Effect of di(2-ethylhexyl) phthalate on the neuroendocrine regulation of reproduction in adult male rats and its relationship to anxiogenic behavior: Participation of GABAergic system.

The endocrine disruptor di-(2-ethylhexyl) phthalate (DEHP) is used in a variety of consumer products made with polyvinyl chloride and also in the manufacture of medical devices. DEHP disrupts reproductive tract development in an antiandrogenic manner and also may induce neurobehavioral changes. The aim of this study was to investigate the effects of chronic postnatal exposure to DEHP (30 mg/kg body weight/day, orally from birth to day 60) on the neuroendocrine regulation of the gonadal axis and its impact on the anxiety-like behavior in adult male rats, as well as the probable participation of the GABAergic system in these effects. DEHP produced a significant increase in plasmatic luteinizing hormone and follicle stimulating hormone, as well as significant testosterone decrease, accompanied with a decrease in hypothalamic gamma-aminobutyric acid (GABA) concentration. On the other hand, DEHP increased the anxiety-like behavior in the elevated plus maze test, evidenced by a significant decrease in the percentages of time spent in the open arms and the frequency in the open arm entries and a significant increase in the percentage of time spent in closed arms. Neuroendocrine and behavioral effects were reversed by GABA agonists, muscimol (2 mg/kg i.p. ) and baclofen (10 mg/kg i.p.). In conclusion, chronic DEHP postnatal exposure induced a disruption in the neuroendocrine regulation of the testicular axis in young adult male rats, and this effect was correlated with an anxiety-like behavior. Since GABA agonists reversed these effects, the results suggest that GABA could participate in the modulation of reproductive and behavioral DEHP effects.

Antiandrogenic effect of perinatal exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate increases anxiety-like behavior in male rats during sexual maturation.

Di-2-ethylhexyl phthalate (DEHP) is the most widely used phthalate to convey flexibility and transparency to plastic products made of polyvinyl chloride. It has been recognized as endocrine disruptor and associated with reproductive toxic effects. We examined the effects of perinatal exposure to DEHP on anxiety-like behavior, using the Elevated Plus Maze (EPM) test, in male and female rats at different stages of sexual development. Anxiety-like behavior was expressed as a) frequency of open arm entries over the total arm entries (% FEO); b) time spent in them compared with total time the animal stayed in the EPM (% TSO) and c) time spent in closed arms (TSC). Because DEHP has anti-androgenic action we also tested control and exposed immature male rats pretreated with testosterone. We found sex differences in behavior induced by DEHP; while male rats of 45 and 60 days of age showed a significant decrease in FEO and TSO percentages, as well as an increase in TSC, no changes were observed in anxiety-like behavior in perinatal DEHP exposed females at these ages of sexual maturation. In 60-day-old male rats, DEHP exposure produced a significant decrease in serum testosterone levels. Testosterone replacement was able to antagonize the adverse effects of DEHP exposure on LH, activating the negative feed-back mechanism of this steroid on reproductive axis, as well as increasing FEO and TSO percentages to similar values observed in the control group. These findings suggest that the anti-androgenic action of this chemical could be one possible mechanism underlie anxiogenic-like behavior produced by perinatal DEHP exposure in 60-day-old male rats.

Different effects by sex on hypothalamic-pituitary axis of prepubertal offspring rats produced by in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP).

This study investigated the effect of pre and perinatal exposure to di-(2-ethylhexyl) phthalate (DEHP) on the neuroendocrine parameters that regulate reproduction in prepubertal male and female rats. DEHP at doses of 3 and 30mg/kgbw/day was administered orally in the drinking water to dam rats since pregnancy onset until the moment of pups sacrifice at 15 days of age. In these animals gonadotropin serum level and the hypothalamic contents of the amino acids aspartate, glutamate and gamma-aminobutyric acid were determined. No changes in gonadotropin levels and amino acid neurotransmitters were detected at the low dose in both sexes. However, DEHP administered at high dose (30mg/kgbw/day) to dams produced a significant decrease in the inhibitory neurotransmitter GABA and an increase in the stimulatory neurotransmitter aspartate in prepubertal male offspring rats. These modifications were accompanied by gonadotropin serum levels increase. On the contrary, in treated female rats this chemical increased both, aspartate and GABA, which exert a characteristic stimulatory action on gonadotropin in 15-day-old normal females. This study provides new data about changes produced by DEHP on the hypothalamic amino acid neurotransmitters involved in the neuroendocrine reproductive regulation, in prepubertal male and female rat offspring from dams exposed during gestational and lactational periods. These alterations induced by DEHP exposure could be related to the gonadotropin modifications also described in this work, and with changes in the production of sexual hormones previously reported by other authors.

In vitro effect of octyl - methoxycinnamate (OMC) on the release of Gn-RH and amino acid neurotransmitters by hypothalamus of adult rats.

OMC (octyl-methoxycinnamate), an endocrine disruptor having estrogenic activity, is used in sunscreen creams as UV filter. We studied its "in vitro" effects on the hypothalamic release of Gn-RH as well as on the amino acid neurotransmitter system. OMC significantly decreased Gn-RH release in normal male and female rats as well as in castrated rats with substitutive therapy. No effects were observed in castrated rats without substitutive therapy. In males OMC increases the release of GABA, decreasing the production of glutamate (GLU) while in the female decreases the excitatory amino acid aspartate (ASP) and GLU without modifications in the hypothalamic GABA release. These results suggest that OMC acting as endocrine disruptor could alter the sex hormone-neurotransmitter-Gn-RH axis relationships in adult rats.

Impact of 4-methylbenzylidene-camphor (4-MBC) during embryonic and fetal development in the neuroendocrine regulation of testicular axis in prepubertal and peripubertal male rats.

4-Methylbenzylidene-camphor (4-MBC), an UV-B ray filter, belongs to the endocrine disrupters involved with alterations in the reproductive axis. Our target was to study the effect of 4-MBC on the neuroendocrine parameters that regulate reproduction in prepubertal and peripubertal male rats, which received this disrupter during embryonic and fetal development. 4-MBC was administered (sc) to female rats since pregnancy onset in doses of 20, 100 and 500 mg/kg/day. The litters were sacrificed at 15 or 30 days old to determine testicular weight, gonadotropin and prolactin serum levels and also GnRH and amino acids release from the hypothalamus. The exposure to 20 mg/kg/day only increased the LH serum levels in 30-day-old males. Doses of 100 and 500 mg/kg/day caused a decrease in testicular weight and in LH, GnRH and glutamate levels, in prepubertal rats (15-day-old specimens), and an increase in, gonadotropin (LH and FSH) con-centration and aspartate levels in peripubertal rats (30-day-old specimens), without changes in testicular weight. Prolactinaemia remained unaltered in all groups. Results obtained show that the administration of high doses of 4-MBC during embryonic and fetal stage inhibits the testicular axis in male rats during the prepubertal stage and stimulates it during peripubertad stage. On the other hand in the case of low doses no significant effects were observed.

Impact of the UV-B filter 4-(Methylbenzylidene)-camphor (4-MBC) during prenatal development in the neuroendocrine regulation of gonadal axis in male and female adult rats.

4-(Methylbenzylidene)-camphor (4-MBC), a UV-B ray filter, is an endocrine disruptors (ED). Our goal was to study the effect of 4-MBC on the neuroendocrine parameters that regulate reproduction in adult female and male rats that received this disrupter during prenatal development. The 4-MBC was administered (sc) to female rats (FO) since pregnancy onset, in doses of 100mg/kg every other day. The litters (F1) were sacrificed at 70 days to determine gonadotrophin serum levels and also GnRH and the amino acids glutamate, aspartate and GABA release from the hypothalamus. The male litter rats (F1) present at adult age a decrease in serum LH and FSH concentration and so also GnRH, excitatory amino acids and GABA hypothalamic secretion. The female litters (F1) rats present at adult age an increase in serum LH and FSH concentration, whereas hypothalamic GnRH release was not modified. In these animals a significant increase of hypothalamic aspartate release as well as GABA secretion decrease were observed. Glutamate secretion was not modified. All these changes were accompanied by an advance (3 days) on the vaginal opening in 4-MBC rats group. In conclusion, prenatal administration of 4-MBC disrupts the gonadal axis in a sexual dimorphic mode that could be connected with the physiological sexual differences in the development of gonadotrophin secretion hypothalamic control mechanisms.

Nitric oxide synthase inhibition prevents leptin induced Gn-RH release in prepubertal and peripubertal female rats.

The aim of the present paper was to study the role of NO as a mediator of leptin action at the hypothalamic level during sexual maturation. First, we analyzed the effect of different leptin concentrations (10 (-13), 10 (-11) and 10 (-9) M) on Gn-RH release from anterior preoptic area and medio basal hypothalamus (APOA-MBH) of prepubertal (15 days old) and peripubertal (30 days old) female rats. Leptin 10 (-13) M was the most effective concentration in releasing Gn-RH in both groups of animals. Since glutamate (GLU) and GABA are involved in the hypothalamic control of Gn-RH neurons and also in the neuroendocrine mechanism of puberty, in a second serie of experiments, we evaluated the effect of a competitive inhibitor of nitric oxide synthase (NOS), N-monomethyl-L-arginine (NMMA) on Gn-RH, GLU and GABA release in response to leptin. Co incubation of APOA-MBH with NMMA 0.5 mM, completely blocked Gn-RH and GLU release induced by leptin 10 (-13) M in prepubertal and peripubertal rats. NMMA also blocked the stimulation of GABA release in prepubertal rats, as well as the inhibition of GABA release induced by leptin in peripubertal rats. It can be proposed that the different effect of NO on GABA release, could be related to ontogenic changes, e.g, maturation of receptors and/or interneuronal connections during sexual development. Present results provide evidence that leptin acts at the hypothalamic level to stimulate NO release, which in turn modifies the release of amino acid neurotransmitters involved in Gn-RH control.

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.

Effect of leptin on hypothalamic release of GnRH and neurotransmitter amino acids during sexual maturation in female rats.

The purpose of the present study was to analyse the effect of leptin treatment on the hypothalamic release of GnRH, GABA, and the excitatory amino acids (EAA), aspartate (ASP) and glutamate (GLU) involved in NMDA neurotransmission in prepubertal (15 day old) and peripubertal (30 day old) female rats. The animals were treated with a single dose of leptin (30 microg/kg i.p.) and sacrificed 60 min later. Hypothalamic samples were incubated in Earle's medium; GnRH was determined by RIA and GLU, ASP and GABA by HPLC by UV detection. The hypothalamic release of GnRH was increased by leptin at both ages, the release being significantly higher in peripubertal than in prepubertal rats. The levels of hypothalamic GABA release were different in the two groups; whereas in prepubertal rats the hypothalamic release of GABA increased with leptin administration, the neurotransmitter release decreased in the peripubertal group. On the other hand, the release of ASP was modified only in the peripubertal group, where leptin significantly increased its hypothalamic release. No modifications in leptin-induced hypothalamic release of GLU were observed at the two ages studied. In conclusion, the results showed that leptin increased GnRH release by the hypothalamus of prepubertal and peripubertal rats. In peripubertal rats this increase was accompanied by a significant decrease in the hypothalamic release of GABA as well as an enhanced release of ASP. These results and previous reports suggest that at this stage of sexual maturation, leptin exerts an stimulatory effect on GnRH by inducing release of excitatory amino acids (ASP) and reducing release of inhibitory amino acids (GABA) involved in GnRH control. In prepubertal rats the stimulating effect of the adipocyte hormone on GnRH appears to be related to its stimulative action on GABA which at this age increases GnRH release.

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 the sensitivity of gonadotrophin axis to leptin during sexual maturation in female rats.

OBJECTIVES: The aim of the present paper was to determine the sensitivity of the GnRH-LH axis to leptin administration during sexual maturation in female rats. METHODS: For this purpose the hypothalamic concentration of GnRH, the pituitary content and the plasmatic levels of LH were determined in prepubertal (15 days of age) and peripubertal female rats (30 days of age), treated with leptin at a dose of 30 microg/kg. i.p. in a single injection, 90 min before sacrifice. RESULTS: The results indicate that leptin significantly increased the GnRH concentration at 15 days of age (p <0.01). At 30 days of age the hormone did not significantly modify the hypothalamic GnRH content. Leptin increased the pituitary LH levels, both in prepubertal and peripubertal rats. Nevertheless, while the increase at 15 days of age was around 180%, in peripubertal rats it was about 51,2 %. In spite that leptin significantly increased LH plasmatic levels at both ages (p < 0.01 ), in rats of 15 days of age leptin increased LH in about 244%, at 30 days of age this increase was only about 102%. CONCLUSION: These results clearly demonstrated that leptin has stimulatory effect on gonadotrophin axis been higher in prepubertal than in peripubertal rats. On these basis, and on the results of previous papers, (in which it has been demonstrated that the hypothalamic control of gonadotrophins by neurotransmitters and neuromodulators also showed qualitative and quantitative changes during sexual maturation), it is proposed that these differences are connected with the maturation of the neuroendocrine mechanisms involved in the regulatory action of leptin on the gonadotrophins axis.

Interrelationships of GABAergic, serotoninergic and excitatory amino acid systems in its regulatory effect on prolactin secretion in prepubertal rats.

GABAergic, serotoninergic and excitatory amino acid systems (EAAs) regulate the prolactin (PROL) secretion in prepubertal female rats. The aim of the present paper was to determine the interrelationships of these systems on the control of this pituitary hormone. It was carried out through the following scheme: 1. The participation of the EAAs and serotonin in the effect of GABAergic system on PROL release, determined by evaluating the GABA A and GABA B receptor agonists. It was carried out on animals that were previously treated with AAEs receptor antagonist or p-chlorophenylamphetamine (PCA), this one depleting serotonin in the hypothalamus. 2. The participation of GABAergic system in the effect of serotonin and EAAs systems, determined by the evaluation of the effects of EAAs receptor agonists and of 5-HTP, a serotonin precursor. With this purpose the rats were previously treated with GABA A and GABA B receptor antagonists. 3. The interrelationships between the EAAs and the serotoninergic systems in the control of PROL secretion, determined (a) by using EAAs agonists (in rats depleted of serotonin by PCA) and (b) using EAAs antagonists (in rats treated with 5-HTP, a serotonin precursor). The administration of GABAergic agonists significantly increased PROL secretion in prepubertal female rats. Neither EAAs antagonists nor the depletion of serotonin in the brain, modified the stimulatory effects of the GABAergic system on PROL levels. This is a clear indication that the activity of the GABAergic system is independent of the serotoninergic and of the EAAs system effects on the pituitary hormone. The EAAs neurotransmitter system agonists significantly increase PROL levels. This effect was blocked by the GABAergic system antagonists but was not modified by serotonin depletion. Taking into account these facts it may be considered that the GABAergic system is involved in the stimulatory effect of EAAs on PROL secretion, this effect being independent of the serotoninergic system. 5-HTP significantly increased PROL plasma levels, and this effect was modified neither by the GABAergic nor by the EAAs receptor antagonists. These results indicate that the stimulatory effect of serotonin on PROL release is independent of the GABAergic and EAAs systems. In conclusion it may be considered that in prepubertal female rats, the GABAergic and serotoninergic systems stimulate PROL secretion by independent mechanisms that do not include EAAs. On the other hand, the effects of EAAs neurotransmission are exerted via the GABAergic system.

[Effect prolonged GABAergic activation on the pubertal development of female rats]. / Efecto de la activación GABAérgica sobre la maduración sexual en ratas hembras peripuberales.

We evaluated, in immature female rats, the effect of the GABAergic system on the reproductive axis and on pubertal development. Initially, using a prolonged treatment with aminooxyacetic acid (AOAA), increasing hypothalamic GABA (p < 0.002), and decreasing GnRH and glutamate content (p < 0.05 and < 0.02). Treated rats showed diminished serum LH (p < 0.05) and estradiol (p < 0.005) levels. Vaginal opening occurred at 30.8 +/- 0.6 days in controls, and at 36.7 +/- 0.98 days in AOAA-treated rats. Acute treatment with AOAA resulted in a decreased GnRH and glutamate output, and in an increased taurine release from superfused hypothalamic fragments. This effect was mimicked by the GABA-A and GABA-B agonists. The activation of the GABAergic system during postnatal days 23-29 significantly restrains the hypothalamo-pituitary-ovaric axis and delays the onset of puberty. The existence of a physiological cross-talk between excitatory and inhibitory amino acid neurotransmitters regulating GnRH release during the onset of puberty is postulated.

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.

Efecto de la activación GABAérgica sobre la maduración sexual en ratas hembras peripuberales. / [Effect prolonged GABAergic activation on the pubertal development of female rats]

We evaluated, in immature female rats, the effect of the GABAergic system on the reproductive axis and on pubertal development. Initially, using a prolonged treatment with aminooxyacetic acid (AOAA), increasing hypothalamic GABA (p < 0.002), and decreasing GnRH and glutamate content (p < 0.05 and < 0.02). Treated rats showed diminished serum LH (p < 0.05) and estradiol (p < 0.005) levels. Vaginal opening occurred at 30.8 +/- 0.6 days in controls, and at 36.7 +/- 0.98 days in AOAA-treated rats. Acute treatment with AOAA resulted in a decreased GnRH and glutamate output, and in an increased taurine release from superfused hypothalamic fragments. This effect was mimicked by the GABA-A and GABA-B agonists. The activation of the GABAergic system during postnatal days 23-29 significantly restrains the hypothalamo-pituitary-ovaric axis and delays the onset of puberty. The existence of a physiological cross-talk between excitatory and inhibitory amino acid neurotransmitters regulating GnRH release during the onset of puberty is postulated.

Effect of an aproteic diet on gonadotropin release response to GnRH and estrogen-progesterone in rats.

The fasting-induced gonadotropin function decrease is unspecific, because in this situation there is a lack of all nutrients. We report here the effect of specific protein lack in the diet during 21 days, on pituitary gonadotropin synthesis and response to exogenous GnRH in adult male rats. We also studied the effect of the aproteic diet (AP) on the positive feedback mechanism in adult female castrated rats. The AP diet decreased significantly, both LH and FSH pituitary concentration and also basal gonadotropin plasma levels in male rats. GnRH produced a significantly increment in LH secretion in both treated and control groups, reaching similar levels after stimulation. Nevertheless, the percentile increment from basal levels in the aproteic group was almost four times the controls, suggesting an increased sensitivity in pituitary response to GnRH in rats fed with AP diet. In female castrated rats, the aproteic diet imposed 3 weeks after the surgery was unable to reduce basal gonadotropin secretion, and so also prolactin secretion. Estradiol/progesterone (EP) administration produced the activation of positive feedback mechanism, increasing significantly LH and FSH secretion in both controls and AP groups. Nevertheless, both gonadotropin responses to EP were significantly greater in rats fed with AP diet. Basal prolactin levels and response to EP were not different between both groups. This results suggest that selective protein lack in a diet, reduced pituitary LH and FSH synthesis and secretion. This type of diet also increments pituitary sensitivity to GnRH administration in male rats, and gonadotropin response to positive feedback mechanism in female rats.

GABAergic activation inhibits the hypothalamic-pituitary-ovaric axis and sexual development in the immature female rat. Associated changes in hypothalamic glutamatergic and taurinergic systems.

The aim of the present studies was to assess, in immature female rats, the effect of the GABAergic system on the reproductive axis and on pubertal development. With this purpose we initially evaluated, in 30-day-old female rats, the effect of persistently enhanced GABAergic activity (aminooxyacetic acid (AOAA) 10 mg/kg per day i.p., during postnatal days 23-29) on hypothalamic gonadotropin-releasing hormone (GnRH) and amino acid neurotransmitter (AANT; glutamate or GLU, and taurine or TAU) concentrations, on circulating luteinizing hormone (LH) and estradiol levels, and on ovaric weight. In a second group of similarly treated rats, the date of vaginal opening (VO) was recorded. Complementary in vitro experiments (superfusion of anterior/mediobasal hypothalamic fragments obtained from rats aged 30 days) were performed to evaluate the effect of the short-term activation of the GABAergic system (by means of AOAA, muscimol or baclofen) on hypothalamic GnRH and AANT release. Prolonged treatment with AOAA led to a marked increase in hypothalamic gamma-aminobutyric-acid (GABA) concentrations (p<0.002), and to a significant decrease in hypothalamic GnRH and GLU content (p<0.05 and <0.02, respectively). Furthermore, treated animals showed diminished serum LH (p<0.05) and estradiol (p<0.005) levels, and a clear reduction in ovaric weight (p<0.002). Mean age at VO was 30. 8+/-0.6 days in control animals (range: 29-34 days), and 36.7+/-0.98 days in AOAA-treated rats (range: 33-40 days; p<0.0001). Acute treatment with AOAA resulted in a decreased GnRH and GLU output, and in an increased TAU release from superfused hypothalamic fragments. This effect was mimicked by the GABA-A and GABA-B agonists. Our results show that the activation of the GABAergic system during postnatal days 23-29 significantly restrains the hypothalamic-pituitary-ovaric axis, resulting in a clear-cut delay in sexual development. This can be attributed to the inhibitory effect exerted by GABA (acting on both GABA-A and GABA-B receptor subtypes) on GnRH release. Furthermore, the pharmacologic manipulation of the GABAergic system induces significant changes in the release of GLU and TAU, giving biochemical support to the existence of a physiological cross-talk between the excitatory and inhibitory AANT regulating GnRH release during the onset of puberty.

Effects of the GABA-A receptor agonist and antagonist on the in vitro release of hypothalamic catecholamines: apparent parallelism between these effects and the LHRH secretion in adult male rats.

GABA (gamma-aminobutyric acid) has a well-known inhibitory effect on the luteinizing hormone-releasing hormone (LHRH) secretion. In order to evaluate the contribution of the catecholaminergic neurotransmitters on the inhibitory effect produced by GABA on the LHRH release, we measured in adult male rats the in vitro hypothalamic output of LHRH, epinephrine (E), norepinephrine (NE) and dopamine (DA); after the administration of, either muscimol 1 microM (GABA-A agonist), and/or 1 microM bicuculline (GABA-A antagonist). The following results were obtained: muscimol inhibited LHRH secretion, and this effect was accompanied by a decrease of NE, E and DA output. The opposite effects were observed after the addition of bicuculline, i.e, stimulation of LHRH, NE, E and DA release. In conclusion, our results show that, in the adult male rats, GABA has an inhibitory effect on the in vitro release of LHRH, acting on the GABA-A receptor. This effect on LHRH secretion might be exerted directly, or indirectly, by altering the release of either NE,E, and/or DA.

Interleukin-1 stimulates hypothalamic inhibitory amino acid neurotransmitter release.

Interleukin-1 (IL-1), a polypeptide cytokine, has been postulated as a chemical messenger between the immune and the neuroendocrine system. IL-1 receptors and immunopositive neurons have been visualized in the human and rat hypothalamus, suggesting that IL-1 can act as a neurotransmitter within the brain. In the hypothalamus IL-1 and the amino acid neurotransmitters are known to modulate several functions, such as fever, anorexia and the gonadal and adrenal axis. Since the hypothalamic actions of IL-1 on the amino acid neurotransmitter output are unknown, the aim of the present paper was to evaluate the effects of IL-1 on the hypothalamic release of both, the inhibitory taurine, glycine and GABA and the excitatory glutamate, amino acid neurotransmitters. Intact adult male rats were employed. The preoptic/mediobasal hypothalamic area was dissected and superfused with Earle's balanced salt solution. Superfusate fractions were collected after a 60-min stabilization period. Following 60 min of basal release, IL-1 was added to the superfusion medium over 30 min. GABA, taurine and glycine release were significantly (p < 0.05) increased in the superfusion medium, while glutamate was not modified compared with the control group. These observations show that IL-1 increased GABA, taurine and glycine release. These effects indicate that this cytokine can affect the hypothalamic inhibitory amino acid output, which may help us to understand the mechanism by which IL-1 exerts its effects.

Hypothalamic relationships between interleukin-6 and LHRH release affected by bacterial endotoxin in adult male rats. Involvement of the inhibitory amino acid system.

Immune system alterations coexist with modifications in the reproductive axis. The bacterial endotoxin lipopolysaccharide (LPS) has inflammatory effects and stimulates cytokine release in the hypothalamus where LHRH neurons are located. LPS inhibition of LHRH release at hypothalamic level appears to be associated with modifications in the cerebral immune system. Central and peripheral LPS administration induces the expression and release of several cytokines in the central nervous system. Hence the present study was designed to investigate a possible function of the interleukin-6 (IL-6) stimulated by LPS in the regulation of LHRH secretion. Male rats were decapitated, and the preoptic mediobasal hypothalamic area (PO/MBH) was dissected and superfused with Earle's balanced salt solution. Superfusate fractions were collected at 15-min intervals after a 60-min stabilization superfusion period. LPS (100 ng/ml) and IL-6 receptor antagonist (IL-6ra) were then added to the superfusion medium over 1 h in two different experimental designs: (1) LPS only and (2) LPS followed by IL-6ra, performed in different experiments. This was followed by a washout period. The PO/MBH fragments were then subjected to a 56 mM K+ stimulus. Control PO/MBH fragments were continuously superfused with Earle's solution. As expected, LHRH release was significantly reduced (p < 0.05) during and following exposure to LPS. At the same time, IL-6 concentrations significantly increased in the superfusion medium compared with the control group. IL-6ra significantly (p < 0.01) potentiated the inhibitory effect of LPS on LHRH secretion. On the bases of previous papers indicating a stimulatory effect of IL-6 on LHRH release it could be considered that the potentiation of IL-6ra of the inhibitory effect of LPS on LHRH could be the consequence of the lack of the stimulatory effect of IL-6 on LHRH produced by the receptor antagonist. IL-6ra also increased IL-6 levels measured in medium probably due to a decrease in the metabolization induced by the blockage of the receptors and the consequent accumulation of IL-6 in the media. These results could indicate that IL-6 partly attenuates the inhibitory effect of LPS on LHRH release. These observations indicate that there is an increase in IL-6 release that becomes significant at the same time when LHRH release is decreased. Also, depolarizing concentrations of K+ (56 mM) did not increase IL-6 release, while LHRH release from the hypothalamic fragments was significantly increased. These data suggest that the inhibitory effect of LPS on LHRH release may be explained by the stimulation of other cytokines than IL-6, meanwhile the augmented levels of IL-6 probably released via a nonneuronal source was shown to be higher when LHRH was decreased. This could confirm the stimulatory role of IL-6 on LHRH release.