Hypothalamus Of Female Rats Research Articles

Insulin-like growth factor I of peripheral origin acts centrally to accelerate the initiation of female puberty

In several species, including humans, circulating insulin-like growth factor I (IGF-I) levels increase during the onset of puberty, suggesting that this peptide contributes to attaining sexual maturity. Because IGF-I elicits LHRH release from the median eminence (ME) of immature female rats in vitro, we hypothesized that it may represent one of the peripheral signals suspected to link somatic development to the LHRH-releasing system at puberty. We now present evidence in support of this concept. Quantitation of IGF-I messenger RNA (mRNA) levels by ribonuclease protection assay revealed that expression of the IGF-I gene did not change in the medial basal hypothalamus or preoptic area of female rats during peripubertal development. In contrast, the contents of both IGF-Ia and IGF-Ib mRNA, the two alternatively spliced forms of the IGF-I gene, increased significantly in the liver during the early proestrous phase of puberty. This change was followed by an elevation in serum IGF-I levels during the late proestrous phase of puberty along with a concomitant increase is serum gonadotropin levels. The proestrous change in serum IGF-I levels was accompanied by a selective increase in IGF-I receptor (IGF-IR) mRNA in the ME. Small doses of IGF-I (2-200 ng), administered intraventricularly, effectively induced LH release in both juvenile and peripubertal female rats, an increase prevented by prior immunoneutralization of LHRH actions. Importantly, intraventricular injections of IGF-I (20 ng), administered twice daily in the afternoon to immature animals, significantly advanced puberty. Thus, these results suggest that IGF-I of peripheral origin contributes to the initiation of female puberty by stimulating LHRH release from the hypothalamus, an effect that appears to be amplified by the increased synthesis of IGF-I receptors in the ME during first proestrus.

Insulin-like growth factor I of peripheral origin acts centrally to accelerate the initiation of female puberty.

In several species, including humans, circulating insulin-like growth factor I (IGF-I) levels increase during the onset of puberty, suggesting that this peptide contributes to attaining sexual maturity. Because IGF-I elicits LHRH release from the median eminence (ME) of immature female rats in vitro, we hypothesized that it may represent one of the peripheral signals suspected to link somatic development to the LHRH-releasing system at puberty. We now present evidence in support of this concept. Quantitation of IGF-I messenger RNA (mRNA) levels by ribonuclease protection assay revealed that expression of the IGF-I gene did not change in the medial basal hypothalamus or preoptic area of female rats during peripubertal development. In contrast, the contents of both IGF-Ia and IGF-Ib mRNA, the two alternatively spliced forms of the IGF-I gene, increased significantly in the liver during the early proestrous phase of puberty. This change was followed by an elevation in serum IGF-I levels during the late proestrous phase of puberty along with a concomitant increase is serum gonadotropin levels. The proestrous change in serum IGF-I levels was accompanied by a selective increase in IGF-I receptor (IGF-IR) mRNA in the ME. Small doses of IGF-I (2-200 ng), administered intraventricularly, effectively induced LH release in both juvenile and peripubertal female rats, an increase prevented by prior immunoneutralization of LHRH actions. Importantly, intraventricular injections of IGF-I (20 ng), administered twice daily in the afternoon to immature animals, significantly advanced puberty. Thus, these results suggest that IGF-I of peripheral origin contributes to the initiation of female puberty by stimulating LHRH release from the hypothalamus, an effect that appears to be amplified by the increased synthesis of IGF-I receptors in the ME during first proestrus.

Exogenous estrogen acts differently on production of estrogen receptor in the preoptic area and the mediobasal hypothalamic nuclei in the newborn rat

Expression of the estrogen receptor (ER) in the preoptic area (POA) and the mediobasal hypothalamus (MBH) in newborn female rats was studied by immunohistochemistry (IHC) and in situ hybridization histochemistry (ISHH). The number of ER immunoreactive (ER-IR) cells decreased and expression of ER mRNA was suppressed in the arcuate (ARH) and the ventromedial (VMH) hypothalamic nuclei by daily injections of estradiol benzoate (EB) for ten consecutive days. In contrast, in the POA, expression of ER mRNA was not suppressed by EB treatment, while the ER immunoreactivity and the number of ER-IR cells was decreased by EB treatment. Results of quantification of ER mRNA by reverse transcription-polymerase chain reaction correlated well with results from ISHH: that is, ER mRNA expression decreased in the MBH but not in the POA. Thus, estrogen affects ER gene expression differently in these two brain regions.

Exogenous estrogen acts differently on production of estrogen receptor in the preoptic area and the mediobasal hypothalamic nuclei in the newborn rat

Expression of the estrogen receptor (ER) in the preoptic area (POA) and the mediobasal hypothalamus (MBH) in newborn female rats was studied by immunohistochemistry (IHC) and in situ hybridization histochemistry (ISHH). The number of ER immunoreactive (ER-IR) cells decreased and expression of ER mRNA was suppressed in the arcuate (ARH) and the ventromedial (VMH) hypothalamic nuclei by daily injections of estradiol benzoate (EB) for ten consecutive days. In contrast, in the POA, expression of ER mRNA was not suppressed by EB treatment, while the ER immunoreactivity and the number of ER-IR cells was decreased by EB treatment. Results of quantification of ER mRNA by reverse transcription-polymerase chain reaction correlated well with results from ISHH: that is, ER mRNA expression decreased in the MBH but not in the POA. Thus, estrogen affects ER gene expression differently in these two brain regions.

Morphological and pharmacological evidence for neuropeptide Y-galanin interaction in the rat hypothalamus.

Galanin (GAL) and neuropeptide Y (NPY) have been shown to play important roles in the regulation of pituitary hormone secretion, as well as ingestive and sexual behaviors, by acting within the hypothalamus. While the mechanism of action of these regulatory peptides is under intensive investigation, less attention has been paid to the possible interaction between them in influencing these central regulatory processes. Because NPY and GAL augment pituitary gonadotropin release, the present study was undertaken to evaluate the nature of morphological and functional relationships between these excitatory hypothalamic peptidergic systems. Double immunolabeling for NPY and GAL was carried out on vibratome sections taken from the hypothalamus of colchicine-pretreated female rats. Avidinbiotin peroxidase technique and a dark blue diaminobenzidine reaction was used to visualize NPY profiles, while the GAL neurons were labeled with a light brown diaminobenzidine reaction using either the avidin-biotin peroxidase or the peroxidase antiperoxidase technique. Light microscopic examination of the immunostained material showed that in the arcuate nucleus, paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, and medial preoptic area, an abundant network of NPY-immunoreactive axons surrounded GAL-immunostained cells. Numerous dark blue NPY-containing putative boutons were observed in close proximity to GAL-immunolabeled cell bodies and dendrites. Correlated light and electron microscopic examination revealed that most of the immunoreactive NPY axon terminals established synaptic connections with GAL-expressing cells. Synaptic connections were most frequently found in the medial preoptic area and in the magnocellular region of the paraventricular nucleus and arcuate nucleus. Fewer connections were observed in the supraoptic nucleus. These morphological observations demonstrate the existence of a strong NPY input to hypothalamic GAL neurons, thereby suggesting a modulatory role for NPY in monitoring GAL release. To evaluate the functional relevance of this anatomical relationship, the effects of intraventricular injection of a GAL receptor antagonist, galantide, were examined on NPY-induced LH release in ovarian steroid-primed ovariectomized rats. As expected, intraventricular injection of NPY readily stimulated LH release. Although, while on its own, galantide was ineffective in altering basal LH release, it markedly attenuated the NPY-induced LH response, thereby suggesting that GAL released in response to NPY administration may, in part, mediate the excitatory effects of NPY. These experimental results, taken together with the morphological observations, document the involvement of an NPY --> GAL signaling modality in the release of gonadotropins and, likewise, raise the possibility of a similar signaling process in the release of other pituitary hormones and elicitation of behavioral effects attributed to NPY and GAL.

The perinatal ontogeny of estrogen receptor-immunoreactivity in the developing male and female rat hypothalamus

Developmental expression of the estrogen receptor (ER) in rat hypothalamus was examined using immunohistochemistry. In the medial preoptic nucleus and ventromedial nucleus ER-immunoreactivity was detected as early as E17, whereas ER protein expression in the periventricular preoptic nucleus and arcuate nucleus was delayed until E19. These results show that following a region specific onset of the ER protein expression sex differences in ER levels are already detectable during the perinatal period.

Effects of prenatal morphine and adult estrogen administration on mu-opioid inhibition of norepinephrine release from hypothalamic slices.

The present study tested the hypotheses that exposure to morphine in utero (10 mg/kg twice a day on gestation days 11-18) and acute estrogen treatment in adulthood alter mu-opioid regulation of hypothalamic norepinephrine (NE) release in sexually mature rats. Both basal and KCl-stimulated NE releases were measured in superfused hypothalamic and preoptic area (POA) slices preloaded with 3H-NE. The mu-opioid receptor agonist D-Ala2, MePhe4-Gly-ol5-enkephalin (DAMGO; 10 or 100 nM) or the opioid antagonist naloxone (1 microM) was applied to some slices 15 min prior to KCl stimulation. Prenatal morphine had no effect on basal or KCl-stimulated release of preloaded 3H-NE from hypothalamic and POA slices from adult male progeny. Neither the mu-opioid agonist DAMGO nor the nonspecific antagonist naloxone significantly affected KCl-evoked overflow of 3H-NE in slices from either brain region of male rats. Adult female offspring were ovariectomized (OVX), and some were injected with a replacement dose of estrogen 48 h prior to sacrifice. Prenatal morphine had no effect on basal or KCl-stimulated release of 3H-NE from hypothalamic or POA slices or on the response of slices to opioid drugs. Estrogen treatment modestly increased KCl-evoked release of 3H-NE from POA slices from females. Moreover, there was a significant interaction between opioid drugs and estrogen treatment on KCl-evoked overflow of 3H-NE. In hypothalamic and POA slices from OVX females, DAMGO reduced KCl-evoked efflux of 3H-NE. This inhibitory effect of DAMGO was not apparent in slices from estrogen-treated females. In addition, naloxone increased KCl-stimulated NE release in slices from both hypothalamus and POA of estrogen-treated female rats but not control OVX females. Thus, prenatal exposure to morphine does not alter basal, KCl-evoked or mu-opioid modulation of NE efflux from hypothalamic or POA slices in adult progeny of either sex. However, treatment of adult, OVX females with estrogen attenuates mu-opioid inhibition and promotes the appearance of naloxone facilitation of KCl-evoked 3H-NE release from hypothalamic and POA slices.

Sex-related alterations in hypothalamic growth hormone-releasing hormone mRNA-but not somatostatin mRNA-expressing cells in genetically obese Zucker rats.

The possibility that the growth hormone (GH) suppression associated with obesity is due to alterations in hypothalamic GH-releasing hormone (GHRH) and/or somatostatin (SRIH) has been considered, but the data are not consistent. In the present study, we sought to clarify the roles of GHRH and SRIH in obesity by using in situ hybridization to localize and quantify the level of expression of GHRH mRNA- and SRIH mRNA-containing neurons in the hypothalamus of male and female lean and obese Zucker rats (12 weeks of age; n = 6 per group). In lean animals, the number of GHRH mRNA-expressing cells in the arcuate nucleus and SRIH mRNA-containing neurons in the periventricular nucleus was 2- to 3-fold higher in males compared to females. The obese phenotype in the male was associated with a striking reduction in arcuate GHRH mRNA expression, both in terms of number of cells (-71%; p < 0.01) and grains/cell (-44%; p < 0.05). In contrast, in obese females, there was a marked augmentation (+ 175%; p < 0.05) in the number of GHRH mRNA-containing cells in the arcuate nucleus compared to their lean littermates. The small population of GHRH mRNA-containing neurons of the ventromedial nucleus was not modified in male obese rats, while it was considerably increased (p < 0.05) in obese females. Neither the number of labeling density of SRIH mRNA-containing neurons in the periventricular and arcuate nuclei of obese rats of either sex was changed when compared to their sex-matched lean counterparts. These results demonstrate that: (1) the obese male Zucker rat exhibits a marked diminution in hypothalamic GHRH mRNA expression, while a reverse pattern is evident in the obese female; (2) hypothalamic SRIH mRNA-containing neurons are not significantly altered in obese rats of both sexes. Our findings suggest that the impaired GH secretion of the obese Zucker rat is due, at least in part, to alterations in hypothalamic GHRH gene expression and that SRIH does not play a major role.

Birthdates of the tyrosine hydroxylase immunoreactive neurons in the hypothalamus of male and female rats.

This study determined the birthdates of the tyrosine hydroxylase-(TH) immunoreactive (IR) neurons in the zona incerta (ZI), periventricular nucleus (PeVN) and arcuate nucleus (AN) of male and female rats. 'Long-survival' [3H]thymidine autoradiography combined with TH immunocytochemistry, the first enzyme of catecholamine synthesis, was used. In males, TH-IR neurons originate in the ZI between embryonic days (E) 12 and 13, while in the PeVN and AN this process is prolonged until E16. The majority of TH-IR neurons became postmitotic at E12 in the ZI, between E12 and E14 in the PeVN and at E15 in the AN. The birthdate of TH-IR neurons was sexually dimorphic with (a) generation of the majority of TH-IR neurons in the ZI in males proceeding that in females, (b) generation of TH-IR neurons in the AN of males delayed as compared to females, and (c) average daily fractions of the newborn TH-IR neurons in each hypothalamic region of females exceeding that seen in males. This sexual dimorphism was observed prior to E16, i.e. before the onset of sex difference in androgen levels, implying a hormone-independent mechanism, determined at the genetic level.

Anesthesia during hormone administration abolishes the estrogen induction of preproenkephalin mRNA in ventromedial hypothalamus of female rats

Estrogen treatment increases preproenkephalin (PPE) mRNA levels in the ventromedial nucleus of the hypothalamus (VMH). Roy et al. (Brain Res., 337 (1985) 163-166) discovered that anesthesia during estrogen priming could reduce female rat sexual receptivity. In the present study we tested whether the action of estrogen to induce PPE gene expression in the VMH could be similarly affected by anesthesia. By quantitative in situ hybridization and slot-blot analysis techniques we found a 1.8-fold increase in PPE mRNA levels in the VMH after 1 hour of estrogen treatment in ovariectomized (OVX) Sprague-Dawley female rats. Anesthetizing the rats with pentobarbital for 1 h during the exposure to estrogen blocked the estrogen induction of PPE mRNA in the VMH. By way of contrast no changes in the PPE mRNA levels were observed in the caudate putamen. A similar trend was seen using chloral hydrate. It appears that neuronal activity is required for the early phase of estrogen induction of PPE mRNA levels in the VMH. This in turn could be correlated with changes in female sociosexual behaviors.

The effect of acute MDMA administration on body temperature, serum corticosterone and neurotransmitter concentrations in male and female rats

AbstractThe aim of this study was to examine the effect of acute administration of (±)3,4‐methylenedioxymethamphetamine (MDMA) (20 mg/kg, i.p.) on colonic temperature, serum corticosterone and brain biogenic amine concentrations in group‐housed male and female rats and to determine if there are any gender differences in susceptibility to MDMA‐induced changes. Prior to treatment, basal colonic temperatures were higher in female than in male rats (p &lt;0·05). Temperatures were significantly increased 30 min following MDMA (20 mg/kg, i.p.) in male rats (p &lt;0·05), and 120 min following MDMA in female rats (p &lt;0·05), and were still elevated 240 min following MDMA administration in both sexes. Basal corticosterone concentrations were also higher in female than in male rats (p &lt;0·05), prior to treatment. Serum corticosterone concentrations were significantly elevated in both male and female rates 15 min after MDMA administration (p &lt;0·001), and remained elevated for up to 4 h following MDMA (p &lt;0·001 and p &lt;0·05 respectively). 5‐Hydroxytryptamine (5‐HT) and 5‐hydroxy‐indole‐3‐acetic acid (5‐HIAA) concentrations were significantly depleted in the frontal cortex, hypothalamus and striatum of both male and female rats. However, pineal 5‐HT was resistant to depletion by MDMA in both male and female rats. The 5‐HIAA concentration was significantly reduced in the pineal gland of female rats, probably due to decreased catabolism of pineal 5‐HT. There was a profound decrease in the ratio 5‐HT/5‐HIAA in the frontal cortex but no changes occurred in these ratios in the striatum. hypothalamus or pineal gland in either male or female rats. Dopamine (DA) concentrations were increased and 3,4‐dihydroxyphenyl‐acetic acid (DOPAC) levels decreased in the striatum in both male and female rats 4 h after MDMA administration. The noradrenaline (NA) content of the hypothalamus was increased in male rats 30, 60 and 120 min post MDMA (p &lt;0·05) but no changes were noted in the hypothalamus of female rats. This study indicates that there are slight gender differences in susceptibility to MDMA induced changes in rats.

Ovulatory cycle influences the stimulatory effect of stress on the expression of corticotropin-releasing factor receptor messenger ribonucleic acid in the paraventricular nucleus of the female rat hypothalamus

Ovulatory cycle influences the stimulatory effect of stress on the expression of corticotropin-releasing factor receptor messenger ribonucleic acid in the paraventricular nucleus of the female rat hypothalamus

Ovulatory cycle influences the stimulatory effect of stress on the expression of corticotropin-releasing factor receptor messenger ribonucleic acid in the paraventricular nucleus of the female rat hypothalamus.

The purpose of this study was to investigate whether the ovulatory cycle interferes with the effect of immobilization exposure on the expression of the type I CRF receptor (CRF-R) gene in the brains of female rats. The reproductive stages of adult female rats (200-250 g; 14 h of light; lights on at 0600 h) were verified by daily vaginal smears taken every morning for a minimum of three or four cycles before the experiment. Three hours after beginning the 90-min immobilization session, rats were deeply anesthetized and transcardially perfused with a solution of 4% paraformaldehyde in the morning (1100 h) and the afternoon (1700 h) of proestrus and diestrous day 2. Frozen brains were mounted on a microtome, cut in 30-microns slices, and processed for the detection of CRF-R messenger RNA (mRNA) by in situ hybridization histochemistry. Strand-specific antisense riboprobe was generated by in vitro transcription using T7 RNA polymerase and [35S]UTP. Basal levels of CRF-R transcripts were observed in several defined regions of the brain, such as the medial septal nucleus, nucleus of the diagonal band, basolateral and medial nuclei of the amygdala, red nucleus, pontine gray, and various layers of the cerebral cortex. Although control animals displayed almost undetectable levels of CRF-R mRNA in the paraventricular nucleus (PVN), immobilization stress induced a marked expression of the gene encoding CRF-R in the dorsomedial parvocellular division of this hypothalamic structure in immobilized female rats. The large majority of CRF-R-positive cells were colocalized in CRF-immunoreactive neurons. Interestingly, stress-induced CRF-R transcription was significantly higher in the morning of proestrus than in the afternoon and on diestrous day 2. Apart from the PVN, none of the other sites exhibiting basal signals of CRF-R mRNA in the brain was affected by acute exposure to immobilization. These results provide evidence that neurogenic stress can stimulate the transcription of the type I CRF-R selectively in the parvocellular PVN, a phenomenon influenced by the neuroendocrine events regulating the reproductive cyclicity in female rats.

Morphological evidence for a galanin-opiate interaction in the rat mediobasal hypothalamus.

It is well established that hypothalamic galanin- and beta-endorphin-containing circuits play important roles in the neuroendocrine regulation of pituitary hormone secretion and sexual behaviors, as well as in feeding. Recent experimental evidence suggests that an opiate-galanin interaction may be involved in these neuroendocrine responses. In particular, galanin and beta-endorphin have been shown to stimulate prolactin release from the pituitary, and concurrently, evoke feeding in the rat. The present study was designed to elucidate the morphological component underlying these responses in the hypothalamus. Sections of the mediobasal hypothalamus of colchicine-pretreated female rats were double immunostained for galanin and beta-endorphin. A dark blue nickel ammonium sulfate-intensified diaminobenzidine reaction was used to visualize galanin profiles, while beta-endorphin neurons were labeled with a light brown diaminobenzidine reaction. Light microscopy revealed putative connections between galanin boutons and beta-endorphin cells. Electron microscopic examination showed that galanin boutons form axo-somatic and axo-dendritic synaptic connections with beta-endorphin neurons. The vast majority (89.6%) of the beta-endorphin-immunoreactive neurons were found to be contacted by galanin-immunopositive fibers in the hypothalamus. To determine the origin of the galanin fibers innervating this region, the arcuate nuclei of additional rats were isolated unilaterally using a Halász-knife. After a ten day survival period, immunostaining was carried out for galanin. The relative surface occupied by galanin immunoreactive profiles on the ipsi- and contralateral sides were compared using an image analyzer. This analysis revealed that deafferentation of the arcuate nucleus did not decrease the density of galanin immunoreactive profiles on the isolated side of the arcuate nucleus compared to the control side, thus, indicating that the galanin boutons contacting beta-endorphin cells are most probably of local origin. These studies support the proposal that galanin-evoked prolactin secretion and feeding behavior may, in part, be mediated by enhanced beta-endorphin release and raises the possibility that a hypothalamic galanin-beta-endorphin axis may operate in the control of other pituitary hormones.

N-methyl-D-aspartic acid receptor messenger ribonucleic acid levels and luteinizing hormone release in immature female rats: effects of stage of pubertal development and exposure to ethanol

This research was designed to determine 1) whether changes occur in the levels of N-methyl-D-aspartic acid (NMDA) receptor (NMDA-R) messenger RNA (mRNA) in the reproductive hypothalamus of female rats as they approach puberty, 2) whether NMDA-R stimulation would promote differential LH responses during the specific stages of peripubertal development, and 3) whether ethanol (ETOH), which is known to affect the NMDA-R in other brain systems, can alter NMDA-R-activated LH secretion at puberty. In the first experiment, female rats were killed at 15, 20, 25, and 34-36 days of age to determine the levels of mRNA that code for the NMDA-R, specifically NMDA-R1, in the arcuate nucleus-median eminence (AN-ME) and preoptic area (POA) during pre- and peripubertal development by a ribonuclease protection assay. Results indicate that in juvenile animals, NMDA-R mRNA levels in the AN-ME increased at 25 days (P < 0.01). In the POA, the levels increased at 20 days (P < 0.05), but were unchanged at 25 days. During the peripubertal period, NMDA-R gene expression in the AN-ME did not change; however, gene expression in the POA increased (P < 0.05) during first proestrus, then declined during first estrus. In the second experiment, NMDA-R stimulation with N-methyl-D,L-aspartic acid (NMA; 2.5 mg/kg) produced differential stimulatory effects on LH release depending upon the stage of pubertal development. In this regard, significant post-NMA percent increases in LH released over pre-NMA (basal) levels occurred during anestrus (46%; P < 0.01) and first proestrus (95%; P < 0.01), with nonsignificant increases of 18% and 28% during first estrus and diestrus, respectively. Finally, a 3 g/kg dose of ETOH given intragastrically 90 min before the NMA challenge blocked (P < 0.05) NMA-induced LH release during first proestrus. In conclusion, these findings demonstrate regional differences in the timing of NMDA-R gene expression in the reproductive hypothalamus during pubertal development, show differential responses of LH to NMDA-R activation during the peripubertal period, and continue to demonstrate the vulnerability of the hypothalamic-pituitary axis to the detrimental effects of ETOH at this critical time of development.

N-methyl-D-aspartic acid receptor messenger ribonucleic acid levels and luteinizing hormone release in immature female rats: effects of stage of pubertal development and exposure to ethanol.

This research was designed to determine 1) whether changes occur in the levels of N-methyl-D-aspartic acid (NMDA) receptor (NMDA-R) messenger RNA (mRNA) in the reproductive hypothalamus of female rats as they approach puberty, 2) whether NMDA-R stimulation would promote differential LH responses during the specific stages of peripubertal development, and 3) whether ethanol (ETOH), which is known to affect the NMDA-R in other brain systems, can alter NMDA-R-activated LH secretion at puberty. In the first experiment, female rats were killed at 15, 20, 25, and 34-36 days of age to determine the levels of mRNA that code for the NMDA-R, specifically NMDA-R1, in the arcuate nucleus-median eminence (AN-ME) and preoptic area (POA) during pre- and peripubertal development by a ribonuclease protection assay. Results indicate that in juvenile animals, NMDA-R mRNA levels in the AN-ME increased at 25 days (P < 0.01). In the POA, the levels increased at 20 days (P < 0.05), but were unchanged at 25 days. During the peripubertal period, NMDA-R gene expression in the AN-ME did not change; however, gene expression in the POA increased (P < 0.05) during first proestrus, then declined during first estrus. In the second experiment, NMDA-R stimulation with N-methyl-D,L-aspartic acid (NMA; 2.5 mg/kg) produced differential stimulatory effects on LH release depending upon the stage of pubertal development. In this regard, significant post-NMA percent increases in LH released over pre-NMA (basal) levels occurred during anestrus (46%; P < 0.01) and first proestrus (95%; P < 0.01), with nonsignificant increases of 18% and 28% during first estrus and diestrus, respectively. Finally, a 3 g/kg dose of ETOH given intragastrically 90 min before the NMA challenge blocked (P < 0.05) NMA-induced LH release during first proestrus. In conclusion, these findings demonstrate regional differences in the timing of NMDA-R gene expression in the reproductive hypothalamus during pubertal development, show differential responses of LH to NMDA-R activation during the peripubertal period, and continue to demonstrate the vulnerability of the hypothalamic-pituitary axis to the detrimental effects of ETOH at this critical time of development.

Sex-specific changes in preoptic regulatory factor-1 and preoptic regulatory factor-2 mRNA expression in the rat brain during development

Preoptic regulatory factor-1 (Porf-1) and Preoptic regulatory factor-2 (Porf-2) are two novel peptide genes which are expressed in the central nervous system. Expression is modified by age and by hormones of the reproductive system. In this study nuclease protection assays were employed to investigate Porf-1 and Porf-2 mRNA expression in the cerebral cortex (CC), hippocampus (HIPP), preoptic area (POA) and medial basal hypothalamus (MBH) of male and female rats, aged 15, 30 and 60 days. Porf-1 and Porf-2 mRNA expression tended to decrease from 15 to 60 days, with two exceptions. Porf-2 in the hippocampus of female rats, and Porf-1 in the MBH of the male rats, were found instead to increase with age. There were distinctive sex differences inporf-1 andporf-2 gene expression. Consistently higher mRNA levels were measured for both genes in the POA of female rats at all ages examined, and this difference reached statistical significance for Porf-1 at the age of 60 days. In contrast, levels of Porf-2 mRNA were higher in MBH of male than female rat MBH at 15 days, and Porf-1 mRNA was substantially more abundant in male than in female rat MBH at 30 and 60 days of age. These results indicate that there is sexual dimorphism and regional specificity in the developmental expression of these genes.

Progesterone attenuation of alpha 1-adrenergic receptor stimulation of phosphoinositol hydrolysis in hypothalamus of estrogen-primed female rats

These experiments examined whether the previously observed abolition by progesterone (P) of alpha 1-adrenergic potentiation of adenylyl cyclase activity in brain slices of estrogen-primed female rats is attributable to a reduced capacity of alpha 1-adrenoceptors to stimulate phosphoinositol hydrolysis. In preoptic area and hypothalamic slices from ovariectomized (OVX) female rats, both norepinephrine (NE) and the alpha 1-adrenergic agonist phenylephrine (PHE) were robust stimulators of inositol phosphate (IP) formation. The NE response was completely blocked by the alpha 1-adrenergic antagonist prazosin. PHE-induced IP formation in tissue from OVX females exposed for 48 h to estrogen alone was comparable to that in OVX controls. In hypothalamic tissue from OVX rats given estrogen plus P, NE and PHE activation of phosphoinositol hydrolysis was attenuated. Both chlorethylclonidine, an irreversible antagonist of alpha 1b-adrenoceptors, and 5-methylurapidil, an alpha 1a-selective antagonist, reduced NE-induced IP formation regardless of the hormonal condition of the animals. Analysis of PHE competition for [3H]prazosin binding indicated that agonist binding was of high affinity (Ki, 16-30 microM) and was unaffected by hormonal status. Therefore, P abolition of alpha 1-adrenergic augmentation of adenylyl cyclase is correlated with reduced phosphoinositol hydrolysis, but not with changes in alpha 1-adrenoceptor agonist binding affinity. Moreover, both alpha 1a- and alpha 1b-adrenoceptors appear to work together to stimulate this second messenger system in the female rat hypothalamus.

Progesterone attenuation of alpha 1-adrenergic receptor stimulation of phosphoinositol hydrolysis in hypothalamus of estrogen-primed female rats.

These experiments examined whether the previously observed abolition by progesterone (P) of alpha 1-adrenergic potentiation of adenylyl cyclase activity in brain slices of estrogen-primed female rats is attributable to a reduced capacity of alpha 1-adrenoceptors to stimulate phosphoinositol hydrolysis. In preoptic area and hypothalamic slices from ovariectomized (OVX) female rats, both norepinephrine (NE) and the alpha 1-adrenergic agonist phenylephrine (PHE) were robust stimulators of inositol phosphate (IP) formation. The NE response was completely blocked by the alpha 1-adrenergic antagonist prazosin. PHE-induced IP formation in tissue from OVX females exposed for 48 h to estrogen alone was comparable to that in OVX controls. In hypothalamic tissue from OVX rats given estrogen plus P, NE and PHE activation of phosphoinositol hydrolysis was attenuated. Both chlorethylclonidine, an irreversible antagonist of alpha 1b-adrenoceptors, and 5-methylurapidil, an alpha 1a-selective antagonist, reduced NE-induced IP formation regardless of the hormonal condition of the animals. Analysis of PHE competition for [3H]prazosin binding indicated that agonist binding was of high affinity (Ki, 16-30 microM) and was unaffected by hormonal status. Therefore, P abolition of alpha 1-adrenergic augmentation of adenylyl cyclase is correlated with reduced phosphoinositol hydrolysis, but not with changes in alpha 1-adrenoceptor agonist binding affinity. Moreover, both alpha 1a- and alpha 1b-adrenoceptors appear to work together to stimulate this second messenger system in the female rat hypothalamus.

Effects of dehydration and salt-loading on hypothalamic vasopressin mRNA level in male and female rats

Experiments were carried out on 9- to 11-week-old male and female Sprague-Dawley rats. Dot-blot analysis and 3′-end digoxigenin-labeled 26mer oligonucleotide probe were used in the investigation of vasopressin (AVP) mRNA level in the hypothalamus of male and female rats. The normal hypothalamic AVP-mRNA level in males was 48% higher than that in females ( P < 0.05). Plasma osmolality was also higher in males than in females ( P < 0.05). In dehydrated rats, the hypothalamic AVP-mRNA level was 2.47 and 1.98 times in females ( P < 0.001) and males ( P < 0.01), respectively, as much as in their normal controls; the difference in hypothalamic AVP-mRNA level between dehydrated females and males was statistically insignificant. Plasma osmolality was higher in dehydrated females than in dehydrated males ( P < 0.01). In salt-loaded rats, hypothalamic AVP-mRNA level was 2.47 and 2.17 times in females ( P < 0.001) and males ( P < 0.01), respectively, as much as in their controls. The difference in hypothalamic AVP-mRNA level between salt-loaded males and females was not statistically significant. Plasma osmolality in salt-loaded females was also higher than that in salt-loaded males ( P < 0.001). These findings indicate that there is sex difference in hypothalamic AVP mRNA level and plasma osmolality under normal conditions; during dehydration and salt-loading AVP mRNA level increases and the difference in AVP mRNA level between males and females becomes insignificant.