Arcuate-median Eminence Region Research Articles

Intermittent Fasting Dietary Restriction Regimen Negatively Influences Reproduction in Young Rats: A Study of Hypothalamo-Hypophysial-Gonadal Axis

Nutritional infertility is very common in societies where women fail to eat enough to match their energy expenditure and such females often present as clinical cases of anorexia nervosa. The cellular and molecular mechanisms that link energy balance and central regulation of reproduction are still not well understood. Peripheral hormones such as estradiol, testosterone and leptin, as well as neuropeptides like kisspeptin and neuropeptides Y (NPY) play a potential role in regulation of reproduction and energy balance with their primary target converging on the hypothalamic median eminence-arcuate region. The present study was aimed to explore the effects of negative energy state resulting from intermittent fasting dietary restriction (IF-DR) regimen on complete hypothalamo-hypophysial-gonadal axis in Wistar strain young female and male rats. Significant changes in body weight, blood glucose, estrous cyclicity and serum estradiol, testosterone and LH level indicated the negative role of IF-DR regimen on reproduction in these young animals. Further, it was elucidated whether serum level of metabolic hormone, leptin plays a mechanistic role in suppressing hypothalamo-hypophysial-gonadal (HPG) axis via energy regulators, kisspeptin and NPY in rats on IF-DR regimen. We also studied the effect of IF-DR regimen on structural remodeling of GnRH axon terminals in median eminence region of hypothalamus along with the glial cell marker, GFAP and neuronal plasticity marker, PSA-NCAM using immunostaining, Western blotting and RT-PCR. Together these data suggest that IF-DR regimen negatively influences reproduction in young animals due to its adverse effects on complete hypothalamus-hypophysial-gonadal axis and may explain underlying mechanism(s) to understand the clinical basis of nutritional infertility.

Valproic Acid Alters GnRH-GABA Interactions in Cycling Female Rats

SUMMARY OF THE AIMS: Women with epilepsy using antiepileptic drug valproic acid (VPA) often suffer from reproductive endocrine disorders, menstrual disorders and polycystic ovaries. Valproic acid exerts anticonvulsive effects via gamma amino butyric acid (GABA) neurotransmitter system, which also acts as a neurochemical regulator of gonadotropin-releasing hormone (GnRH) neurons and suggests possibility of valproic acid mediated interruption in gonadotropin releasing hormone pulse generator in hypothalamus. The aim of this study was to investigate the effects of valproic acid treatment on the expression of gonadotropin releasing hormone, gamma amino butyric acid and polysialylated form of neural cell adhesion molecule (PSA-NCAM) a marker of neuronal plasticity in the median preoptic area (mPOA) and median eminence-arcuate (ME-ARC) region having GnRH neuron cell bodies and axon terminals, respectively. Three-month-old virgin Wistar strain female rats received VPA (i.p.) at a dose of 300 mg/kg once a day for 12 weeks; control group received an equivalent volume of vehicle. GnRH, GABA and PSA-NCAM expressions were studied by immunohistofluorescence technique from mPOA and ME-ARC region of hypothalamus. Ovarian histology was also studied using Mayer's Haematoxylin-Eosin staining method. GnRH and PSA-NCAM staining was much higher in mPOA and ME-ARC region from vehicle treated control proestrous rats, whereas VPA treatment significantly enhanced GABA expression, and reduced both GnRH and PSA-NCAM expression. Mayer's Haematoxylin-Eosin staining of mid-ovarian sections revealed significantly higher number of ovarian follicular cysts in VPA treated rats. Our findings of alterations in GnRH and GABA expression and GnRH neuronal plasticity marker PSA-NCAM as well as changes in ovarian histology suggest that treatment with VPA disrupts hypothalamo-hypophyseal-gonadal axis (HPG) at the level of GnRH pulse generator in hypothalamus.

Potential of PSA-NCAM in neuron-glial plasticity in the adult hypothalamus: Role of noradrenergic and GABAergic neurotransmitters

The present study was designed to establish the dynamic regulation of polysialylated form of neural cell adhesion molecule (PSA-NCAM) expression by neurotransmitters controlling gonadotropin releasing hormone (GnRH) secretion. The expression of PSA-NCAM and glial fibrillary acidic protein (GFAP) on GnRH cell bodies and axon terminals in the medial preoptic area (mPOA) and median eminence-arcuate (ME-ARC) region of hypothalamus was studied in the proestrous phase of cycling rats treated with α-adrenergic receptor blocker phenoxybenzamine (PBZ) and γ-aminobutyric acid (GABA) by using dual immunohistofluorescent staining and Western blot hybridization. To further elucidate whether activity mediated regulation of PSA-NCAM in GnRH neuron is via regulation of PSA biosynthesis by polysialytransferase (PST) enzyme, the expression of PST-1 enzyme was studied by using fluorescent in situ hybridization (FISH). Both GnRH and PSA-NCAM immunostaining was much higher in the mPOA and ME-ARC region from proestrous phase rats, whereas, PBZ and GABA treatments significantly reduced their expression, GFAP-ir and its content were increased in the PBZ and GABA treated proestrous rats. Taken together, our observations add to the growing evidence that PSA-NCAM plays permissive role for neuronal-glial remodeling and further suggest a functional link between activity dependent structural remodeling in GnRH neurons. Further, enhanced mRNA expression of PST suggests that the biosynthesis of PSA on NCAM is regulated at the transcriptional level.

Neuroendocrine plasticity in GnRH release during rat estrous cycle: correlation with molecular markers of synaptic remodeling

Morphological changes in the gonadotropin releasing hormone (GnRH) neurons in the preoptic area (POA) and their terminals in the median eminence-arcuate (ME-ARC) region are reported to occur during ovarian cycle that may be involved in the GnRH release into the portal blood during preovulatory surge. However, the neuronal substrates participating in altered GnRH neuronal plasticity are poorly understood. The present study was designed to determine whether morphological changes occurring in the GnRH neuron cell bodies in the POA and their terminals in the ME-ARC region of hypothalamus with pulsatile GnRH release in cycling female rats are associated with expression of intrinsic determinants of neuronal plasticity. The plasticity markers studied are polysialylated neural cell adhesion molecule (PSA-NCAM), high molecular weight isoforms of NCAM, growth associated protein (GAP-43), glial fibrillary acidic protein (GFAP) and synaptophysin. Regularly cycling female rats were sacrificed at diestrous, i.e., when GnRH release is low, and at proestrous, i.e., when preovulatory GnRH surge occurs, using perfusion fixation method for immunohistochemical staining of GnRH cells. GnRH cell bodies and their terminals from the POA and ME-ARC region respectively, were localized using immunohistochemical technique in proestrous and diestrous phase of estrous cycle and our results showed a marked increase in the GnRH nerve terminals length and immunoreactivity in the ME-ARC region from proestrous phase rats as compared to diestrous rats. Immunoblot analyses of the POA and ME-ARC region of the hypothalamus revealed a significant increase in the content of PSA-NCAM, NCAM-180, NCAM-140, GAP-43 and synaptophysin from proestrous phase rats as compared to diestrous phase rats. The ME-ARC region showed more pronounced increase in the protein expression of these markers of neuronal plasticity as compared to the POA, whereas, hippocampal region did not show any significant change in the content of these markers showing specificity of the changes to the GnRH system. GFAP content was significantly decreased in the POA with a marginal increase in the GFAP level from the ME-ARC region. These results demonstrate the involvement of synaptic proteins in the dynamic plasticity of the ME-ARC region of hypothalamus, allowing GnRH nerve terminals to release the neurohormone into the pituitary portal blood on the day of proestrous.

Role of opioidergic and monoaminergic neurotransmission in the GnRH release mechanism of EBP-primed OVX rats

We examined the effect of intracerebroventricular (i.c.v.) administration of μ-opioid agonist, morphine, and its antagonist naloxone followed by morphine on the activities of monoamine-metabolizing enzymes, namely tyrosine hydroxylase (TH) and monoamine oxidase (MAO) along with adenosinetriphosphatase (Na +, K + -ATPase), the enzyme responsible for the maintenance of ionic gradients across the membrane, in seven discrete regions of brain from estrogen- and progesterone-primed ovariectomized rats. TH activity decreased after morphine treatment in some areas such as the median eminence-arcuate region (ME-ARC), the amygdala, and the thalamus, showing statistically significant change. MAO activity increased in all the areas studied, but more appreciable change was observed in medial preoptic area (mPOA), the ME-ARC region, and the cortex. Pronounced increase in Na +, K + -ATPase enzyme activity was observed after the drug treatment. Naloxone given prior to morphine injection resulted in recovery of the enzyme activities in most of the areas studied. Our study may provide insights into the precise opioidergic modulation of gonadotropin releasing hormone (GnRH) release mechanisms through the involvement of monoaminergic system, elucidating the basis of various neuronal dysfunctions and their management in opioid addicts.

17β-estradiol inhibits the production of dopamine by the tuberoinfundibular dopaminergic neurons of the male rat

Previous work in our laboratory suggests that the indirect stimulation of prolactin gene transcription observed in male rats between 3 and 6 h following estrogen injection results from an inhibition of dopamine release from the tuberoinfundibular dopaminergic neurons. To evaluate further the role of dopamine in the regulation of prolactin gene transcription by estrogens, we have examined the acute effects of 17β-estradiol (E 2) on dihydroxyphenylalanine accumulation in the arcuate median eminence region and the amount of dopamine associated with the arcuate median eminence region. Dihydroxyphenylalanine accumulation in the arcuate median eminence region was unaffected when examined 2 h following a single injection of E 2 (10 μg), and was reduced by 60% when examined 4 h following E 2 treatment. When examined 4 h after E 2 injection, dopamine content in the arcuate median eminence region was significantly increased. These data suggest that estrogens exert acute inhibitory effects on the tuberoinfundibular dopaminergic neurons of the male rat, and further support the assertion that the indirect stimulatory actions of estrogens on prolactin gene transcription result from inhibition of hypothalamic dopamine production.

Androgen receptor-like immunoreactivity in the Brazilian opossum brain and pituitary: distribution and effects of castration and testosterone replacement in the adult male

Androgens are involved in a variety of centrally mediated functions after binding to their intracellular receptors. In the present report, we have employed the androgen receptor antibody, PG-21, and indirect immunuhistochemistry to examine the distribution of cells containing androgen receptor-like immunoreactivity (AR-IR) in the intact adult male Brazilian opossum brain and pituitary. Additional adult males were castrated to examine the effects of withdrawal of circulating androgens and testosterone replacement on AR-IR. Immunoblots and immunohistochemical controls demonstrated that the androgen receptor in the opossum brain and peripheral tissues are of a similar molecular mass as to has been reported for the rat. Cells containing AR-IR were widely distributed throughout the brain of intact adult males. The highest number of immunoreactive cells were present in the dorsal and ventral nuclei of the lateral septum, medial division of the bed nucleus of the stria terminalis, medial preoptic area, median preoptic nucleus, nucleus of the lateral olfactory tubercle, central amygdaloid nucleus, anterior cortical amygdaloid nucleus, posterior amygdaloid nucleus, subiculum, ventromedial hypothalamic nucleus, arcuate-median eminence region, and ventral premammillary nucleus. The anterior pituitary gland also contained a high number of cells containing AR-IR. The general distribution of AR-IR both in the brain and anterior pituitary gland resembled that reported for other mammalian species. Castration of the adult males four days prior to perfusion eliminated androgen receptor immunostaining throughout the brain except for a few lightly immunostained cells in the ventral nucleus of the lateral septum and stria terminalis. Androgen receptor immunostaining was decreased in the anterior pituitary gland following castration and became cytoplasmic. Testosterone administration 2 h before perfusion restored AR-IR both in the brain and anterior pituitary gland. These data suggested that immunohistochemscal detection of bound (nuclear) androgen receptors as seen with PG-21 antibody in the brain and anterior pituitary gland of the opossum is dependent upon circulating androgens. Further, the wide distribution and similarity in localization of androgen receptors in the opossum brain and anterior pituitary gland to that of other species suggests that androgen receptors might be involved in similar functions in the opossum as has been reported for other species.

LHRH pulse generator is stimulated by naloxone in the pentobarbital-blocked proestrous rat.

Previous studies by us and others led us to hypothesize that there are separate LHRH pulse and surge generators in the rat brain. The present study was designed to detect the activity of LHRH pulse generator by checking changes in LH secretion and the multiunit activity (MUA) of the arcuate-median eminence region of the hypothalamus during infusions of naloxone (NAL, 2 mg/h) in the proestrous rat in which the LHRH surge generator activity was blocked by pentobarbital sodium (PB, 32 mg/kg bw, ip). The animals were subjected to blood sampling in the morning (1000-1300 h) or afternoon (1400-1700), and injected with PB at 09.45 or 13.45, respectively. During saline infusions in the rat given PB injection at either 09.45 or 13.45, serum LH levels were low but fluctuated significantly, suggesting a pulsatile secretion in either the morning or the afternoon period. The pulse intervals were an average 28.2 min in the morning and 42.2 min in the afternoon. NAL infusions decreased the pulse interval significantly, to 22.0 min in the morning and to 27.0 min in the afternoon. In the electrophysiological experiment, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and therefore are considered to represent an increased activity of the LHRH pulse generator, appeared during NAL (5 mg/h) infusions in either the morning or the afternoon. These results strongly suggest that separate LHRH pulse and surge generators exist in the brain, and that, even during the critical period of proestrus, the activity of LHRH pulse generator is disclosed by PB, which, on the other hand, arrests the surge generator.

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.

Glutamic acid induces luteinizing hormone releasing hormone release via alpha receptors

Glutamic acid (GA) and norepinephrine (NE) stimulate luteinizing hormone-releasing hormone (LHRH) release via release of nitric oxide (NO) from NOergic neurons in the arcuatemedian eminence region. To determine if GA releases LHRH via direct stimulation of NOergic neurons, or via stimulation of noradrenergic terminals, arcuate median eminence explants from male rats were incubated with various compounds, and the LHRH release into the medium was measured. GA-induced release of LHRH was completely blocked by phentolamine (1 μM), an alpha receptor blocker, which, by itself, had no effect on the release. Nitroprusside (NP), which spontaneously releases NO, more than doubled LHRH release. To determine if alpha receptors on the LHRH neuron are required for the action of NP, the tissue was incubated with phentolamine, plus NP. Phentolamine had no effect on the LHRH-releasing action of NP. The results are interpreted to mean that GA activates the release of NE from the noradrenergic terminals. This acts on alpha receptors on the NOergic neuron to produce the release of NO. This NO diffuses to the LHRH terminals and induces release of LHRH.

Neonatal exposure to estradiol prevents the expression of ovarian hormone-induced luteinizing hormone and prolactin surges in adulthood but not antecedent changes in neuropeptide Y or adrenergic transmitter activity: Implications for sexual differentiation of gonadotropin secretion

Sex differences in adult patterns of mating behavior and gonadotropin secretion in rats are determined in part by the presence or absence of gonadal steroids during a perinatal critical period. For example, male rats and female rats exposed neonatally to androgen do not exhibit LH surge patterns when treated appropriately with ovarian hormones in adulthood, and there is evidence that this may be due to a failure of ovarian hormones to activate the hypothalamic neuronal systems that stimulate LH secretion in such animals. Because considerable evidence suggests that estradiol formed centrally from testosterone is responsible for the permanent defeminization of mating behavior and gonadotropin secretion, the present studies compared normal females with normal males and with females treated neonatally with estradiol on the ability of ovarian hormones to induce several important neurochemical changes antecedent to the LH surge, including changes in neuropeptide Y (NPY) and LH-releasing hormone (LHRH) concentrations in the median eminence, as well as changes in turnover rates for catecholamine transmitters in the medial basal hypothalamus and medial preoptic area. Normal ovariectomized female rats responded to sequential treatment with estradiol followed by progesterone with afternoon LH and prolactin (PRL) surges, and with sequential accumulation followed by decline in concentrations of LHRH and NPY in the median eminence prior to the LH surge. In addition, administration of progesterone increased the turnover rates of norepinephrine (NE) and epinephrine (EPI) in the arcuate-median eminence region of normal females. Gonadectomized male rats receiving the same ovarian hormone treatment failed to exhibit LH or PRL surges and displayed none of the changes in neurotransmitter turnover or peptide concentrations characteristically seen in the normal female. Unexpectedly however, when females that were treated with estradiol benzoate on days 1–3 postpartum were ovariectomized and treated with ovarian hormones in adulthood, they showed the same accumulation/decline in median eminence NPY concentrations and the same activation of NE and EPI turnover in the arcuate-median eminence region as normal females, even though they showed no LH or PRL surges or changes in median eminence LHRH concentrations. These results suggest that estradiol may not mediate all of the defeminizing actions of androgen exerted during the early neonatal period, and particularly those actions that result in a lack of responsiveness in central noradrenergic, adrenergic and NPY systems in adulthood. However, an action of neonatal estradiol may result in uncoupling of the LHRH neurosecretory system from normal excitatory neurochemical influences.

Effect of alloxan-induced diabetes on acetylcholinesterase activity from discrete areas of rat brain

The effect of experimental diabetes induced in rats was examined in brain areas like the septum, medial preoptic area, median eminence-arcuate region, amygdala, thalamus, hippocampus, pons and medulla. In all the areas studied, acute hyperglycemia caused an increase in the activity of acetylcholinesterase, the degradative enzyme of cholinergic system, whereas insulin administration reversed this effect. These findings suggest that the dysfunction of cholinergic system may also be involved in the diabetes associated CNS complications.

Changes in glucose metabolism from discrete regions of rat brain and its relationship to reproductive failure during experimental diabetes.

This study reports the effects of alloxan induced diabetes on glucose metabolism enzymes viz. Hexokinase, Lactate dehydrogenase, and Glucose-6-phosphate dehydrogenase from discrete brain regions. Enzymes activity was assayed from hypothalamic areas such as medial preoptic area and median eminence-arcuate region which have gonadotropin releasing hormone cell bodies and their terminals, respectively and other brain regions like septum, amygdala, hippocampus, and thalamus. In all the areas studied, induction of diabetes resulted in a significant decrease in particulate bound HK activity, whereas soluble HK, LDH and G6PDH activity showed increase at 3, 8, 15 and 28 days intervals. Insulin treatment of diabetic rats led to recovery in enzyme activity. Blood glucose levels increased significantly after induction of diabetes and recovery was seen after insulin treatment. The present results suggest that altered cerebral glucose metabolism may also be responsible for reproductive failure observed in diabetic rats.

Ablation of the hypothalamic arcuate-median eminence region reduces the concentration of vasoactive intestinal peptide in the anterior pituitary gland of male rats

This study was designed to determine the influence of the hypothalamus on the content of vasoactive intestinal peptide (VIP) in the anterior pituitary. Disruption of the hypothalamic-pituitary connection was performed by ablating the arcuate-median eminence (ARC-ME) region in adult male rats. Fifteen days later, there was a significant reduction in pituitary mass, adrenal and testicular weight and an increase in water consumption and serum prolactin levels indicating the elimination of hypothalamic influence on the pituitary gland in the ARC-ME group when compared to controls. Anterior pituitary VIP content was also significantly reduced in the lesion group. These data suggest that the hypothalamus is involved in the regulation of pituitary VIP.

Diurnal modulation of rat hypothalamic gonadotropin-releasing hormone release by melatonin in vitro

The mechanism(s) by which melatonin (MEL) can regulate gonadotropin secretion remains unresolved. Accordingly, we used acute in vitro incubations of male rat hypothalamic tissues to investigate effects of MEL on hypothalamic gonadotropin-releasing hormone (GnRH) secretion. At 10:00 h (3.5 after lights on), addition of 1 nM MEL to the medium inhibited (p = 0.028) GnRH release from the median eminence (ME) by 24%, 100 nM MEL had no significant effect on GnRH release, and 10 microM MEL tended to inhibit (p = 0.056, 21%) GnRH release. At 15:00 h, none of these MEL dosages significantly altered GnRH release from the ME. When the effect of a lower range of MEL dosages was evaluated, 1 nM MEL again inhibited (24%, p = 0.032) GnRH release from the ME at 10:00 h, and a linear dose-response relationship between initially increasing dosages (0, 0.01, 0.1, 1 nM) of MEL treatment and decreasing GnRH release was evident (r = 0.88), although a further 10 fold increase in MEL dosage (10 nM) resulted in a loss of suppression by MEL. Treatment with these dosages of MEL did not significantly alter ME GnRH release at 15:00 h. In contrast to the results with the ME alone, treatment with 0.1 microM MEL increased (p = 0.018) GnRH release from the arcuate-median eminence region (ARC-ME) by 60.2% at 10:00 h, whereas treatment with either 0.001 or 10 microM MEL did not significantly affect GnRH release. Treatment with these dosages of MEL did not significantly alter ARC-ME GnRH release at 15:00 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of c- fos protein by [formula omitted] in hypothalamus of immature female rats: blockade by MK-801 or neonatal treatment with monosodium glutamate

Significant NMDA-induced cellular activity in the median eminence-arcuate region of immature female rats was immunocytochemically localized via c- fos protein-like immunoreactivity. The production of this protein was completely prevented by pre-injection of MK-801 or APV, antagonists of the NMDA receptor-coupled ion channel. Treatment of newborn rats with monosodium glutamate (MSG), which destroys glutamate-sensitive neurons, significantly attenuated NMDA-induced c- fos when tested in the peripubertal period. These results implicate NMDA receptors in the expression of c- fos protein-like immunoreactivity in cells of the arcuate nucleus-median eminence of immature female rats.

Comparison of Age- and Sex-Related Changes in Cell Nuclear Estrogen-Binding Capacity and Progestin Receptor Induction in the Rat Brain*

Previous studies have suggested parallels between the mechanisms underlying sexual differentiation and age-related loss of reproductive cyclicity in the female rat. Both appear to involve the actions of estrogen on the brain and are associated with a reduction in hypothalamic estrogen sensitivity. In this study the effects of sex and aging on cell nuclear estrogen receptor-binding capacity and cytosol progestin receptor induction in the pituitary gland, periventricular preoptic area (PVP), medial preoptic area (mPO), bed nucleus of the stria terminalis, arcuate median eminence region (ARC), ventromedial nucleus (VMN), and corticomedial amygdala were directly compared. Young (2.5 months old) and middle-aged (8-10 months old) male and female and old (19 months old) female rats were gonadectomized 14 days before adrenalectomy (ADX). For comparison of cell nuclear estrogen receptor-binding capacity, animals received a saturating dose of estradiol (36.0 micrograms/kg BW) 3 days after ADX and 1 h before death. Cell nuclear estrogen binding was measured by an in vitro exchange assay. For comparison of estrogen-induced progestin receptors, animals received a sc placed Silastic capsule containing 10% 17 beta-estradiol at the time of ADX and were killed 3 days later. Cytosol progestin binding was measured by an in vitro binding assay. Cell nuclear estrogen-binding capacities and cytosol progestin receptor induction were lower in the PVP, mPO, and VMN of the young male than in the young female. In the female, the level of progestin receptor induction in the pituitary and brain was unaffected by age; however, cell nuclear estrogen-binding capacity in the mPO, VMN, ARC, and pituitary gland was lower in old than in middle-aged females. These results demonstrate that the effects of sexual differentiation and aging on the hypothalamus involve similar, but not identical, region-specific reductions in cell nuclear estrogen receptor-binding capacity. The consequences of these reduced estrogen receptor binding levels in terms of the induction of progestin receptor in response to estrogen exposure are, however, very different in the male compared to those in old female rats.

Morphine-induced inhibition of ovulation in normally cycling rats: Neural site of action

Intracerebral injection of morphine ( 3 μg 0.2 μl of solvent) on the day of proestrus at 1200 hr bilaterally in the medial preoptic area (mPOA) and median eminence-arcuate (ME-ARC) region inhibited ovulation in the cycling female rats. The morphine antagonist, lethidrone ( 2 μg 0.2 μl of solvent) when injected in the mPOA and ME-ARC region followed by intraperitoneal morphine ( 3 mg 100 g body weight) administration, was found to reverse the ovulation blocking action of this opiate. Implantation sites at which morphine was effective in blocking ovulation were restricted to a narrow medial band encompassing the mPOA and ME-ARC region. Our data are consistent with the view that the endogenous opioid peptides in mPOA and ME-ARC region of the hypothalamus may be involved in the physiological regulation of the central neural events which lead to ovulation in the rat.

Concomitant changes in the in vitro and in vivo release of opioid peptides and luteinizing hormone-releasing hormone from the hypothalamus following blockade of receptors for corticotropin-releasing factor.

In vitro and in vivo perfusion techniques were used to examine the changes in the release of beta-endorphin, methionine-enkephalin (met-enkephalin), dynorphin and luteinizing hormone releasing hormone (LHRH) in response to the corticotropin releasing factor (CRF) receptor antagonist, alpha-helical CRF9-41. All four peptides were measured in the same sample collected at each time interval by specific radioimmunoassay methods. In vitro release experiments were conducted using slices of hypothalami obtained from male rats whereas the in vivo release of these peptides was assessed in push-pull perfusates of the arcuate-median eminence (ARC-ME) region of the medial basal hypothalamus of chloral hydrate-anaesthetized male rats. Treatment of rat hypothalamic slices in vitro with alpha-helical CRF9-41 (10(-6) M) resulted in a significant suppression of the release of beta-endorphin and met-enkephalin within 10 min of application of the antagonist and a coincident significant increase in the release of LHRH. The levels of dynorphin were reduced but these changes were not significant. Within 10 min of withdrawal of the receptor antagonist and perfusion with normal (antagonist-free) medium the levels of these peptides returned to pretreatment values, i.e. the levels of beta-endorphin, met-enkephalin and dynorphin rose while those of LHRH fell. Comparable results were obtained in vivo during push-pull perfusion of the ARC-ME region with alpha-helical CRF9-41.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of progesterone on monoamine turnover in the brain of the estrogen-primed rat

The effect of progesterone (P) on monoamine levels and turnover was evaluated in 8 brain nuclei in estrogen-primed rats. Animals were subcutaneously (SC) injected with P or vehicle 21 hours after SC treatment with 5 μg of estradiol benzoate (EB). EB-primed animals treated with P showed high levels of lordosis behavior and an LH surge three hours later. Initial concentrations of norepinephrine (NE), dopamine (DA), serotonin (5HT) and 5-hydroxyindole acetic acid were determined in EB-saline treated controls 3 hours after P or vehicle. NE and DA turnover was estimated from the exponential decline of these amines 2 hours after IP injection of alpha-methyl-p-tyrosine (5 hours after P or vehicle). The accumulation of 5HT 20 min following IP injection of pargyline was used as an index of 5HT turnover. P did not affect the initial NE, 5HT or 5HIAA concentrations in any of the brain nuclei studied, but decreased DA content in the arcuate-median eminence region (Ar-ME). The DA rate constant was elevated in the nucleus of the diagonal band of Broca and the DA turnover rate was decreased in the Ar-ME. In the periventricular region (PVE, anterior hypothalamic level) the NE turnover rate (K, pg/μg protein/hr) and rate constant (k, hr −1) decreased following P treatment. Progesterone treatment decreased the accumulation of 5HT in the ventromedial hypothalamus (VMN, pars lateralis) and the dorsal midbrain central grey (MCG). Progesterone effects on monoamine turnover were not found in the lateral septal, medial preoptic, anterior hypothalamic or dorsal raphe nuclei. These results suggest that progesterone influences monoamine activity in a few discrete brain nuclei in EB-primed rats. Decreased 5HT turnover in the VMN and MCG after P is consistent with the hypothesis that 5HT exerts an inhibitory modulation of lordosis behavior and suggests that a component of the facilitatory action of progesterone is the suppression of 5HT activity at these brain sites. Changes in catecholamine turnover are discussed in relation to possible contributions in regulating gonadotropin secretion.