Arcuate Nucleus-median Eminence Research Articles

Role of galanin in stimulation of pituitary luteinizing hormone secretion as revealed by a specific receptor antagonist, galantide.

In this study, a specific galanin (GAL) receptor antagonist, galantide, was employed to evaluate the role of endogenous GAL in episodic basal and phasic LH release in rats. To assess the specificity of galantide, a series of experiments was performed. In the first experiment, we observed that administration of GAL (0.62 nm) intracerebroventricularly (icv) in ovarian steroid-primed ovariectomized (ovx) rats rapidly increased plasma LH levels between 10-30 min, and prior injection of galantide icv (5 nm) blocked the GAL-induced LH release. In the second experiment, galantide inhibited the GAL-evoked in vitro release of LHRH from the median eminence-arcuate nucleus (ME-ARC) of ovarian steroid-primed ovx rats. In addition, galantide on its own significantly decreased the basal efflux of LHRH from the ME-ARC of similarly treated rats, thereby suggesting that even the basal LHRH secretion may be a GAL-dependent event. In the third experiment, the effects of galantide on the phasic LH surge elicited by progesterone (P) in estradiol benzoate-primed ovx rats and that occurring spontaneously on proestrus were examined. Ovx rats bearing icv cannulae were primed with estradiol benzoate (30 micrograms/rat, sc) and 2 days later received a P (2 mg/rat, sc) injection at 1000 h to evoke a LH surge in the afternoon. Galantide (1 or 5 nm) in 3 microliters saline or saline was injected icv at 1300, 1400, and 1500 h. The results showed that the two dosages of galantide suppressed the LH surge in the afternoon. On the other hand, only a very high dose of galantide (15 nm) injected iv at 1300, 1400, and 1500 h blunted the P-induced LH hypersecretion. Central injections of galantide (1 or 5 nm) at 1300, 1400, and 1500 h on proestrus also inhibited the preovulatory LH surge and significantly reduced the numbers of rats ovulating the following day. In the final experiment, the role of GAL receptors in modulation of episodic LH release was analyzed in ovx rats. The results showed that an injection of galantide (5 nm, icv) significantly decreased both the mean LH levels and the amplitude of LH episodes during the 3-h observation period. Cumulatively, these results show that normally GAL stimulates LHRH release by activation of a specific receptor located in the ME-ARC, and that GAL may be a key excitatory signal in the hypothalamic neural circuitry involved in the regulation of basal episodic and phasic LHRH secretion in cycling female rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of galanin in stimulation of pituitary luteinizing hormone secretion as revealed by a specific receptor antagonist, galantide

In this study, a specific galanin (GAL) receptor antagonist, galantide, was employed to evaluate the role of endogenous GAL in episodic basal and phasic LH release in rats. To assess the specificity of galantide, a series of experiments was performed. In the first experiment, we observed that administration of GAL (0.62 nm) intracerebroventricularly (icv) in ovarian steroid-primed ovariectomized (ovx) rats rapidly increased plasma LH levels between 10-30 min, and prior injection of galantide icv (5 nm) blocked the GAL-induced LH release. In the second experiment, galantide inhibited the GAL-evoked in vitro release of LHRH from the median eminence-arcuate nucleus (ME-ARC) of ovarian steroid-primed ovx rats. In addition, galantide on its own significantly decreased the basal efflux of LHRH from the ME-ARC of similarly treated rats, thereby suggesting that even the basal LHRH secretion may be a GAL-dependent event. In the third experiment, the effects of galantide on the phasic LH surge elicited by progesterone (P) in estradiol benzoate-primed ovx rats and that occurring spontaneously on proestrus were examined. Ovx rats bearing icv cannulae were primed with estradiol benzoate (30 micrograms/rat, sc) and 2 days later received a P (2 mg/rat, sc) injection at 1000 h to evoke a LH surge in the afternoon. Galantide (1 or 5 nm) in 3 microliters saline or saline was injected icv at 1300, 1400, and 1500 h. The results showed that the two dosages of galantide suppressed the LH surge in the afternoon. On the other hand, only a very high dose of galantide (15 nm) injected iv at 1300, 1400, and 1500 h blunted the P-induced LH hypersecretion. Central injections of galantide (1 or 5 nm) at 1300, 1400, and 1500 h on proestrus also inhibited the preovulatory LH surge and significantly reduced the numbers of rats ovulating the following day. In the final experiment, the role of GAL receptors in modulation of episodic LH release was analyzed in ovx rats. The results showed that an injection of galantide (5 nm, icv) significantly decreased both the mean LH levels and the amplitude of LH episodes during the 3-h observation period. Cumulatively, these results show that normally GAL stimulates LHRH release by activation of a specific receptor located in the ME-ARC, and that GAL may be a key excitatory signal in the hypothalamic neural circuitry involved in the regulation of basal episodic and phasic LHRH secretion in cycling female rats.

Increased neuropeptide Y concentrations in specific hypothalamic regions of lactating rats: Possible relationship ot hyperphagia and adaptive changes in energy balance

Lactation is accompanied by hyperphagia and a reduction in brown adipose tissue (BAT) thermogenesis, which are unexplained. Neuropeptide Y (NPY) powerfully stimulates feeding and inhibits BAT thermogenesis when injected into the paraventricular nucleus and other specific regions of the rat hypothalamus. We have tested the hypothesis that hypothalamic NPY activity is increased in lactating rats. Lactating rats consumed over four times as much food as nonlactating controls ( n = 10; p < 0.001). Final plasma insulin concentrations in lactating rats were lower than in controls ( 6.8 ± 0.8 vs. 11.7 ± 2.1 pmol/l ; p < 0.05) although plasma glucose and corticosterone concentrations were comparable ( p > 0.05). Lactating rats showed significantly higher NPY levels than controls in specific hypothalamic regions, namely the arcuate nucleus-median eminence complex (a 41% rise; p < 0.001), paraventricular nucleus (35%; p < 0.001), ventromedial nucleus (66%; p = 0.003), and dorsomedial nucleus (78%; p < 0.001). Other hypothalamic regions showed no significant differences between groups. Increased NPY concentrations in specific hypothalamic regions, particularly the arcuate nucleus where NPY is synthesized, suggest increased activity of the hypothalamic NPYergic system in lactation. Neuropeptide Y may mediate hyperphagia and reduced BAT thermogenesis in lactation. Hypoinsulinemia may be a stimulus to hypothalamic NPY in lactation, as has been postulated in other conditions of negative energy balance.

Sexually dimorphic effects of aging on tuberoinfundibular dopaminergic neurons and lactotropes of rats

The effect of aging on plasma prolactin (PRL) levels, hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons and pituitary lactotropes was evaluated in prolactinoma-free young (5-month-old) and old (23- to 24-month-old) Long-Evans rats of either sex. The young female rats were in diestrus, the old ones in recurrent pseudo-pregnancy. The tyrosine hydroxylase (TH)-labelled neurons in the arcuate nucleus (AN) and axons in the median eminence (ME) as well as the PRL-labelled lactotropes in the pituitary gland were studied by morphometry and densitometric immunohistochemistry. Further, we investigated the secretory function of isolated lactotropes by reverse hemolytic plaque assay (RHPA) and by cell culture in comparable animal groups. Compared with young animals, the plasma PRL levels of old rats of both sexes were similar or reduced. All morphometric and densitometric measurements of the AN neurons, ME axons (except number) and pituitary lactotropes were comparable in young and old female rats. In old male rats the AN and ME measurements were mostly decreased, while the lactotropes remained almost unchanged. The RHPA generally showed a reduced PRL release from lactotropes of old animals of both sexes. The PRL release from the cultured lactotropes, on the contrary, was greatly increased in old female rats and unchanged in old male rats. Our functional and morphological data suggest that the in vivo function of lactotropes in old prolactinoma-free female and male rats does not seem to be strongly influenced by the mildly reduced TIDA neuron activity, yet emphasize the differences of the aging process in the two sexes

Role of Multiple Voltage-Sensitive Calcium Channels in Depolarization-Induced Release of Neuropeptide Y and Luteinizing Hormone-Releasing Hormone from Rat Median Eminence-Arcuate Nucleus

The objective of the present studies was to identify the subtypes of voltage-sensitive Ca2+ channels (VSCC) that regulate the basal and depolarization-induced release of neuropeptide Y (NPY) and LH-releasing hormone (LHRH) from rat hypothalamus. Tissues containing median eminence-arcuate nucleus (ME-ARC) were dissected from male rats and incubated in vitro in the presence and absence of agents selectively affecting N- or L-type VSCC. KCl depolarization-induced release of NPY, but not basal release, was abolished by Ca2+-free/EGTA medium and was significantly reduced by cobalt. The depolarization-induced release of both NPY and LHRH from ME-ARC was significantly augmented by BAY K 8644, an agonist at L-type VSCC, and this effect was blocked by nitrendipine, an antagonist at these channels. Neither agent modified basal release of NPY or LHRH. Nitrendipine alone blocked the release of NPY and LHRH evoked by a low concentration (23 mM) of KCl, but not by a higher concentration (45 mM). Omega-conotoxin (CgTX), which primarily blocks N-type VSCC, significantly blunted, but did not abolish, depolarization-induced release of NPY and LHRH without altering basal release. These findings suggest that multiple VSCC subtypes participate in the regulation of depolarization-induced release of NPY and LHRH. Similar to other systems, the N-type channel may be predominantly involved in the process by which entry of extracellular Ca2+ triggered by depolarization leads to the release of these two peptides. The inability of nitrendipine and CgTX to completely block depolarization-induced NPY and LHRH release suggests the involvement of at least one other VSCC in this process. Although L-type VSCC may not be of primary importance in depolarization-induced release of NPY and LHRH, activation of this channel subtype may enhance Ca2+ entry and therefore modulate release of these neuropeptides.

Evidence that nitric oxide may mediate the ovarian steroid-induced luteinizing hormone surge: involvement of excitatory amino acids.

The involvement of excitatory N-methyl-D-aspartate (NMDA) receptors in the hypothalamic control of pituitary LH secretion is well recognized. Recent evidence shows that nitric oxide (NO), a free radical gas, may act as neurotransmitter in the brain, and its efflux is stimulated by activation of NMDA receptors. Studies were undertaken to determine whether NO is involved in the hypothalamic release of LHRH and in the LH surge induced by progesterone (P) in estrogen-primed ovariectomized rats. Rats were ovariectomized and 2 weeks later received estradiol benzoate (30 micrograms sc) at 1000 h. Two days later, P was injected at 1000 h to potentiate the estradiol benzoate-induced LH surge in the afternoon. Serial blood samples were collected at hourly intervals from 1400-1800 h via an intraatrial cannula implanted the day before P injection. Additionally, at various times before onset of the LH surge at 1400 h, the rats were injected sc with one of three inhibitors of NO synthase, the enzyme that generates NO. Control, saline-injected rats showed unambiguous LH surges in the afternoon. However, either a single injection at 1000 h of NG-methyl-L-arginine (20 mg/kg) or three injections at 1000, 1200, and 1400 h of either Nw-nitro-L-arginine methyl ester (NAME, 40 mg/kg) or Nw-nitro-L-arginine (60 mg/kg) to inhibit NO efflux markedly suppressed the P-induced LH surge in the afternoon. To ascertain whether suppression of LH surge was due to blockade of hypothalamic LHRH release, a series of in vitro studies were performed in steroid-primed rats. First we examined the effects of sodium nitroprusside (NPS), a compound that spontaneously generates and releases NO. NPS increased basal and KCl-induced LHRH release in vitro from the medial basal hypothalamus-preoptic area and median eminence fragments. No direct effect of NO at the pituitary level was seen, since NPS did not alter basal or LHRH-induced LH in vitro release from hemipituitaries. In addition, we tested the effects of NAME on NMDA-induced LHRH release in vitro from the median eminence-arcuate nucleus fragments. As expected, NMDA alone (50 mM) induced a significant increase in LHRH release. Addition of NO synthase inhibitor, NAME (1 or 10 mM) to suppress NO efflux, significantly diminished the NMDA-induced LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Pargyline-induced increase in serotonin levels: Correlation with inhibition of lordosis in rats

The effect of intrahypothalamic infusion of the monoamine oxidase inhibitor pargyline on lordosis behavior and monoamine levels in the preoptic area and hypothalamus was examined. Progesterone-facilitated lordosis was blocked by pargyline in half the treated rats. The inhibition of lordosis was correlated with increases in serotonin and dopamine levels in the ventromedial nucleus of the hypothalamus and serotonin levels in the arcuate nucleus-median eminence when compared to controls or pargyline-treated rats with high levels of lordosis responding. Changes in norepinephrine levels were not correlated with changes in behavior. The results provide further evidence for an inhibitory role of basomedial hypothalamic serotonin in the control of female sexual behavior.

Abstracts of Communications

Neuropeptide Y (NPY), a thirty-six amino acid peptide, is a potent centrally-acting appetite-stimulating agent which is believed to regulate eating behaviour and body weight (Williams et al. 1991). Lactation in rodents is associated with increased nutrient requirements which are met by extraordinary increases in food intake (Vernon, 1989). The aim of the present study was to investigate whether hyperphagia in lactating rats is associated with increased hypothalamic NPY levels, as in the case of other hyperphagic states such as starvation and diabetes. Twenty female Wistar rats, initially matched for age and weight, were studied. The experiment began when the lactating group (n 10) was on average at day 16 of lactation (range, 13-17). The lactating rats’ food intake was over 330% that of the controls (P 0.05). Four of the eight hypothalamic areas showed significantly increased NPY levels in the lactating rats compared with controls, namely: the arcuate nucleus-median eminence (+41%; P<0.001); the paraventricular nucleus (+35%; P<0.00l); the ventromedial nucleus (+66% ; P=0.003); the dorsomedial nucleus (+78%; P<0.00l). Other hypothalamic regions showed no differences between the two groups. Increased NPY levels in the arcuate nucleus (its principal hypothalamic site of synthesis) and in the paraventricular and dorsomedial nuclei (NPY-sensitive sites to which the arcuate nucleus projects) suggest increased activity of the NPY-ergic system in lactation. NPY may therefore mediate the markedly increased food intake in lactation.

Endothelin-3 stimulates luteinizing hormone-releasing hormone (LHRH) secretion from LHRH neurons by a prostaglandin-dependent mechanism.

Endothelin (ET) peptides have recently been recognized as putative regulators of the endocrine system. Particularly in the gonadal system, ET-3 stimulates LH secretion from anterior pituitary cells cultured in vitro. In these studies, we evaluate the actions of ET-3, the most abundant species of the ET family in the central nervous system, on LHRH release from arcuate nucleus-median eminence (AN-ME) fragments and an LHRH-secreting neuronal cell line (GT1 cells) in vitro. ET-3 exhibited a stimulatory effect on LHRH secretion from AN-ME fragments and GT1 cells incubated in a static system as well as in a dynamic perifusion paradigm. In all the systems used, the effects of ET-3 on LHRH secretion showed a dose dependency. The increase in LHRH secretion induced by ET-3 was accompanied by an increased secretion of prostaglandin E2 (PGE2), not only in the AN-ME incubations, but also in the GT1 incubation and perifusion systems. Blockade of arachidonic acid and/or PG synthesis significantly reduced the ET-3-induced LHRH and concomitant PGE2 release from both AN-ME fragments and GT1 cells incubated in vitro. In AN-ME incubations, ET-3 effects were enhanced by potassium-induced depolarization. This suggests that activation of other transmitter system(s) may be needed for obtaining a physiological activation of the LHRH neuronal system. In summary, in these studies we provide evidence for a direct action of ET-3 on the LHRH neuronal system. This action is exerted directly on LHRH neurons either at the level of the nerve terminals, the perikaryon, or both. In addition, the effects of ET-3 on LHRH release require a functional arachidonic acid metabolic pathway, particularly involving PG synthesis, in order to obtain stimulation, indicating that PGs are involved in the intracellular events leading to ET-3-evoked LHRH secretion.

Acute hyperinsulinemia increases neuropeptide Y concentrations in the hypothalamic arcuate nucleus of fasted rats

Neuropeptide Y, a major hypothalamic peptide, stimulates feeding, insulin secretion and weight gain when injected intrahypothalamically. Hypothalamic NPY may be regulated by insulin availability at hypothalamic level, as its activity is apparently inhibited by intrahypothalamic insulin administration and is stimulated under insulin-deficient conditions. To determine the effects of acute physiological hyperinsulinemia, we measured regional hypothalamic NPY levels in rats during a hyperinsulinemic, euglycemic clamp. Seven male Wistar rats with implanted jugular cannulae, fasted for 24 h, were infused with insulin at 100 mU/h together with variable-rate glucose to maintain euglycemia (3.9±0.1 mmmol/l), for 150 min. Controls were infused for the same period with polygeline vehicle alone (n=8), and had blood glucose concentrations of 4.0±0.5 mmol/l. Insulin levels were 80.2±3.9 mU/l in insulin-infused rats and 15.2±1.4 mU/l in polygeline-treated controls (p<0.001). NPY levels, measured by radioimmunoassay, were significantly higher in the arcuate nucleus/median eminence (ARC/ME) of hyperinsulinemic rats than in controls (4.8±1.2 vs 2.5±0.6 fmol/ μg protein; p< 0.001), but were comparable with controls in 7 other hypothalamic regions. Acute physiological hyperinsulinemia therefore increases NPY levels selectively in the ARC/ME. Insulin could cause NPY accumulation in the ARC by blocking its transport to NPY-sensitive areas. This would be consistent with the suggestions that insulin inhibits hypothalamic NPY activity and also acts as a central satiety factor.

Ethanol alters N-methyl-DL-aspartic acid-induced secretion of luteinizing hormone releasing hormone and the onset of puberty in the female rat.

In the present study, we have evaluated the effects of ethanol (ETOH) on luteinizing hormone releasing hormone (LHRH) secretion induced by N-methyl-DL-aspartic acid (NMA) in vitro and the ability of NMA to induce precocious puberty in vivo. For the in vitro experiments, the basal and NMA-stimulated release of LHRH from arcuate nucleus-median eminence (AN-ME) fragments obtained from immature female rats was measured by RIA following static incubation in medium consisting of Krebs-Ringer bicarbonate glucose buffer. Control vials contained medium only and the test vials contained medium plus ETOH doses of 30, 50, and 70 mM. These data demonstrate that ETOH did not alter basal LHRH release, but dose-dependently blocked (p < 0.01) the NMA-induced release of the peptide during anestrus, as well as first proestrus and estrus. For the in vivo experiment, 26-day-old females began receiving saline or saline-ETOH (3 g/kg) solution by gastric gavage daily at 12.30 p.m. Each day at 2.00 and 4.00 p.m., each of the animals received subcutaneous injections of a 40-mg/kg solution of NMA in saline. Other control animals received saline gastrically, as well as subcutaneously. The timing of puberty was assessed in all animals by monitoring vaginal opening (VO) and first diestrus (D1). The mean (+/- SEM) age at VO for animals in the non-ETOH group, which received NMA, was 31.7 +/- 0.40 days. Vaginal smears revealed that D1 occurred at a mean (+/- SEM) age of 33.0 +/- 0.42 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y release from the paraventricular nucleus increases in association with hyperphagia in streptozotocin-induced diabetic rats.

We tested the hypothesis that the hyperphagia observed in streptozotocin (STZ)-induced diabetic rats is due to increased release of neuropeptide Y (NPY) in the paraventricular nucleus (PVN) of the hypothalamus. In the first experiment, male rats were injected with STZ or vehicle (control) via the tail vein and 18-20 days later, NPY levels in seven hypothalamic sites and release in vitro from selected hypothalamic sites were evaluated. The results showed that in association with STZ-produced marked hyperglycemia and hyperphagia, NPY concentrations were increased in four hypothalamic sites, including the PVN. Evaluation of NPY release in vitro showed that both basal and KCl-induced release was significantly higher from the micro-dissected PVN of STZ-treated than control rats. A similar augmentation in the NPY efflux in vitro was detected from the median eminence arcuate nucleus, but not from the neighboring ventromedial nucleus of STZ-treated rats. In the second experiment, rats were treated with STZ or vehicle and received permanent push-pull cannula (PPC) in the PVN for evaluation of NPY release in vivo 18-21 days after STZ treatment. The results showed that mean NPY levels in the perfusates collected from the PVN of diabetic rats were significantly higher as compared to control rats. Since NPY is the most potent naturally occurring orexigenic signal and the PVN is an important initial site of NPY action in the stimulatory pathway regulating feeding, our findings of augmented PVN NPY release in vivo and in vitro are in accord with the hypothesis that increased NPY secretion in the PVN may be responsible for hyperphagia in diabetic rats.

Neurokinin A in the anterior pituitary of female rats: Effects of ovariectomy and estradiol

The effect of acute and chronic ovariectomy and the substitutive treatment with 17-β estradiol and/or progesterone on anterior pituitary levels of neurokinin A (NKA) was studied in female rats. Acute ovariectomy did not result in significant changes of NKA in the anterior pituitary gland as compared with the levels in diestrous intact rats, but a single injection of 5 μg of estradiol in ovariectomized rats significantly decreased NKA levels in the anterior pituitary gland. Progesterone was without effect and did not modify the decrease of NKA in the anterior pituitary gland induced by estradiol. In rats examined 11 to 17 days after ovariectomy, NKA in the anterior pituitary gland was significantly higher than in diestrous intact rats. In the hypothalamus, ovariectomy resulted in decreased levels of NKA in the median eminence-arcuate nucleus. Estradiol significantly reduced NKA stores in the anterior pituitary gland but increased them in the whole hypothalamus and in the median eminence-arcuate nucleus. Thus, estradiol seems to be a powerful regulator of NKA stores in the adenohypophysis and also in the hypothalamus.

Cross-talk between excitatory and inhibitory amino acids in the regulation of luteinizing hormone-releasing hormone secretion

Inhibitory (IAA) and excitatory amino acid (EAA) neurotransmitters appear to play an important role in regulating reproductive functions. L-Glutamic acid (GLU), the major representative of the EAA system, stimulates LHRH release from arcuate nucleus-median eminence (AN-ME) fragments in vitro. Several studies have provided evidence for considering gamma-aminobutyric acid (GABA), a major IAA neurotransmitter, as another regulator of LHRH secretion. Recent reports have indicated that a cross-talk between GABA and GLU participates in the regulation of synaptic transmission in the brain. In concert with this notion, we present evidence indicating that this cross-talk between GABA and GLU appears to be also involved in neuroendocrinological paradigms. In this respect, bicuculline, a GABA-A receptor antagonist, blocked GLU-evoked LHRH secretion from AN-ME fragments in vitro without affecting basal LHRH release. In addition, activation of GABA-A receptors by muscimol (MUS) stimulated basal LHRH secretion. Interestingly, when MUS and GLU were added together to the incubation medium, an additive, stimulatory effect was observed. These observations clearly indicate that a GABAergic mechanism participates, via GABA-A receptors, in GLU-induced LHRH secretion from terminals of the ME. Furthermore, GABA-B receptors appear to negatively modulate the effects of GLU. Activation of GABA-B receptors by baclofen (BAC) blocked GLU-induced LHRH secretion, while phaclofen, a GABA-B receptor antagonist, reversed this effect. In summary, our data provide evidence for a cross-talk between EAA and IAA systems in the regulation of LHRH release, and, therefore, in the control of gonadal function.

Cross-talk between excitatory and inhibitory amino acids in the regulation of luteinizing hormone-releasing hormone secretion.

Inhibitory (IAA) and excitatory amino acid (EAA) neurotransmitters appear to play an important role in regulating reproductive functions. L-Glutamic acid (GLU), the major representative of the EAA system, stimulates LHRH release from arcuate nucleus-median eminence (AN-ME) fragments in vitro. Several studies have provided evidence for considering gamma-aminobutyric acid (GABA), a major IAA neurotransmitter, as another regulator of LHRH secretion. Recent reports have indicated that a cross-talk between GABA and GLU participates in the regulation of synaptic transmission in the brain. In concert with this notion, we present evidence indicating that this cross-talk between GABA and GLU appears to be also involved in neuroendocrinological paradigms. In this respect, bicuculline, a GABA-A receptor antagonist, blocked GLU-evoked LHRH secretion from AN-ME fragments in vitro without affecting basal LHRH release. In addition, activation of GABA-A receptors by muscimol (MUS) stimulated basal LHRH secretion. Interestingly, when MUS and GLU were added together to the incubation medium, an additive, stimulatory effect was observed. These observations clearly indicate that a GABAergic mechanism participates, via GABA-A receptors, in GLU-induced LHRH secretion from terminals of the ME. Furthermore, GABA-B receptors appear to negatively modulate the effects of GLU. Activation of GABA-B receptors by baclofen (BAC) blocked GLU-induced LHRH secretion, while phaclofen, a GABA-B receptor antagonist, reversed this effect. In summary, our data provide evidence for a cross-talk between EAA and IAA systems in the regulation of LHRH release, and, therefore, in the control of gonadal function.

Hypothalamic electrical activity that relates to the pulsatile release of luteinizing hormone exhibits diurnal variation in ovariectomized rats

The multiunit activity (MUA) was recorded in the arcuate nucleus-median eminence region in freely moving ovariectomized rats maintained under controlled lighting conditions (lights on 05.00–19.00 h) over a period of 24 h. The MUA of each rat had been confirmed to exhibit characteristic increases (volleys) in association with pulses of luteinizing hormone. In 6 MUAs examined, the frequency of MUA volleys was significantly greater in the dark period than in the light period, suggesting a diurnal variation in the release of luteinizing hormone-releasing hormone in the ovariectomized rat.

Endogenous excitatory amino acids and glutamate receptor subtypes involved in the control of hypothalamic luteinizing hormone-releasing hormone secretion.

These studies were designed to evaluate the actions and relative potencies of different endogenous and excitatory amino acid (EAA) selective analogs on EAA-induced neuropeptide secretion as well as to analyze the receptor subtypes involved. For this purpose, different glutamate agonists were tested for their ability to evoke release of the hypothalamic neuropeptide LHRH from arcuate nucleus-median eminence (AN-ME) fragments incubated in vitro. Different glutamate agonists, i.e. 3-amino-3-hydroxy-5-methyl-isoxazole-4-propionic (AMPA), kainic, quisqualic, homocysteic (HCA), quinolinic (QUIN), N-methyl-D-aspartic (NMDA), and pyroglutamic (PYR) acids, elicited LHRH release from AN-ME fragments in vitro. Further evaluation of the range of activity of several of these compounds, both in terms of the dose inducing a half-maximal response and the LHRH-releasing effect at that particular dose, indicated that AMPA greater than HCA greater than QUIN greater than PYR, suggesting that non-NMDA receptors are primarily involved in EAA-induced LHRH release at the level of the AN-ME. Evaluation of the receptor types involved using two specific antagonists for NMDA and non-NMDA receptors, D,L-2-amino-7-phosphoheptanoic acid and 6,7-cyanoquinoxaline-2,3-dione, respectively, showed that the effects of AMPA and HCA on LHRH release can be completely blocked by 6,7-cyanoquinoxaline-2,3-dione, whereas QUIN activity was blocked by D,L-2-amino-7-phosphoheptanoic acid. The effects of PYR on LHRH release were abolished by both receptor blockers. The metabotropic receptor agonist trans-1-amino-cyclopentyl-1,1,3-dicarboxylic acid was not active in eliciting LHRH secretion. The data indicate that endogenous substances active at EAA receptor sites, such as HCA, QUIN, and PYR, can significantly increase the secretion of the neuropeptide LHRH and, thus, may participate in the physiological regulation of the activity of this important neuroendocrine neuronal system. In addition, the results suggest that non-NMDA receptor sites may be preferentially activated at lower ligand concentrations, although NMDA receptors may also be involved in the response to certain endogenous agonists.

Effects of estrous cycle, estrogen and progesterone administration on the antidromic and orthodromic reaction threshold of medial preoptic neurons in the rat hypothalamus.

In the present study, attempts were made to evaluate the effects of the estrous cycle and the administration of estradiol (Ovx-E) or progesterone (Ovx-P) after ovariectomy on the threshold of antidromic (AD) and orthodromic (OD) responses of neurons in the medial preoptic area (POA) induced by electrical stimulation of the median eminence-arcuate nucleus (ARC) of the hypothalamus. Single units were evaluated with extracellular recording. The threshold of AD and OD response in POA neurons declined during estrus and proestrus of the estrous cycle and in the Ovx-E group, but increased in the Ovx-P group and during diestrus. The number of neurons with the inhibitory OD response increased during proestrus and in the Ovx-E group, but decreased during diestrus. The neurons with the excitatory OD response increased in the Ovx-P group and during diestrus, and decreased during proestrus. The threshold of the inhibitory and excitatory OD response decreased during proestrus, estrus and in the Ovx-E group, but increased in the Ovx-P group. It is apparent from these studies that estrogen appears to activate the neurons of the POA receiving inhibitory synaptic input via the axonal collateral branches of the ARC neurons, and that progesterone seems to activate the neurons of the POA receiving excitatory synaptic input.

Androgen‐Dependent and ‐Independent Aromatase Activity Coexists with Androgen Receptors in Male Guinea‐Pig Brain

Abstract Using a microdissection technique we localized androgen receptors and aromatase activity (AA) in the brain of male guinea-pigs. In addition, we evaluated the effects of castration and androgen replacement on androgen receptor dynamics and induction of AA. In the castrate animal, cytosolic androgen receptor content was highest in the basal hypothalamus, specifically in the median eminence-arcuate nucleus (> 15 fmol mg protein (1)), while lesser levels were found in the preoptic regions and amygdala. Nuclear receptor content was highest (> 150 fmol mg DNA (-1)) in the median eminence-arcuate nucleus, periventricular region of the preoptic area and cortical amygdala. All regions investigated showed a significant decrease in nuclear receptors following castration and an increase with androgen replacement. However, reciprocal changes in cytosolic androgen receptors were not always evident. Aromatase activity was high in the cortical amygdala, medial amygdala, periventricular region of the preoptic area and bed nucleus of the stria terminalis. Castration and androgen replacement had significant stimulatory effects on AA in the ventral medial hypothalamus, median eminence-arcuate nucleus, cortical amygdala and periventricular regions of the preoptic area and anterior hypothalamus. Thus, androgen receptors and AA are unevenly distributed throughout the subcortical regions of the male guinea-pig brain and respond differently to endocrine stimuli. Our data demonstrate that AA is androgen-dependent in some subcortical regions which contain androgen receptors. Even though nuclear receptors in all brain regions were affected by castration and dihydrotestosterone treatment, the events were not always linked to AA regulation. Due to this difference in regulation, AA may serve divergent functions in guinea-pig brain.

A potential mechanism for copper amplification of prostaglandin E 2 action: Attenuation of prostaglandin E 2-induced efflux of cyclic AMP from median eminance explants

It is known that prostaglandin E 2 (PGE 2) stimulation of LHRH release from the median eminence-arcuate nucleus (MEA) is mediated by the cAMP pathway, and a short pretreatment with copper markedly amplifies this release process. Because stimulation of cAMP accumulation is accompanied by cAMP efflux in many tissues, we considered the possibility that attenuation of cAMP efflux is one mechanism by which copper can enhance PGE 2 action. When rat MEA explants were incubated in vitro, PGE 2 induced a rapid (<2.5 min) and sustained (15 min) increase in cAMP efflux, the degree of which was a function of [PGE 2]: by 5 min exposure to 10 μM PGE 2, efflux was 8-fold greater than the control (no PGE 2) and it accounted for 12.4% of the total (tissue + medium) cAMP. Unlike the dramatic increase in cAMP efflux, PGE 2 induced a moderate increase in cAMP content (49%) and in the incorporation of [ 3H] adenine into [ 3H] cAMP (78%); this increase was transient: it was evident after a 2.5 but not after a 5 min period of PGE 2 exposure. Copper pretreatment did not alter this PGE 2-induced increase in tissue cAMP content. In contrast, copper markedly inhibited (by 49%–66%) PGE 2-induced cAMP efflux and this inhibition was noted regardless of the length of PGE 2 exposure and PGE 2 concentration. There was no evidence for hydrolysis of [ 3H]3′, 5′-cAMP included in the medium during the incubation with PGE 2 with and without copper pretreatment. In summary, copper attenuated PGE 2-induced cAMP efflux from MEA explants and this attenuation is not a consequence of a reduction in the availability of intracellular cAMP nor of hydrolysis of cAMP extracellularly,