Gamma-aminobutyric acid neurons in the preoptic/anterior hypothalamic area synchronize the phasic activity of the gonadotropin-releasing hormone pulse generator in ovariectomized rats

To achieve a bolus-type release of gonadotropin-releasing hormone (GnRH) into the portal vessels it is required that GnRH neurons exert phasic and synchronous activity. The activity of GnRH neurons appears to be under an inhibitory influence of the amino acid neurotransmitter gamma-aminobutyric acid (GABA). Preoptic GABA concentrations in ovariectomized (OVX) rats decrease prior to a luteinizing hormone (LH) episode. This reduction of GABAergic activity in the preoptic/anterior hypothalamic area (PO/AH) may be the synchronizing signal for the simultaneous release of GnRH from the hypothalamus. To further study the role of GABA in controlling the GnRH pulse generator we applied GABA, 3-mercaptopropionic acid (MPA) or bicuculline (BIC) locally into the PO/AH by means of push-pull cannulae (PPC). PPC were implanted into the PO/AH of OVX rats and the contralateral, not PPC-implanted PO/AH was lesioned electrochemically. The effects of GABA, MPA or BIC on the GnRH pulse generator were determined by measuring LH levels in blood samples collected in 5-min intervals. Local application of GABA into the PO/AH caused a pronounced reduction of average LH secretion and abolished LH pulsatility. This inhibitory effect was completely reversible. Results of intrapreoptic MPA application on GABA secretion were variable. In only 45% of treated rats MPA caused a reduction of GABA secretion which was associated with a cessation of pulsatile LH release. A pronounced reduction of LH secretion and pulsatility was observed upon local application of the GABA antagonist BIC. Based on these data we propose that oscillating GABA levels in the PO/AH may be the synchronizing signal which triggers bolus release of GnRH into the portal vessels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further evidence that preoptic anterior hypothalamic GABAergic neurons are part of the GnRH pulse and surge generator

The in vivo release rates of GABA from the preoptic/anterior hypothalamic area (PO/AH) of ovariectomized rats were assessed by means of a focal perfusion cannula system. Seven days after surgery all animals received a sc silastic capsule implant containing either estradiol-17 beta (E2) or corn oil, and they were perfused 3 days later. Perfusate fractions were sampled at 5-min intervals and blood was collected every 10 min over a period of 5 h. In ovariectomized animals PO/AH GABA release was pulsatile without any diurnal rhythm. Prior to frequency analysis by means of the pulsar-programme, LH and GABA values were z-transformed. Significant LH peaks of all examined ovariectomized rats were superimposed and GABA data were arranged accordingly. It became evident that LH episodes are preceded by a significant reduction of preoptic anterior hypothalamic GABA release. The secretion patterns of GABA and LH were profoundly affected by E2 replacement. During early noon when LH levels were low, constantly elevated hypothalamic GABA release rates were observed in E2-substituted rats in comparison to ovariectomized rats. GABA release rates fell significantly during the E2-induced LH surge. Our previous demonstration of the existence of a large number of estrogen-respective GABAergic neurons in the PO/AH is suggestive of these neurons changing their activity in response to estrogen treatment. We conclude that these estrogen-respective GABAergic neurons are involved in the generation of GnRH pulses as well as in the generation of the so-called positive feedback effect of E2 on LH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

A norepinephrine-dependent mechanism in the preoptic/anterior hypothalamic area but not in the mediobasal hypothalamus is involved in the regulation of the gonadotropin-releasing hormone pulse generator in ovariectomized rats.

Pulsatile gonadotropin secretion from the pituitary is dependent upon the gonadotropin-releasing hormone (Gn-RH) pulse generator producing intermittent release of the neuropeptide into the portal vessels. Various neurotransmitters seem to be involved in the regulation of pulsatile Gn-RH release. The present study was an attempt to determine in vivo the temporal relation of preoptic/anterior hypothalamic area (PO/AH) norepinephrine (NE) release and pulsatile luteinizing hormone (LH) secretion in ovariectomized rats. To assess whether NE acts in the PO/AH to maintain pulsatile Gn-RH release, we applied locally an alpha 1-receptor antagonist into this structure. Push-pull cannulae (PPC) were implanted into the PO/AH of ovariectomized rats. The contralateral, not PPC-implanted PO/AH was lesioned electrochemically. Another group of ovariectomized rats was implanted with a PPC into the mediobasal hypothalamus. Two experiments were performed: (1) To determine whether the PO/AH or the mediobasal hypothalamus is the site where NE exerts its stimulatory effect on LH secretion, we applied doxazosine, a new specific alpha 1-receptor antagonist, locally into these structures by means of PPC. The effect of this adrenergic drug on the Gn-RH pulse generator was examined by measuring blood LH levels. (2) To study the temporal relation between in vivo release rates of NE and amine metabolites in the preoptic area and pulsatile pituitary LH secretion, preoptic perfusates and blood samples were collected at 5-min intervals. Brain perfusates were subjected to high-performance liquid chromatography-electrochemical analysis. In blood samples LH concentrations were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active transport of gamma-aminobutyric acid and glycine into synaptic vesicles.

Although gamma-aminobutyric acid (GABA) and glycine are recognized as major amino acid inhibitory neurotransmitters in the central nervous system, their storage is poorly understood. In this study we have characterized vesicular GABA and glycine uptakes in the cerebrum and spinal cord, respectively. We present evidence that GABA and glycine are each taken up into isolated synaptic vesicles in an ATP-dependent manner and that the uptake is driven by an electrochemical proton gradient. Uptake for both amino acids exhibited kinetics with low affinity (Km in the millimolar range) similar to vesicular glutamate uptake. The ATP-dependent GABA uptake was not inhibited by the putative amino acid neurotransmitters glycine, taurine, glutamate, or aspartate or by GABA analogs, agonists, and antagonists. Similarly, ATP-dependent glycine uptake was hardly affected by GABA, taurine, glutamate, or aspartate or by glycine analogs or antagonists. The GABA uptake was not affected by chloride, which is in contrast to the uptake of the excitatory neurotransmitter glutamate, whereas the glycine uptake was slightly stimulated by low concentrations of chloride. Tissue distribution studies indicate that the vesicular uptake systems for GABA, glycine, and glutamate are distributed in different proportions in the cerebrum and spinal cord. These results suggest that the vesicular uptake systems for GABA, glycine, and glutamate are distinct from each other.     

Preoptic catecholamine, GABA, and glutamate release in ovariectomized and ovariectomized estrogen-primed rats utilizing a push-pull cannula technique

The push-pull cannula technique was used to evaluate the role of the medial preoptic/anterior hypothalamic area (MPO) in regulating pituitary luteinizing hormone (LH) and prolactin release. The concentrations of the three catecholamines--dopamine, norepinephrine (NE), epinephrine (E)--and gamma-aminobutyric acid (GABA) and glutamate could be measured in 15-min fractions at which interval blood samples for LH and prolactin determination were also collected. Comparison of neurotransmitter release rates into the MPO were made between ovariectomized and ovariectomized estradiol benzoate treated rats. Release of the neurotransmitters occurred in a pulsatile manner, the release episodes for each transmitter appeared to be independent of the others. No direct correlation between neurotransmitter release episodes and blood LH or prolactin levels could be established. The release of GABA was significantly lower and that of NE and E higher in ovariectomized animals in comparison to estrogen-primed ovariectomized animals under negative feedback conditions. In the afternoon, however, when the estrogen stimulated LH and prolactin release, preoptic GABA release was low, whereas preoptic NE and particularly E release rates were high. Conspicuously high dopamine and NE release episodes were observed in estrogen-primed animals at noon, i.e., prior to the expression of the positive feedback signal. This may reflect a biochemical correlate to the so-called critical period. No consistent differences between ovariectomized and ovariectomized estradiol-17 beta benzoate treated animals were observed for preoptic glutamate release rates. The data show that preoptic GABA release rates show generally an inverse pattern to NE and E release and therefore also to blood LH and prolactin levels. No direct mathematical correlation between any of the neurotransmitter release rates and blood hormone levels could be established.     

Changes in the control of gonadotrophin secretion by neurotransmitters during sexual development in rats.

Gonadotrophin hormone releasing hormone (GnRH) is the primary messenger involved in sexual maturation and the onset of puberty. The activity of these neurons are controlled by several neurotransmitters systems. The onset of puberty implies changes from a prepubertal type of gonadotrophin secretion, characterized by a low activity of GnRH neurons, to an adult pattern of secretion with phasic and synchronous activation of GnRH neurons resulting in an increase in the amplitute and frequency of GnRH pulses. Neurotransmitter systems are involved in these changes of GnRH secretion during the onset of puberty by quantitative and qualitative modifications in the effect on GnRH secretion. Serotonin (5-HT), GABA and catecholamines (CA) have qualitative differences in the effects on GnRH and LH secretion in early prepubertal than in late prepubertal and adult female rats. The administration of 5-hydroxytryptophan a precursor of serotonin (5-HT) which increases 5-HT hypothalamic levels induces GnRH and LH release in early prepubertal female rats, these effects dissapear in late prepubertal stage having an inhibitory action in adult female rats. GABAergic system also stimulates GnRH and LH secretion in early prepubertal female rats and has an inhibitory action on this axis in late prepubertal period and in adult female rats. On the contrary the inhibition of catecholamines synthesis by alpha-methyl-p-tyrosine induced an increase of LH secretion in early prepubertal female rats and inhibitory effect in late prepubertal and adult stage. These effects indicate tha CA has an inhibitory effects on GnRH-LH secretion in early prepubertal female rats changing to an stimulatory action in the late puberty and adult rats. These qualitative modifications were observed only in female rats and are probably connected with the hypothalamic differentiation into a female type of gonadotrophin control. Opiadergic and excitatory amino acid systems have quantitative differences on GnRH-LH secretion during prepubertal and peripubertal and adult stages. Opiates has an high inhibitory tone in early prepubertal rats that is decreasing during sexual maturation to reach puberty. On the contrary EAA increases its stimulatory activity on GnRH-LH secretion during sexual maturation by increasing the hypothalamic release of aspartate and glutamate, the excitatory amino acids involved in GnRH release, and the sensibility of NMDA receptors to these amino acids. In conclusion sexual maturation and the onset of puberty in the female rats involve qualitative and quantitative modifications in the effects of neurotrasmitters system on GnRH secretion.     

Changes in brain amino acids and nitric oxide after melatonin administration in rats with pentylenetetrazole-induced seizures

We examined the effect of melatonin on brain levels of amino acids and nitric oxide (NO) after pentylenetetrazole (PTZ)-induced seizures in rats. Animals were treated with melatonin (10-160 mg/kg, i.p.) 30 min before PTZ administration (100 mg/kg, s.c.), and were killed 3 hr later. At the dose of 80 mg/kg, melatonin significantly increased the latency (5.7-12.7 min) and decreased the duration (31.2-18.4 s) of the first seizure, reducing PTZ induced mortality from 87.5 to 25%. After kill, brains were removed and neurotransmitters and nitrite levels measured in prefrontal cortex (PF), parieto-temporal cortex (PF), striatum (ST), hippocampus (HP) and brain stem (BS) by high performance liquid chromatography. PTZ treatment increased glutamine levels in all brain areas studied, without changes in glutamate, gamma-amino butyric acid (GABA) and glycine. Aspartate and taurine increased in PF and PT and in HS and PT, respectively. Melatonin administration displayed a dose-dependent effect. At doses of 10-40 mg/kg, melatonin counteracted the PTZ-induced glutamine increase and reduced both glutamate and aspartate levels in the studied areas, with minor changes in GABA and glycine content. At doses of 80 and 160 mg/kg, the levels of glutamine, and glutamate, and to a lesser extent aspartate increased, whereas serine levels did not change. These two doses of melatonin also increased taurine, GABA and glycine in most brain areas studied. Treatment with melatonin (40-160 mg/kg) significantly decreased nitrite content in PT cortex, ST and BS areas of epileptic rats, without changes in the other brain regions. The results suggest that the anticonvulsant property of melatonin involves a modulation of both brain amino acids and NO production.     

Regulatory role of excitatory amino acids in reproduction

Glutamate, the major excitatory amino acid (EAA) transmitter in the central nervous system, has been implicated as a critical mediator in brain function. Glutamate and its receptors are found in all key hypothalamic areas critically involved in reproduction. Administration of glutamate and its agonists can bring about LH release in animals with a steroid background. Antagonists of the ionotropic glutamate receptors inhibited LH release and abolished the steroid-induced and the preovulatory LH surge. Both NMDA and non-NMDA receptor antagonists can also inhibit pulsatile LH release in castrated animals. The preoptic area has been implicated as a primary site of action of NMDA, while non-NMDA agonists have been suggested to act primarily at the arcuate/median eminence level. While EAAs may act directly on GnRH neurons to enhance GnRH release, the majority of evidence suggests that an indirect mechanism, involving EAA activation of nitric oxide and/or catecholamines, plays a major role in the GnRH-releasing effects of EAAs. Furthermore, there is also some evidence that the tonic inhibitory effect of opioids on GnRH may also involve, at least in part, a suppression of glutamate. Finally, EAA stimulation of GnRH/LH release is markedly attenuated in middle-aged rats, suggesting that a defect in glutamate neurotransmission may underlie the attenuated LH surge observed in aging.     

In vitro gonadotropin-releasing hormone release from hypothalamic tissues of ovariectomized estrogen-treated cynomolgus macaques

The effects of in vivo 17 beta-estradiol (E2) treatment on in vitro GnRH release and serum LH levels were studied to determine the loci of E2 feedback actions and to examine the hypothalamic mechanisms by which this steroid may regulate LH secretion in monkeys. Ovariectomized cynomolgus macaques received sc Silastic capsule implants containing E2 and were killed 12, 36, 42, or 48 h later. At least one control (CTL) animal received a blank implant and was killed concurrently with each E2-treated monkey. Three untreated animals were used in validation experiments. Before death, each animal was anesthetized with ketamine (15 mg/kg, im), and blood samples were drawn for subsequent LH analysis by Leydig cell bioassay. A diencephalic tissue block was obtained at autopsy and immediately immersed in Krebs-Ringer-phosphate medium (KRP). Mediobasal hypothalamic (MBH) and anterior hypothalamic/preoptic (AH/POA) fragments were quickly dissected from the block and placed in separate superfusion chambers maintained at 37 C. Tissues were superfused at 50 microliter/min with KRP, and 10-min fractions were collected, acidified, and stored at -20 C for subsequent GnRH RIA. Basal immunoreactive GnRH (IR-GnRH) release was measurable from MBH (0.367 +/- 0.063 pg/min) and AH/POA (0.176 +/- 0.065 pg/min) fragments from CTL monkeys. In validation experiments, IR-GnRH release was increased 3- to 7-fold by superfusion with 60 mM K+-KRP only in the presence of Ca+2. Superfusate IR-GnRH coeluted with synthetic GnRH from a Sephadex G-25 chromatographic column, and superfusate and tissue extract GnRH showed appropriate LH-releasing capacities, as determined by rat pituitary cell culture assay. IR-GnRH release rates from MBH or AH/POA tissues varied as a function of in vivo estrogen treatment. GnRH release from both tissues was increased in the E2-treated group killed at 12 h when LH levels were suppressed. Thirty-six hours after E2 treatment, in vitro GnRH release was not significantly different from CTL values. GnRH release rates from MBH and AH/POA tissues obtained 42 h after E2 treatment were significantly greater than CTL release rates (P less than 0.01). This increased in vitro GnRH release at 42 h occurred during the apparent rising phase of the LH surge. Elevated GnRH release was not sustained at 48 h, when surge levels of LH were apparent.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

In vivo release of prolactin-releasing peptide in rat hypothalamus in association with luteinizing hormone and prolactin surges.

Prolactin (PRL)-releasing peptide (PrRP) is a novel hypothalamic peptide reported to be a potent and specific stimulator of PRL secretion. This author recently reported that PrRP might play a significant role in mediating the steroid-induced PRL surge in the rat. In order to examine the secretory profile of PrRP in the rat hypothalamus before and during the luteinizing hormone (LH) and PRL surges, this study employed the push-pull perfusion technique and determined the in vivo release of PrRP and also of gonadotropin-releasing hormone (GnRH) in ovariectomized rats primed with estradiol and progesterone. In the medial preoptic area (MPOA) where the GnRH neuronal perikarya exist, GnRH release was increased prior to the initiation of the LH surge, and PrRP also started rising even earlier than GnRH. In the median eminence-arcuate nucleus complex (ME-ARC), where GnRH neuronal fibers terminate, GnRH secretion started increasing before the commencement of the LH surge, but the release of PrRP did not change significantly. These results suggest that PrRP may play a role in mediating the steroid-induced LH surge by activating GnRH neurons in the MPOA. A possible involvement of PrRP in the PRL surge was not suggested from the present data. The lack of a significant alteration in PrRP release in the ME-ARC may argue against a direct hypophysiotropic action of the peptide.     

Effects of ovarian steroids on the gonadotropin response to N-methyl-D-aspartate and on hypothalamic excitatory amino acid levels during sexual maturation in female rats.

In order to evaluate the involvement of estrogen-progesterone (EP) in the effects of N-methyl-D-aspartate (NMDA) receptor stimulation on gonadotropin secretion during sexual development in female rats, NMDA (30 mg/kg sc) was administered to 16- and 30-day-old female rats pretreated with EP. NMDA administration induced increases in plasma LH concentration that were 13.6-fold and 94.5-fold higher, respectively, than those found after NMDA alone. The increase of LH levels induced by NMDA was accompanied by a significant enhancement of the content of GnRH in the anterior and preoptic hypothalamic areas and in the medial basal hypothalamus (APOA/MBH). EP potentiated this increase of GnRH induced by NMDA. NMDA increased plasma FSH levels at 16 days of age, and this increase was inhibited by EP treatment. In 30-day-old rats EP induced FSH release in response to NMDA. This release was not observed in rats treated only with NMDA. In 16-day-old rats EP induced an increase in the concentrations of aspartate, glutamate, and glycine in the anterior and preoptic hypothalamic areas and in the medial basal hypothalamus, the excitatory amino acids involved in NMDA neurotransmission. This effect was not observed in rats of 30 days of age. In summary, the present results show that during sexual maturation ovarian steroids potentiated the LH-releasing response to NMDA probably by acting at the hypothalamic level; furthermore, during sexual maturation there are changes in the response to EP of the hypothalamic concentrations of excitatory amino acids. These findings could be related to the neuroendocrine mechanisms regulating the onset of puberty and the sexual cycle in female rats.     

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.     

Pup removal suppresses estrogen-induced surges of LH secretion and activation of GnRH neurons in lactating rats

During lactation, the suckling stimulus exerts profound influences on neuroendocrine regulation in nursing rats. We examined the acute effect of pup removal on the estrogen-induced surge of LH secretion in ovariectomized lactating rats. Lactating and nonlactating cyclic female rats were given an estradiol-containing capsule after ovariectomy, and blood samples were collected through an indwelling catheter for serum LH determinations. In lactating, freely suckled ovariectomized rats, estrogen treatment induced an afternoon LH surge with a magnitude and timing comparable to those seen in nonlactating rats. Removal of pups from the lactating rats at 0900, 1100, or 1300 h, but not at 1500 h, suppressed the estrogen-induced surge that normally occurs in the afternoon of the same day. The suppressive effect of pup removal at 0900 h was completely abolished when the pups were returned by 1400 h. In contrast, pup removal was ineffective in abolishing the stimulatory effect of progesterone on LH surges. Double immunohistochemical staining for gonadotropin-releasing hormone (GnRH) and c-Fos, a marker for neuronal activation, revealed a decrease, concomitantly with the suppression of LH surges, in the number of c-Fos-immunoreactive GnRH neurons in the preoptic regions of nonsuckled rats. An LH surge was restored in nonsuckled rats when 0.1 microg oxytocin was injected into the third ventricle three times at 1-h intervals during pup removal. These results suggest that the GnRH surge generator of lactating rats requires the suckling stimulus that is not involved in nonlactating cyclic female rats.     

Age-related differences in the effects of bacterial endotoxin (LPS) upon the release of LHRH, gonadotropins and hypothalamic inhibitory amino acid neurotransmitters measured in tissues explanted from intact male rats.

In adult rats, bacterial endotoxin (lipopolysaccharide or LPS) is known to diminish the activity of the reproductive axis, mainly by inhibiting luteinizing hormone-releasing hormone (LHRH) secretion; until now, this effect has not been studied in immature rats. The aim of the present study was to evaluate the effect of LPS 1) on LHRH output (and associated changes in the release of inhibitory amino acid neurotransmitters such as gamma-aminobutyric acid (GABA) and taurine) by superfused hypothalamic fragments, and 2) on gonadotropin secretion by incubated hemipituitaries, obtained from young adult (60-day-old) and peripubertal (30-day-old) intact male rats. In adult animals, LPS induced a significant inhibition (50% of basal values) of LHRH release, accompanied by an increase in GABA and taurine output. In juvenile rats the inhibition of LHRH secretion by LPS attained 90% of basal values (p<0.0001 versus adult rats), and the concurrent increase in GABA release was significantly greater (p<0.0001 versus adult rats). LPS did not affect in vitro gonadotropin secretion in adult animals. Conversely, the release of these hormones was significantly (p<0.001 and <0.02 for LH and FSH, respectively) reduced in 30-day-old rats. Our results demonstrate the existence of age-related differences in the effect of LPS on LHRH and gonadotropin secretion. These differences might well be attributed to an increased activity of the hypothalamic GABAergic system. Furthermore, the participation of other factors known to play a role in immune-neuroendocrine relationships (e.g., corticotropin-releasing hormone, testosterone) is discussed.     

Leptin stimulates the reproductive male axis in rats during sexual maturation by acting on hypothalamic excitatory amino acids.

The purpose of the present study was to determine the effect of treatment with leptin on gonadotrophin secretion and hypothalamic GnRH, excitatory and inhibitory amino acids release, in prepubertal (15 days old) and peripubertal (30 days old) male rats. Rats of both ages received a single (ip) injection of 30 microg/kg leptin 60 minutes previous to sacrifice. Serum LH was determined, and the hypothalamus dissected and incubated in Earle's medium. GnRH and amino acids release were determined in the media. LH and GnRH were measured by RIA. Amino acids were assessed by HPLC-UV detection. In the two prepubertal stages, (prepubertal and peripubertal, 15 and 30 days of age respectively) leptin increased plasmatic LH levels (p < 0.01) and hypothalamic GnRH release (p < 0.01). Glutamate (GLU) release showed an increment in leptin-treated rats (p < 0.01) at both ages, while only the 30 days old rats showed an increment of the aspartate (ASP) release. GABA secretion was not modified by leptin treatment. In conclusion, the results demonstrated that leptin stimulates the LH-GnRH axis during sexual development in male rats, increasing the secretion of both hormones. The hypothalamic excitatory amino acid neurotransmitter system appears to be involved in this change.     

The morphometry of astrocytes in the rostral preoptic area exhibits a diurnal rhythm on proestrus: relationship to the luteinizing hormone surge and effects of age

The morphometry of astrocytes in the arcuate nucleus exhibits cyclic changes during the estrous cycle leading to dynamic changes in the communication between neurotransmitters and neuropeptides that regulate pituitary hormone secretion. Data suggest that remodeling of direct and/or indirect inputs into GnRH neurons may influence the timing and/or amplitude of the preovulatory LH surge in young rats. We have previously found that aging alters the timing and amplitude of the LH surge. Therefore, the purpose of this study was to focus on the rostral preoptic area where GnRH cell bodies reside. We assessed the possibility that the morphometry of astrocytes in the rostral preoptic area displays time-related and age-dependent changes on proestrus. Our results demonstrate that, in young rats, astrocyte cell surface area decreases between 0800 h and 1200 h, before the initiation of the LH surge. Changes in surface area over the cycle were specific to astrocytes in close apposition to GnRH neurons. In contrast, in middle-aged rats astrocyte surface area was significantly less than in young rats and did not change during the day. These findings suggest that a loss of astrocyte plasticity could lead to the delayed and attenuated LH surge that has been previously observed in middle-aged rats.     

A decrease in opioid tone amplifies the luteinizing hormone surge in estrogen-treated ovariectomized rats: comparisons with progesterone effects

It is well known that the estrogen-induced LH surge in ovariectomized (ovx) rats is invariably far less in magnitude than the preovulatory LH surge or that induced by progesterone (P) in estrogen-primed ovx rats. Recent studies show that a decrease in hypothalamic inhibitory opioid tone by the neural clock (NC) is responsible for the induction of the preovulatory LH surge on proestrus. Therefore, we hypothesized that the diminished LH response in estrogen-treated ovx rats may be due to an inadequate reduction in opioid tone. To test this hypothesis the effects of transiently decreasing the opioid tone with an opiate receptor antagonist, naloxone (NAL), on LH secretion in estrogen-primed, short term (5 days) and long term (4 weeks) ovx rats were examined. NAL (2 mg/h) was infused iv from 1100-1400 h on day 2 in rats receiving either sc implants (two, 15 mm each) filled with 17 beta-estradiol (300 micrograms/ml in oil) or sc estradiol benzoate (EB; 10 micrograms/rat) injections at 1000 h on day 0. For comparison of NAL- and P-induced LH responses, EB-primed short and long term ovx rats received P injection (2 mg/rat, sc) instead of NAL infusion at 1100 h. Estrogen treatment alone induced a spontaneous rise in plasma LH on the afternoon of day 2, with peak LH levels ranging between 1.5-2.4 ng/ml. NAL infusion markedly enhanced the LH surge in both groups of ovx rats. In short term ovx rats NAL-induced peak LH levels (5-6 ng/ml) were less than those observed in rats receiving supplemental P treatment or that observed previously on proestrus (10-15 ng/ml). However, in long term EB-primed ovx rats, NAL infusion evoked LH surges equivalent to those observed after P injection. In addition, analysis of the episodic LH secretion pattern showed that NAL infusion accelerated the frequency and amplitude of LH discharge and significantly changed the contour of LH episodes. These results show that a transient decrease in inhibitory opioid tone before a spontaneous LH rise in estrogen-treated ovx rats can accelerate episodic LH secretion to culminate in LH surges that resemble those induced by P and the preovulatory LH surge. Therefore, these observations are in accord with the view that the NC-induced curtailment in the inhibitory opioid tone may be inadequate in estrogen-treated rats; NAL infusion and P treatment intensify decrements in inhibitory opioid tone to reinstate the preovulatory-type LH surge in these rats.     

Effect of leptin on LH levels and hypothalamic release of GnRH. Its relationship with the hypothalamic neurotransmitter amino acids system in adult male rats

The objective of the present paper was to determine the effect of leptin on the reproductive axis in adult male rats, as well as the hypothalamic mechanisms involved in this effect. For this purpose, we studied the in vivo effect of leptin in adult male rats on serum LH levels, and the in vitro effect on hypothalamic GnRH and amino acid neurotrasmitter release. For in vivo experiments, animals were injected i.p. with leptin at a dose of 30, 100 and 300 microg/kg. In the in vitro experiments, hypothalamic samples were incubated for 60 min in Earle's medium with leptin: 10(-9), 10(-10) and 10(-12) M for GnRH determination, and 10(-10) M for amino acids evaluation. Finally, we studied the effect of the lowest effective leptin dose on plasma LH levels in peripubertal male rats to compare the effect between this group and adults. Leptin induces significant decreases of serum LH levels with the different studied doses (p < 0.01 vs. control) in adult male rats, while in peripubertal male rats, it induced a significant (p < 0.01 vs. control) increment in serum LH levels. On the other hand, in vitro leptin in adult male rats, significantly decreases GnRH release as well as the hypothalamic release of glutamate (GLU). In contrast, leptin increased the GABA release by this hypothalamus in these animals. These results indicate that leptin has an inhibitory effect on the GnRH-LH axis in adult male rats and this effect appears to be connected with an inhibition of hypothalamic release of GLU (the excitatory amino acid) and a stimulatory effect on GABA release (the inhibitory amino acid). On the other hand, in peripubertal male rats, leptin showed a stimulatory effect.