bluuebunny/crossref_metadata_2025 huggingface dataset:
Ontogeny of the Diurnal Rhythm of Tuberoinfundibular Dopaminergic Neuronal Activity in Peripubertal Female Rats: Possible Involvement of Cholinergic and Opioidergic Systems
Authors: Shieh Kun-Ruey , Pan Jenn-Tser
Published: April 2003

The ontogeny of the diurnal change of tuberoinfundibular dopaminergic (TIDA) neuronal activity in female rats and its control mechanism were the foci of this study. Intact pre (28- and 35-day-old)-, peri (39- and 42-day-old)- and post (49- and 56-day-old)-pubertal female, as well as pre (28-day-old)- and post (56-day-old)-pubertal male Sprague-Dawley rats were used. Basal TIDA neuronal activity, determined by measuring 3,4-dihydroxyphenylalanine or 3,4-dihydroxyphenylacetic acid in the median eminence, increased 3- to 4-fold in the female and less than 2-fold in the male rats from day 28 to 49; so did serum prolactin levels. The diurnal rhythm of TIDA neuronal activity was not evident until day 42 in the female, but not male, rats. Neither nigrostriatal, nor mesolimbic DA neurons exhibited any significant change between pre- and postpubertal stages. In postpubertal (56-day-old) female rats, injection of nicotine (10 µg/kg) in the morning, but not in the afternoon, inhibited TIDA neuronal activity while similar injections of mecamylamine (1 mg/kg) or naloxone (2.5 mg/kg) stimulated these neurons in the afternoon, but not in the morning. Serum prolactin levels changed accordingly. In contrast, none of the injections had any effect in prepubertal (28-day-old) female rats. These results indicate that the genesis of the diurnal rhythm of TIDA neuronal activity in female rats occurs during the peripubertal stage, and endogenous cholinergic and opioidergic neurons may play a significant role.
Expression of Hypothalamic Peptides in Mice Lacking Neuronal Nitric Oxide Synthase: Reduced β-END Immunoreactivity in the Arcuate Nucleus
Authors: Bernstein Hans-Gert , Keilhoff Gerburg , Seidel Bertolt , Stanarius Andreas , Huang PaulL. , Fishman MarkC. , Reiser Michael , Bogerts Bernhard , Wolf Gerald
Published: April 2003

The gas nitric oxide (NO) is an important messenger in brain signaling. Along with many other functions, NO is thought to influence the expression and/or release of various hypothalamic hormones (corticotropin-releasing hormone (CRH), gonadotropin-releasing hormone (GnRH) and vasopressin). To learn more about the role of NO in neuroendocrine mechanisms, we studied in mutant mice lacking neuronal isoform of NO synthase (nNOS) the cellular expression of CRH, neurophysin (the carrier protein of vasopressin/oxytocin) and pro-opiomelanocortin (POMC), as well as of the POMC-derived peptides β-endorphin (β-END), α-melanocyte-stimulating hormone (α-MSH) and corticotropin (ACTH) by use of immunohistochemistry and in situ hybridization. Additionally, the remaining NO-generating capacities of the nNOS minus mice were investigated by NADPH-diaphorase histochemistry and citrulline immunohistochemistry as well as by immunohistochemical localization and Western blot analysis of endothelial NOS (eNOS) and nNOS isoforms. Amongst all hypothalamic peptides under investigation, only β-END was found to be altered in mutant mice. A morphometric analysis of β-END producing neurons of the arcuate nucleus revealed that significantly less cells were immunoreactive in mutant mice, whereas the expression of the precursor POMC as well as of other POMC-derived peptides was found to be unchanged. In addition to that, fewer β-END-immunoreactive fibers were found in the paraventricular nucleus of nNOS minus mice in comparison to wild-type animals. Hence, the reduction of hypothalamic β-END is probably a posttranslational event that might reflect a disturbed endorphinergic innervation of those hypothalamic neurons which normally express nNOS.
Estrogens Normalize the Hypothalamic- Pituitary-Adrenal Axis Response to Stress and Increase Glucocorticoid Receptor Immuno- reactivity in Hippocampus of Aging Male Rats
Authors: Ferrini Monica , Piroli Gerardo , Frontera Miriam , Falbo Andrea , Lima Analia , De Nicola AlejandroF.
Published: April 2003

Aging is associated with a disturbance in the regulation of the hypothalamic-pituitary-adrenal axis (HPA) and reduced levels of glucocorticoid receptors (GR) in the hippocampus. To compensate for these effects, we have investigated whether estrogen therapy normalized the HPA response to stress and GR in hippocampus and paraventricular (PVN) nucleus. Young (3–4 months) and old (20 months) male Sprague-Dawley rats were bled by tail cut in the basal state and following ether stress. While basal and ether-stimulated levels of plasma corticosterone (CORT) were similar in the two groups, old animals presented a delayed termination of the response to ether stress. A dexamethasone inhibition test carried out in old animals, showed a failure to completely block plasma CORT after ether stimulation. Furthermore, in old rats GR-immunoreactive levels were reduced in CA1-CA2 hippocampal subfields and subiculum, while normal levels were obtained in CA3-CA4 and PVN. We observed that prolonged estrogen treatment (6 weeks) of old rats normalized the termination of the stress response, restored dexamethasone inhibition of plasma CORT, and increased GR immunoreactivity in CA1 and CA2 hippocampal subfields and subiculum. The results suggest that estrogen treatment enhanced the glucocorticoid feedback signal by increasing GR in hippocampus, and corrected the disturbances in HPA axis regulation. These animal experiments may be important to elucidate the effects of estrogenic on the hippocampal and HPA dysfunction associated with aging and Alzheimer’s disease in humans.
Changes in Hypothalamic Estrogen Receptor-Containing Cell Numbers in Response to Feed Restriction in the Female Lamb
Authors: Hileman Stanley M. , Lubbers Laura S. , Jansen Heiko T. , Lehman Michael N.
Published: April 2003

The mechanism whereby undernutrition enhances the ability of estradiol (E) to inhibit reproductive activity is unknown. This study aimed to determine the effect of feed restriction on E receptor (ER)-containing cell numbers in the female sheep hypothalamus. Ovariectomized lambs at 7 months of age received either ad libitum (AL; n = 5) or restricted (FR; n = 10) levels of feed intake. Lambs were weighted weekly and FR lambs fed to lose approximately 15% of their initial body weights over 7 weeks, at the end of which jugular blood samples were collected at 10-min intervals for 5 h to assess the patterns of LH release. After blood collection, lambs were euthanized and hypothalami collected for immunocytochemical detection of ER. Based on LH secretory profiles, FR lambs were subdivided into two groups. The first group (FR + LH; n = 5) exhibited patterns of LH release similar to AL controls. LH secretion in the second group (FR-LH; n = 5) was obviously suppressed. Numbers of ER-containing cells did not differ significantly (p > 0.10) among treatment groups in the bed nucleus stria terminalis, anterior hypothalamic area and arcuate nucleus. ER-containing cell numbers were greater (p < 0.05) in the preoptic area (POA) but less (p < 0.05) in the ventromedial/ventrolateral hypothalamus (VMH/VLH) for FR-LH lambs compared to AL animals. Notably, for both the POA and VMH/VLH, ER-containing cell numbers in the FR + LH animals were intermediate and did not differ (p > 0.10) from either FR-LH or AL lambs. These results suggest that feed restriction differentially alters ER-containing cell numbers in specific regions of the ovine hypothalamus (numbers increased in the POA but decreased in the VMH/VLH). These changes may, at least in part, represent a mechanism whereby undernutrition enhances the ability of E to inhibit reproduction.
GABAergic Regulation of Lordosis: Influence of Gonadal Hormones on Turnover of GABA and Interaction of GABA with 5-HT
Authors: Luine Victoria N. , Wu Vincent , Hoffman Cynthia S. , Renner Kenneth J.
Published: April 2003

The role of GABAergic neurons in activating female sexual behavior and possible mechanisms for GABAergic effects on behavior were examined in female rats. First, effects of the ovarian hormones estrogen and progesterone (P), at doses which promote lordosis, on levels and turnover/activity of GABA, were examined in brain areas which regulate lordosis. Utilizing AOAA, an inhibitor of GABA degradation, the accumulation rate of GABA (turnover/activity) was assessed in ovariectomized (Ovx), Ovx + estrogen and Ovx + estrogen + P-treated rats. Estradiol increased GABA accumulation rates in the arcuate-median eminence and in the area dorsal to and surrounding the VMN (VMN-S). P administration following estrogen priming enhanced GABA turnover in the medial preoptic area (mPOA) and further increased turnover in the VMN-S while GABA turnover decreased in the dorsomedial nucleus. No effects of hormones were noted in the VMN itself or in the dorsal midbrain central gray. Reverse dialysis of the GABAA antagonist bicuculline into the basomedial hypothalamus was associated with a time-dependent inhibition of lordosis and a 300% increase in 5-HT release in the basomedial hypothalamus as measured by in vivo dialysis. These results provide additional evidence that GABAergic neurons mediate the physiological regulation of female sexual behavior and suggest that such mediation may involve an interaction with 5-HT containing neurons.
Thyroid Hormones Regulate the Expression of Somatostatin Receptor Subtypes in the Rat Pituitary
Authors: Lam Karen S.L. , Wong Rachel L.C.
Published: April 2003

Circulating TSH levels are increased in hypothyroidism and suppressed in hyperthyroidism. On the other hand, the hypothalamic hormone somatostatin suppresses basal and TRH-induced TSH release, an effect which is enhanced by thyroid hormones. To investigate whether the effects of thyroid hormones on TSH secretion may be mediated in part through alterations in the gene expression of pituitary somatostatin receptors (SSTR), 3-week-old male Sprague-Dawley rats were rendered hypothyroid with antithyroid drugs for 3 weeks. Total RNA extracted from anterior pituitaries were analysed for SSTR mRNA levels, using Northern blot hybridization. Compared to controls, hypothyroid rats had significantly lower pituitary mRNA levels of SSTR1 and SSTR2 (p < 0.0001 for both, n = 16); the reductions could be prevented by T4 supplementation (3 µg/100 g body weight/day i.p.). In vitro studies using GH4C1 rat pituitary cells showed that the addition of T3 10–8 M to cells cultured in charcoal-stripped bovine calf serum resulted in significant increases in mRNA levels of SSTR1 (p < 0.0001; n = 7) and the two transcripts of SSTR2 (p < 0.0005; n = 7). The increase for SSTR1 showed no further increase with higher doses of T3, but was time-dependent and could be seen consistently after 8 h of incubation. We conclude that thyroid hormones regulate the gene expression of SSTR subtypes in the pituitary, via a direct action on anterior pituitary cells. Changes in SSTR gene expression may contribute to the increase in circulating TSH levels in hypothyroidism.
Transgenic Mice Overexpressing the Growth-Hormone-Releasing Hormone Gene Have High Concentrations of Tachykinins in the Anterior Pituitary Gland
Authors: Debeljuk Luciano , Wright James C. , Phelps Carol , Bartke Andrzej
Published: April 2003

According to recent reports, substance P (SP) is localized in the anterior pituitary gland within subsets of thyrotropes and somatotropes, although earlier electron-microscopic studies described the presence of this tachykinin in mammotropes and gonadotropes. Transgenic mice overexpressing the growth-hormone-releasing hormone (GHRH) gene have markedly enlarged pituitary glands, due to hyperstimulation of the somatotropes. Therefore, we speculated that if somatotropes are able to synthesize tachykinins, these peptides should be greatly increased in the anterior pituitary of transgenic GHRH mice. We found that, in accordance with our working hypothesis, both SP and neurokinin A (NKA) were markedly increased in the anterior pituitary gland of male and female transgenic mice, compared with their respective normal controls. In male transgenic mice, NKA was 13.6- and SP 20.2-fold higher than in the anterior pituitary from normal mice. In female transgenic mice, NKA was 40- and SP 100-fold higher than in the anterior pituitary from normal female mice. In male transgenic mice, NKA and neuropeptide K (NPK) contents in the anterior pituitary showed no significant changes between 26 and 50 days of age but significantly increased between 50 days and 5 months of age. The concentration of NKA in the anterior pituitary did not show significant differences between 26 days and 5 months of age, but NPK concentrations in the anterior pituitary significantly decreased with age. In female transgenic mice, NKA content and concentration in the anterior pituitary increased after 35 days of age, but NPK concentrations significantly decreased after 26 days of age. Triiodothyronine markedly decreased anterior pituitary tachykinins, but ovariectomy and estrogen administration failed to significantly affect tachykinin concentrations in the anterior pituitary of transgenic mice. Tachykinin immunostaining was detected in some somatotropes, but tachykinins were also present in cells that were not GH positive. These findings indicate that hyperstimulated somatotropes contain increased stores of tachykinins and that these cells are a source of tachykinins in the anterior pituitary. Tachykinin stores in the anterior pituitary of transgenic mice were affected by thyroid hormones but seem to be insensitive to estrogens. The GHRH transgenic mice may be an interesting model to study the regulation of tachykinin stores in the anterior pituitary gland.
Decreased Type 2 Corticotropin-Releasing Hormone Receptor mRNA Expression in the Ventromedial Hypothalamus during Repeated Immobilization Stress
Authors: Makino Shinya , Asaba Koichi , Nishiyama Mitsuru , Hashimoto Kozo
Published: April 2003

Chronic or repeated stress results in reduction of food intake and body weight in rats. Stress-induced anorexia has been attributed to increased corticotropin-releasing hormone (CRH) function in the central nervous system. To explore possible roles of other neuropeptides and peripheral hormones involved in food intake and energy utilization during continuing stress, we examined the impact of repeated immobilization stress on expression of mRNAs coding for CRH, neuropeptide Y (NPY), galanin and pro-opiomelanocortin (POMC) mRNAs in such hypothalamic nuclei as the paraventricular nucleus (PVN), arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH), as well as plasma insulin and leptin concentrations. Changes in type 2 CRH receptor (CRHR-2) mRNA in the ventromedial hypothalamus (VMH), a possible target of anorectic CRH effect, were also examined. Rats were immobilized for 2 h daily for 6 days and sacrificed 24 h after the last immobilization. Immobilized rats had lower food intake and body weight and higher levels of PVN CRH mRNA than controls. Repeated immobiliza tion also lowered plasma insulin and leptin concentrations and VMH CRHR-2 mRNA levels. These results provide additional evidence linking VMH CRHR-2 mRNA levels to plasma leptin concentration. ARC NPY and DMH galanin mRNAs increased following repeated immobilization, while ARC POMC mRNA decreased. DMH NPY mRNA and ARC galanin mRNA were unaltered by immobilization. Since NPY and galanin are considered orexigenic, while the POMC-melanocortin-4 receptor system is apparently anorexigenic, the changes in neuropeptide mRNAs and VMH CRHR-2 mRNA may play counterregulatory roles against anorectic CRH effects.
Activin A Regulation of Gonadotropin-Releasing Hormone Synthesis and Release in vitro
Authors: MacConell Leigh A. , Lawson Mark A. , Mellon Pamela L. , Roberts Veronica J.
Published: April 2003

Activin is essential for the regulation of normal mammalian reproductive function at both the pituitary and gonadal levels. However, its central actions in the control of the hypothalamic-pituitary-gonadal axis remain largely unexplored. The present study aims to determine whether activin could regulate the reproductive axis at the level of the hypothalamus, through control of the GnRH neuroendocrine system. Using the GnRH-secreting GT1-7 neuronal cell line as a model system, we demonstrate expression of mRNAs encoding activin receptor types I, IB, and II. We examined the effects of activin A on GnRH protein secretion and mRNA levels in GT1-7 cells. Treatment with rh-activin A regulated both GnRH protein secretion and GnRH mRNA expression in the GT1-7 cells in a time-dependent fashion. Using transient transfection assays, we explored a potential transcriptional basis for these changes. Activin A increased reporter gene activity driven by minimal GnRH enhancer and promoter elements, suggesting that activin may regulate GnRH gene expression at the level of transcription. Lastly, activin A treatment of male rat hypothalami, in vitro, increased GnRH protein secretion. Collectively, molecular and physiological evidence support the presence of an activin system which might act at a hypothalamic site to regulate mammalian reproduction via activation of GnRH synthesis and release.
On the Gender Differences in Sleep-Endocrine Regulation in Young Normal Humans
Authors: Antonijevic Irina A. , Murck Harald , Frieboes Ralf-Michael , Holsboer Florian , Steiger Axel
Published: April 2003

Sleep-endocrine regulation in humans involves high activity of the somatotropic axis at the beginning of the night and an increase in the hypothalamic-pituitary-adrenocortical (HPA) system during the night. Gender differences were examined with regard to sleep-endocrine regulation in young healthy controls (10 men, 9 women). The sleep EEG was recorded (23:00–07:00 h) and plasma samples were collected and analyzed for GH, cortisol and ACTH at 20-min intervals. Cortisol secretion was significantly higher in females during the first half of the night (F = 9.9, p < 0.05), while ACTH was not different. In women, sleep-EEG analysis showed less slow wave sleep (SWS) during the second half of the night (F = 4.5, p < 0.05) and a significantly greater decrease in SWS and delta activity from the first to the second half of the night (F = 3.7 and 7.4, respectively, p < 0.05). Sigma activity increased during the night in women only (F = 3.7, p < 0.05). Our data are compatible with the hypothesis that in women compared to men activity of hypothalamic CRH neurons and central CRH release is greater, but is not reflected by greater HPA activity.
Muscarinic Regulation of Basal versus Thyrotropin-Releasing Hormone-Induced Prolactin Secretion in Rat AnteriorPituitary Cells
Authors: Pu Hsiao-Fung , Tan Sai-Koong , Chen Hsing-Liang , Jea Jing-Chi , Liu Tsuei-Chu
Published: April 2003

Acetylcholine (ACh), synthesized in the pituitary, can act locally to modulate pituitary function. We used rat primary anterior pituitary (AP) cells to investigate how ACh affects pituitary prolactin (PRL) secretion in the presence or absence of known PRL regulators: thyrotropin-releasing hormone (TRH), 17β-estradiol (E2) and triiodothyronine (T3). Cultured AP cells were prepared from ovariectomized rats and pretreated with diluent, 0.6 nM E2, 10 nM T3, or E2 plus T3 for 5 days, then challenged with various doses of ACh or muscarinic receptor agonists (oxotremorine or carbachol) and TRH (100 nM) for 20 min. Significant ACh (10–5M) suppression of both basal and TRH-induced PRL secretion was not evident in diluent-, E2- or T3-pretreated cells, but observed only in cells pretreated with both E2 and T3. Moreover, in E2 plus T3-pretreated cells, oxotremorine and carbachol, like ACh (10–7–10–5M), suppressed both responses in a dose- related manner. Pertussis toxin (PTX; 100 ng/ml) as well as atropine (a muscarinic receptor antagonist; 1 mM) blocked these effects of cholinomimetics. ACh also inhibited both PRL responses elicited by drugs elevating intracellular cAMP (10 µM forskolin) or Ca2+ (1 µM Bay K-8644) in a PTX-sensitive manner. ACh inhibition of basal PRL secretion was unaltered by intracellular Ca2+ mobilization blockers, TMB-8 (100 µM) and thapsigargin (1 µM), but abrogated by the nitric oxide synthase inhibitor (300 µM L-NAME). ACh inhibition of TRH-induced PRL secretion was accentuated by TMB-8 and alleviated by thapsigargin or L-NAME. In summary, muscarinic inhibition of either basal or TRH-induced PRL secretion was augmented by E2 and T3, and involved the PTX-sensitive cAMP/Ca2+ pathways. Furthermore, nitric oxide mediated the basal rather than TRH-induced PRL response to ACh, whereas the intracellular Ca2+ mobilization concerned the TRH-induced rather than the basal PRL response to ACh. Thus, ACh synthesized in the AP appears to inhibit basal vs. TRH-induced PRL secretion via different mechanisms.
The Insulin-Like Growth Factor System in the GT1-7 GnRH Neuronal Cell Line
Authors: Anderson Richard A. , Zwain Ismail H. , Arroyo Armando , Mellon Pamela L. , Yen Samuel S. C.
Published: April 2003

Evidence suggests that insulin-like growth factors (IGFs; IGF-I and IGF-II) are involved in the regulation of reproductive function including the development of the gonadotropin-releasing hormone (GnRH) neuronal system and the modulation of GnRH secretory activities. To further characterize the regulatory role of the IGF system on GnRH neuronal function, we have examined the gene expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-binding proteins (IGFBPs) in a GnRH neuronal cell line (GT1-7 cells). The relative effects of IGFs and insulin on GnRH secretion by these cells was also investigated. RT-PCR analysis demonstrated IGF-I, IGF-II and IGF-IR mRNAs in GT1-7 cells. The mRNAs for IGFBP-2, -3, -4, -5 and -6 but not IGFBP-1 were also detected. Immunoreactive protein bands for IGFBP-2, -4 and -5 but not for other IGFBPs were demonstrated by Western blot with IGFBP-5 appearing to be the most abundant IGFBP secreted by GT1-7 cells. IGFBP-5 production by GT1-7 cells was stimulated by both IGF-I and IGF-II in a dose-dependent manner with approximately equal potency, whereas insulin caused no significant effect. GnRH secretion by GT1-7 cells treated with IGF-I or IGF-II but not insulin showed an increase (80–100%) at 2 h of treatment followed by a decrease (46%) at 6 h that continued up to 24 h. We conclude that the expression of IGFs, IGF-IR and IGFBPs and their interactions in the regulation of GnRH secretion by GT1-7 cells as demonstrated by our study provide a basis for an autocrine regulatory role for the IGF system in GnRH neuronal secretory activities.
Regional Serotonin Metabolism under Basal and Restraint Stress Conditions in the Brain of Transgenic Mice with Impaired Glucocorticoid Receptor Function
Authors: Farisse Jean , Boulenguez Pascale , Sémont Alexandra , Héry Francis , Barden Nicholas , Faudon Maxime , Héry Micheline
Published: April 2003

Transgenic (TG) mice deficient in glucocorticoid receptors (GR) were used in order to study the effects of a reduced GR function on adrenocorticotropin hormone and corticosterone plasma levels and on serotonin metabolism in different brain areas under basal resting conditions, after a 30-min restraint stress and 60 min after the end of the restraint stress. There was no difference in basal or stress-induced levels of either adrenocorticotropin hormone or corticosterone in control and TG mice, but the return of adrenocorticotropin hormone to basal values after the end of the stress was delayed in TG mice. Under basal conditions, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine was decreased only in the hippocampus of TG mice compared to controls. In the brain stem, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine increased compared to basal values after a 30-min restraint stress and values were still high 60 min after the end of the restraint stress in both control and TG mice. In the hippocampus, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine increased at the end of the stress and returned to basal levels 60 min later in control mice, whereas there was no change at the end of the stress but an increase 60 min later in TG mice. Finally there was no change in serotonin metabolism in the cortex, striatum or hypothalamus in either group or situation. Our results support the hypothesis of a tonic activation of serotonin turnover by corticosterone through GR in the mouse hippocampus. Moreover, stress-induced stimulation of serotonin metabolism in the brain stem and hippocampus appears to be delayed in TG mice compared to control mice. These results are particularly relevant for mood disorders such as depression where alterations of serotoninergic transmission might be secondary to an impairment of GR functions.
Generation of a Recombinant Herpes Simplex Virus Type 1 Expressing the Rat Corticotropin- Releasing Hormone Precursor: Endoproteolytic Processing, Intracellular Targeting and Biological Activity
Authors: Tomasec Peter , Preston Christopher M. , Linton Elizabeth A. , Ahmed Irshad , Lowenstein Pedro R. , Castro Maria G.
Published: April 2003

We describe the generation of a recombinant herpes simplex virus type 1 (HSV1) vector, tsK/CRH10, derived from the temperature-sensitive mutant tsK, expressing rat pre-procorticotropin-releasing hormone (ppCRH). In hypothalamic neurons, within the paraventricular and supraoptic nuclei, this neuropeptide precursor is processed to mature CRH (1–41), the key modulator of the hypothalamic-pituitary-adrenal stress response. We used the recombinant HSV1 tsK/CRH10 to study posttranslational processing, intracellular localization and biological activity of proCRH (pCRH) within neuronal, glial and epithelial cell lines. We showed that CRH-like immunoreactivity expressed in neuronal, glial and epithelial cells infected with tsK/CRH10 was biologically active, could be detected intracellularly and was also secreted. Our data also show that within Neuro2a and NG115 cells, the CRH precursor is cleaved to yield a CRH-like immunoreactive fragment of approximately 4.75 kD which could account for mature CRH (1–41). No endoproteolytic processing of the precursor takes place within the astrocytic 1321 NI cell line. Using immunocytochemistry techniques we detected CRH-like immunoreactivity within the endoplasmic reticulum-Golgi region in all cells and within secretory vesicles of Neuro2a and NG115 cells, suggesting correct targeting to the regulated secretory pathway within these cells. Our results demonstrate that the HSV1 recombinant vector expressing the full-length CRH precursor molecule constitutes an excellent delivery system for both cell lines and postmitotic neurons in vitro, which has enabled the study of targeting, endoproteolytic processing and biological activity of this neuropeptide precursor. Furthermore, it can also be used to generate transient transgenesis of the CRH precursor in vivo, to study neuroendocrine-immune interactions within the mammalian central nervous system.
Activation of Gene Expression of the γ-Aminobutyric Acid Rather than the Glutamatergic System in the Preoptic Area during the Preovulatory Gonadotropin Surge of the Rat
Authors: Leonhardt Sabine , Böning Bianca , Luft Heike , Wuttke Wolfgang , Jarry Hubertus
Published: April 2003

There is increasing evidence that in the rat prior to and during the preovulatory LH surge, release rates of GABA in the preoptic area (POA) are decreased while no such changes occurred in the mediobasal hypothalamus (MBH). In addition, GnRH release appears to be facilitated by an increased preoptic excitation of glutamate (GLU). To investigate whether such changes of secretory activity of intrahypothalamic GABA or GLU neurons are associated with altered gene expression of biosynthetic enzymes or transporter proteins characteristic for either neuronal system, we determined mRNA levels of the two forms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD65 and GAD67), the glutamate-synthesizing enzyme glutaminase (GLS), the GABA transporter type 1 (GAT-1) and the glutamate-aspartate transporter type 1 (GLAST). Competitive RT-PCRs using mutant cRNAs as internal standards were conducted with mRNA extracted from microdissected tissue of POA and MBH from diestrous, proestrous, and estrous rats. Proestrous animals were subgrouped according to their endocrine status as follows: ‘prior to’, on the ‘ascending’ or on the ‘descending’ limb of the LH peak, and ‘after the LH surge (post)’. During the preovulatory LH surge, mRNA concentrations of GAD67 and GAT-1 in the POA were significantly increased compared to those observed on diestrous (2.8-fold for GAD67 and 2.5-fold for GAT-1, p < 0.01), while in the MBH the amount of both mRNAs remained constant. The expression levels of GAD65, GLS and GLAST were without any changes in the POA as well as in the MBH. These findings support the hypothesis that in rats induction of the preovulatory LH surge is controlled at the level of GnRH perikarya, and suggest that altered activities of intrapreoptic GABA neurons at both transcriptional and secretory levels are pivotal for the preovulatory activation of GnRH neurons.
Far Upstream Sequences Regulate the Human Prolactin Promoter Transcription
Authors: van de Weerdt Cécile , Peers Bernard , Belayew Alexandra , Martial Joseph A. , Muller Marc
Published: April 2003

The human prolactin gene is mainly expressed in pituitary lactotrope cells, but transcription from an alternative, far upstream promoter was detected in lymphoid, placental and mammary cells. We describe the transcriptional activity in rat pituitary cells of the complete region separating the two promoters, using transient transfection experiments. A far upstream activating region was only functional in combination with the prolactin promoter. DNaseI protection experiments revealed, in addition to binding sites for the pituitary-specific factor Pit-1, sites (e.g. SD1) for several ubiquitous factors and one lymphoid-specific factor (SD4). A single copy of the ubiquitous site SD1 or the lymphoid-specific site SD4 was unable to activate transcription of a heterologous promoter in pituitary cells. However, SD1 activated transcription in nonpituitary cells and SD4 was functional specifically in lymphoid cells. Five copies of a distal site (D8) activated transcription in each cell type tested. Gel retardation experiments show that this site binds the specific factor C/EBP in liver and a distinct factor in other cell types. Our results suggest that different elements within this large region direct specific expression from each promoter via a complex interplay between cell-specific and ubiquitous transcription factors.
Contribution of Sex and Cellular Context in the Regulation of Brain Corticosteroid Receptors following Restraint Stress
Authors: Karandrea Despoina , Kittas Christos , Kitraki Efthimia
Published: April 2003

The two subtypes of corticosterone receptors in the rat brain play a pivotal role in the modulation of the stress response. Appropriate control of their gene expression is therefore critical for the maintenance of cellular and organism homeostasis. In this study, we investigated the contribution of gender and of the cellular environment of certain brain areas to the expression of both types of corticosteroid receptors, following restraint stress. Adult Wistar rats of both sexes were subjected to acute, chronic or to a combined chronic plus acute stress regimen, and the expression of glucocorticoid and mineralocorticoid receptors was evaluated in their hippocampus, hypothalamus, pituitary and frontal cortex, by using Northern blot analysis. Significant sex differences were observed in the first three brain areas examined as to the stress-induced expression of corticosteroid receptors. Among these, females showed a distinct mechanism of regulating glucocorticoid/mineralocorticoid receptor ratio in the hippocampus upon chronic stress, while the female hypothalamus was more prone than the male to changing corticosteroid receptor expression in response to restraint stress. In another set of experiments, we assessed the influence of ovarian steroids on stress-induced corticosteroid receptor expression in the above brain areas by analyzing ovariectomized rats exposed to short-term restraint. Our results showed that although ovarian steroids affect the stress-induced expression of receptor genes in a region-specific manner, their elimination does not appear to lead to the male pattern of expression. These findings provide further evidence for the existence of both regional and gender specificity in the regulation of brain and pituitary corticosteroid receptors following stress, and support the hypothesis of a distinct male and female neuroendocrine axis in response to stress.
Diurnal Variations in Lipopolysaccharide-Induced Sleep, Sickness Behavior and Changes in Corticosterone Levels in the Rat
Authors: Mathias Stefan , Schiffelholz Thomas , Linthorst Astrid C.E. , Pollmächer Thomas , Lancel Marike
Published: April 2003

Inoculation of rats with microorganisms or microbial constituents that activate host defense promotes non-rapid eye movement sleep (non-REMS) and suppresses REMS. In this study, we evaluated circadian influences on the effects of lipopolysaccharide (LPS) on sleep, sickness behavior and plasma corticosterone levels in the rat. Three sets of experiments were performed. In each, the animals were intraperitoneally injected with vehicle for LPS (30 µg/kg) during 2 consecutive days, at the beginning of either the circadian rest or the activity phase. In experiment 1, sleep-wake behavior and brain temperature were recorded, and in experiment 2, core body temperature, locomotor activity as well as food and water intake. In experiment 3, corticosterone blood levels were measured. The results show that LPS-evoked changes in temperature, sleep and other behavioral parameters depend markedly on the time of day LPS is administered. However, a direct comparison of the LPS data demonstrates that, except for sleep parameters, the absolute time course of the assessed parameters was rather similar between the rest and activity phases. These findings suggest that LPS evokes a state characterized by high temperature and low vigilance, which is reached independently of the circadian phase.
An Endogenous Serotonergic Rhythm Acting on 5-HT2A Receptors May Be Involved in the Diurnal Changes in Tuberoinfundibular Dopaminergic Neuronal Activity and Prolactin Secretion in Female Rats
Authors: Liang Shu-Ling , Pan Jenn-Tser
Published: April 2003

The central serotonergic system has long been known to have a stimulatory role on the secretion of prolactin (PRL). The integrity of serotonergic neurotransmission is essential for the expression of the estrogen-induced afternoon PRL surge. Whether its effect on PRL involves change in the activity of tuberoinfundibular dopaminergic (TIDA) neurons has not been ascertained. In adult ovariectomized rats treated with estrogen, depletion of central serotonin (5-HT) by 5,7-dihydroxytryptamine (5,7-DHT, 200 µg/rat, i.c.v.) effectively prevented the afternoon fall in TIDA neuronal activity (using the levels of 3,4-dihydroxyphenylalanine and 3,4-dihydroxyphenylacetic acid (DOPAC), and the ratio of DOPAC/dopamine in the median eminence as indices), and blunted the afternoon PRL surge. A single injection of a 5-HT2A receptor antagonist, ketanserin (5 mg/kg, i.p. at 12.00 h), also had the same effects on the diurnal changes in TIDA neuronal activity and PRL secretion as the treatment with 5,7-DHT did. Intracerebroventricular injection of a 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) in the morning inhibited the TIDA neuronal activity and stimulated PRL secretion in a dose-dependent manner; while injection of a 5-HT1 agonist, 8-hydroxy-dipropylaminotetralin, was without effect. Injection of DOI in 5,7-DHT-pretreated rats at 14.30 h also lowered the TIDA neuronal activity and reinstated the PRL surge. In all, endogenous 5-HT, acting through the 5-HT2A receptor, appears to exhibit an inhibitory effect on TIDA neuronal activity during the afternoon, which is essential for the PRL surge.
Involvement of Endogenous Opioidergic Neurons in Modulation of Prolactin Secretion in Response to Mating in the Female Rat
Authors: Yang Shu-Ping , Lee Youngsoo , Voogt James L.
Published: April 2003

Mating in female rats induces an acute prolactin (PRL) release within 60 min and twice-daily surges of PRL throughout the first 10 days of pregnancy to maintain luteal function. Little is known about the brain mechanism whereby the vaginocervical stimulation is processed to induce PRL release. Our recent results revealed an increase in Fos expression in the arcuate nucleus (ARC) following mating in the intact estrous rat, suggesting that a neuronal network in the brain area may participate in conveying and integrating the genitosensory stimulation. To further investigate the phenotype of activated neurons in the ARC, the present study examined whether β-endorphin (β-END) and/or dopamine (DA) neurons are activated by mating, and if so, whether activation is involved in the mating-induced acute release of PRL and the establishment of the twice-daily surges of PRL. In experiment 1, proestrous rats receiving intromissions (mated group) from males or mounts without intromission (mounted group) were sacrificed along with rats taken directly from their home cage (control group) 60 min after the beginning of mating or mounting. Expression of Fos in β-END neurons and expression of fos-related antigen (FRA) in DA neurons, which were labeled by tyrosine hydroxylase (TH) antibody in the ARC were examined by double-label immunocytochemistry. In experiment 2, proestrous females with indwelling atrial catheters were mated with males. Naloxone (10 µl/min, 2 mg/10 min), an opiate antagonist, or saline was infused before, during and after mating. Blood samples were collected during the mating session and also at several times 3 days after mating. The results showed that mating induced a significant increase in the percentage of β-END/Fos colabeled neurons and a significant decrease in the number of β-END cells in all subdivisions of the ARC. In contrast, neither the percentage of FRA/TH colabeled cells nor the number of TH cells was influenced by mating. Mating induced an acute increase in PRL release in saline-treated control animals within 30 min and a subsequent diurnal surge (18.00 h) and a nocturnal surge of PRL (2.00 h) 3 days after mating. Naloxone infusion during mating blocked the mating-induced acute PRL response and the diurnal surge of PRL 3 days after mating, but affected neither the nocturnal surge of PRL nor the incidence of pregnancy. These results demonstrate that (1) β-END neurons but not DA neurons in the ARC are activated in response to mating in proestrous rats, and (2) the mating-induced activation of β-END neurons may participate in the acute response of PRL release to mating and the memory mechanism for the establishment of the diurnal PRL surge, but not the nocturnal PRL surge in early pregnancy. These results lead to a conclusion that endogenous opioid peptides may be involved in the neuronal transmission of genitosensory stimulation to induce PRL secretion.
Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase-Activating Polypeptides Stimulate Mitogen-Activated Protein Kinase in the Pituitary Cell Line GH4C1 by a 3′,5′-Cyclic Adenosine Monophosphate Pathway
Authors: Le Péchon-Vallée Caroline , Magalon Karine , Rasolonjanahary Ramahefarizo , Enjalbert Alain , Gérard Corinne
Published: April 2003

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP38) regulate anterior pituitary cell secretion and proliferation. In the somatolactotrope GH4C1 cell line, these effects are mediated through the type-II-like PACAP receptor (VPAC2) coupled to the cAMP pathway. In this study, the control of the extracellularly responsive kinases (ERKs) by VIP and PACAP38 was investigated in GH4C1 cells. VIP and PACAP38 increased ERK1 and ERK2 phosphorylation and were equipotent stimulators of both kinases. ERK activation was mimicked by cholera toxin, forskolin and 8bromo-cAMP. VIP and PACAP38 activation of ERK2 was blocked by the protein kinase A inhibitor H89, whereas the protein kinase C inhibitor GF109203X, or prior PMA-induced depletion of the protein kinases C, failed to inhibit VIP and PACAP38 activation of ERK2. In contrast, thyrotropin-releasing hormone (TRH) elicited ERK activation by a PKC-dependent process. ERK activation by VIP or PACAP38 and TRH were additive and both sensitive to the MEK inhibitors PD98059 and U0126. In parallel, U0126 reduced prolactin (PRL) mRNA levels induced by VIP. These results demonstrate for the first time that VIP and PACAP38 activate ERK in GH4C1 cells. Cyclic AMP increase is sufficient to elicit ERK activation in these cells and thus likely to represent the transduction pathway underlying VIP- and PACAP38-dependent ERK activation. This mechanism seems to be involved in VIP-induced PRL gene regulation.
Sexual Dimorphism in Diethylstilbestrol-Induced Prolactin Pituitary Tumors in F344 Rats
Authors: Piroli Gerardo G. , Torres Alicia , Pietranera Luciana , Grillo Claudia A. , Ferrini Mónica G. , Lux-Lantos Victoria , Aoki Agustin , De Nicola Alejandro F.
Published: April 2003

Female F344 rats treated chronically with diethylstilbestrol (DES) develop prolactin (PRL)-producing pituitary tumors. These tumors are larger in female than in male rats. To investigate gender differences in DES-induced pituitary tumor formation, we employed female and male rats and neonatally androgenized females, which received 100 µg of testosterone propionate (TP) after birth. At 3 months of age, all rats were deprived of their gonads and divided into control and DES-treated groups. Forty days after beginning treatment, control pituitary weight and serum PRL were similar in gonadectomized males (GDX), ovariectomized females (OVX) and androgenized-ovariectomized females (OVX + TP), but weight of DES-induced tumors was 2.5-fold higher and serum PRL 5.6-fold higher in OVX + DES than in GDX + DES or OXV + TP + DES (p < 0.001). At the pituitary level, nuclear estrogen receptors (NE2R) amounted to >100 fmol/mg DNA in all rats receiving DES. However, NE2R were lower in OVX + DES (101.3 ± 9.0 fmol/mg DNA) than in GDX + DES (174.6 ± 16.8; p < 0.05) and in OXV + DES + TP (150.3 ± 27.7; p < 0.05). A similar profile was found for cytosolic progestin receptors. Using electron microscopy (EM), hyperplasia/hypertrophy of lactotropes was found in all DES-stimulated pituitaries. However, tumors of OVX + DES rats were enriched in hyperstimulated typical lactotropes, i.e., cells with high rate of hormonal synthesis, processing and secretion. Instead, tumors from GDX + DES and OVX + TP + DES rats were a mixture of typical and atypical lactotropes, i.e. a cell subpopulation with refractory secretory response and a few gonadotropes. In agreement with these data, immunoreactive pituitary PRL was lower in OVX + DES than in OVX + TP + DES and GDX + DES groups. Thus, differences in the sensitivity to DES, serum and tumor PRL, NE2R and progestin receptors between estrogenized female rats on one side and male and TP-androgenized females on the other, may by due in part to heterogeneity of cell populations. Our data further suggest that neonatal hypothalamic exposure to androgens, as in normal males or androgenized females with masculinization of hypothalamic centers, may condition the response to DES stimulation later in life.
Effects of Estradiol and Raloxifene Analog on Brain, Adrenal and Serum Allopregnanolone Content in Fertile and Ovariectomized Female Rats
Authors: Genazzani Andrea R. , Bernardi Francesca , Stomati Massimo , Monteleone Patrizia , Luisi Stefano , Rubino Silvia , Farzati Angelo , Casarosa Elena , Luisi Michele , Petraglia Felice
Published: April 2003

Allopregnanolone is a neuroactive steroid synthesized in rat gonads, adrenal cortex, and central nervous system. It has been suggested that sex steroid hormones might influence allopregnanolone concentrations but no clear data have ever been reported. The aim of the present study was to investigate the effects of administration of 17β-estradiol (17β-E2), the raloxifene analog LY-117018 or their combination on allopregnanolone levels in fertile and ovariectomized (OVX) rats. Thirteen groups of 12 Wistar female rats each received either 17β-E2 (0.1 or 1 µg/day) or LY-117018 (25, 250, and 1,250 µg/day), or 17β-E2 1 µg/day plus LY-117018: 25, 250, and 1,250 µg/day for 14 days. The rats were then sacrificed and allopregnanolone content was assessed in the hypothalamus, hippocampus, pituitary, adrenals, and serum. Ovariectomy determined a significant decrease in allopregnanolone content in the hypothalamus, hippocampus, pituitary, and serum, while increasing it in the adrenals (p < 0.01). In OVX rats, the administration of either 17β-E2 or LY- 117018 restored ovariectomy-induced allopregnanolone changes. The administration of LY-117018 in addition to 17β-E2 to OVX animals suppressed the increase in allopregnanolone levels determined by 17β-E2 in the hippocampus, hypothalamus, and pituitary, but not in the adrenals and serum. In fertile rats, the administration of LY-117018 reproduced the effects of ovariectomy. This study shows that the raloxifene analog LY-117018 has an estrogen-like action on the central nervous system of OVX rats when administered alone, while it acts as an antiestrogen in the presence of 17β-E2, both in OVX animals treated with 17β-E2 and in fertile rats. A different effect was observed in the adrenal glands. The mechanism of action of this compound has still to be clarified.
Effect of Oxidative Stress on the Uptake of GABA and Glutamate in Synaptosomes Isolated from Diabetic Rat Brain
Authors: Duarte Ana , Santos Maria , Seiça Raquel , Resende de Oliveira Catarina
Published: April 2003

It has been suggested that increased oxidative stress might be involved in the pathophysiology of diabetic complications. In this study, we investigated the effect of diabetes on the susceptibility of synaptosomes to oxidative stress (induced by the oxidizing pair ascorbate/Fe2+) and on the uptake of the amino acid neurotransmitters γ-aminobutyric acid (GABA) and glutamate. We found a lower susceptibility of synaptosomes isolated from Goto-Kakizaki (GK) rats, a model of non-insulin-dependent diabetes mellitus, to lipid peroxidation as compared with synaptosomes isolated from Wistar control rats (6.40 ± 1.05 and 12.14 ± 1.46 nmol thiobarbituric acid reactive substance/mg protein, respectively). The lower susceptibility of GK rat synaptosomes to membrane lipid peroxidation correlates with an increase in synaptosomal vitamin E levels (835 ± 58.04 and 624.26 ± 50.26 pmol/mg protein in diabetic and normal rats, respectively). In the absence of ascorbate/Fe2+, no significant differences were observed between the levels of lipid peroxidation of synaptosomes isolated from diabetic and normal rats. Studies of neurotransmitter uptake show that the [3H]glutamate uptake was decreased by about 30% in diabetic GK rats as compared with control Wistar rats, whereas the [3H]GABA uptake was not significantly different from controls. Under oxidizing conditions, the glutamate uptake in diabetic rats was unaffected, and a decreased GABA uptake (41.39 ± 4.41 and 60.96 ± 6.4% of control in GK and Wistar rats, respectively) was observed. We conclude that the increased resistance to oxidative stress in GK rat synaptosomes may be due to the increased vitamin E content and that diabetic state and oxidative stress conditions differentially affected the uptake of the neurotransmitters GABA and glutamate.