bluuebunny/crossref_metadata_2025 huggingface dataset:
Dexamethasone Rapidly Regulates TRH mRNA Levels in Hypothalamic Cell Cultures: Interaction with the cAMP Pathway
Authors: Pérez-Martínez Leonor , Carreón-Rodríguez Alfonso , González-Alzati Ma. Elena , Morales Claudia , Charli Jean Louis , Joseph-Bravo Patricia
Published: April 2003

The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations. To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1–3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways. TRH mRNA relative changes were determined by the RT-PCR technique. One hour incubation with 10–10–10–4 M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10–10M, an increase above control at 10–8–10–6M and a reduction at higher concentrations (10–5– 10–4M). The stimulatory effect of 10–8M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment. Changes in TRH mRNA levels were reflected by enhanced TRH cell content. Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively. An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells. The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment. The stimulatory effect of 10–7M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10–3M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs. An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone. These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.
Hypothalamic-Pituitary-Gonadal Axis in the Mutant Weaver Mouse
Authors: Schwartz Neena B. , Szabo Marta , Verina Tatyana , Wei Jianjun , Dlouhy Stephen R. , Won Lisa , Heller Alfred , Hodes Marion E. , Ghetti Bernardino
Published: April 2003

The weaver (wv) mutant mouse manifests severe locomotor defects, a deficiency in granule cells of the cerebellum, and cellular deficits in the midbrain dopaminergic system. The wv phenotype is associated with a missense mutation in the pore region of the G-protein-gated inwardly rectifying potassium channel, GIRK2. The homozygous male wv mouse is essentially infertile due to an inadequate level of sperm production. Females are fertile although they also manifest the neurological phenotype. Homozygotes of both sexes have reduced body weight. We have evaluated the hypothalamic-pituitary-gonadal axis in heterozygote and homozygote male and female wv mutants in comparison with wild-type controls. Testicular weight was significantly reduced in the homozygous males, due to degenerative changes of seminiferous epithelium. Serum and pituitary content of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were normal in all groups, and the normal sex differences were noted (FSH and LH higher in males, prolactin higher in females). Pituitary growth hormone (GH) concentration was normal, with control and mutant males showing higher GH than females. Serum testosterone levels were normal in the mutants, as was testicular testosterone. Testicular α-inhibin content was mildly reduced, but high in proportion to testicular weight. The defect in spermatogenesis appeared predominantly in the postmeiotic stages. In situ hybridization was consistent with expression of some GIRK2 mRNA isoforms in seminiferous epithelium. There were no significant differences between genotypes in the levels of dopamine, dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid in the mediobasal and preoptic hypothalamic regions. Homovanillic acid levels in these two areas were, however, reduced in wv homozygotes compared to wild-type animals. In the light of normal pituitary hormone levels, normal hypothalamic monoamine concentrations and normal sex differences in gonadotropins, we conclude that the infertility in the male homozygote wv mouse lies within the tubule and is probably a primary defect in the germ cells. The hormonal data suggest that Leydig cell function, and at least some aspects of Sertoli cell function, are normal in the mutant mice.
Transcription Factor STAT3 in Leptin Target Neurons of the Rat Hypothalamus
Authors: Håkansson Marie-Louise , Meister Björn
Published: April 2003

Leptin is an adipose tissue-derived hormone that regulates body weight via interactions with hypothalamic neuronal circuitries expressing specific leptin receptors (Ob-R). The Ob-Rs act via the JAK-STAT (Janus kinase-signal transducers and activators of transcription) pathway of signal transduction. Recent evidence suggests that primarily the transcription factor STAT3 mediates leptin’s action in the hypothalamus. We have investigated the presence and cellular localization of STAT3 protein in the rat hypothalamus by means of indirect immunofluorescence histochemistry using a rabbit polyclonal STAT3 antiserum. The antiserum identified a 92-kDa protein using Western blotting on rat hypothalamic homogenates, corresponding to the expected size of STAT3. STAT3 immunoreactivity was demonstrated in Ob-R-containing neurons of the paraventricular nucleus (parvocellular part), periventricular nucleus, arcuate nucleus and in the lateral hypothalamic area. Direct double-labeling showed presence of STAT3 immunoreactivity in neuropeptide Y (NPY)-containing neurons of the ventromedial part of the arcuate nucleus and in proopiomelanocortin (POMC)-containing neurons of the ventrolateral part of the arcuate nucleus. The results provide an anatomical basis for a leptin action mediated by STAT3 in Ob-R-containing NPY and POMC neurons of the arcuate nucleus, as well as by Ob-R-containing neurons of the parvocellular paraventricular nucleus and lateral hypothalamic area.
Prohormone Convertases 1 and 2 Process ProPACAP and Generate Matured, Bioactive PACAP38 and PACAP27 in Transfected Rat Pituitary GH4C1 Cells
Authors: Li Min , Shuto Yujin , Somogyvári-Vigh Anikó , Arimura Akira
Published: April 2003

Pituitary adenylate cyclase-activating polypetide (PACAP) exists in two amidated forms, PACAP38 and PACAP27, which are expressed in the magnocellular and parvocellular neurons of the paraventricular nucleus (PVN) and the magnocellular neurons of the supraoptic nucleus (SON) of the hypothalamus. The prohormone convertases PC1 and PC2, subtilisin-like PCs of the Kex2 family, are expressed in neuroendocrine cells. Immunocytochemistry and in situ hybridization of PC1 and PC2 in the hypothalamus have shown that PC1 and PC2 are also present in the PVN and SON. Therefore, it is possible that the precursor of PACAP is processed by PC1 and/or PC2 in the hypothalamic nuclei and then converted to its mature forms. To test this hypothesis, rat pituitary GH4C1 cells were supertransfected with human PACAP cDNA and either rat PC1 or PC2 cDNA. The acid extracts of these cells were analyzed by reversed-phase HPLC for proPACAP, PACAP38 and/or PACAP27 radioimmunoassays using three antibodies with different recognition sites, and then bioassayed for the ability to stimulate adenylate cyclase. The cells transfected with PACAP cDNA alone yielded PACAP-like immunoreactivity (PACAP-li) corresponding to molecular weights between 15 and 20 kDa without PACAP bioactivity. Cotransfection of these cells with PC1 or PC2 generated PACAP-li, which coeluted with synthetic PACAP38 and PACAP27, respectively. Western blot also revealed 4.5- and 3.0-kDa PACAP-li bands, which correspond to the molecular weights of PACAP38 and PACAP27, respectively. The HPLC fractions containing PACAP-li, which were coeluted with synthetic PACAP38 and PACAP27, showed marked bioactivities. These findings suggest that the precursor of PACAP expressed in the PVN and SON of the hypothalamus could be efficiently processed by PC1 and PC2, and then converted to mature, bioactive PACAP38 and PACAP27.
Neurotransmitter Modulation of Glucocorticoid Receptor mRNA Levels in the Rat Hippocampus
Authors: Tritos Nikos , Kitraki Efthimia , Philippidis Helen , Stylianopoulou Fotini
Published: April 2003

Glucocorticoids in the hippocampus mediate adaptive responses elicited by stressful stimuli. In this study we investigated glucocorticoid receptor gene expression in the rat hippocampus following acute stress. A significant decrease in glucocorticoid receptor mRNA levels was observed in the hippocampus less than 1 h after the onset of stress. This decrease was inhibited by administering either MK-801, diazepam or propranolol prior to exposure to stress. The effect of diazepam on the stress-induced decrease in hippocampal glucocorticoid receptor mRNA was reversed by Ro-15-1788, suggesting that it is mediated by central benzodiazepine receptors, i.e. GABA-A. These results indicate that NMDA, GABA-A and β-adrenergic receptors are involved in the mechanism of the stress-induced decrease in glucocorticoid receptor mRNA levels in the rat hippocampus.
Corticotropin-Releasing Effect of Hexarelin, a Peptidyl GH Secretagogue, in Normal Subjects Pretreated with Metyrapone or RU-486, a Glucocorticoid Receptor Antagonist, and in Patients with Addison’s Disease
Authors: Arvat Emanuela , Ramunni Josefina , Maccagno Barbara , Giordano Roberta , Broglio Fabio , Deghenghi Romano , Boscaro Marco , Ghigo Ezio
Published: April 2003

GH secretagogues (GHS) are peptidyl and nonpeptidyl molecules which possess strong GH-releasing activity but also stimulatory effect on hypothalamo-pituitary-adrenal axis. The ACTH and cortisol responses to Hexarelin (HEX), a peptidyl GHS, are abolished by low-dose dexamethasone pretreatment in normal subjects but are exaggerated and higher than those after hCRH in patients with pituitary ACTH-dependent Cushing’s disease, in spite of their hypercortisolism. Based on the foregoing, we studied the ACTH, cortisol and GH responses to HEX (2.0 µg/kg i.v. at 0 min) alone and after metyrapone (2 g p.o. at 23:00 h the night before) or RU-486 (400 mg p.o. at 02:00 h), a glucocorticoid receptor antagonist, in 6 normal women (NS, age 26–34 years). The endocrine responses (mean ± SEM) to HEX alone were also studied in 8 patients with Addison’s disease (AD, 6 males, 2 females, age 30–77 years; last hydrocortisone administration the day before testing). In NS, HEX stimulated basal ACTH (peak, mean ± SEM: 26.0 ± 7.8 vs. 10.7 ± 2.0 pg/ml, p < 0.05), cortisol (163.2 ± 18.3 vs. 137.4 ± 15.4 µg/l, p < 0.05) and GH (72.6 ± 23.5 vs. 3.7 ± 1.3 µg/l, p < 0.01) levels. Metyrapone markedly increased basal ACTH (294.4 ± 61.6 pg/ml, p < 0.05), reduced basal cortisol (19.6 ± 7.2 µg/l, p < 0.05), while it did not modify GH levels. After metyrapone pretreatment the ACTH response to HEX was clearly increased (ΔAUC: 2,857.4 ± 901.9 vs. 367.3 ± 274.0 pg/ml/h, p < 0.05), while the GH response was not modified. HEX did not stimulate the low cortisol levels after metyrapone pretreatment. RU-486 significantly increased basal ACTH (76.6 ± 12.5 pg/ml, p < 0.05) and cortisol (312.7 ± 22.2 µg/l, p < 0.05), while it did not modify basal GH levels. RU-486 pretreatment did not modify the ACTH, cortisol and GH responses to HEX. In AD, HEX elicited a marked ACTH response (6,619.4 ± 3,365.8 pg/ml/h; p < 0.01), which was clearly higher (p < 0.01) than that in NS after HEX alone but not significantly different from that after HEX+MET. The GH response to HEX in AD (1,325.6 ± 284.1 µg/l/h) was similar to that in NS (1,519.7 ± 483.8 µg/l/h). In conclusion, our present data demonstrate that the ACTH-releasing activity of HEX is increased in primary hypoadrenalism as well as in normal subjects after metyrapone but not after RU-486 pretreatment. These findings indicate that in normal subjects as well as in hypocortisolemic patients the ACTH-releasing activity of GHS is enhanced by the lack of negative glucocorticoid feedback.
Leptin116–130 Stimulates Prolactin and Luteinizing Hormone Secretion in Fasted Adult Male Rats
Authors: Gonzalez Lucas C. , Pinilla Leonor , Tena-Sempere Manuel , Aguilar Enrique
Published: April 2003

Leptin is a hormone secreted by adipocytes with an important role in the control of feeding behavior and neuroendocrine function. Leptin stimulates in vivo LH secretion in fasted female rats and in vitro PRL secretion. Recent data indicate that leptin116–130, an active fragment of the native molecule, exerts effects similar to those of the native peptide on body weight and food intake. The present study was carried out to determine whether this fragment is also able to stimulate LH and PRL secretion. Adult male rats fasted for 5 days were injected with saline or leptin116–130 (15 µg i.c.v.) and LH and PRL concentrations were measured thereafter at 15-min intervals during a 150-min period. Administration of leptin116–130 increased the frequency of LH pulses (2.0 ± 0.26 vs. 1.20 ± 0.37/150 min; p ≤ 0.05), mean LH levels (0.24 ± 0.06 vs. 0.07 ± 0.03 ng/ml; p ≤ 0.05), LH pulse amplitude (0.33 ± 0.10 vs. 0.10 ± 0.05 ng/ml; p ≤ 0.05) and the net LH secretion estimated by area under curve (AUC: 36 ± 8.5 vs. 9 ± 3.9 ng/ml/150 min; p ≤ 0.01). In addition leptin116–130 increased the frequency of PRL pulses (2.83 ± 0.48 vs. 1.33 ± 0.21/150 min; p ≤ 0.05), trough PRL levels (5.71 ± 0.99 vs. 2.72 ± 0.32 ng/ml; p ≤ 0.01), mean PRL levels (13.02 ± 0.92 vs. 5.89 ± 0.51 ng/ml; p ≤ 0.01) and net PRL secretion (AUC: 1,625 ± 171 vs. 658 ± 46 ng/ml/150 min; p ≤ 0.05). In conclusion, these data show that leptin116–130 stimulates LH and PRL secretion in fasted adult male rats.
Localization of Estrogen-Receptive Neurons Projecting to the GnRH Neuron-Containing Rostral Preoptic Area of the Ewe
Authors: Goubillon Marie-Laure , Delaleu Bernadette , Tillet Yves , Caraty Alain , Herbison Allan E.
Published: April 2003

Estrogen exerts important feedback effects upon the biosynthetic and secretory behavior of gonadotropin-releasing hormone (GnRH) neurons to control reproductive functioning. The mechanism of estrogen action upon these neurons is unclear and seems likely to involve the transsynaptic regulation of GnRH neurons. The objective of the present study was to identify the estrogen-receptive neural populations which project to the general vicinity of the GnRH perikarya in the rostral preoptic area and diagonal band of Broca (rPOA/DBB) of the ewe. Intact breeding-season ewes received an injection of the retrograde tracer fluorogold (FG) into the rPOA/DBB, and their hypothalami and brainstems examined for the presence of FG and estrogen receptor α (ERα) immunocytochemistry. Retrogradely labeled neurons were identified principally within the lateral septum (LS), lamina terminalis, bed nucleus of the stria terminalis, POA, arcuate nucleus (ARN), ventromedial nucleus (VMN) and median eminence. Smaller numbers of FG-immonoreactive cells were found in the caudal brainstem where they resided mostly in the ventrolateral medulla (VLM). Dual-labeled cells exhibiting both FG and ERα staining were prominent in the POA, LS and at all rostrocaudal levels of the VMN and ARN. Small numbers of dual-labeled cells were found in the VLM. These observations indicate that a number of distinct ERα-expressing neural populations project to the rPOA/DBB where the majority of the GnRH perikarya are found in the ewe. Although it is not possible to determine the direct connectivity of these projections with GnRH neurons, the findings provide an initial neuroanatomical framework through which the transsynaptic actions of estrogen on ovine GnRH neurons may be tested.
Androgen Regulation of Immunological and Biological Activities of Pituitary Follicle-Stimulating Hormone Isoforms inMale Rats
Authors: Rulli Susana B. , Creus Silvina , Pellizzari Eliana , Cigorraga Selva B. , Calandra Ricardo S. , Campo Stella
Published: April 2003

Follicle-stimulating hormone (FSH) is involved in the regulation and maintenance of gametogenesis. It exists in multiple molecular forms with different oligosaccharide structures which in turn are influenced by the hormonal milieu. Previous studies from our laboratory demonstrated that antiandrogen administration to immature male rats altered the biological activity and the distribution profile of pituitary FSH isoforms. The aim of this study was to examine possible modifications in pituitary FSH polymorphism throughout sexual development (10-, 32- and 75-day-old rats). In addition, the effect of androgen deprivation by castration (32-day-old rats) and its replacement with a nonaromatizable androgen – dihydrotestosterone – on pituitary FSH polymorphism was determined. Concanavalin A affinity chromatography was used to isolate groups of FSH isoforms according to their carbohydrate inner structure. Radioimmunoassay and Sertoli cell bioassay were used to evaluate FSH immuno- and bioactivities. Androgen rise in serum was accompanied by a marked increase in pituitary bio- and immuno-FSH content in 32- and 75-day-old rats. However, FSH pituitary content did not vary despite the significant increment observed in serum FSH levels after castration and decrease to control levels after androgen replacement. The distribution profile of immuno- and bioactive FSH changed throughout sexual maturation. The proportion of pituitary FSH isoforms bearing complex oligosaccharide structures (triantennary, bisecting, complete and truncated biantennary) increased with age, with a concomitant decrease in the proportion of isoforms bearing incomplete carbohydrate chains. The distribution profile observed in castrated 32-day-old rats was similar to that determined in 10-day-old animals. Androgen replacement restored the distribution profile to normal. These results suggest that androgens regulate the incorporation of sugar residues to the carbohydrate chains of pituitary FSH favoring the biosynthesis of complex-type oligosaccharide structures.
Patch-Clamp-Induced Perturbations of [Ca2+]i Activity in Somatotropes
Authors: Robert Christophe , Tseeb Vadim , Kordon Claude , Hammond Constance
Published: April 2003

Somatotropes and GC cells, a GH-producing cell line, exhibit [Ca2+]i oscillations that result from rhythmic Ca2+ action potentials. Determination of this operating mode required simultaneous recording of both parameters by fura-2 imaging and patch-clamp techniques. In order to test whether patch recording induces artificial alteration of the [Ca2+]i oscillatory pattern, we recorded separately or simultaneously [Ca2+]i and membrane potential. In the absence of any other stimulation, seal formation in patch-clamp recording evoked by itself a 2.5- to 4-fold persistent increase in basal [Ca2+]i, speeded up their frequency (from 0.03–0.17 to 0.4 Hz) and changed their pattern to a tonic mode. Patch-induced [Ca2+]i increase was reproduced by mechanical contact between the pipette and the membrane. It was reduced by nifedipine, a blocker of L-type Ca2+ channels, as well as by removal of external Na+. It was fully blocked by external Ca2+ removal or gadolinium. All patch-clamp-induced perturbations were reversed by membrane hyperpolarization. We propose that patch-clamp recording evokes Ca2+ entry through L-type Ca2+ channels either directly, or indirectly via membrane depolarization. This shows that patch recordings in endocrine cells showing mechanosensitivity have to be interpreted with caution, and explains why long-lasting patch recordings are so difficult to obtain.
Expression of Vasoactive Intestinal Peptide/Peptide Histidine Isoleucine in Several Hypothalamic Areas during the Turkey Reproductive Cycle: Relationship to Prolactin Secretion
Authors: Chaiseha Yupaporn , El Halawani Mohamed E.
Published: April 2003

The present study examined the changes in vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) gene expression in the hypothalamus and compared their level of expression to prolactin (PRL) secretion throughout the turkey reproductive cycle. Both VIP and PHI expression were determined using in situ hybridization histochemistry and reverse transcription-polymerase chain reaction/Southern blot analysis. Plasma PRL levels were determined by radioimmunoassay. The changes in total hypothalamic VIP and PHI mRNA steady-state levels mirrored the changes seen in plasma PRL across the reproductive stages. In situ hybridization revealed that VIP mRNA was highly expressed throughout the hypothalamus and predominated within the ventromedial nucleus (VMN), inferior hypothalamus, and infundibular nuclear complex (INF). PHI mRNA was expressed in very low abundance within the same areas expressing VIP mRNA. VIP mRNA abundance within the VMN and INF was lowest in nonphotostimulated hens (VMN, 13.8 ± 1.7; INF, 17.0 ± 1.8 arbitrary densitometric units (ADU)), intermediate in laying hens (VMN, 29.6 ± 3.3; INF, 35.4 ± 4.3 ADU), and highest in incubating hens (VMN, 76.4 ± 10.2; INF, 119.2 ± 3.4 ADU). Levels decreased when birds shifted from incubation to photorefractoriness (VMN, 75%; INF, 82%). This relationship was not observed within other areas of the hypothalamus. The expression of PHI mRNA was also highest in the VMN and INF of incubating hens but no correlation between PHI mRNA and the other reproductive states was observed. This study provides additional evidence that VIP is the avian PRL-releasing factor, and suggests that the central site for avian PRL regulation lies within the INF of the hypothalamus.
Galanin’s Functional Significance in the Regulation of the Neuroendocrine Reproductive Axis of the Monkey
Authors: Finn Patricia D. , Pau K.-Y. Francis , Spies Harold G. , Cunningham Matthew J. , Clifton Donald K. , Steiner Robert A.
Published: April 2003

Galanin stimulates the neuroendocrine reproductive axis in the rat, but whether galanin acts similarly in primate species is unknown. To test the hypothesis that galanin acts within the hypothalamo-hypophyseal axis to stimulate luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH) secretion in the primate, galanin was administered either systemically or directly into the arcuate nucleus-median eminence of ovariectomized macaques (pigtailed or rhesus, respectively) that were maintained on estradiol. The mean plasma levels of LH were significantly elevated in pigtailed macaques after peripheral injection of galanin (2 mg) as compared with vehicle treatment. In rhesus monkeys, galanin (80 µM) administered by push-pull perfusion into the arcuate nucleus-median eminence did not significantly alter either GnRH or LH release. To determine whether in the monkey, as in the rat, subpopulations of medial forebrain GnRH neurons coexpress galanin mRNA, we used single- and double-label in situ hybridization and computerized imaging techniques. GnRH mRNA-containing cells were identified in both the medial and lateral forebrain of the female pigtailed macaque. No galanin mRNA expression was detectable in GnRH neurons located in either the medial preoptic area or mediobasal hypothalamus; however, within the substantia innominata a subset of GnRH mRNA-expressing neurons did coexpress galanin mRNA. Taken together, these results suggest that galanin induces LH release in primates, but galanin may not act directly on hypothalamic GnRH neurons. Presently, we have confirmed in another primate species the existence of GnRH gene expression in the lateral forebrain and discovered that a small subset of these neurons coexpress galanin. These particular cells may have a unique and as of yet undefined physiological function that is distinct from those GnRH neurons serving a hypophysiotropic function.
Acute Effects of Estradiol Pretreatment on the Response to d-Amphetamine in Women
Authors: Justice Angela J.H. , de Wit Harriet
Published: April 2003

Little is known about the interactions between ovarian hormones and responses to psychoactive drugs in humans. Preclinical studies suggest that ovarian hormones such as estrogen and progesterone have direct and indirect central nervous system actions and that these hormones can influence behavioral responses to psychoactive drugs. In the present study, we assessed the subjective and physiological effects of d-amphetamine (AMPH; 10 mg p.o.) after pretreatment with estradiol. Two groups of healthy, regularly cycling women participated in two sessions scheduled during the early follicular phases of two menstrual cycles. One group received estradiol patches (Estraderm TTS; 0.8 mg) which elevated plasma estradiol levels to approximately 750 pg/ml on both sessions; the other group received placebo patches on both sessions. Both groups received AMPH (10.0 mg) and placebo in a randomized and counterbalanced order on the two sessions. Dependent measures included self-report questionnaires, physiological measures, and plasma hormone levels. Most of the subjective and physiological effects of AMPH were not affected by acute estradiol treatment. Nevertheless, estradiol pretreatment increased the magnitude of the effects of AMPH on subjective ratings of ‘pleasant stimulation’ and decreased ratings of ‘want more’. Also, estradiol produced some subjective effects when administered alone: It increased subjective ratings of ‘feel drug’, ‘energy and intellectual efficiency’, and ‘pleasant stimulation’. These results provide limited evidence that the stimulating effects of AMPH are increased by acute estradiol pretreatment.
Potentiated Response of Corticotropin (ACTH) to Repeated Moderate Hemorrhage Requires Amygdalar Neuronal Processing1
Authors: Lilly Michael P. , Putney Dennis J. , Carlson Drew E.
Published: April 2003

We examined the role of the amygdala in the potentiation of the corticotropin (ACTH) response to a 10 mg/kg hemorrhage by a 1-hour episode of equivalent hypovolemia done 24 h earlier. Unanesthetized rats were studied on the fourth (D1) and fifth (D2) day after chronic implantation of arterial and venous catheters. Immunocytochemistry for Fos protein indicated that neurons in the central and medial nuclei of the caudal amygdala were activated by hemorrhage. We then tested the effect of excitotoxic destruction of the neurons in these areas by bilateral injections of ibotenic acid 10 days prior to catheter placement. In rats that were hemorrhaged on both D1 and D2, the responses of ACTH and corticosterone increased significantly from the first (H1) to the second hemorrhage (H2) in a control group injected with saline (p < 0.05) and in lesioned groups without bilateral damage of the Fos-responsive areas (p < 0.01). In the group with bilateral damage to these sites, the responses to H1 and H2 did not differ. Additional rats had H1 on D2 to control for the long-term effects of the chronic cannulation. The responses of ACTH to H1 on either D1 or D2 did not differ between the saline-injected controls and any of the lesioned groups. In contrast, the response of ACTH to H2 on D2 in rats with bilateral damage of the caudal amygdala was not significant and was less than the response of ACTH to H2 in both rats with unilateral damage of this area (p < 0.05) and those injected with saline (p < 0.05). We conclude that bilateral neuronal processing within the caudal amygdala is required for the potentiation of the response of ACTH to H2 by H1.
Effect of Suckling on Prolactin Receptor Immunoreactivity in the Hypothalamus of the Rat
Authors: Pi Xiujun , Voogt James L.
Published: April 2003

Previous studies showed that expression of prolactin (PRL) receptor is increased in numerous hypothalamic nuclei in mid-lactating rats. The increase in PRL receptor expression could be initated by neurohormonal changes during proestrus or pregnancy, or by the suckling stimulus during lactation. The present study investigated whether the PRL receptor expression in numerous hypothalamic nuclei is altered by the suckling stimulus. Three groups (n = 4) of rats on lactation day 10 were used: a continuously suckled group, a nonsuckled group (pups removed for 12 h) and a resuckled group (pups removed for 12 and then resuckled for 9 h). Animals were perfused with 2% paraformaldehyde and brains were sectioned (20 µm) for the immunofluorescence study. Immunoreactivity was semiquantitatively analyzed by counting the immunoreactive cells and measuring the immunostaining intensity in a specific area. Neurons expressing PRL receptors were observed in numerous hypothalamic areas with the highest number being in the arcuate, paraventricular and supraoptic nuclei. The PRL receptor immunofluorescence in several nuclei was significantly decreased in the nonsuckled group, and recovered in the resuckled group. These areas included the ventromedial preoptic, ventrolateral preoptic, lateroanterior hypothalamic, ventrolateral hypothalamic and ventromedial hypothalamic nuclei. PRL receptor immunoreactivity in other areas was not significantly altered by the suckling stimulus. These results demonstrate that expression of PRL receptor in hypothalamic nuclei was differentially affected by the suckling stimulus. PRL receptors in those nuclei which were significantly altered by suckling stimulus may play more critical roles during lactation than those areas which were less sensitive to the suckling stimulus.
Induction of Pituitary Adenylate Cyclase-Activating Polypeptide mRNA in the Medial Parvocellular Part of the Paraventricular Nucleus of Rats following Kainic-Acid-Induced Seizure
Authors: Nomura Masayoshi , Ueta Yoichi , Hannibal Jens , Serino Ryota , Yamamoto Yukiyo , Shibuya Izumi , Matsumoto Tetsuro , Yamashita Hiroshi
Published: April 2003

We examined the effects of kainic acid (KA)-induced seizure on the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) gene in the paraventricular nucleus (PVN) of rats using in situ hybridization histochemistry. Subcutaneous administration of KA (12 mg/kg) in adult male Sprague-Dawley rats caused a progressive development of seizure behavior. An induction of the PACAP gene expression in the medial parvocellular part of the PVN (mpPVN) was observed 3, 6, 12, 24 and 48 h after subcutaneous administration of KA. From a nearly undetectable level, PACAP gene expression increased in the mpPVN and reached maximum 12 h after subcutaneous administration of KA. PACAP gene expression returned to near basal level 48 h after stimulation with KA. Using a specific monoclonal PACAP antibody, PACAP immunoreactivity (-IR) gradually increased during the following 24 h after KA administration. In controls, PACAP-IR was located exclusively in nerve fibers of the mpPVN, whereas KA administration induced PACAP-IR in cell bodies of the mpPVN, and a dense accumulation of PACAP-IR nerve fibers in the external zone of the median eminence was observed. Induction of the PACAP gene expression following KA-induced seizure was significantly reduced by pretreatment with diazepam or MK-801 (nonselective N-methly-D-aspartate receptor antagonist). These results suggest that PACAP in the hypothalamo-adenohypophysial system may have a hypophysiotropic role during KA-induced seizure.
Galanin-Like Peptide (GALP) mRNA Expression Is Restricted to Arcuate Nucleus of Hypothalamus in Adult Male Rat Brain
Authors: Larm Jari A. , Gundlach Andrew L.
Published: April 2003

Galanin-like peptide (GALP) is a novel 60-amino acid neuropeptide, isolated from porcine hypothalamus and subsequently identified in rats and humans, which has reported selectivity for the Gal-R2 galanin receptor [Ohtaki T et al: J Biol Chem 1999; 274: 37041–37045]. In the current study, the regional and cellular distribution of GALP mRNA in rat brain has been investigated by in situ hybridization of [35S]-labelled oligonucleotide probes. In a thorough screening of adult male rat brain, GALP mRNA expression was detected only throughout the rostrocaudal extent of the arcuate nucleus (ARC) with the most abundant hybridization signal in the posterior, periventricular zones. GALP mRNA-positive neurons were mostly localized in the ventromedial division of the ARC, with many closely adjacent to the wall of the third ventricle. Smaller numbers of labelled neurons were also found in ventrolateral areas. The distribution of GALP mRNA was somewhat complementary to that for galanin (GAL) mRNA in the ARC, but contrasted with the broad distribution of this transcript throughout the hypothalamus. GAL mRNA was also distributed along the rostrocaudal extent of the ARC, but was most abundant in the anterior to middle levels and in ventrolateral regions. Interestingly, somatostatin mRNA expression appeared to overlap the distribution of GALP mRNA in posterior, ventromedial regions of the ARC. Thus, in adult rat brain GALP mRNA expression was restricted to a discrete subpopulation of neurons in the ARC, with a unique localization pattern unlike GAL or many other known peptide- and transmitter-containing cells in this region. GALP could, however, be co-expressed in sub-populations of other neuronal phenotypes (e.g. somatostatin neurons) or within cells that express Gal-R2 receptors. In view of the established anatomy and function of the ARC and the restricted localization of GALP mRNA, this novel peptide is likely to play a role in regulation of anterior pituitary hormone secretion, or in regulation of other hypothalamic peptide and transmitter systems.
Adrenergic and Cholinergic Regulation of in vitro Melatonin Release during Ontogeny in the Pineal Gland of Long Evans Rats
Authors: Wagner Gabriela , Brandstätter Roland , Hermann Anton
Published: April 2003

Melatonin, produced by the pineal gland, plays an important role in a great variety of neuroendocrine functions. The rhythmic release of melatonin by the mammalian pineal gland is regulated by norepinephrine (NE) acting via α- and β-adrenergic receptors utilizing distinct signal transduction pathways. Acetylcholine has been demonstrated to exert various effects in the mammalian pineal gland, including an inhibitory action on the NE-induced stimulation of melatonin production. However, data obtained by different laboratories on the interaction of adrenergic receptors are not consistent and whether muscarinic and/or nicotinic receptors participate in the various effects of acetylcholine is still contradictory. To investigate noradrenergic as well as cholinergic mechanisms during ontogeny, we have investigated in vitro melatonin release from isolated pineal glands of Long Evans rats of different ages. NE as well as the β-adrenergic receptor agonist isoproterenol (ISO) significantly elevated the melatonin release in pineal glands from postnatal week 2 on. In pineal glands originating from 2- to 4-week-old rats, simultaneous activation of α- and β-adrenergic receptors by ISO and the α-adrenergic receptor agonist methoxamine (MET) or NE resulted in significantly weaker stimulation of melatonin production than β-receptor activation alone. Acetylcholine evoked a significant increase in melatonin release in pineal glands from 2- to 4-week-old rats. In pineal glands from 8- to 20-week-old animals, ISO, ISO + MET or NE stimulated pineal melatonin release to comparable maxima, whereas acetylcholine was without effect. Our data indicate (1) that the adrenergic stimulation of pineal melatonin production in Long Evans rats is dominated by a β-adrenergic mechanism, (2) that additional α-adrenergic receptor activation is inhibitory and (3) dependent on the developmental status of the animal, and (4) that acetylcholine acting via muscarinic receptors has the capacity to stimulate melatonin release during early ontogeny. These data suggest that the melatonin-generating system of the pineal gland of Long Evans rats undergoes substantial functional changes during early postnatal development, including adrenergic as well as cholinergic mechanisms.
Identification of Angiotensin IV Binding Sites in the Mouse Brain by a Fluorescent Binding Study
Authors: von Bohlen und Halbach Oliver , Albrecht Doris
Published: April 2003

In the brain the renin-angiotensin system has been implicated in the regulation of the cardiovascular system as well as of regulation of reproductive hormones, water balance and behavior. Angiotensin II (Ang II) binds to two distinct receptors called AT1 and AT2. Recently a binding site has been discovered which selectively binds a fragment of Ang II. This biologically active Ang II 3–8 fragment is referred to as angiotensin IV (Ang IV) and has been reported to participate in memory acquisition and recall as well as in the regulation of blood flow, neurite outgrowth, angiogenesis and kidney function. The distribution of binding sites for Ang IV within the mouse brain is unknown. Using a nonradioactive binding assay with a fluorescence-coupled Ang IV, we were able to demonstrate specific Ang IV binding sites in the forebrain of mice, in particular in the cortex, the hippocampus, the amygdala, the thalamus and the hypothalamus.
Serotonin Inhibits Luteinizing Hormone Release via 5-HT1A Receptors in the Zona incerta of Ovariectomised, Anaesthetised Rats Primed with Steroids
Authors: Siddiqui Arif , Kotecha Krishna , Salicioni Ana-Maria , Kalia Veena , Murray Joanne F. , Wilson Catherine A.
Published: April 2003

The zona incerta (ZI), an area in the dorsal hypothalamus, contains neuronal systems that appear to control gonadotropin release. Previous findings show that there is an inverse relationship between serotonin (5-HT) activity in the ZI and plasma luteinizing hormone (LH) levels, indicating that the 5-HT system in this area has an inhibitory effect on LH release. Employing anaesthetised, ovariectomised rats primed with 5 µg oestradiol benzoate followed at 48 h by 0.5 mg progesterone, we have shown that 2 µg/side 5-HT in the ZI inhibits the LH surge that normally occurs 4 h after the progesterone treatment. This effect was mimicked by 2 µg/side 8-OH-DPAT, a 5-HT1A agonist, but not by DOI, a 5-HT2 agonist, BMY7378, a presynaptic 5-HT1A agonist or MCPP, a 2B & 2C agonist. The inhibitory effect of 5-HT and 8-OH-DPAT was prevented by pretreatment, 1 h before, with either 2 mg/kg i.p. WAY100135, a 5-HT1A antagonist or 0.25 mg/kg i.p. ritanserin, a 5-HT2 antagonist. These results indicate that 5-HT in the ZI exerts its inhibitory effect on LH release via 5-HT1A receptors but that another 5-HT subtype may also be involved.
Responsiveness of Irradiated Rat Anterior Pituitary Cells to Hypothalamic Releasing Hormones Is Restored by Treatment with Growth Hormone
Authors: Chiarenza Andrea , Lempereur Laurence , Palmucci Tullio , Cantarella Giuseppina , Amico-Roxas Matilde , Goffin Vincent , Murabito Paolo , Magro Gaetano , Bernardini Renato
Published: April 2003

Hypopituitarism is a common sequela of irradiation in cancer patients. Here we report that recombinant human growth hormone (r-hGH) prevents cell death and restores secretory capacity of irradiated rat pituitary cells in vitro. Dispersed rat pituitary cells from male Sprague-Dawley rats, irradiated with a 9-Gy sublethal dose, were incubated with r-hGH before, after, or before and after irradiation. Treatment with GH resulted in increased cell survival, which reached its maximum at the concentration of 5 nM, with an EC50 of 3.5 nM. Protective effects of GH on pituitary cells were more pronounced in cultures treated before and after irradiation. Similarly, beneficial effects of GH were observed on the secretory capacity of surviving cells. In fact, irradiated pituitary cells treated with GH secreted substantial amounts of GH, luteinizing hormone, follicle-stimulating hormone, prolactin, thyroid-stimulating hormone and adrenocorticotropic hormone in response to specific releasing hormones. Such effects of GH were prevented in the presence of the specific GH receptor antagonists B2036 and G120K. Our results show that r-hGH exerts a specific protective effect on irradiated rat pituitary cells and suggest possible use of GH as an adjuvant agent for prevention of postirradiation hypopituitarism.
Cloning, Pharmacological Characterization and Brain Distribution of the Rat Apelin Receptor
Authors: De Mota Nadia , Lenkei Zsolt , Llorens-Cortès Catherine
Published: April 2003

The peptide apelin, recently isolated from bovine stomach tissue extracts, has been identified as an endogenous ligand of the human putative receptor protein related to the angiotensin receptor AT1 (APJ). In this article, we report cloning of the rat apelin receptor cDNA. The sequence shares 90% identity with the human APJ receptor and 31% with the rat AT1A angiotensin receptor. Subsequently a stable CHO cell line expressing the receptor fused at its C-terminal part with the enhanced green fluorescent protein (EGFP) was established, allowing to verify its cell surface distribution and to determine the affinity of various apelin and angiotensin fragments on the cloned receptor. As shown for the human APJ receptor, the rat apelin receptor expressed in the cell line was negatively coupled to adenylate cyclase. The apelin fragment K17F (Lys1-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) inhibited forskolin-stimulated cAMP production at sub-nanomolar concentrations whereas angiotensin II and angiotensin III were inactive. N-terminal elongation of K17F with a tyrosine or the N-terminal deletion of the first four amino acids did not modify the inhibitory action of K17F on cAMP production. In contrast, deletion of the first seven amino acids of K17F or substitution of phenylalanine by an alanine residue at the C-terminus completely abolished the activity of the peptide. In situ hybridization analysis of apelin receptor mRNA expression in the adult rat brain showed intense labeling in the hypothalamus, especially in the supraoptic and the paraventricular nuclei. The anterior and intermediate lobes of the pituitary were also highly labeled, as well as the pineal gland. Labeling was also found in extrahypothalamic structures such as the piriform cortex, the nucleus of the lateral olfactory tract, the central grey matter, the pars compacta of the substantia nigra, the dorsal raphe nucleus, the entorhinal cortex, the dentate gyrus and the Ammon’s horn. The hypothalamic and hypophyseal distribution of the receptor suggests an involvement of apelin in the control of neuro- and adenohypophyseal hormone release, whereas its presence in the pineal gland and in discrete higher brain structures points out to possible roles in the regulation of circadian rhythms and of water and food intake behavior.
Galanin-Like Peptide mRNA in Neural Lobe of Rat Pituitary
Authors: Shen Jun , Larm Jari A. , Gundlach Andrew L.
Published: April 2003

Galanin-like peptide (GALP) was recently identified in the porcine hypothalamus, pituitary gland and gut, and has reported selectivity for the GalR2, c.f. the GalR1 receptor. GALP cDNAs have been cloned from pig, rat and human, and GALP mRNA expression is restricted to the arcuate nucleus in normal rat brain. This study examined the regional and cellular distribution of GALP mRNA in the rat pituitary gland, and subsequently determined the effect of osmotic stimulation on GALP transcript levels. GALP mRNA was not detected in the anterior or intermediate lobes, but moderate levels of GALP mRNA were present in the neural (posterior) lobe, in presumed pituicytes, of normal male and female rats. Osmotic stimulation by dehydration or salt loading produced a time-dependent increase in GALP mRNA levels in the neural lobe. Thus, dehydration for 4 days increased GALP mRNA 40-fold, while salt loading for 4, 7 or 10 days increased GALP levels 14-, 21- and 25-fold, respectively (p ≤ 0.001). Levels of vasopressin (VP) mRNA in the neural lobe were also increased by these treatments, consistent with previous reports. Galanin (GAL) and GalR2 receptor mRNAs were not detected in the neural lobe, under normal or osmotic stimulation conditions. In addition, GALP mRNA levels in the arcuate nucleus were not altered in dehydrated or salt-loaded rats; and GALP mRNA was not detected in magnocellular neurons of the supraoptic or paraventricular nucleus, despite the characteristic up-regulation of VP and GAL mRNA in these cells. In view of the established anatomy and function of VP/oxytocin neurons in the hypothalamo-neurohypophysial system and the role played by pituicytes in their regulation, the likely synthesis/release of GALP by these specialized astrocytes strongly suggests a role for this novel peptide in regulation of pituicyte morphology/function and/or neurohormone release.
Estrogen Modulates α1/β-Adrenoceptor- Induced Signaling and Melatonin Production in Female Rat Pinealocytes
Authors: Hernández-Díaz Francisco J. , Sánchez Juan J. , Abreu Pedro , López-Coviella Ignacio , Tabares Lucía , Prieto Luis , Alonso Rafael
Published: April 2003

Nocturnal rise in pineal melatonin output is due to the night-induced acceleration of noradrenergic transmission and α1- and β-adrenoceptor activation. In addition, in female animals, cyclic oscillations in circulating levels of sex steroid hormones are accompanied by changes in the rate of pineal melatonin secretion. To investigate whether estrogen directly affects pineal adrenoceptor responsiveness, pinealocytes from 21-day-old ovariectomized rats were exposed to physiological concentrations of 17β-estradiol (17β-E2) and treated with noradrenergic agonists. Direct exposure to 17β-E2 reduced α1/β-adrenoceptor-induced stimulation of melatonin synthesis and release. This effect was mediated by an estrogen-dependent inhibition of both β-adrenoceptor-induced accumulation of cAMP and α1-adrenoceptor-induced phosphoinositide hydrolysis. Furthermore, estrogen reduced transient Ca2+ signals elicited in single pinealocytes by α1-adrenoceptor activation or by potassium-induced depolarization. In the case of β-adrenoceptor responsiveness, neither forskolin- nor cholera toxin-induced accumulation of cAMP were affected by previous exposure to 17β-E2. This indicates that estrogen effects must be exerted upstream from adenylylcyclase activation, and independent of modifications in G protein expression, therefore suggesting changes in either adrenoceptor expression or receptor-effector coupling mechanisms. Since estrogen effects upon adrenoceptor responsiveness in pineal cells was not mimicked by 17β-E2 coupled to bovine serum albumin and showed a latency of 48 h, this effect could be compatible with a genomic action mechanism. This is also consistent with the presence of two estrogen receptor proteins, α- and β-subtypes, in female rat pinealocytes under the present experimental conditions.
Central Bombesin Activates the Hypothalamic-Pituitary-Adrenal Axis
Authors: Kent Pam , Anisman Hymie , Merali Zul
Published: April 2003

While corticotropin-releasing hormone (CRH) is a primary regulator of the hypothalamic-pituitary-adrenal (HPA) axis, the mechanism(s) triggering the release of this corticotropin (ACTH) secretagogue remains unknown. Stressful and appetitive events evoke the release of not only CRH but also of bombesin (BN)-like peptides. Furthermore, CRH antagonists attenuate the endocrine and behavioral effects of BN, suggesting that BN-like peptides may mediate their effects via CRH release. An initial (mapping) study revealed that centrally administered BN (0.25 or 0.5 µg i.c.v.) increased circulating corticosterone and ACTH levels and decreased immunoreactive (ir)-CRH at the nucleus of the solitary tract, ventromedial (VMH) and anterior hypothalamic nuclei, and the central amygdaloid nucleus. Whereas BN treatment decreased ir-vasopressin (AVP) at the VMH, it elevated levels of this peptide in the hypothalamic paraventricular and median eminence/arcuate (Me/Arc) regions. Dynamic, in vivo release experiments (using push-pull perfusion) revealed that BN evoked the release of ir-CRH and ir-AVP from the Me/Arc and increased interstitial levels of these secretagogues at the anterior pituitary. These results suggest that BN-like peptides may regulate certain hypothalamic and extrahypothalamic circuits, including the HPA axis, by affecting regional utilization of ir-CRH and ir-AVP, and/or by provoking the release of these peptides at the Me/Arc, thus increasing their availability downstream at the anterior pituitary and increasing circulating ACTH and corticosterone levels.