Excitatory amino acid neurotransmission evidence for a role in neuroendocrine regulation.

There is compelling evidence that endogenous excitatory amino acid neurotransmission is an important component of the neuroendocrine transmission line that regulates anterior pituitary-hormone release and, thus, reproduction. Excitatory amino acids (EAAs), such as glutamate and aspartate, are found in large quantities in neuroendocrine tissues such as the hypothalamus, and neurons from a variety of hypothalamic nuclei respond with marked excitation to EAA application. Exogenous EAA administration rapidly increases the release of GnRH, LH, and prolactin secretion in vivo and in vitro. Antagonist studies demonstrate that EAA-receptor activation is involved in a number of reproductive-endocrine events, such as the induction of puberty, seasonal breeding, steroid-induced LH secretion, and the preovulatory surge of LH and prolactin in the female. EAA regulation of these neuroendocrine events appears to be achieved through modulation and regulation of hypothalamic GnRH secretion.     

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.     

Endogenous excitatory amino acid neurotransmission regulates thyroid-stimulating hormone and thyroid hormone secretion in conscious freely moving male rats

The role of neurotransmission of endogenous excitatory amino acid (EAA) on serum thyroid hormones and thyroid-stimulating hormone (TSH) levels was examined in conscious and freely moving adult male Sprague-Dawley rats. The rats were cannulated at the third ventricle 2 d before the experiments. Several glutamate receptor agonists, such as kainic acid and domoic acid, and antagonists, such as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and dizocilpine (MK-801) were administered into the third ventricle. Serum TSH levels were assesed by radioimmunoassay, and serum thyroid hormone levels were assessed by enzyme immunoassay. The results showed that the administration of CNQX and MK-801 produced a decrease in serum levels of TSH and thyroid hormones. The administration of kainic acid and domoic acid increased TSH concentrations, whereas CNQX completely blocked the release of TSH induced by kainic acid and domoic acid. These results suggest the importance of endogenous EAA in the regulation of hormone secretion from the pituitary-thyroid axis, as well as the role of the N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the stimulatory effect of EAAs on the pituitary-thyroid axis.     

Role of excitatory amino acids in the control of growth hormone secretion

Excitatory amino acids (EEAs), such as glutamate, are pivotal elements in the hypothalamic circuitry involved in the control of pituitary function. The actions of EEAs are mediated by different postsynaptic receptor subtypes, which include the ionotropic receptors N-methyl-D-aspartate (NMDA), kainate (KA), 2-amino-3-hydroxy- 5 methyl-4-isoxazol propionic acid (AMPA), as well as metabotropic receptors. In this review, we summarize the experimental work on the role of EAA neurotransmission in the control of GH secretion in the rat. Detailed characterization of the effects of agonists and antagonists of glutamate receptors on GH release revealed that activation of NMDA, KA, and AMPA receptors at different age-points resulted in clear-cut stimulation of GH secretion, although age- and sex-dependent differences were detected in the pattern of response to the different agonists. This stimulatory action was proven nitric oxide (NO)-dependent and not exerted at the pituitary level. Moreover, the effects of NMDA on GH release likely involve additional mediators other than hypothalamic GH-releasing hormone (GHRH). In contrast, the role of metabotropic receptors seems to be marginal, and only inhibitory actions were observed after activation of different receptor subtypes. Furthermore, evidence was obtained on the modulation of the EEA system by gonadal factors in the control of GH secretion, and on the physiological relevance of EEA pathways in the regulation of pulsatile GH release. The analysis of interactions between EAA receptors and other neuronal pathways evidenced the close interactions between different systems involved in the control of GH secretion. Blockade of glutamate receptors abolished the stimulatory effect of GABA and ghrelin on GH secretion and, inversely, blockade of ghrelin or GABA receptors abolished the stimulatory effect of EAAs. In conclusion, our data using the rat as animal model provide evidence for a pivotal role of glutamate pathways in the regulation of GH secretion throughout the life-span.     

Attenuation of preoptic area glutamate release correlates with reduced luteinizing hormone secretion in middle-aged female rats

Glutamate (Glu) and its receptors are involved in the maturation and maintenance of the neural mechanisms governing the preovulatory LH surge of young, reproductive-aged rodents and nonhuman primates. Little is known about the role of Glu in the delayed onset and reduced peak amplitude of the LH surge that characterizes female rodents during early reproductive senescence. The present study tested the hypothesis that the delayed and attenuated LH surge observed in middle-aged female rats is associated with altered hypothalamic Glu release. We used intracerebral microdialysis in young (3-4 months) and middle-aged (9-11 months) female rats to monitor changes in medial preoptic area Glu release and jugular vein catheters to monitor changes in serum LH levels. All animals were ovariectomized and injected with estradiol and progesterone in doses sufficient to produce a robust LH surge in most (approximately 70%) young rats. In both young and middle-aged females that surged, extracellular Glu levels were higher than in those that did not surge. Among animals that surged, the onset of the LH surge was significantly delayed, and the amplitude of the surge was significantly reduced in middle-aged compared with young rats. Middle-aged females also had significantly reduced extracellular Glu levels throughout the day of the LH surge when compared with young females. These data strongly suggest that age-related hypothalamic dysfunction contributes to reproductive aging independent of gonadal failure. We propose that reduced medial preoptic area Glu transmission contributes to reproductive aging by attenuating excitatory input to GnRH neurons.     

Endogenous excitatory amino acid neurotransmission regulates the estradiol-induced LH surge in ovariectomized rats

The present study was designed to evaluate the relative contribution of endogenous excitatory amino acids to the control of the estradiol-induced LH surge using specific blockers for N-methyl-D-aspartic acid (NMDA) and non-NMDA receptor types. Adult female rats ovariectomized for 2-3 weeks were implanted with third ventricular cannulae one week before the experiments. Silastic capsules (3 cm active surface) containing estradiol benzoate (250 micrograms/ml dissolved in sesame oil) were implanted subcutaneously two days prior to bleeding. Blood samples were collected at hourly intervals (from 1300 to 2100 h) through indwelling atrial cannulae implanted the day before the bleeding. (+) 2-amino-7-phosphoheptanoic acid (AP-7), a NMDA receptor antagonist, and 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, were administered (10 and 20 nmole dissolved in 10 microliters 0.9% sodium chloride, respectively) at 1300 and 1400 h into the third ventricle. LH, FSH and PRL levels were assayed by RIA in plasma samples. AP-7 and DNQX administration completely blocked the estradiol-induced LH surge, whereas PRL and FSH secretion was not affected by the treatments. These results indicate that endogenous EAA play an important role in controlling LH secretion. Furthermore, the study reveals that both EAA receptor types; i.e. NMDA and non-NMDA, appear to be necessary for the physiological mechanism(s) triggering the estradiol-induced LH surge.     

Differences in the luteinizing hormone and prolactin responses to multiple injections of kainate, as compared to N-methyl-D,L-aspartate, in cycling rats.

We have previously reported that repetitive iv injections of NMA [N-methyl-D,L-aspartate, the mixed analog acting on the N-methyl-D-aspartate (NMDA) receptor] can induce a consistent increase in LH and PRL secretion in cycling rats, but not in lactating rats. To further explore the use of excitatory amino acids (EAAs) as tools for understanding the regulation of the neuroendocrine reproductive axis, we have examined the effects of multiple injections of kainate, an agonist to another subclass of EAA receptor, on LH and PRL secretion in cycling rats. Recent studies suggest that kainate receptors may be more abundant than NMDA receptors in the hypothalamus. Five iv injections of kainate were administered at 50-min intervals to diestrous or estrous rats. Blood samples were collected every 10 min and assayed for LH and PRL. LH, but not PRL secretion, was stimulated by this regimen of kainate treatment. Surprisingly, the LH response to kainate, unlike NMA, decreased with repetitive injections of the drug. The response to the last pulse of kainate was approximately 30-40% of the first pulse. This decline in LH responsiveness to kainate was not due to desensitization at the level of the pituitary or to refractoriness of GnRH neurons, since further stimulation of LH release could be obtained by the administration of GnRH or NMA. The mechanisms responsible for the diminishing GnRH response to kainate remain unclear. However, we speculate that it might be due to the delayed activation of inhibitory inputs to GnRH neurons or to the desensitization of kainate receptors. On the other hand, the absence of a PRL response to kainate, in contrast to the stimulatory effect of NMA, most likely reflects differences in the distribution of kainate and NMDA receptors on dopamine neurons and neurons containing PRL-releasing factors, or on extrahypothalamic afferent neuronal populations projecting to the hypothalamus. In conclusion, the effects of systemic injections of kainate on LH and PRL secretion differed from NMA in that the LH response could not be sustained with multiple injections and PRL was unresponsive to kainate stimulation.     

Differential effects of hypothalamic IGF-I on gonadotropin releasing hormone neuronal activation during steroid-induced LH surges in young and middle-aged female rats

Interactions between brain IGF-I receptors and estrogen receptors regulate female reproductive physiology and behavior. The present study investigated potential mechanisms by which IGF-I receptors in the neuroendocrine hypothalamus regulate GnRH neuronal activation and LH release in young and middle-aged female rats under estradiol (E2) positive feedback conditions. We infused vehicle, IGF-I, or JB-1, a selective antagonist of IGF-I receptors, into the third ventricle of ovariectomized female rats primed with E2 and progesterone or vehicle. In young females, blockade of IGF-I receptors attenuated the steroid hormone-induced LH surge, reduced the percent of GnRH neurons expressing c-fos on the day of the LH surge, and decreased the total number of neurons expressing c-fos in the preoptic area. Middle-aged females had fewer GnRH neurons expressing c-fos during the LH surge than young females, and the LH surge amplitude was attenuated. Infusion of an IGF-I dose previously shown to increase LH surge amplitude did not increase the percent of GnRH neurons expressing c-fos in middle-aged females. Brain IGF-I receptor blockade did not modify E2 induction of progestin receptor-immunoreactive neurons in the preoptic area, arcuate, or ventromedial hypothalamus of young rats. These findings indicate that brain IGF-I receptors are required for E2 activation of GnRH neurons in young rats and for robust GnRH release from axon terminals in middle-aged females. IGF-I likely exerts its effects by actions on E2-sensitive neurons that are upstream of GnRH neurons and terminals.     

Involvement of endogeneous glutamate in the stimulatory effect of norepinephrine and serotonin on the hypothalamo-pituitary-adrenocortical axis

The effects of ionotropic glutamate receptor antagonists on the pituitary adrenal responses following injections of norepinephrine (NE) and serotonin (5-HT) receptor agonists into the hypothalamic paraventricular nucleus (PVN) or electrical stimulation of central NE and 5-HT pathways were studied in anesthetized male rats. PVN injections of an alpha(1)-adrenergic receptor agonist or a serotonergic 5-HT(1A) receptor agonist markedly increased both adrenocorticotropin (ACTH) and corticosterone (CS) serum levels. These responses were significantly inhibited by separate pre-injection of the selective non-NMDA and NMDA glutamate receptor subtype antagonists into the PVN in a dose-dependent manner. Electrical stimulation of either the ventral noradrenergic bundle or the dorsal raphe nucleus markedly increased serum ACTH and CS. These responses were also significantly attenuated by pre-injection of the above glutamate ionotropic receptor antagonists in a dose-dependent manner. These results suggest that glutamatergic interneurons in the PVN, acting via non-NMDA and NMDA receptors, may act as an excitatory mechanism in the NE and 5-HT control of hypothalamic ACTH secretagogues.     

Control of puberty by excitatory amino acid neurotransmitters and its clinical implications

Excitatory amino acids, glutamate in particular, have a marked stimulatory effect on the reproductive axis, particulary at puberty. Glutamate, N-methyl-d-aspartate (NMDA), and kainate stimulate gonadotropin-releasing hormone (GnRH) secretion in immature mammals and NMDA receptor stimulation results in precocious puberty in rats and monkeys. Puberty is characterized by an increased sensitivity of GnRH to glutamate as well as an increase in glutaminase activity in the hypothalamus. Glutamatergic and GABAergic regulation of GnRH secretion seem strongly interdependent around puberty. In addition to the transsynaptic glutamatergic regulation of GnRH secretion, a coordinated activity of glutamatergic neurons and astroglial cells has been shown to play an active role in puberty. The participation of kainate receptors in the estradiol-induced advancement of puberty suggest that these receptors may be involved in the estradiol-mediated activation of GnRH secretion at puberty. A case of precocious puberty associated with hyperglycinemia illustrates the NMDA involvement in puberty in humans. In this patient, the occurrence of precocious puberty was thought to result from excessive stimulation by glycine of the NMDA receptors linked to the GnRH neurons. Glutamate plays several roles in the hypothalamic mechanism of puberty as it has been shown in animal models, but there are still few clinical data supporting the role of glutamate in human puberty.     

Neuromodulatory actions of dopamine in the neostriatum are dependent upon the excitatory amino acid receptor subtypes activated.

In the mammalian neostriatum, dopamine modulates neuronal responses mediated by activation of excitatory amino acid receptors. The direction of this modulation varies with the specific subtype of excitatory amino acid receptor activated. Responses evoked by iontophoretic application of glutamate (Glu) and the non-N-methyl-D-aspartate (NMDA) agonists quisqualate and alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid were significantly attenuated when dopamine was applied. In contrast, responses evoked by NMDA were markedly potentiated. The enhancement of NMDA-evoked excitations was mimicked by bath application of SKF 38393, a D1 receptor agonist. The D1 receptor antagonist SCH 23390 blocked the dopamine enhancement of NMDA-induced excitations. Quinpirole, a D2 receptor agonist, attenuated responses evoked by both NMDA and non-NMDA receptor agonists. These results indicate that the complex modulatory actions of dopamine in the neostriatum are a function of the excitatory amino acid receptor as well as the specific dopamine receptor subtype activated. These findings are of clinical relevance since the actions of dopamine and excitatory amino acids have been implicated in neurological and affective disorders.     

Evolving role of gonadotropin-releasing hormone antagonists.

GnRH antagonists, unlike GnRH agonists, do not act via "downregulation." Instead, GnRH antagonists monopolize the GnRH receptors to such an extent that endogenous GnRH is unable to bind to sufficient numbers of GnRH receptors to provoke release of LH/FSH. This fundamental difference in the mechanism of action of GnRH antagonists versus GnRH agonists is anticipated to result in clinical benefits for certain applications.     

Interactions between GABAergic and aminoacidergic pathways in the control of gonadotropin and GH secretion in pre-pubertal female rats

Present experiments were carried out in 23-day-old female rats to analyze the interaction between excitatory amino acids (EAAs) and gamma-aminobutyric acid (GABA) in the control of gonadotropin and GH secretion. For this purpose, serum concentrations of LH, FSH and GH were measured after injection of different agonists of EAA receptor subtypes [N-methyl-D-aspartate (NMDA); kainic acid (KA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)], antagonists of GABA receptors (bicuculline, phaclofen) or the combined administration of both types of drugs. The results obtained indicated that: 1) GABA has a minor physiological role in the control of LH and GH secretion, since neither LH nor GH serum concentrations changed after administration of bicuculline (antagonist of GABA(A) receptors) or phaclofen (antagonist of GABA(B) receptors); 2) GABA has a sex-specific physiological role in the control of FSH secretion in female rats, in which FSH secretion increases after phaclofen administration; 3) GH secretion was enhanced after administration of NMDA, KA and AMPA, while LH increased only after activation of NMDA receptors; 4) the stimulatory effect of NMDA on LH secretion was counteracted by administration of phaclofen; and 5) bicuculline and phaclofen reduced the ability of NMDA and AMPA to stimulate GH secretion. In conclusion, present experiments evidenced a physiological role of GABA, mediated by GABA(B) receptors, in the control of FSH secretion and a cross-talk between excitatory and inhibitory amino acids in the control of anterior pituitary secretion.     

Glutamate stimulates somatostatin release from diencephalic neurons in primary culture

The action of excitatory amino acid agonists on endogenous somatostatin release was examined in primary cultures of rat diencephalic neurons. Increasing concentrations of glutamate stimulated somatostatin release in a dose-dependent manner. Since this effect was decreased by Mg2+, all experiments were performed in Mg2+-free media. We found that excitatory amino acid agonists evoked somatostatin release in the following order of potency: quisqualate greater than glutamate = N-methyl-D-aspartate (NMDA) greater than kainate, as calculated from the dose-response curves. The increase in somatostatin release elicited by glutamate or NMDA was selectively antagonized by DL-2-amino-5-phosphonovaleric acid and by thyenyl-phencyclidine, two specific antagonists of NMDA receptors. The NMDA effect was strongly inhibited: in a competitive manner by APV and in a noncompetitive manner by TCP with IC50 of 90 microM and 0.2 microM, respectively. Glutamate-induced somatostatin release was not blocked by tetrodotoxin (1 microM) suggesting that tetrodotoxin-sensitive sodium-dependent action potentials are not involved in this effect. Our data suggest the presence of functionally active excitatory amino acid receptors in somatostatinergic neurons. Glutamate seems to exert its stimulatory action on somatostatin release essentially through NMDA type receptor sites.     

The Involvement of GABAA receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the preoptic area in anestrous ewes.

This study examined role of GABA A receptors in the control of GnRH, beta-endorphin release and catecholaminergic system activity in the preoptic area and LH secretion in anestrous ewes. Stimulation of GABA A receptors in the medial preoptic area (MPOA) by muscimol attenuated GnRH release and dopaminergic system activity and increased extracellular noradrenaline (NE) and MHPG concentration. Muscimol has no evident effect on the extracellular concentration of beta-endorphin-like immunoreactivity (B-END-LI) in the MPOA. The decrease of LH pulse frequency and concentration of this hormone in blood plasma suggests that GABA A receptor agonist applied in the MPOA suppresses GnRH release from the GnRH axon terminals in the ventromedial hypothalamus-nucleus infundibularis region (VEN/NI) into the hypophyseal vascular system. Blockade of GABA A receptors with bicuculline did not change GnRH release, catecholaminergic activity, B-END-LI concentration in the MPOA, and LH release. The presented data indicate that activation of GABA A receptors in the MPOA decreases extracellular concentration of GnRH in this structure and LH level in the blood plasma thus suggesting that GABA may act in the MPOA to inhibit GnRH release in the VEN/NI. These results suggest that suppression of GnRH/LH release during muscimol treatment may result from activation of GABA A receptors on the GnRH perikarya and/or through GABA A receptor mechanism on the dopaminergic and noradrenergic system in the MPOA. Lack of changes in B-END-LI concentration during stimulation or blocking GABA A receptors suggests, that beta-endorphinergic system in the MPOA does not participate in the GABA A receptors mechanism modulating GnRH release.     

The role of GabaA receptors in the neural systems of the ventromedial hypothalamus-nucleus infundibular region in the control of GnRH release in ewes during follicular phase.

To examine the role of the GABAA receptor mediating systems in the control of gonadotropin releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/NI) of the hypothalamus of ewes during the follicular phase of the estrous cycle, the extracellular concentrations of GnRH, beta-endorphin (B-END), noradrenaline (NE), dopamine (DA), and their metabolites MHPG, DOPAC and concentration of luteinizing hormone (LH) in blood plasma were quantified during local stimulation or blockade of GABAA receptors with muscimol and bicuculline, respectively. Stimulation of GABAA receptors attenuated GnRH and LH release, increased beta-endorphin outflow and dopaminergic activity but had no evident effect on noradrenergic activity. Blockade of GABAA receptors decreased beta-endorphin release but had no evident effect on the extracellular concentration of GnRH, LH levels in the blood and catecholaminergic activity. It is suggested that suppression of GnRH/LH release under muscimol treatment may result from activation of GABAA receptors on GnRH nerve terminals and through GABAA receptor mechanism activated beta-endorphinergic and dopaminergic neurons in the VEN/NI. Lack of changes in NE and MHPG concentration during stimulation or blockade of GABAA receptors suggests, that during the follicular phase of the estrous cycle the noradrenergic system in the VEN/NI is not involved in the control of GnRH/LH release by GABA.     

Direct activation of gonadotropin-releasing hormone secretion through different receptors to neuroexcitatory amino acids

In order to evaluate the involvement of gonadotropin-releasing hormone (GnRH) in the effects of neuroexcitatory amino acids on luteinizing hormone (LH) secretion, N-methyl-D,L-aspartate (NMDA; 30 mg/kg s.c.) was administered to 50-day-old male rats. The in vitro release of GnRH from the hypothalamus showed a maximal increase (4.6-fold) in animals sacrificed 7.5 min after NMDA injection, while serum LH levels rose concomitantly. Incubation of rat hypothalami in vitro with kainate or NMDA concentrations greater than 0.1 mM resulted in a dose-related release of GnRH, NMDA being twofold more potent than kainate. Quisqualate (10 mM) did not affect the release of GnRH. On a molar basis, quinolinate (50 mM), a possible endogenous ligand for NMDA receptors, was the most effective in inducing GnRH secretion (34.9 +/- 4.9 pg/7.5 min, mean increment +/- SEM, n = 10). The effects of kainate and NMDA were mediated through different types of receptors, since GnRH response to kainate was unchanged in the absence of glycine or in the presence of increased concentrations of Mg2+ (2 mM) or Ca2+ (5.8 mM). In contrast, the GnRH response to NMDA was reduced by Ca2+ (5.8 mM) and abolished in the absence of glycine or in the presence of Mg2+ (2 mM). In addition, D,L-amino-5-phosphonopentanoic acid (AP5), a competitive antagonist of NMDA receptors, prevented the NMDA-induced release of GnRH. The permissive effect of glycine on GnRH response to NMDA was 2.7-fold more important using glycine concentrations of 0.01 microM than when concentrations greater than or equal to 100 microM were used.(ABSTRACT TRUNCATED AT 250 WORDS)     

The neuroactive steroid pregnenolone sulfate stimulates the release of gonadotropin-releasing hormone from GT1-7 hypothalamic neurons, through N-methyl-D-aspartate receptors

Immortalized hypothalamic GT1-7 neurons represent a good model system to investigate the control of GnRH secretion. Using these cells, we observed that the neuroactive steroid, pregnenolone sulfate (PREGS), is able to stimulate the release of GnRH in a dose-dependent manner through N-methyl-D-aspartate (NMDA) receptors, because its action is completely blocked by a specific NMDA receptor antagonist and magnesium. GT1-7 neurons express mRNAs for various mouse NMDA receptor subunits (zeta,1, epsilon3, epsilon4, and epsilon2, corresponding to the NR1, NR2C, NR2D, and NR2B rat subunits) and increase their spontaneous release of GnRH when incubated in the presence of exogenous glutamate or NMDA. In addition, we found that these neurons are able to release and synthesize glutamate, as demonstrated by the presence of glutamate accumulated in the defined incubation medium of the neurons, during the experiment and the expression of mRNA coding for vesicular glutamate transporter 2, a specific marker of glutamatergic neurons. The potentiating effect of PREGS on the secretion of GnRH induced by glutamate is consistent with the role of the steroid as a positive allosteric modulator of NMDA receptors. Together these results point to a novel mechanism by which the neuroactive steroid PREGS may potentiate an autocrine excitatory loop in GnRH neurons.     

Endogenous excitatory amino acid involvement in the preovulatory and steroid-induced surge of gonadotropins in the female rat

The physiological role of N-methyl-D-aspartate (NMDA) receptors in the regulation of preovulatory and steroid-induced surges of gonadotropins in the female rat was examined. The specific and potent noncompetitive NMDA receptor antagonist MK801 was used for blockade of NMDA neurotransmission. MK801 treatment completely inhibited the ability of progesterone to induce LH and FSH surges in the estrogen-primed ovariectomized rat. Administration of MK801 on proestrus in the immature female rat primed with PMSG resulted in a significant attenuation of the proestrous LH, FSH, and PRL surge and a corresponding attenuation of ovulation. Similarly, in the adult cycling female rat, MK801 administration on proestrus led to a significant attenuation of the proestrous LH and PRL surges. Mean FSH levels were lower in MK801-treated adult rats than in vehicle-treated rats, but this effect was not significant. In the estrogen-primed ovariectomized immature rat, the agonist NMDA caused a rapid (less than 10 min) elevation of LH and FSH in vivo. The gonadotropin-releasing effect of NMDA may be mediated at the level of the hypothalamus, since the medial basal hypothalamus/preoptic area of NMDA-treated rats killed 3 and 5 min post-NMDA had a significantly greater release of GnRH in vitro than that of vehicle-treated rats. In conclusion, these findings demonstrate that the preovulatory gonadotropin surge in the female rat is dependent on NMDA neurotransmission for its expression and add further evidence for a critically important role for NMDA receptors in the physiological regulation of gonadotropin secretion in the female rat.     

Effect of ionotropic and metabotropic glutamate agonists and D-aspartate on prolactin release from anterior pituitary cells.

Although the presence of ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptors has been demonstrated in the anterior pituitary, recent reports on the direct effect of glutamate on prolactin (PRL) secretion by anterior pituitary cells have presented contradictory results. Hence, the aim of this study was to determine the effect of ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptor agonists on prolactin (PRL) release. In addition, since D-Aspartate (D-Asp) is found in the pituitary and is involved in neuroendocrine regulation, we also studied the direct action of D-Asp on PRL secretion. Finally, since the posterior pituitary participates in the regulation of PRL secretion, we examined the influence of the posterior pituitary on the effects of NMDA and D-Asp on PRL release. Glutamate (1000 microM) increased PRL secretion from cultured anterior pituitary cells. Both NMDA (100 microM) and kainate (100 microM) increased PRL secretion and these effects were blocked by a specific NMDA receptor antagonist. AMPA did not modify PRL release in these cultures. The group I and II mGluR agonist, trans-ACPD (1000 microM), and a specific group II mGluR agonist, L-CCG-I (100-1000 microM), inhibited whereas specific group I and III mGluR agonists, 3-HPG and L-AP4 respectively, had no effect on PRL release. Finally, D-Asp (100-1000 microM) stimulated PRL secretion and this effect was reduced by a NMDA receptor antagonist. When anterior pituitary cells were cultured in the presence of posterior pituitary cells, NMDA did not modify PRL or GABA release, while D-Asp increased PRL secretion and decreased GABA release in these cocultures. In conclusion, our results show that L-glutamate has a differential direct effect on PRL release: it exerts a stimulatory action via iGluRs and an inhibitory effect via mGluRs. D-Asp could directly stimulate PRL release through NMDA receptors. D-Asp may also stimulate PRL release by decreasing GABA release from the posterior pituitary.