bluuebunny/crossref_metadata_2025 huggingface dataset:
Fos Expression in the Female Rat Brain during the Proestrous Prolactin Surge and following Mating
Authors: Yang Shu-Ping , Lee Youngsoo , Voogt JamesL.
Published: April 2003

A prolactin (PRL) surge occurs in the female rat during proestrus in response to elevated estradiol levels. The elevated release of ovarian steroids on the day of proestrus is also associated with sexual receptivity. Mating triggers twice-daily PRL surges that supplant the proestrous PRL surge and are responsible for maintaining luteal function during the first half of pregnancy. In order to understand the neuronal mechanisms controlling the proestrous- and mating-induced PRL surges, we examined patterns of Fos expression by immunocytochemistry in specific brain regions as a measure of neuronal activity. Intact female rats were sacrificed at 09.00, 15.00, and 18.00 h on the day of proestrus and the day of diestrus. Brain tissues were also collected at 21.00 h on the day of proestrus from rats receiving intromissions or mounts from males or taken directly from their homecage. On the day of proestrus, the number of neurons expressing Fos in the medial preoptic area (mPOA), medial amygdaloid nucleus (mAMYG), and ventromedial nucleus of the hypothalamus (VMH) was few and was associated with low plasma PRL levels at 09.00 h; however, the number of Fos-positive cells in these brain regions significantly increased at 15.00 and 18.00 h when the proestrous PRL surge occurred. Mating during the evening of proestrus resulted in a dramatic increase in the number of Fos-positive cells in the mPOA, mAMYG, and VMH as well as in the arcuate nucleus (ARC). Nonmated animals in diestrus showed low and unchanged PRL levels and Fos expression in all the brain areas throughout the day. These results suggest that the mPOA, mAMYG, VMH and ARC may be important brain sites for the integration of stimuli associated with proestrous- and mating-induced PRL surges.
Sex Differences in Male-Typical Copulatory Behaviors in Response to Androgen and Estrogen Treatment in Rats
Authors: Roselli CharlesE. , Chambers Kathleen
Published: April 2003

The present experiment was performed to test the hypothesis that gender differences in the capacity for brain estrogen synthesis could constitute a sexually dimorphic mechanism that limits the activational effects of testosterone (T) in females, and enhances them in males. We determined the effects of treatments with equivalent levels of either T or estradiol (E2) on olfactory behavior and mounting in age-matched heterosexually naive gonadectomized male and female rats that were genitally ansthetized with lidocaine paste in order to minimize the contribution of sexually dimorphic somatosensory inputs to the expression of copulatory behavior. We found that T stimulated mounting to a greater extent in males than in females, but had equivalent effects on mount latency and genital investigation in the two sexes. On the other hand, E2 stimulated equivalent levels of mounting in males and females and reduced mount latency to a similar extent in males and females. However, E2 had a pronounced effect on the levels of genital investigation in males but not in females. Serum steroid levels and the levels of nuclear steroid receptor occupation in the brain were not different between males and females, suggesting that the behavioral differences between males and females cannot be attributed to differences in peripheral steroid metabolism or brain uptake. The results obtained corroborate previous studies suggesting that female rats normally undergo considerable male-typical behavioral masculinization during fetal development. However, such male-typical features of normal development in female rats do not extend to the regulation of preoptic aromatase activity or to the capacity of females to display olfactory behaviors in response to adult E2 exposure, functions which are sexually dimorphic even in the rat. The present results support the view that gender differences in the capacity for brain estrogen synthesis contribute to the sexually dimorphic display of T-stimulated male-typical sexual motivation and copulatory behavior in rats.
PACAP Gene Expression in Neurons of the Rat Hypothalamo-Pituitary-Adrenocortical Axis Is Induced by Endotoxin and Interleukin-1β
Authors: Hannibal Jens , Jessop David S. , Fahrenkrug Jan , Harbuz Michael S. , Larsen Philip J.
Published: April 2003

Inflammatory stress due to infection by various micro-organisms is known to activate the hypothalamo-pituitary-adrenocortical (HPA) axis through inflammatory mediators. Recently, pituitary-adenylate-cyclase-activating polypeptide (PACAP) was shown to be located in corticotropin-releasing factor containing neurons of the medial parvocellular part of the hypothalamic paraventricular nucleus (mpPVN). In the present study, we demonstrate that PACAP gene expression is induced in neurons of the mpPVN after intraperitoneal administration of bacterial lipopolysaccharide (LPS) which was accompanied by a marked increase in PACAP immunoreactivity in the external zone of the median eminence. As determined by quantitative in situ hybridization, PACAP gene expression was rapidly induced after 4 h and was elevated for 48 h, declining to normal levels after 72 h. A significant increase in PACAP mRNA was also observed following intraperitoneal injection of interleukin-1β. PACAP gene expression was not induced by LPS in vagotomized animals, suggesting that the increase in PACAP mRNA following immune activation by LPS is mediated via the vagus nerve. The findings suggest that PACAP may function as a hypothalamo-pituitary-releasing factor during acute inflammation.
Effect of Exogenous Nitric Oxide and Inhibitors of Nitric Oxide Synthase on the Hypothalamic Pituitary Adrenal Axis Responses to Neural Stimuli
Authors: Weidenfeld Joseph , Feldman Shaul , DeKeyser Freda G. , Ovadia Haim
Published: April 2003

It has been shown that the hypothalamic-pituitary-adrenal (HPA) axis responses to immune-derived stimuli in particular can be modulated by nitric oxide (NO). In the present study we examined the effect of endogenous and exogenous NO on the HPA axis responses to neural stimuli which are not related to immune functions. Intracerebroventricular injection of NOR-3, a donor of NO, had no effect on basal HPA axis activity but significantly attenuated the secretion of median eminence (ME) CRH-41 as well as the serum ACTH and corticosterone (CS) in response to acute photic stimulation in a dose-dependent manner. Intracerebroventricular administration of N-ω-nitro-L-arginine methyl ester (L-NAME), a general NOS inhibitor, significantly enhanced ACTH and CS responses to this stress but did not change the basal levels of these hormones. On the other hand, i.c.v. injection of aminoguanidine, an inhibitor of inducible NO synthase (NOS) but not of neuronal NOS, did not affect the HPA axis responses to photic stimulation. These results suggest that: (1) NO is involved in modulation of the HPA axis responses to neural stimuli which are not dependent on immune factors, (2) the effect of NO is mediated by inhibition of hypothalamic ME CRH-41 secretion, and (3) this effect is probably mediated by neuronal NOS and not by inducible NOS.
Central LPS-Induced c-fos Expression in the PVN and the A1/A2 Brainstem Noradrenergic Cell Groups Is Altered by Adrenalectomy
Authors: Condé Gillian L. , Renshaw Derek , Zubelewicz Barbara , Lightman Stafford L. , Harbuz Michael S.
Published: April 2003

The A1 and A2 brainstem noradrenergic cell groups project to the hypothalamic paraventricular nucleus (PVN), which is involved in integrating the stress response. Bi-directional communication between the brain and immune system is well established, with both neuroendocrine and immune responses being activated by lipopolysaccharide (LPS). The mechanisms underlying such activation and differences between alternative routes of administration remain unclear. We examined activation of the PVN and A1/A2 cell groups, by assessing c-fos mRNA, or counting Fos-positive neurons in either the PVN or in brainstem A1/A2 cell groups 3 h after intracerebroventricular (i.c.v.) LPS, in control and adrenalectomized (ADX) rats. We also measured corticotropin-releasing hormone (CRH) mRNA in the PVN, and plasma corticosterone (CORT) levels. A group of ADX/CORT-replaced animals received i.c.v. LPS, and CRH mRNA and Fos peptide in the PVN were analysed. ADX increased CRH mRNA in the PVN, as did LPS, but no enhancement of this response was seen in LPS/ADX animals. C-fos mRNA also increased in both the PVN and the A2 cell group following LPS, but this response was potentiated by ADX. Fos peptide-containing cells increased in the PVN and A2 following LPS, and this change was amplified by ADX. Only 11.25% of Fos was found in DBH-positive (putative noradrenergic) neurons, suggesting activation of neurons containing other transmitters. ADX/LPS/CORT animals showed numbers of Fos neurons in the brainstem, and CRH mRNA levels in the PVN which were comparable to intact/LPS animals. Central LPS activates the hypothalamo-pituitary-adrenal axis, a process mediated partly by brainstem noradrenergic neurons, suggesting the involvement of afferent/efferent pathways within the brain. Peripheral administration of LPS involves activation of vagal inputs leading to the nucleus tractus solitarius. We suggest that centrally administered LPS activates the A2 cell group by a mechanism independent of the vagus. In the absence of CORT, despite the lack of a CRH mRNA response, an exaggerated c-fos and peptide response to LPS is observed, which is reversed following CORT pretreatment.
Suppression of Tonic Luteinizing Hormone Secretion and Norepinephrine Release near the GnRH Neurons by Estradiol in Ovariectomized Rats
Authors: Legan Sandra J. , Callahan W. Hunter
Published: April 2003

One of the major neurotransmitters that controls pulsatile luteinizing hormone (LH) secretion is norepinephrine (NE). NE pulses detected in the median eminence of ovariectomized rhesus monkeys are highly correlated with both GnRH and LH pulses. In contrast, previous reports suggest that this is not the case in rats, thus it remains to be determined whether NE stimulates LH release on a pulse-by-pulse basis in that species. Further, a variety of indirect evidence supports the hypothesis that in rats, estradiol exerts its negative feedback action on LH secretion in part by inhibiting noradrenergic neurotransmission that is stimulatory to LH release, but there is no direct evidence to support this hypothesis. Therefore the following study was designed to test the hypothesis that estradiol suppresses NE release in the vicinity of the GnRH neurons after ovariectomy. In addition, we examined whether episodes of NE release are correlated with LH pulses in ovariectomized rats. Blood samples and microdialysates of the diagonal band of Broca/medial preoptic area (DBB/MPOA) were collected every 5 min from 09:00 to 14:00 h from untreated or estradiol-treated (4–5 days), long-term ovariectomized (1–4 months) rats for determination of plasma LH by RIA and NE release by HPLC. The results indicate that in both untreated and estradiol-treated ovariectomized rats, LH pulses are not correlated with episodes of NE. Thus, NE may play a permissive role in the control of pulsatile LH secretion in rats. Further, estradiol treatment leads to a suppression of both plasma LH levels and NE release in the DBB/MPOA, supporting the hypothesis that a decrease in NE neurotransmission that is stimulatory to LH release mediates the negative feedback action of estradiol on tonic LH secretion.
Isoform-Specific Exocytotic Protein mRNA Expression in Hypothalamic Magnocellular Neurons: Regulation after Osmotic Challenge
Authors: Jacobsson Gunilla , Bean Andrew J. , Meister Björn
Published: April 2003

Exocytosis is regulated by proteins which interact to promote docking and fusion of secretory granules with the plasma membrane. We have used in situ hybridization to study the mRNA expression for vesicle-associated membrane protein (VAMP) isoforms VAMP-1 and VAMP-2, synaptosomal-associated protein of 25-kDa (SNAP-25) isoforms SNAP-25a and SNAP-25b, mammalian homologue of unc-18 (munc-18) and Hrs-2 in neurosecretory neurons of the magnocellular paraventricular (PVN) and supraoptic (SON) nuclei of normal and osmotically challenged animals. In PVN and SON neurons of normal animals, strong labeling was demonstrated for VAMP-2 and SNAP-25a mRNA, whereas VAMP-1 or SNAP-25b mRNA could not be detected. Salt-loading (2% NaCl as drinking water), an animal model which increases the expression and secretion of hormones from hypothalamic magnocellular neurons, resulted in significantly increased mRNA levels for VAMP-2 (36%, 28%), munc-18 (74%, 68%) and SNAP-25a (59%, 77%) in the PVN and SON, respectively. There was no significant increase in Hrs-2 mRNA levels in the PVN, whereas a significant increase (22%) was observed in the SON. In the posterior pituitary, immunohistochemistry showed a marked decrease in numbers and intensity of vasopressin-immunoreactive (-IR) nerve endings after salt-loading. There were no obvious changes in numbers or intensity of VAMP-2-, munc-18-, Hrs-2- or SNAP-25-IR fibers. Large varicosities containing VAMP-2- and Hrs-2 immunocreactivity were seen in salt-loaded animals. The results show isoform-specific mRNA expression in neurosecretory neurons and an increased mRNA expression of proteins participating in the molecular regulation of exocytosis during an experimental situation characterized by increased secretion.
Psychological and Endocrine Responses to Psychosocial Stress and Dexamethasone/ Corticotropin-Releasing Hormone in Healthy Postmenopausal Women and Young Controls: The Impact of Age and a Two-Week Estradiol Treatment
Authors: Kudielka Brigitte M. , Schmidt-Reinwald Astrid K. , Hellhammer Dirk H. , Kirschbaum Clemens
Published: April 2003

In this placebo-controlled double-blind study, psychological and endocrine stress responses were investigated in healthy postmenopausal placebo-treated women (n = 15; 60–75 years; placebo via transdermal patches), healthy postmenopausal estradiol-treated women (n = 13; 60–79 years; 0.1 mg 17β-estradiol daily via transdermal patches) and young controls (n = 15; 20–31 years; untreated). The aged subjects received estradiol or placebo treatment for 14 days. All subjects were then exposed to the ‘Trier Social Stress Test’ (TSST) and the dexamethasone (Dex)-human corticotropin-releasing hormone (hCRH) test (100 µg hCRH after premedication with 1.5 mg Dex). Psychological parameters including perceived stressfulness, mood and subjective well-being were measured by visual analog scales, a mood questionnaire and a mood diary, respectively. Results show that the TSST induced significant increases in adrenocorticotropin (ACTH), free salivary cortisol, total plasma cortisol and heart rates (all p < 0.0001). Regardless of age, comparable hormonal response patterns were observed in the TSST as indicated by similar peak levels and recovery phases. Visual analog scales confirmed that the same amount of stress was experienced by young and elderly subjects. In both age groups, hCRH injection after Dex premedication provoked significant increases in ACTH, free salivary cortisol and total plasma cortisol (all p < 0.0001). In contrast to the psychosocial stressor, elderly women were found to respond with a markedly enhanced cortisol response compared to young controls in the Dex-CRH test (p < 0.025). Additional investigation of morning cortisol profiles could not reveal any age-related differences in basal hypothalamus-pituitary-adrenal (HPA) axis activity. Following estradiol treatment, estradiol levels significantly increased only in substituted postmenopausal women (p < 0.001) reaching concentrations typically found in younger women during the follicular phase of the menstrual cycle. Corticosteroid-binding globulin levels did not differ significantly between groups. When confronted with the TSST, no response differences emerged between the three groups. However, estradiol treatment appeared to blunt the total plasma cortisol response in the Dex-CRH test, resulting in smaller increases in untreated premenopausal women and estradiol-treated postmenopausal women compared to placebo-treated postmenopausal women (p < 0.02). In sum, no response differences were observed after confrontation with a psychosocial stress test in our sample of healthy elderly subjects. As shown with the Dex-CRH test, our data suggest that the negative feedback of the HPA axis in elderly women is altered. Moreover, the current data suggest that estradiol replacement may modulate HPA feedback sensitivity in humans.
The Transcription of Corticotropin-Releasing Hormone in Human Endometrial Cells Is Regulated by Cytokines
Authors: Makrigiannakis A. , Margioris A.N. , Zoumakis E. , Stournaras C. , Gravanis A.
Published: April 2003

Corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide, is also produced in the human endometrium where it participates in local inflammatory phenomena associated with the decidualization of endometrial stroma and the implantation of the fertilized egg. The inflammatory cytokines interleukin 1 (IL-1), IL-6 and leukemia inhibitory factor (LIF) appear to be the dominant local regulators of these intrauterine inflammatory processes. In the present study we have examined the direct interactions between cytokines and CRH in the endometrium. For this purpose we have measured the effects of IL-1, IL-6 and LIF on the activity of CRH promoter inserted in human endometrial cells in culture. Homologous transient transfection experiments were conducted employing a 0.9-kb fragment of the 5′ flanking region of the human CRH gene coupled to the luciferase reporter gene, using Ishikawa human endometrial cells. We have found that IL-1β increased the activity of CRH gene promoter, in a time- and dose-dependent manner. This effect was antagonized by the IL-1 receptor antagonist IL-1ra and blocked completely by the cyclo-oxygenase inhibitor indomethacin. Similarly, IL-6 increased the activity of CRH promoter in a dose-dependent fashion, an effect partially reversed by indomethacin. LIF did not have any apparent effect. In conclusion, our data suggest that IL-1 and IL-6 exert a strong stimulatory effect on the expression of endometrial CRH. This effect is most probably mediated via prostaglandins. Based on these data we hypothesize that in the human endometrium interleukins, prostaglandins and CRH form a local network regulating the inflammatory phenomena taking place within the uterine cavity.
Chronic Ventromedial Hypothalamic Infusion of Norepinephrine and Serotonin Promotes Insulin Resistance and Glucose Intolerance
Authors: Luo Shuqin , Luo Jing , Cincotta Anthony H.
Published: April 2003

The ventromedial hypothalamus (VMH) is involved in the regulation of peripheral metabolism. We and others have shown that activities, or extracellular metabolites of norepinephrine (NE) and serotonin (5-HT) are elevated in the VMH of both genetically and seasonally insulin-resistant and glucose-intolerant animals. This study examined whether chronic increases in VMH NE and 5-HT concentration of normal animals might lead to insulin-resistant and glucose-intolerant conditions in hamsters. Euinsulinemic, glucose-tolerant hamsters were infused continuously for 5 weeks into the right VMH with either vehicle, NE (5 or 25 nmol/h), 5-HT (2.5 nmol/h), or NE (5 or 25 nmol/h) plus 5-HT (2.5 nmol/h) through osmotic minipumps. Compared to vehicle, NE (25 nmol/h) significantly increased the glucose total area under the curve (TAUC) by 32% during glucose tolerance tests (GTT) conducted after 5 weeks’ infusion. 5-HT alone significantly increased the GTT insulin TAUC (131%) and basal plasma insulin level (116%) but not glucose TAUC. NE (5 nmol/h) plus 5-HT infusion significantly increased insulin TAUC (129%) and basal plasma insulin (120%), whereas NE (25 nmol/h) plus 5-HT infusion significantly increased both the GTT glucose and insulin TAUC (43 and 113%, respectively), as well as basal plasma insulin level (158%), relative to vehicle infusion. Our findings demonstrate for the first time the differential and, more importantly, interactive effects of increased VMH NE and 5-HT in producing hyperinsulinemia, insulin resistance and glucose intolerance.
Melatonin Regulates the mRNA Expression of the mt1 Melatonin Receptor in the Rat Pars tuberalis
Authors: Guerrero Hilda Y. , Gauer François , Schuster Carole , Pévet Paul , Masson-Pévet Mireille
Published: April 2003

The pars tuberalis (PT) of the pituitary is a major neuroendocrine target site for melatonin as it contains a large number of high-affinity melatonin receptors. We have previously shown that melatonin autoregulates the density of its own receptors in the PT. However, whether melatonin regulation includes mRNA expression in vivo is unclear. In the present study we have used quantitative in situ hybridization to (1) follow the daily profile of mt1 mRNA expression in the rat PT and (2) investigate whether mt1 mRNA expression could be regulated in vivo by melatonin. We found clear diurnal variations of mt1 mRNA expression that persist in constant darkness. We also showed, on pinealectomized animals, that the rhythmic pineal melatonin secretion is necessary for the expression of these daily variations. In a second step, we studied the effect of an acute suppression of endogenous melatonin synthesis on mt1 melatonin receptors by applying a 1-hour light pulse during the night. We found that light induced a dramatic increase in mt1 mRNA which was totally prevented by a melatonin injection showing that the acute effect of melatonin on the receptor mRNA is strongly inhibitory. A light pulse applied to animals with a chronic absence of melatonin was ineffective showing that light only affects melatonin receptors via the light-induced plasma melatonin suppression. Altogether our results show that melatonin regulates mt1 melatonin receptor mRNA expression. However, this regulation seems to be complex: acute changes in plasma melatonin concentration regulate negatively the gene transcription, even if the daily endogenous nocturnal melatonin peak seems a prerequisite for variations in its receptor expression.
Neuroendocrine Consequences of Fasting in Adult Male Macaques: Effects of Recombinant Rhesus Macaque Leptin Infusion
Authors: Lado-Abeal Joaquin , Hickox John R. , Cheung Tony L. , Veldhuis Johannes D. , Hardy Daniel M. , Norman Reid L.
Published: April 2003

Fasting inhibits the gonadotropic axis and stimulates the corticotropic and somatotropic axes. Since leptin is a product of fat cells that has been implicated in the control of both reproduction and metabolism, we hypothesized that the decrease in leptin observed during fasting was responsible for these effects on reproductive and metabolic hormones. Recombinant rhesus leptin (rrhLep) produced in our laboratory was infused (100 µg/h) into fasted adult male rhesus macaques (6–9 kg) beginning at midnight after the first missed meal and continuing until the end of the study. Bioactive luteinizing hormone (LH), testosterone, cortisol and growth hormone (GH) were measured in plasma from samples collected at 15-min intervals for the last 15 h (42–57 h) of the fast. We analyzed pulsatile LH and GH secretion by deconvolution analysis and the orderliness of pulsatile LH and GH release by the approximate entropy (ApEn) statistic. There was no difference in LH pulse frequency between control and fasted groups, but there was a significant decrease in the mean concentration of LH released (7.6 ± 1.4 ng/ml control vs. 2.7 ± 0.65 ng/ml fasted) that was not relieved with rrhLep infusions (2.8 ± 0.83 ng/ml). Model-free Cluster analysis confirmed these inferences and also indicated that the peak height was lower in the fasted (4.6 ± 1.0 ng/ml) and the fasted + rrhLep (2.85 ± 1.0 ng/ml) groups compared to controls (16.3 ± 1.4 ng/ml). Testosterone levels reflected those of LH. Fasting resulted in an increase in GH secretory pulse frequency (5.3 ± 0.95 pulses/15 h control vs. 12.8 ± 1.4 pulses/15 h fasted) and this increase was not affected by rrhLep infusion (12.5 ± 1.4 pulses/15 h). In addition, fasting also increased the ApEn (decreased the orderliness) of pulsatile GH secretion, and this characteristic was not relieved with rrhLep infusions. Cortisol levels in fasted animals were 2- to 3-fold higher than those observed in control studies, and this increase was particularly pronounced at the time when the animals expected their first meal of the day. The increase in circulating cortisol observed in fasted animals was not affected by rrhLep infusion. Glucose levels at the end of the sampling period were 80 mg/dl in controls, 48 mg/dl in fasted animals and 58 mg/dl in the fasted + rrhLep group. Circulating leptin levels averaged 1.2 ± 0.37 ng/ml in control animals, 0.7 ± 0.2 ng/ml in fasted animals and 10.1 ± 5.6 ng/ml in fasted animals infused with rrhLep. These studies suggest that intravenous replacement with homologous leptin does not reverse the acute changes in GH, LH and cortisol secretion observed with fasting in the adult male macaque.
Variations in Plasma Levels of Substance P and Effects of a Specific Substance P Antagonist of the NK1 Receptor on Preovulatory LH and FSH Surges and Progesterone Secretion in the Cycling Cynomolgus Monkey
Authors: Kerdelhué Bernard , Williams Robert F. , Lenoir Véronique , Fardin Véronique , Kolm Paul , Hodgen Gary D. , Jones Georgeanna Seegar , Scholler Robert , Jones Jr. Howard W.
Published: April 2003

These studies investigated the role of substance P (SP) in the regulation of the hypothalamic-pituitary-ovarian axis in cynomolgus monkeys with normal menstrual cycles. Plasma concentrations of SP were determined in blood samples taken every morning in normally menstruating cynomolgus monkeys throughout the menstrual cycle. There was a significant decreasing linear trend of SP during the follicular phase (cycle day –13 to day 0) and a significant inverse relationship between SP plasma values and plasma 17β-estradiol (E2) values from day –13 to day 0 of the adjusted cycle. Correspondingly, SP area under the curve was significantly greater during the follicular phase than the luteal phase. In a second experiment, plasma concentrations of E2, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone and length of cycles were measured after five daily intragastric administrations (10 mg/kg) of an NK1 receptor (SP receptor) antagonist (RPR 100893; 10 mg/kg) initiated after serum E2 concentrations had exceeded 125 pg/ml. There was a statistically significant reduction in the amplitude (41% of control) and the area under the curve (37% of control) of the preovulatory LH surge. In addition, there was a reduction of the duration of the LH surge (3 ± 0.1 days in controls vs. 2.1 ± 0.2 days in treated animals). The present results show for the first time that there are significant variations in plasma levels of SP, with a strong negative correlation with serum levels of E2 during the follicular phase of the cynomolgus monkey, and that endogenous SP has a potentiating role in the interactive hypothalamo-anterior-pituitary mechanisms which lead to the preovulatory LH and FSH surges during the menstrual cycle in the monkey.
Prolactin-Releasing Peptides Do Not Stimulate Prolactin Release in vivo
Authors: Jarry Hubertus , Heuer Heike , Schomburg Lutz , Bauer Karl
Published: April 2003

The prolactin (PRL)-releasing activity of the novel prolactin-releasing peptides (PrRPs) was studied in vivo using male and lactating female rats. Whereas thyrotropin-releasing hormone effectively stimulated PRL and thyrotropin release as expected, PrRP in both animal models neither stimulated PRL secretion nor affected the release of other pituitary hormones. At the anterior pituitary level, in situ hybridization (ISH) histochemistry and Northern blot analysis revealed significantly higher expression levels of PrRP receptor (UHR-1) transcripts in female compared to male rats but not between lactating and nonlactating animals. By ISH, expression of UHR-1 mRNA was also detected in the intermediate lobe but not in the posterior pituitary. UHR-1 transcripts were also readily detectable in various hypothalamic brain areas whereas expression of PrRP mRNA was restricted to the ventral part of the dorsomedial hypothalamic nucleus but was not detected in neuroendocrine hypothalamic nuclei (e.g. PVN, SON). We thus assume that in the central nervous system, PrRP may likely have functions as a neuromodulator. However, together with the detailed cytochemical studies of various investigators that failed to detect PrRP-immunopositive nerve endings in the median eminence, our results strongly suggest that the hypothalamic PrRPs cannot be classified as hypophysiotrophic factors.
Salt Appetite in Salt-Replete Rats: Involvement of Mesolimbic Structures in Deoxycorticosterone-Induced Salt Craving Behavior
Authors: Lucas Louis R. , Pompei Pierluigi , McEwen Bruce S.
Published: April 2003

Chronic administration of the mineralocorticoid deoxycorticosterone acetate (DOCA) induces a steady and robust increase in salt appetite and plasma Na+ over the course of treatment. Interestingly, salt appetite behavior persists in rats even with elevated plasma Na+ levels. Since there is evidence that the pathways normally associated with salt and water homeostasis are relatively unaffected in the DOCA-treated rat, we hypothesized that other regulatory systems may be hyperactive giving rise to this dysfunctional condition. The mesolimbic dopaminergic system has long been associated with orienting and reward-seeking behaviors such as those observed in reproduction, drug abuse, and appetite. Furthermore, we have previously shown that chronic DOCA administration results in an increase in mRNA levels of the endogenous opiate enkephalin in male rats given 24-hour access to tap water and 2% NaCl (two-bottle choice). Thus, in the present study, we tested the hypothesis that the mesolimbic dopaminergic system is dysfunctionally sensitized to the presence of a salt stimulus in DOCA-treated animals. Four groups of rats were injected with DOCA (5 mg/rat/day, 11 days) and one with vehicle (all were given access to water but access to salt was regulated). Two DOCA groups were given 2 h of 2% NaCl access/day and on the last day, one group was not given access (2hX). One of the two remaining DOCA groups was given 24-hour access to salt (24h) and the other no access at all (24hX). Consistent with our hypothesis, in the shell of the nucleus accumbens (AcbSh) we found relatively higher enkephalin- and tachykinin-mRNA abundance in the 2h vs. 2hX and dynorphin-mRNA in the 24h vs. 24hX groups. In addition, there were decreases in dopamine transporter binding in the AcbSh and decreases in tyrosine hydroxylase immunoreactivity throughout the striatum in the 24h vs. 24hX group. Furthermore, rats denied access to salt (2hX and 24hX) had higher cholecystokinin-mRNA levels in the ventral tegmental area compared to the 2h and 24h groups, respectively. These results suggest that basal ganglia structures associated with reward and goal-seeking behavior may be activated to elicit salt craving behavior in the DOCA-induced salt-appetitive rat.
Adrenocorticotropin Inhibition by Restoration of Normal Evening Cortisol Levels: A Measure of Putative Hippocampus-Mediated Glucocorticoid Feedback in Humans
Authors: Goldman Morris B. , Wood Gordon John
Published: April 2003

Hippocampus-mediated glucocorticoid negative feedback is thought to be relevant to the pathophysiology of neuropsychiatric disorders, but no reliable method of measuring it in humans has been developed. Converging lines of evidence indicate that basal hypothalamic-pituitary-adrenal axis activity during the unstressed circadian trough is primarily regulated by this feedback process. To assess whether negative feedback can be demonstrated under these circumstances, we studied normal controls (n = 5) who were pretreated with metyrapone to lower their basal evening cortisol levels. On two separate occasions, in double-blind randomized order, subjects received an infusion of cortisol or of saline. Restoration of normal evening plasma cortisol by the cortisol infusion produced a drop in plasma adrenocorticotropin (ACTH) apparent in the last sample obtained at +200 min (p < 0.05). The ACTH response in schizophrenic patients (n = 4), whose mental illness may arise from hippocampal dysfunction, was relatively blunted compared to that seen in normals (p < 0.02). 11-Desoxycortisol levels paralleled the ACTH responses across conditions and subject groups. These preliminary data suggest that hippocampus-mediated glucocorticoid feedback can be measured in normal subjects and may provide an index of hippocampal dysfunction in neuropsychiatric patients.
Disproportionate Inhibition of Feeding in Ay Mice by Certain Stressors: A Cautionary Note
Authors: De Souza Jose , Butler Andrew A. , Cone Roger D.
Published: April 2003

A study of the effects of insulin-induced hypoglycemia in the obese yellow agouti Ay mouse was initiated to test the hypothesis that the central melanocortin pathways are required for a normal sympathetic response to hypoglycemia. An experimental protocol was performed in which young nonobese male mice were isolated and fasted beginning on day 1, then tested for glucose responses to insulin-induced hypoglycemia on day 2. Normal mice demonstrated the expected glucose rebound to hypoglycemia, exceeding baseline glucose levels by 2–3 times as a consequence of increased gluconeogenesis and glycogenolysis before returning to baseline levels. Ay animals lacked the rebound, exhibiting instead a gradual restoration of baseline glucose levels. The results suggested a defective sympathetic response to hypoglycemia in the Ay mouse. However, a more detailed analysis demonstrated that the lack of a hyperglycemic rebound was due to an acute inhibition of feeding specifically in the Ay mouse, which resulted not from the hypoglycemia stressor, but rather from the stress of isolation. Handling and intraperitoneal administration of saline also specifically inhibited food intake in the Ay but not the wild-type mouse, while restraint stress had an equivalent inhibitory effect on food intake on wild-type and Ay mice. Since the Ay mouse has defective hypothalamic melanocortin-4 receptor (MC4-R) signaling, these data imply that the central melanocortin pathway is necessary for regulating the effects of stress on feeding behavior. Furthermore, these data demonstrate the need for exercising extreme caution in designing experiments to analyze feeding behavior and metabolism in genetic or pharmacological models involving perturbation of the melanocortin system.
Electrical Activity of the Pulse Generator of Gonadotropin-Releasing Hormone in 26-Month-Old Female Rats
Authors: Sano Akane , Kimura Fukuko
Published: April 2003

To know whether age-related changes occur in the activity of the pulse generator of gonadotropin-releasing hormone (GnRH), old (26 months) and young (3 months) female rats were examined by recording multiunit activity (MUA) in the median eminence region of the hypothalamus, concurrently with blood samplings through an intra-atrial cannula at 6-min intervals to determine serum luteinizing hormone (LH) concentrations. We have regarded the MUA showing intermittent increases (volleys) at 20–30 min intervals, followed by LH pulses, as the electrical activity of the GnRH pulse generator. We were successful in recording MUA in 18 (26%) of 69 old ovariectomized rats and in 8 (32%) of 25 young ovariectomized rats. The overall mean (±SE) of the interval between MUA volleys in old ovariectomized rats was 35.1 ± 2.0 min (n = 18), which was significantly longer than that of 22.5 ± 1.5 min (n = 8) in young ovariectomized rats. The mean interval between LH pulses in old ovariectomized rats was 32.2 ± 3.6 (n = 10), also being significantly longer than that of 23.3 ± 1.0 (n = 8) in young ovariectomized rats. Further, the LH pulse amplitude in old rats (0.95 ± 0.07 ng/ml) was significantly smaller than in young rats (3.40 ± 0.36 ng/ml). The present study also confirmed that the increase in serum LH after intravenous injection of 50 ng GnRH was much smaller in old ovariectomized rats. These results show that the electrical activity of the GnRH pulse generator is certainly reduced with age. Taken together with findings suggesting an age-dependent decrease in stimulated transmitter release, attenuation in both frequency and amplitude of GnRH pulses as well as in pituitary responsiveness to GnRH pulses may account for the decreased pulsatile LH secretion observed in aging rats.
Effect of the μ-Opioid Agonist DAMGO on Medial Basal Hypothalamic Neurons in Beta-Endorphin Knockout Mice
Authors: Slugg Robert M. , Hayward Michael D. , Rønnekleiv Oline K. , Low Malcolm J. , Kelly Martin J.
Published: April 2003

The endogenous opioid neurotransmitter β-endorphin (β-END), a product of the proopiomelanocortin (POMC) gene, is strongly implicated in the control of the female reproductive cycle, stress responses, and antinociception. Using selective gene targeting, we have generated a strain of mice that do not express any β-END. These mice exhibit both normal reproduction and normal basal and stress-induced hypothalamic-pituitary-axis activity, but exhibit a significantly attenuated opioid-mediated stress-induced analgesia. To further understand the cellular bases of these responses, we have studied mediobasal hypothalamic (MBH) neurons, including POMC neurons, using whole-cell patch recording in an in vitro slice preparation. Twenty-seven MBH cells were recorded in wild-type and 25 MBH cells were recorded in β-END knockout mice. Neurons from both genotypes showed a significant positive correlation between DAMGO concentration (from 30 nM to 10 µM) and the induced outward K+ current. The genotypes did not differ, however, in either the DAMGO-induced maximum outward current response or EC50, or for the maximal response to the GABAB agonist baclofen. Furthermore, quantitative receptor autoradiography utilizing 3H-DAMGO did not reveal any differences in total µ-opioid receptor binding between genotypes. Therefore, we conclude that the complete absence of β-END throughout development did not alter either the expression of µ-opioid receptors or their coupling to K+ channels in MBH neurons.
Essential Contribution of Intron Sequences to Ca2+-Dependent Activation of c-fos Transcription in Pituitary Cells
Authors: van Haasteren Goedele , Li Senlin , Ryser Stephan , Schlegel Werner
Published: April 2003

In pituitary cells, c-fos transcription induced by releasing hormones and growth factors results from enhanced initiation of transcription, and sustained elongation of transcripts beyond the first intron. We studied the regulatory role of the first intron of the mouse c-fos gene for the control of its transcription in rat pituitary cells. We showed that the intron contains a block to elongation which is relieved by physiological activators TRH and EGF. By expressing luciferase under the control of the c-fos promoter including the first intron in reporter gene constructs, we demonstrate enhancement of TRH and EGF transcriptional stimulation by intron sequences. Further analysis of Ca2+ signalling-depending transcription showed that the intron contains control elements in addition to the block to elongation, and that sequences in the first intron can mediate Ca2+-stimulated transcription also with a minimal or the SV40 promoter, irrespective of the presence or absence of the intronic block site. Within the c-fos promoter the serum response element and the cAMP response element play a permissive role in Ca2+- and cAMP-enhanced transcription of intron containing reporter genes. Specific binding of nuclear proteins to a consensus enhancer binding site (Sp1) within the first intron of c-fos was demonstrated, which might reflect one of the mechanisms that link Ca2+ and intron sequences to c-fos expression. These findings point towards important functions of intronic sequences in gene transcription control.
C-Type Natriuretic Peptide Expression in Olfactory Regions of Rat Brain Is Modulated by Acute Water Deprivation, Salt Loading and Central Angiotensin II
Authors: Cameron Vicky A. , Cumming Sarah A. , Espiner Eric A. , Nicholls Gary , Richards Mark
Published: April 2003

To elucidate the central role of c-type natriuretic peptide (CNP), levels of CNP mRNA in control rat brain were compared with levels following acute water deprivation, salt loading and central administration of angiotensin II (AII), using Northern blot and in situ hybridisation. Rats with water deprivation (WD) had no access to water for 48 h, rats with salt loading (SL) had access to 2% saline for 48 h, and control rats had free access to water. Both WD and SL significantly raised plasma sodium (Na). Levels of CNP mRNA in olfactory regions were significantly decreased in WD and increased in SL. In the medulla, WD and SL both increased CNP mRNA, but levels of CNP mRNA elsewhere in the brain were not significantly altered. Intracerebroventricular AII (500 ng) increased water intake, and induced a significant increase in CNP expression at 4 h in olfactory regions, but not in other brain sites. In summary, CNP expression is regulated in olfactory regions of the rat brain in response to acute challenges to water and salt balance and by central AII.
A Confocal Microscopic Study of Synaptic Inputs to Gonadotropin-Releasing Hormone Cells in Mouse Brain: Regional Differences and Enhancement by Estrogen
Authors: Rajendren Gopalan , Gibson Marie J.
Published: April 2003

Even though the cells producing gonadotropin-releasing hormone (GnRH) are scattered in the basal forebrain, a large proportion of them is present in the organum vasculosum of the lamina terminalis (OVLT) and in the preoptic area. The present studies were undertaken to investigate whether there is any difference in the number of synaptic inputs between GnRH cells located in the OVLT and those located at more anterior levels of the brain. Immunohistochemical staining for the synaptic marker synaptophysin coupled with confocal microscopy was employed to analyze synaptic inputs to GnRH cells located at the two levels examined. The results indicate that GnRH cells in the OVLT region receive a greater number of synaptophysin-immunoreactive appositions as compared with those located in the anterior septum. This supports the existence of subsets among the GnRH cells located in the basal forebrain. The effect of estradiol on the number of synaptophysin-immunoreactive appositions onto GnRH cells was also studied. Treatment of ovariectomized mice with estradiol significantly enhanced the number of synaptophysin-immunoreactive appositions to GnRH cells located at both levels examined. Thus the effect of estrogen on GnRH cells may be mediated in part by changes in the number of synaptic contacts.
Regulation of Gonadotropin-Releasing Hormone (GnRH) Gene Expression during GnRH Neuron Migration in the Mouse
Authors: Simonian Sharon X. , Herbison Allan E.
Published: April 2003

The mechanisms underlying the migration of the gonadotropin-releasing hormone (GnRH) neurons from the nose into the forebrain are not resolved. In an attempt to characterize further the migrating GnRH neurons, we have employed in situ hybridization techniques and transgenic mouse models to examine levels of GnRH mRNA and GnRH gene transcription in GnRH neurons during migration in the mouse. In the first experiment, cellular levels of GnRH mRNA in neurons located throughout the nose and forebrain were examined in embryonic day (E) 12.5, 14.5, 16.5 and 19.5 mice using in situ hybridization. The GnRH mRNA content of cells located in both the nose (p < 0.01) and forebrain (p < 0.05) was found to increase significantly from E12.5 to E19.5 and from E14.5 to E19.5, respectively. However, cellular levels of GnRH mRNA were not significantly different in neurons located in the nose compared with the brain at each developmental age. In the second experiment, levels of GnRH gene transcription were investigated at E14.5 using two different GNLZ transgenic mouse lines in which 13.5 kb of GnRH gene sequences direct the expression of the LacZ reporter to the nucleus of GnRH neurons. Migrating GnRH neurons displayed up to a 3-fold increase (p < 0.01) in transgene expression, an index of GnRH transcription, precisely as they approached and entered the forebrain. These results indicate that GnRH gene expression in migrating GnRH neurons is likely regulated by temporal as well as spatial factors and that, as found postnatally, this may involve both transcriptional and post-transcriptional regulatory mechanisms.
Distribution and Stimulation by Gastrin-Releasing Peptide of Protein Kinase C Subfamilies in Insulin-Secreting Cells
Authors: Bihlmayer Andrea , Ammon Hermann P.T. , Wahl Martin A.
Published: April 2003

The role of the different isoforms of protein kinase C (PKC) in modulating insulin secretion is still widely unknown. The aim of our studies was to investigate which isoforms are influenced by gastrin-releasing peptide (GRP), a neuropeptide which has been shown to modulate insulin secretion by activating PKC. Presence of PKC isoforms α, β, γ, δ, Ε and ζ was tested by immunoblot analysis in whole pancreatic islets of mouse and rat and in the insulinoma cell line RINm5F. Effects of GRP, the truncated peptide GRP1–16 and KCl were also measured on translocation of PKC isoforms. In pancreatic islets of mouse and rat, the PKC isoforms α, β, γ, δ, Ε and ζ could be detected. No PKCγ activity was present in the pancreatic tumor cell line RINm5F. Incubation of mouse or rat islets or of RINm5F cells with GRP induced translocation of the PKC isoforms α, β and ζ. The N-terminal portion of the peptide GRP1–16 induced partial translocation only of the PKC isoforms α, β and ζ in mouse and rat islets in 4 out of 10 cases, but failed to show any effect on PKC isoforms in RINm5F cells. Depolarization of the islets by KCl did not translocate any tested PKC isoform. However, incubation with GRP followed by depolarization with KCl led to translocation of the PKC isoforms α, β and ζ. It is suggested that PKC α, β and/or ζ may play a role in the modulation of insulin secretion by GRP.