Our longstanding interest in the functional anatomy of the ARC led us to investigate whether the suckling stimulus would trigger an expression of Fos‐ir in specific arcuate neuronal populations and to possibly characterize responsive neurons by using double‐labeling immunohistochemistry. Freely nursing lactating females expressed strong Fos‐ir in neurons of the ARC compared to diestrous females. Fos‐ir was encountered in neurons not belonging to the TIDA system and that was for a large proportion identical to the POMCergic neurons.We showed that, in lactating females submitted to suppression of the suckling stimulus by removal of the pups, the pattern of expression of Fos‐ir is similar to that seen in diestrous females and that, a pattern of expression of Fos‐ir indistinguishable from that observed during free lactation is reinstated a short time after the return of the pups and restoration of the suckling stimulus, suggesting that this expression of Fos‐ir strictly depends upon the presence of the newborns and the suckling stimulus. By lowering circulating levels of the PRL with bromocryptine‐or PRL antiserum‐treatment, we noticed a decrease in the number of (β‐endorphin+Fos)‐ir neurons compared to non‐injected freely nursing lactating females. By maintaining high levels of circulating PRL with haloperidol‐treatment, we observed a number of colocalizations close to that observed in freely nursing lactating females. Our results suggest that during lactation a rostral subgroup of the arcuate POMCergic neuronal population is activated at least partially in response to the suckling‐induced secretion of PRL and that this activation participates in maintaining the endocrine and/or metabolic demands of the lactational status.
Glucocorticoid Resistant Syndromes—Molecular Basis andClinical Presentations
Authors: Brönnegård Mikael , Stierna Pontus , Marcus Claude
Published: November 2003

The mechanisms of action of glucocorticoid hormones are mediated via specific intracellular receptor proteins. The glucocorticoid receptor (GR) regulates expression of specific target genes or gene networks by ligand‐dependent transcriptional activation, i.e. ligand‐dependent activation of the receptor with subsequent dimer formation and DNA binding. There are a number of factors, such as the receptor concentration, receptor associated proteins, receptor alterations and the effects on the gene network including hormonal regulation of transcription, mRNA splicing and translation, that might influence glucocorticoid responsiveness in a normal and healthy population as well as in different diseases. Several categories of glucocorticoid resistance have been described including inherited GR resistance which has been explained in terms of specific mutations and offers an important model for genetic and clinical studies of steroid sensitivity, and relative glucocorticoid resistance, which occurs naturally in the course of cellular differentiation, cell to cell or tissue to tissue, since all cells possess receptors for glucocorticoids but do not show the same response to them. From a clinical point of view, it is also interesting to consider preexisting genetic susceptibility to glucocorticoids, acquired changes in the GR gene structure and organization, including alterations of noncoding sequences, and the importance of mutations, deletions and other changes in the GR gene affecting receptor function. Analysis of mutations within the receptor resulting in relative glucocorticoid resistance, both generalized inherited glucocorticoid resistance (GIGR) and directed mutagenesis, has identified two regions of clustered mutations in the proximity of previosuly identified affinity labeled residues directly affecting the steroid binding function. Finally, studies of New Words primates and cell lines derived from hematologic malignancies constitute animal and human models for the molecular basis of glucocorticoid resistance where a number of inherited and aquired mutations in the GR gene have been demonstrated.
Neonatal Maternally Deprived Rats have as Adults Elevated Basal Pituitary‐Adrenal Activity and Enhanced Susceptibility to Apomorphine
Authors: Rots Nynke Y. , de Jong Jeannette , Workel Judith O. , Levine Seymour , Cools Alexander R. , De Kloet E. Ronald
Published: November 2003

Maternal deprivation of neonatal rats for 24 h enhances the adrenocortical response to stress and/or adrenocorticotropin hormone (ACTH) stimulation during the stress hyporesponsive period (SHRP). The present study tests the hypothesis that such maternally deprived neonatal male rats show altered hypothalamic‐pituitary‐adrenal (HPA) regulation not only immediately after deprivation but also in later life. In addition, we found previously that neonatal changes in HPA activity preceded modulation of nigrostriatal dopamine function. Therefore, we also measured dopamine responsiveness in adult rats which were deprived of their mother during infancy.
Some Acute Effects of Intracerebroventricular Neuropeptide Y on Insulin Secretion and Glucose Metabolism in the Rat
Authors: Marks Jonathan L. , Waite Kay
Published: November 2003

Acute administration of neuropeptide Y(NPY) into the hypothalamus and cerebral ventricles can stimulate insulin secretion in the absence of available food. However, the relationship of this effect to blood glucose and other hormones which regulate glucose metabolism remains unclear. The purpose of this study was to compare the effects of NPY injected into the third ventricle (ICV) on serum insulin, glucose, glucagon, corticosterone and non‐esterified fatty acids. Studies were performed on conscious, unrestrained female rats, not given access to food. ICV NPY, 2 and 5 μg produced an increase in serum insulin and glucagon, while the 5 μg dose only increased plasma glucose transiently and increased non‐esterified fatty acids for a longer period. Corticosterone was not affected by ICV NPY. The insulinaemic response to i.v. glucose, 0.5 g/kg was doubled by ICV NPY, 4 μg. The maximal insulin levels were 113±18 for ICV NPY versus 67±8 μU/ml for ICV saline‐treated animals. The glycaemic response was not altered. The hypoglycaemic response to i.v. insulin, 0.15 U/kg was significantly attenuated by ICV NPY, 5 μg. We concluded that ICV NPY promotes insulin secretion in the absence of available food and may potentiate the insulinaemic response to hyperglycaemia. Furthermore, possibly through its effects on glucagon and non‐esterified fatty acids, ICV NPY may decrease the ability of insulin to control glucose metabolism.
Interrelations between Sympathoadrenal System andHypothalamo‐Pituitary‐Adrenocortical/Thyroid Systemsin Rats Exposed to Cold Stress
Authors: Fukuhara Koki , Kvetnansky Richard , Cizza Giovanni , Pacak Karel , Ohara Hiroyuki , Goldstein David S. , Kopin Irwin J.
Published: November 2003

The interrelations between sympathoadrenal (SA) system and hypothalamo‐pituitary‐adrenocortical (HPA) or hypothalamo‐pituitary‐thyroid (HPT) system during cold stress were examined by measuring plasma levels of dihydroxyphenylalanine (DOPA), catecholamine and their metabolites in adrenalectomized (ADX) and thyroidectomized (TX) rats exposed to cold stress (−3 °C). Plasma levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), thyroid‐stimulating hormone (TSH) and thyroid hormones in cold‐stressed rats were measured also. Plasma ACTH levels were increased transiently after 1 h of cold exposure, after which the circadian rhythm and plasma levels of ACTH were similar to those of normal rats. Plasma CORT levels were also elevated after 1 h of cold exposure; the increased levels of CORT tended to return to normal levels after 9 h of cold, but remained higher than those of normal rats during at least 24 h of cold exposure. Plasma ACTH levels of 5 day cold‐stressed rats were no longer elevated above those of control rats and plasma CORT levels were only slightly higher than in control animals. However, plasma levels of TSH and free thyroid hormones were elevated after 1 day and remained elevated after 5 days of cold exposure. Thus, cold stress appears to activate chronically the HPT system, but only transiently activates the HPA system. ADX rats had higher basal plasma levels of dihydroxyphenylglycol (DHPG), methoxyhydroxyphenylglycol (MHPG), DOPA and homovanillic acid (HVA) than those of sham‐operated (SHAM) rats, but norepinephrine (NE) levels were not significantly greater than in SHAM animals. TX rats had higher basal plasma levels of NE, epinephrine (EPI) and dopamine (DA), as well as much higher plasma levels of the metabolites. Exposure to cold increased plasma NE levels in both ADX and TX rats, but the increments in TX rats were much greater than in SHAM and ADX groups. Plasma EPI levels were not significantly elevated during cold exposure in SHAM rats, but were highly elevated in TX rats exposed to cold. TX rats had much larger increments in plasma levels of DHPG, MHPG, DA, dihydroxyphenylacetic acid (DOPAC) and HVA during cold exposure than those of SHAM and ADX rats. These results are consistent with the view that endogenous glucocorticoids restrain responses of catecholamine synthesis, release, reuptake, and metabolism in sympathetic nervous system of cold‐stressed animals, but that in the absence of an effective HPT system, there is enhanced sympathoadrenal medullary function and augmentation of their responses to cold as a means for maintaining body temperature when the HPT thermogenesis system is impaired.
Androgenic Regulation of Androgen Receptor Immunoreactivityin Motoneurons of the Spinal Nucleus of the Bulbocavernosusof Male Rats
Authors: Matsumoto Akira , Arai Yasumasa , Prins Gail S.
Published: November 2003

Androgenic regulation of androgen receptor (AR) immunoreactivity was examined in androgen‐sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by immunohistochemistry using the polyclonal antibody, PG21. In intact controls, intense AR immunoreactivity was confined to the cell nucleus, but not in the nucleolus of SNB motoneurons, whereas cytoplasmic AR immunoreactivity was weak. Androgen withdrawal significantly reduced both the intensity of AR immunoreactivity in the nuclei and number of AR immunoreactive nuclei of the SNB motoneurons within 1 day of castration. AR immunostaining in the nucleus and cytoplasm was completely eliminated 5 or 10 days following castration. These changes were prevented by replacement of testosterone propionate (TP). The number of AR immunoreactive nuclei recovered to about half of the control levels within 20 min or 1 hr of TP administration to males 5 days after castration, although the intensity of AR immunoreactivity was almost the same as that of males 1 day following castration. Both the intensity of nuclear and cytoplasmic AR immunoreactivity and number of AR immunoreactive nuclei recovered to the control levels 2 or 6 hr after TP injection. These results suggest that androgen causes a significant up‐regulation in AR expression of SNB motoneurons.
Osmoregulation of Plasma Vasopressin in Diabetes Mellitus with Sustained Hyperglycemia
Authors: Iwasaki Yasumasa , Kondo Kunikazu , Murase Takashi , Hasegawa Haruhiko , Oiso Yutaka
Published: November 2003

We studied osmoregulation of plasma vasopressin in 5 patients with newly diagnosed diabetes mellitus. All patients showed typical symptoms of uncontrolled diabetes mellitus such as marked hyperglycemia, polyuria, and polydipsia, but did not have advanced diabetic complications. Vasopressin release was studied using 5% hypertonic saline infusion test twice: before treatment when the patient was hyperglycemic, and after treatment 1 to 2 months later when the patient was euglycemic. Plasma vasopressin was measured by a sensitive and specific radioimmunoassay. The mean basal plasma vasopressin value in the patients was significantly higher in the hyperglycemic compared with the euglycemic state (3.75±0.70 vs 1.18±0.46 pmol/l, respectively; P<0.05). The relationship of plasma vasopressin with serum sodium, but not plasma osmolality, during hyperglycemia showed an apparent hypersecretion of vasopressin. In both cases, the sensitivity of the vasopressin response to osmotic stimuli was significantly decreased. During euglycemia, the sensitivity of vasopressin secretion to either sodium or osmolality was almost normal, although a slight rise in the osmostat was observed compared with normal subjects. Together, we found that the positive correlation of vasopressin with sodium or osmolality is maintained but significantly altered in patients with untreated diabetes mellitus. Especially noteworthy is the lowered threshold and decreased sensitivity of osmotically‐induced vasopressin secretion during hyperglycemia, which may be caused by multiple factors such as diabetes‐associated hypovolemia, osmogenic effects of glucose and other osmoles, depletion of the pool of vasopressin available for release, and the metabolic derangement of osmoreceptor/magnocellular neurons.
Involvement of Medullary Catecholamine Cells in Neuroendocrine Responses to Systemic Cholecystokinin
Authors: Buller Kathryn M. , Day Trevor A.
Published: November 2003

Systemic administration of cholecystokinin (CCK) stimulates neurosecretory oxytocin (OT) and tuberoinfundibular corticotrophin releasing factor (CRF) cells of the hypothalamus. Data from previous studies suggest that A2 noradrenergic neurons of the dorsomedial medulla contribute to the OT cell response, but the role of other medullary catecholamine cells remains unclear. Using c‐fos expression as a marker for cellular activity, we have found that CCK (100 μg/kg, ip) activates substantial populations of tyrosine hydroxylase and phenyl‐N‐methyl‐transferase immunoreactive cells in the medulla, consistent with recruitment of overlapped noradrenergic and adrenergic cell populations in both the ventrolateral and dorsomedial medulla. In the ventrolateral medulla there was a particularly prominent activation of C1 adrenergic neurons at the level of the obex. To directly test the contribution of VLM catecholamine cells to hypothalamic neuroendocrine cell responses to CCK, animals were prepared with unilateral VLM lesions corresponding to those areas that had displayed the most marked response to CCK. VLM lesioned animals treated with CCK displayed a significant although small reduction in paraventricular nucleus (PVN) OT cell c‐fos expression ipsilateral to the lesion, but no change in the responses of supraoptic nucleus OT cells or in cells of the medial parvocellular PVN, many of which are CRF cells. These findings indicate that VLM catecholamine cells make little contribution to hypothalamic neuroendocrine cell responses to CCK and thus serve to further highlight the role of dorsomedial catecholamine cells. However, it is now apparent that, in addition to A2 noradrenergic cells, CCK treatment also recruits C2 adrenergic cells of the dorsomedial medulla, many of which have previously been shown to project to the PVN.
Sequential Exposure to Estrogen and Testosterone (T) and Subsequent Withdrawal of T Increases the Level of Arginine Vasopressin Messenger Ribonucleic Acid in the Hypothalamic Paraventricular Nucleus of the Female Rat
Authors: Thomas Abraham , Kim Norma B. , Amico Janet A.
Published: November 2003

The hypothalamic peptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated as mediators of socio‐sexual behaviors in addition to their roles in osmolar homeostasis (AVP), milk ejection and uterine contractility (OT). Within 24 h of parturition, OT and AVP messenger ribonucleic acid (mRNA) levels increase in the hypothalamic paraventricular, and to a lesser degree, the supraoptic nucleus (PVN and SON) of the rat. We previously reported that the prepartum increase in OT mRNA is related to the spontaneous decline in progesterone levels prior to parturition. We also reported that increases in PVN and SON OT mRNA can be induced by exposing the ovariectomized rat to a steroid regimen that mimics the steroid milieu of pregnancy, namely sequential estrogen and progesterone and subsequent progesterone withdrawal. Levels of PVN and SON AVP mRNAs were not affected by progesterone withdrawal in late pregnant rats or the steroid regimen that increased OT mRNA in ovariectomized rats. These observations suggest that other factors, perhaps hormonal, may influence AVP mRNA levels. A decline in testosterone coincident with waning progesterone levels also occurs prepartum. Since peak levels of AVP mRNA prepartum coincide with the prepartum decline in testosterone, we questioned whether declining testosterone levels are important for the increase in AVP mRNA levels.
Evidence for Multiple Opioid Receptors in the Human Posterior Pituitary
Authors: Jordan D. , Tafani J. A. M. , Ries C. , Zajac J. M. , Simonnet G. , Martin D. , Kopp N. , Allard M.
Published: November 2003

The distribution and quantification of opioid receptor types in post‐mortem human pituitary cryostat sections was determined by quantitative in vitro receptor autoradiography. Highly specific radioligands were used for each opioid receptor type, i.e. [125I]‐FK‐33‐824 for μ‐opioid sites, [125I][D.Ala2]‐Deltorphin‐I for δ‐opioid sites and 3H‐U69,593 for κ‐opioid sites.

None of the five specimens tested exhibited any labelling in the anterior lobe of the pituitary for the three radioligands. As for the posterior pituitary, all 5 specimens contained both μ and κ‐opioid binding sites whereas none of them showed δ‐binding sites.

The presence of both μ‐ and κ‐opioid binding sites in the human posterior pituitary contrasts with previous findings in the rat where only κ‐sites have been found. The present study could contribute to understanding of the functional action of opioids in the human pituitary.
The Involvement of Dopaminergic and Opioidergic Neuronal Systems in the Control of the Early Rise in LH Secretionin Bull Calves
Authors: Chandolia R. K. , Evans A. C. O. , Rawlings N. C.
Published: November 2003

There is an early rise in secretion of luteinizing hormone (LH) in bull calves between 6 and 20 weeks of age. This study was designed to examine the involvement of opioidergic and dopaminergic neuronal systems in the control of this early rise in gonadotrophin secretion.
Co‐localization of α1D Adrenergic Receptor mRNA withMineralocorticoid and Glucocorticoid ReceptormRNA in Rat Hippocampus
Authors: Williams Amelia M. , Nguyen My Linh D. , Morilak David A.
Published: November 2003

Many adaptive changes occur in response to chronic or repeated stress, involving complex regulatory interactions between central stress‐related afferents and the central components of the hypothalamo‐pituitary‐adrenal (HPA) axis. One change associated with chronic stress is an attenuation of corticosteroid receptor‐mediated feedback inhibition of the HPA axis, a process thought to involve corticosteroid receptors in the hippocampus. A prominent stress‐related central afferent that innervates the hippocampus and that may participate in the regulation of the HPA axis is the central noradrenergic system. Previous evidence suggests that α1 adrenergic receptors may down‐regulate hippocampal corticosteroid receptors, and may thus contribute to stress‐induced facilitation of HPA responses. In the present study, we used combined nonisotopic and radioisotopic in situ hybridization to examine the overlapping expression and co‐localization of mRNA encoding the post‐synaptic α1D adrenergic receptor subtype, the major α1 subtype expressed in hippocampus, with mRNA for the two corticosteroid receptor subtypes, type I (mineralocorticoid receptor, MR) and type II (glucocorticoid receptor, GR) in rat hippocampal neurons. We observed overlapping distributions and an extensive degree of co‐localization of α1D receptor mRNA with both corticosteroid receptor subtype messages, establishing an anatomical substrate by which these two receptor systems may directly regulate each other. The potential interaction between co‐localized adrenergic and corticosteroid receptors in hippocampus may contribute to stress‐induced alterations in the HPA response to subsequent stress.
Effect of Chronic Prolactin Infusion on Pituitary Prolactin and Hypothalamic Proopiomelanocortin
Authors: Wardlaw Sharon L. , Kim Jennie , Matera Cristina
Published: November 2003

Although there is evidence that endogenous opioids, and in particular β‐endorphin (β‐EP), may mediate some of the suppressive effects of hyperprolactinemia on the hypothalamic‐pituitary‐gonadal (HPG) axis, there is controversy about the effects of prolactin (PRL) on β‐EP and its precursor, proopiomelanocortin (POMC), in the hypothalamus. In this study we have therefore examined the effects of chronic peripheral and intracerebroventricular (i.c.v.) infusion of ovine PRL on POMC gene expression and β ‐EP levels in the medial basal hypothalamus (MBH) of castrated male and female rats. Endogenous pituitary and plasma PRL levels were determined by RIA with an antiserum to rat PRL which does not crossreact with oPRL. Suppression of endogenous rPRL levels was used as a confirmation of the biological effectiveness of the infused oPRL. POMC mRNA was measured in the MBH by solution hybridization assay. In the first experiment oPRL (5 μg/μl/h) or vehicle was infused for 2 weeks by osmotic minipump into the right lateral ventricle of ovariectomized rats. The mean plasma concentration of rPRL declined from 3.7±1.0 ng/ml in the controls to 1.4±0.13 ng/ml in the oPRL infused animals (P<0.05); pituitary rPRL content similarly decreased from 39.1±4.6 μg to 20.4±3.7 μg (P<0.02). There was no significant change in the concentration of POMC mRNA or β‐EP in the MBH of the oPRL treated animals. In the second experiment oPRL was infused for 1 week into the third ventricle of orchiectomized rats. Again despite a fall in endogenous PRL levels, there was no significant change in POMC or β‐EP in the MBH. In the third experiment oPRL was infused subcutaneously into orchiectomized rats for 2 weeks. Mean plasma oPRL levels were 150±7.3 ng/ml after 1 week and 58±7.5 ng/ml after 2 weeks. Pituitary rPRL content was again suppressed in the oPRL treated animals but no change in POMC or β‐EP was detected in the MBH. We conclude that oPRL can be infused both peripherally and centrally for up to 2 weeks with resulting suppression of endogenous pituitary PRL content and release. Under these conditions no effects on the concentrations of POMC mRNA or β‐EP could be demonstrated in the hypothalamus. These results suggest that either PRL has nongenomic effects on hypothalamic β‐EP or that endogenous opioids other than β‐EP mediate the suppressive effects of PRL on the HPG axis.
Intracerebroventricular Neuropeptide Y Acutely Influences Glucose Metabolism and Insulin Sensitivity in the Rat
Authors: Marks Jonathan L. , Waite Kay
Published: November 2003

Acute administration of neuropeptide Y(NPY) into the hypothalamus and third cerebral ventricle (ICV) increases respiratory quotient, reduces energy expenditure and increases circulating, insulin, glucagon and corticosterone. Therefore, it is likely that hypothalamic NPY has acute effects on the metabolism of fuels, such as glucose. To test this hypothesis, we determined if ICV infusion of NPY influences glucose metabolism and its sensitivity to insulin in conscious, unrestrained rats, not given access to food. Glucose turnover was 4.7±0.3 mg/min, 45–55 min after ICV NPY was administered at 3 μg/h vs 3.7±0.2 (P<0.05) for ICV saline. In a time course study, glucose turnover was significantly increased 30 min, and remained elevated at 50 min after starting a similar ICV NPY infusion. In neither study was plasma glucose, insulin, glucagon or corticosterone significantly affected by ICV NPY. During an hyperinsulinaemic euglycaemic clamp, the glucose infusion rate corrected for body weight and insulin concentration, M/I was 0.22±0.03 for NPY vs 0.36±0.05 mg min−1 kg−1 μU−1 ml (P<0.05) for saline. NPY treatment prevented the decline in glucose production rate but did not influence the rise in glucose disposal rate resulting from hyperinsulinaemia. It was concluded that ICV NPY rapidly stimulates glucose turnover by a mechanism that does not depend on changes in insulin, glucagon or corticosterone secretion. Furthermore, ICV NPY decreased insulin sensitivity by reducing the effect of insulin on glucose production but not on whole body glucose disposal.
Localization of Androgen Receptor in Nitric Oxide Synthase‐ and Vasoactive Intestinal Peptide‐Containing Neurons of the Major Pelvic Ganglion Innervating the Rat Penis
Authors: Schirar A. , Chang C. , Rousseau J. P.
Published: November 2003

Neurons of the rat major pelvic ganglia provide innervation to the pelvic organs and external genitalia. In these ganglia, a subpopulation of neurons containing either nitric oxide synthase or vasoactive intestinal peptide or both molecules, is involved in penile erection. The androgen dependence of penile erection is a well established fact. After castration, decreased testosterone levels have been documented to produce erectile dysfunction possibly resulting from functional alterations in major pelvic ganglion neurons. It was therefore of interest to investigate the presence of androgen receptor within these ganglionic neurons.
Immunolocalization of Type I or Type II Activin Receptors in the Rat Brain
Authors: Funaba Masayuki , Murata Takuya , Fujimura Hisako , Murata Eri , Abe Matanobu , Torii Kunio
Published: November 2003

We have studied immunolocalization of activin receptors in the central nervous system using polyclonal antibodies (IgG) to type I (50–55 kDa, ActRI), type II (70–75 kDa, ActRII) or a subtype of type II known as type IIB (ActRIIB) receptors of activin. A total of 7 antisera to rat activin receptors was generated, i.e. 3 kinds of antisera to the extracellular domain (ActRI(81–89), ActRII(91–100), or ActRIIB(90–99)) and 4 antisera to the kinase domain (ActRI(323–333), ActRII(307–319), ActRII(407–420) or ActRIIB(306–319)). The region of aa 407–420 of ActRII is identical with that of ActRIIB. At first, we characterized these antibodies by Western blot analysis using ovarian proteins fractionated by preparative SDS‐PAGE. All antibodies to ActRII and ActRIIB specifically reacted with 75 kDa‐proteins which could also bind to activin‐A. Anti‐ActRII(91–100) antibody also reacted with 62 kDa‐proteins which were capable of binding with activin‐A. Although no positive reactions to anti‐ActRI(81–89) antibody were seen in ovarian proteins, a positive reaction was detected at 52 kDa only when the proteins were deglycosylated. By use of these antibodies, immunolocalization of activin receptors was examined in the rat brain. The patterns of expression of activin type I and type II receptors were different. Positive reactions to anti‐ActRII(91–100) antibody were detected in neurons of the cerebral cortex, hippocampus, medial amygdala and thalamus. In the hypothalamus, some neurons of the supraoptic nucleus were weakly stained, and widely scattered neurons of the lateral hypothalamic area were moderately stained. On the contrary, the most intense reactions to anti‐ActRI(81–89) antibody were detected in neurons of the lateral hypothalamic area. In addition, many neurons of the cerebral cortex were also stained, but neurons of the hippocampus and the amygdala were not stained. These results suggest that activin may have physiological roles not only for hypothalamic neuroendocrinological and feeding‐related systems as suggested previously but may also have functions in cortical and limbic pathways as a neuromodulator or for maintenance of neurons.
Cyclic Feeding Behaviour and Changes in Hypothalamic Galanin and Neuropeptide Y Gene Expression Induced by Zinc Deficiency in the Rat
Authors: Selvais Philippe L. , Labuche Catherine , Ninh Nguyen X. , Ketelslegers Jean‐Marie , Denef Jean‐François , Maiter Dominique M.
Published: November 2003

Dietary zinc‐deficiency induces a striking reduction and a cyclic pattern of food intake in rodents. To elucidate the mechanisms for these effects, we studied the hypothalamic content, synthesis, and distribution of galanin (GAL) and neuropeptide Y (NPY) during zinc deficiency and refeeding in the rat. In Wistar rats, three weeks of zinc‐deprivation consistently induced a reduction and a cyclic pattern of night‐and day‐time food intake, as well as of water intake. This was accompanied in zinc‐deficient (ZD) rats, and to a lesser extent in pair‐fed (PF) rats, by a decrease of hypothalamic GAL mRNA concentration (CTR: 100±8, ZD: 61±4, PF: 78±2 arbitrary densitometric units, ADU, P<0.01) and an increase of hypothalamic NPY (CTR: 100±11, ZD: 154±10, PF: 126±4 ADU, P<0.05), without peptide modification. The two neuropeptidergic systems were not affected by the cycles of feeding, with the exception of the NPY‐immunoreactivity in the suprachiasmatic nuclei (geniculo‐hypothalamic tract), that was inversely correlated to the food intake in both ZD and PF animals. In a second experiment, we showed that zinc‐repletion for 4 days suppressed the behaviour induced by a two‐week zinc‐deprivation, and reversed the increase of NPY mRNA in ZD animals. We finally demonstrated that zinc‐deficiency induced a similar behaviour in Zucker rats. However, in these rats whose synthesis of NPY is constitutively up‐regulated, no change of NPY synthesis was observed in ZD rats, suggesting that the increase observed in Wistar is adaptative rather than instrumental to the abnormal food intake. In conclusion, we have further characterized the cyclic feeding behaviour of the zinc‐deficient Wistar rats, and shown in these animals a decreased activity of the GAL system and an increased activity of the NPY system, likely corresponding to a compensatory response of the two neuropeptidergic systems, as observed in food‐deprived animals. As spontaneous food intake of ZD rats does not increase, a resistance to NPY could also be present. These behavioural and neuropeptidergic changes were partially reversed by reintroduction of zinc in the diet. In Zucker rats, the same behaviour occurred despite an insensitivity of the NPY system to the zinc‐deficiency. In addition, we describe a nutritional regulation of the NPY‐immunoreactivity in the geniculo‐hypothalamic tract, that could constitute the substrate of circadian rhythm modulation by timed feeding.
Duality of Glutamatergic and Gabaergic Control of PulsatileGnRH Secretion by Rat Hypothalamic Explants: II. ReducedNR2C‐ and GABAA‐Receptor‐Mediated Inhibition atInitiation of Sexual Maturation
Authors: Bourguignon Jean‐Pierre , Gérard Arlette , Purnelle Gentiane , Czajkowski Vincent , Yamanaka Chutaro , Lemaître Marc , Rigo Jean‐Michel , Moonen Gustave , Franchimont Paul
Published: November 2003

N‐methyl‐D‐aspartate (NMDA) receptors and γ‐aminobutyric acid (GABA) receptors are involved in the mechanism of pulsatile gonadotrophin‐releasing hormone (GnRH) secretion. The aim of this study was to elucidate the role of those receptors in the acceleration of pulsatile GnRH secretion seen at onset of puberty. Using hypothalamic explants from prepubertal (15 days), early pubertal (25 days) and adult (50 days) male rats, we studied the effects of pharmacological antagonists and antisense oligodeoxynucleotides on GnRH release evoked by NMDA and GABA receptor agonists as well as the interval between spontaneous GnRH secretory pulses. At the three studied ages, the muscimol‐evoked release of GnRH is similarly inhibited by the GABAA receptor antagonist bicuculline. In contrast, the frequency of pulsatility is stimulated by bicuculline as indicated by a reduction of the mean GnRH interpulse interval from 60 to 40 min and such an effect is seen at 15 days only. The GnRH interpulse interval is also reduced by GABAA receptor antisense oligodeoxynucleotides at 15 days while no effects are seen at 25 days. At the three studied ages, the NMDA‐evoked release of GnRH and the GnRH interpulse interval are similarly inhibited by 100 or 500 μM of the NMDA receptor antagonist 7‐chlorokynurenic acid (7CK). These effects are consistent with the increase of GnRH interpulse interval caused by NR2A antisense oligodeoxynucleotides at 15 days (86 vs 64 min in controls) as well as 25 days (44 vs 36 min). A low (5 μM) concentration of 7CK does not result in any effect except a reduction of GnRH interpulse interval which is seen at 15 days only. A similar reduction of GnRH interpulse interval is obtained using NR2C antisense oligodeoxynucleotides at 15 days (50 vs 64 min in controls) while no effects are seen at 25 days (35 vs 36 min). At 25 days, muscimol can prevent the developmental increase in frequency of pulsatile GnRH secretion. In summary, pulsatile GnRH secretion by the prepubertal hypothalamus characteristically involves an inhibition mediated through GABAA receptors and the NR2C subunit of NMDA receptors. Based on these data, we propose a model for the mechanism of the onset of puberty which involves the disappearance or inactivation of GABAergic neurons located in the retrochiasmatic hypothalamus and expressing the NR2C subtype of NMDA receptors.
Extracellular Sodium Dependence of GnRH‐Stimulated Growth Hormone Release in Goldfish Pituitary Cells
Authors: Van Goor Fredrick , Goldberg Jeffrey I. , Chang John P.
Published: November 2003

In goldfish, gonadotropin‐releasing hormone (GnRH) stimulation of growth hormone (GH) release has been shown to involve extracellular Ca2+ entry through voltage‐sensitive Ca2+ channels and the activation of protein kinase C (PKC). In this study, the possible involvement of extracellular Na+ in mediating the GH response to GnRH was examined using dispersed pituitary cells. Perifusion with Na+‐depleted medium reversibly reduced the acute GH response to 5‐min pulses of either 10 nM salmon (s)GnRH or 10 nM chicken (c)GnRH‐II. Similarly, replacement of normal medium with Na+‐depleted medium attenuated the long‐term GH release response to sGnRH and cGnRH‐II under static incubation conditions. These results suggest that GnRH‐induced GH release requires the presence of extracellular Na+. Treatment with 5‐min pulses of the Na+‐channel agonist veratridine (10 μM) increased GH release in an extracellular Ca2+‐dependent manner, presumably due to activation of voltage‐sensitive Ca2+ channels resulting from the depolarizing effect of increased Na+ influx. On the other hand, Na+ entry through tetrodotoxin (TTX)‐sensitive, voltage‐dependent Na+ channels is not involved in GnRH‐induced GH release. Application of 250 nM TTX, which abolished the voltage‐sensitive Na+ currents in identified goldfish somatotropes, did not affect the acute GH responses to 5‐min pulses of sGnRH and cGnRH‐II. The possible participation of Na+/H+ antiport in mediating the extracellular Na+‐dependent GnRH action on GH release was then examined. In static incubation experiments, sGnRH‐ and cGnRH‐II‐induced GH secretion were reduced by inhibitors of the Na+/H+ antiport, amiloride and dimethylamiloride (DMA). Likewise, the GH response to the PKC activator, tetradecanoyl phorbol acetate, was attenuated by treatment with Na+‐depleted medium, amiloride, and DMA. The inhibitory actions of amiloride and DMA were selective as these drugs did not affect the GH response elicited by the Ca2+ ionophore ionomycin and the voltage‐sensitive Ca2+ channel agonist, Bay K 8644. Taken together, these results indicate that extracellular Na+ and the Na+/H+ exchanger are involved in the mediation of GnRH‐stimulated GH release in goldfish. Furthermore, this dependence on Na+ and Na+/H+ antiport probably occurs distal to the activation of PKC by GnRH.
Immunocytochemical Localization of GnRH Precursor in the Hypothalamus of European Starlings during Sexual Maturationand Photorefractoriness
Authors: Parry Dilys M. , Goldsmith Arthur R. , Millar Robert P. , Glennie Linda M.
Published: November 2003

Immunocytochemistry with quantitative image analysis, for both GnRH and its precursor proGnRH‐GAP, was used in male European starlings (Sturnus vulgaris) to investigate four stages of a photoperiodically‐induced reproductive cycle. Four different groups of birds were examined: photosensitive but sexually immature, sexually mature, undergoing gonadal regression, and after the completion of regression and fully photorefractory.
Concomitant Up‐Regulation of Proopiomelanocortin and Dopamine D2‐Receptor Gene Expression in the Pituitary Intermediate Lobe of the Spontaneously Hypertensive Rat
Authors: Autelitano Dominic J. , Van Den Buuse Maarten
Published: November 2003

Alterations in central dopaminergic mechanisms in the Spontaneously Hypertensive Rat (SHR) have been previously implicated in the development of the hypertensive phenotype in this rat strain. We have examined the expression and regulation of the dopamine‐responsive gene proopiomelanocortin (POMC) in the neurointermediate lobe (NIL) of the pituitary in both SHR and normotensive Wista Kyoto (WKY) rats. Solution hybridization/nuclease protection analysis showed that adult SHR express POMC mRNA in the NIL at approximately 2–4 times the level seen in normotensive WKY controls, associated with a concomitant 2‐fold increase in dopamine D2‐receptor (D2‐R) mRNA expression. Despite the obvious difference in D2‐R gene expression, NIL POMC mRNA in both rat strains was regulated in an identical manner following 4 d in vivo bromocriptine or haloperidol treatment. In contrast, though D2‐R mRNA expression in the WKY NIL was significantly up‐regulated by D2‐R blockade with haloperidol, the elevated levels of D2‐R mRNA in the NIL of the hypertensive strain were not altered by D2‐R antagonism. Following isolation from all hypothalamic input by 5 d in vitro culture, SHR melanotrophs exhibited a 2–3 fold higher rate of βEP secretion and POMC mRNA expression than melanotrophs derived from normotensive WKY rats, though βEP secretion was inhibited in a similar fashion by the D2‐R agonist quinpirole in both cultures. The current data demonstrate changes in expression of both POMC and D2‐R mRNA in the SHR NIL which may be a consequence of altered dopaminergic input and/or alterations in D2‐R regulation in this tissue, possibly enabling other factors in addition to dopamine to maintain the NIL of the SHR in a relatively hyperactive state. Whether or not POMC‐derived peptides or other factors secreted from the melanotroph cell play any role in the development or maintenance of hypertension in this strain is yet to be established.
Post‐Transcriptional Regulation of the Gonadotropin‐Releasing Hormone Gene in GT1–7 Cells
Authors: Gore Andrea C. , Yeo Tracy T. , Ho Angela , Roberts James L.
Published: November 2003

GT1–7 cells respond to treatment with the phorbol ester, phorbol 12‐myristate 13‐acetate (PMA), with an inhibition of transcription of the proGnRH gene and decreases in GnRH mRNA levels. However, the timing of this decrease in GnRH mRNA levels suggests that a decrease in GnRH mRNA stability may be involved in addition to an inhibition of transcription of the proGnRH gene. To address this possibility, we treated GT1–7 cells with 100 nM PMA for 4 h and then monitored GnRH mRNA levels over time after blockade of GnRH gene transcription with DRB. PMA treatment caused GnRH mRNA half‐life to decrease from 30 to 11 h. Then, to verify this observation, we examined changes in GnRH mRNA poly (A) tail length, which may be a reflection of mRNA turnover, following treatment of GT1–7 cells with PMA or vehicle for 0, 4, 8 or 24 h. The poly (A) tail was removed from half of the GT1 cytoplasmic RNA sample by digestion with RNase H and the difference in GnRH mRNA size with and without RNase H treatment was determined by Northern hybridization. PMA treatment (4 and 8 h) resulted in a significant decrease in the length of the GnRH mRNA poly (A) tail, consistent with a decrease in GnRH mRNA stability. This finding suggests that GnRH mRNA turnover is inducible by substances such as PMA. Our study indicates that a change in mRNA stability is one of a multiplicity of levels at which GnRH gene expression is regulated.
Hypophysiotropic CRF Neurons Display a Sustained Immediate‐Early Gene Response to Chronic Stress but not to Adrenalectomy
Authors: Brown E. R. , Sawchenko P. E.
Published: November 2003

Immediate‐early genes (IEGs) are now widely used to identify neurons acutely activated by extracellular stimuli. Though challenge‐induced IEG expression is typically transient, examples of sustained elevation have been reported, including in hypothalamic magnocellular neurosecretory neurons of osmotically challenged rats. Another chronic stimulus, adrenalectomy (ADX), targets parvocellular neurosecretory neurons in the paraventricular nucleus of the hypothalamus (PVH), resulting in a persistent elevation in the synthesis and secretion of corticotropin‐releasing factor (CRF). In the present study we used hybridization histochemical methods to compare the effects of ADX on the induction over time of two independent IEG markers, c‐fos and NGFI‐B, in hypophysiotropic CRF neurons. The induction of both c‐fos and NGFI‐B mRNA was greatest 3 to 6 h after ADX, diminished by 12 h, and no longer detectable at 24, 48, 72 h, or 6 days after surgery. Immunohistochemical analyses using a Fos‐specific N‐terminally directed antiserum also revealed Fos immunoreactivity (‐ir) in the PVH at early time points (3 h), but not later than 12 h after ADX. Combined immuno‐ and hybridization histochemistry on tissue from 3 h ADX rats localized Fos‐ir and NGFI‐B mRNA to parvocellular CRF‐expressing neurons, the majority of which expressed both Fos and NGFI‐B. These early IEG responses, however, were not paralleled by increases in CRF mRNA, which was not significantly elevated until 48 h after ADX. In the chronically ADX rat, CRF neurons are capable of c‐fos expression since animals subjected to an acute injection of hypertonic saline 5 days after ADX displayed a robust induction of Fos‐ir in the parvocellular PVH. Furthermore, a chronic challenge, insulin‐induced hypoglycemia, provoked comparable c‐fos mRNA and protein expression in CRF neurons in the PVH of ADX and sham‐operated rats, which was observed both acutely (2 h) and chronically (5 days) after the onset of hypoglycemia. The maintenance of Fos expression in parvocellular CRF neurons following chronic hypoglycemia, but not ADX, suggests an involvement of distinct signaling pathways in the maintenance of hypophysiotropic neuron responses to chronic steroid withdrawal and stress.
Electrophysiological Evidence for Retinal Projections to the Hypothalamic Supraoptic Nucleus and its Perinuclear Zone
Authors: Cui L.‐N. , Jolley C. J. , Dyball R. E. J.
Published: November 2003

Secretion of vasopressin (VP) and oxytocin (OT) displays a daily rhythm. Using electrophysiological methods, we investigated the projections from the optic nerve to the supraoptic nucleus (SON) and its perinuclear zone (PNZ) which might underlie the rhythm. Extracellular recordings were made from magnocellular cells in the SON and its PNZ in 22 urethane‐anaesthetized female Wistar rats while stimulating the optic nerve. The responses of magnocellular and PNZ cells were classified as orthodromic excitatory (OD+) or inhibitory (OD‐) after creating peri‐stimulus time histograms (PSTHs). Twenty‐six of 73 (35.6%) VP and OT cells and 16 of 42 (38.1%) PNZ cells were excited by optic nerve stimulation. PNZ cells displayed both short (for 7 cells 30 ms or less) and long (>60 ms) latency responses. Most (6/7) short latency responses had a short duration but longer latency responses were longer. No magnocellular cells showed responses with both short latency and short duration. Short latency responses with a short duration probably reflect direct monosynaptic inputs whereas longer latency responses with longer duration may reflect complex inputs. Thus the retina projects to the PNZ and to the SON but the PNZ receives a stronger direct input. Such projections might provide a light‐related input to SON cells and suggest a role for the PNZ in this input.