Serotonin inhibits luteinizing hormone release via 5-HT1A receptors in the zona incerta of ovariectomised, anaesthetised rats primed with steroids

The zona incerta (ZI), an area in the dorsal hypothalamus, contains neuronal systems that appear to control gonadotropin release. Previous findings show that there is an inverse relationship between serotonin (5-HT) activity in the ZI and plasma luteinizing hormone (LH) levels, indicating that the 5-HT system in this area has an inhibitory effect on LH release. Employing anaesthetised, ovariectomised rats primed with 5 microg oestradiol benzoate followed at 48 h by 0.5 mg progesterone, we have shown that 2 microg/side 5-HT in the ZI inhibits the LH surge that normally occurs 4 h after the progesterone treatment. This effect was mimicked by 2 microg/side 8-OH-DPAT, a 5-HT1A agonist, but not by DOI, a 5-HT2 agonist, BMY7378, a presynaptic 5-HT1A agonist or MCPP, a 2B & 2C agonist. The inhibitory effect of 5-HT and 8-OH-DPAT was prevented by pretreatment, 1 h before, with either 2 mg/kg i.p. WAY100135, a 5-HT1A antagonist or 0.25 mg/kg i.p. ritanserin, a 5-HT2 antagonist. These results indicate that 5-HT in the ZI exerts its inhibitory effect on LH release via 5-HT1A receptors but that another 5-HT subtype may also be involved.     

5-HT1 and 5-HT2 receptor activation reduces N-methyl-D-aspartate (NMDA)-stimulated LH secretion in prepubertal male and female rats

OBJECTIVE: Excitatory amino acids and serotonin are involved in the control of gonadotropin secretion. The actions of these neurotransmitters are interconnected and recently we have reported that 5-HT(1) and 5-HT(2) receptor agonists blunted (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-stimulated GH secretion in prepubertal rats. The present experiments were carried out to analyze the effects of activation of different 5-hydroxytryptamine (5-HT) receptor subtypes on gonadotropin secretion and their role in the N-methyl-d-aspartate (NMDA)-stimulated LH release. DESIGN AND METHODS: We analyzed the gonadotropin secretion after manipulation of serotoninergic and aminoacidergic systems and their interactions in 5-, 16- and 23-day-old male and female rats. To this end, serum LH and FSH concentrations were measured in rats treated with 5-hydroxytryptophan methyl ester (5-HTP) (a precursor of 5-HT synthesis) plus Fluoxetine (Fx, a blocker of 5-HT reuptake), d,l-p-chlorophenyl-alanine methyl ester (PCPA, a blocker of 5-HT synthesis), R-(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, an agonist of 5-HT(1A) receptors), (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT, agonists of 5-HT(2) receptors), and 1-Phenylbiguanide (1-PHE an agonist of 5-HT(3) receptors). In addition, the effects of 8-OH-DPAT and DOI on NMDA-stimulated LH secretion were analyzed. RESULTS: Neither the activation nor blockade of the serotoninergic system modified LH secretion. Basal gonadotropin secretion remained unchanged in 23-day-old male and female rats after activation of 5-HT(1A), 5-HT(2) and 5-HT(3) receptors. The stimulatory effect of NMDA on LH secretion was blocked in both sexes after activation of the serotoninergic system, through specific 5-HT(1) and 5-HT(2) receptor agonists. CONCLUSIONS: Activation of serotoninergic receptors decreased the stimulatory effect of NMDA on LH secretion in prepubertal male and female rats.     

Mechanisms of serotonin receptor agonist-induced activation of the hypothalamic-pituitary-adrenal axis in the rat

A substantial body of experimental evidence indicates that serotonin (5-HT) and several synthetic 5-HT receptor agonists activate the hypothalamic-pituitary-adrenal (HPA) axis. To explore the mechanism(s) by which 5-HT or 5-HT agonists enhance the activity of the HPA axis in vitro, we examined the stimulatory effects of the 5-HT1a agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5-HT1c/5-HT1b agonist m-chlorophenylpiperazine (m-CPP), and the 5-HT2/5-HT1c agonist 1-(2,5-dimethoxy-4-iodophenyl)2-amino-propane (DOI) on plasma ACTH and corticosterone secretion in the rat. To test whether 8-OH-DPAT, m-CPP, or DOI increase plasma ACTH levels by stimulating the release of endogenous CRH, catheterized conscious male Sprague-Dawley rats were pretreated with hyperimmune CRH rabbit serum (TS-6) or normal rabbit serum and subsequently challenged with a maximally stimulatory dose of the above 5-HT agonists. Pretreatment with TS-6 completely suppressed the ACTH response to m-CPP and significantly blunted the responses to 8-OH-DPAT or DOI. To examine whether the remaining ACTH response to 8-OH-DPAT or DOI was also mediated by a pituitary site of action, we administered each of these agents to pituitary stalk-transected or sham-operated rats. The ACTH responses to 8-OH-DPAT and DOI in stalk-transected rats were preserved, although significantly blunted, compared to those in sham-operated rats. This suggested that both of these 5-HT agonists may also act at the pituitary level to stimulate ACTH release in vivo. Although the ACTH responses to 8-OH-DPAT, m-CPP, and DOI were blunted after both TS-6 pretreatment and pituitary stalk transection, corticosterone responses were only slightly affected, suggesting that some of these compounds may cause corticosterone release in the rat through another mechanism. To evaluate this hypothesis, ACTH and corticosterone responses to 8-OH-DPAT, m-CPP, and DOI were examined in rats whose HPA axis had been suppressed by a single high dose injection of dexamethasone. The corticosterone responses to 8-OH-DPAT and DOI were blunted compared to those of saline-pretreated rats, but were inappropriately high compared to the ACTH responses observed in these rats. On the other hand, both ACTH and corticosterone responses to m-CPP were completely abolished by dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)     

5-HT1 and 5-HT2 receptor agonists blunt +/- -alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-stimulated GH secretion in prepubertal male rats

OBJECTIVE: Excitatory amino acids, gamma-amino butyric acid (GABA), serotonin and catecholamines are involved in the control of GH secretion. The actions of these neurotransmitters are interconnected, and recently we showed that the stimulatory effect of GABA was blocked by MK-801, an antagonist of N-methyl-D-aspartate receptors. The present experiments were carried out to analyze the interrelationships between +/- -alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and serotoninergic and catecholaminergic pathways in the control of GH secretion in prepubertal (16-23-day-old) male rats. DESIGN AND METHODS: The GH response to AMPA was analyzed in animals pretreated with 5-hydroxytryptophan methyl ester (5-HTP) plus fluoxetine (a precursor of 5-hydroxytryptamine (5-HT) synthesis and a blocker of 5-HT re-uptake), R (+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, an agonist of the 5-HT1 receptors), +/- -2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and alpha-methyl-5-hydroxytryptamine (agonists of 5-HT2 receptors), I-phenylbiguanide (an agonist of 5-HT3 receptors), or alpha-methyl-p-tyrosine (alpha-MPT) and diethyldithiocarbamate (DDC) (blockers of catecholamine synthesis). RESULTS: Basal GH secretion remained unchanged in prepubertal rats after activation of the serotoninergic system or blockade of catecholamine synthesis. The stimulatory effect of AMPA on GH secretion was blocked after activation of the serotoninergic system, through specific 5-HT1 and 5-HT2 receptor agonists. In contrast, activation of 5-HT3 receptors potentiated AMPA-stimulated GH secretion. CONCLUSIONS: Serotoninergic receptors modulate the stimulatory effect of AMPA on GH secretion in prepubertal male rats.     

Effects of 5-hydroxytryptamine uptake blockers on the release of LH and prolactin in several different experimental steroid models in the rat

The effect of the 5-hydroxytryptamine (5-HT) uptake blockers on the surges of LH and prolactin has been investigated in pro-oestrous rats and various experimental models used frequently to study the effects of steroids on LH and prolactin secretion in female rats. The steroid models were: rats ovariectomized on dioestrus, injected immediately with oestradiol benzoate (OB) and at 12.00 h on the next day (presumptive pro-oestrus) with progesterone (model 1); long-term ovariectomized rats injected with a single injection of OB and 72 h later with either progesterone (model 2) or OB (model 3); long-term ovariectomized rats injected daily with OB (model 4). The uptake blockers alaproclate (3-30 mg/kg) and zimelidine (20 mg/kg) were injected and blood samples withdrawn from previously implanted intra-atrial cannulae. Plasma LH and prolactin concentrations were determined by radioimmunoassay. The present study confirmed that a surge of LH occurs at about 17.00-18.00 h of the presumptive day of pro-oestrus in model 1, at about 5 h after (approximately 17.00 h) the injection of either progesterone or the second injection of OB in models 2 and 3, and diurnally in model 4, and the simultaneous occurrence of a prolactin surge in models 2 and 4. A surge of prolactin at the same time as the LH surge was shown to occur also in models 1 and 3. Alaproclate (30 mg/kg) administered at 15.00 h delayed significantly the peak of the prolactin surge in the pro-oestrous rat and models 1, 3 and 4, and in the latter the magnitude of the prolactin surge was also significantly reduced. By contrast, the peak of the prolactin surge in model 2 was significantly prolonged by alaproclate. Alaproclate had no significant effect on either the timing or the magnitude of the LH surge in the pro-oestrous rat, and models 3 and 4. The peak of the LH surge was delayed by alaproclate in model 1 and abolished in model 2, providing further evidence for the possible importance of interactions between 5-HT and progesterone in neuroendocrine control. Zimelidine had no significant effect on either the LH or prolactin surge in the pro-oestrous rat and in models 1 and 2. These results show that normal 5-HT uptake is necessary for the normal timing and/or magnitude of the spontaneous and steroid-induced prolactin surge but is not essential for the normal timing and magnitude of the spontaneous surge of LH and the LH surge in some but not all steroid models.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of endogenous opioid influence on luteinizing hormone secretion by progesterone and estrogen

Studies were undertaken using the opiate receptor antagonist naloxone (NAL) to evaluate the relative influence of endogenous opioid peptides (EOP) on LH secretion in cycling and ovariectomized, steroid-treated adult rats. Intact animals received NAL (2 mg/kg, sc) or saline (control vehicle) at 0800 and 1400 h on estrus, 0800 h on diestrus day 1,2000 h on diestrus day 2, and before (at 0800, 1200, and 1400 h) and during the preovulatory LH surge (at 1600 and 1800 h) on proestrus. NAL stimulated LH release by 2- to 3-fold at all stages of the estrous cycle, including during the proestrous gonadotropin surge. Ovariectomized rats were treated with estradiol benzoate (EB; 7.5 micrograms/rat, sc) and 2 days later received NAL (2 mg/kg, sc) or saline at 1000, 1200, 1400, 1600, and 2000 h. NAL induced a relatively small (44-73%), but significant, increase in LH release before (1000, 1200, and 1400 h), during (1600 h), and after (2000 h) the afternoon LH rise. While progesterone (P) treatment (5 mg, sc, day 2 at 1000 h) of EB-primed ovariectomized rats augmented NAL-induced LH release before the LH surge (1200 h), it abolished the LH secretory response to NAL during the LH surge (1400, 1600, and 1800 h). The NAL-induced LH response returned after the LH surge at 2000 h. Likewise, administration of P on proestrus morning (0900 h) abolished the LH secretory response to NAL during the LH surge. These studies indicate that central opioid neurons participate in the tonic inhibition of LH secretion at all stages of the estrous cycle of the rat. The ability of exogenous P to advance and amplify the LH surge on proestrus and in EB-primed ovariectomized rats appears to result in part from a reduction in the EOP inhibitory influence on LH secretion and may indicate a role for EOP in mediating the stimulatory effects of endogenous steroids on LH secretion in the female rat.     

An endogenous serotonergic rhythm acting on 5-HT(2A) receptors may be involved in the diurnal changes in tuberoinfundibular dopaminergic neuronal activity and prolactin secretion in female rats

The central serotonergic system has long been known to have a stimulatory role on the secretion of prolactin (PRL). The integrity of serotonergic neurotransmission is essential for the expression of the estrogen-induced afternoon PRL surge. Whether its effect on PRL involves change in the activity of tuberoinfundibular dopaminergic (TIDA) neurons has not been ascertained. In adult ovariectomized rats treated with estrogen, depletion of central serotonin (5-HT) by 5,7-dihydroxytryptamine (5,7-DHT, 200 microg/rat, i.c.v.) effectively prevented the afternoon fall in TIDA neuronal activity (using the levels of 3,4-dihydroxyphenylalanine and 3, 4-dihydroxyphenylacetic acid (DOPAC), and the ratio of DOPAC/dopamine in the median eminence as indices), and blunted the afternoon PRL surge. A single injection of a 5-HT(2A) receptor antagonist, ketanserin (5 mg/kg, i.p. at 12.00 h), also had the same effects on the diurnal changes in TIDA neuronal activity and PRL secretion as the treatment with 5,7-DHT did. Intracerebroventricular injection of a 5-HT(2) receptor agonist 2, 5-dimethoxy-4-iodoamphetamine (DOI) in the morning inhibited the TIDA neuronal activity and stimulated PRL secretion in a dose-dependent manner; while injection of a 5-HT(1) agonist, 8-hydroxy-dipropylaminotetralin, was without effect. Injection of DOI in 5,7-DHT-pretreated rats at 14.30 h also lowered the TIDA neuronal activity and reinstated the PRL surge. In all, endogenous 5-HT, acting through the 5-HT(2A) receptor, appears to exhibit an inhibitory effect on TIDA neuronal activity during the afternoon, which is essential for the PRL surge.     

Role of central serotonin systems in the stimulatory effects of ovarian hormones and naloxone on luteinizing hormone release in female rats

The present experiments tested whether serotonergic neurons that innervate discrete areas of the hypothalamus are involved in the stimulation of LH release by ovarian hormones or the opiate antagonist naloxone in female rats. Ovariectomized rats received oil vehicle, estradiol benzoate (EB) alone, or EB followed by progesterone (P) 2 days later. Serotonin (5-HT) activity was assessed from the accumulation observed after the administration of the monoamine oxidase inhibitor pargyline. Two days after EB treatment, LH concentrations were reduced in the morning and rose by later afternoon. Administration of P to EB-primed rats stimulated a LH surge. This latter treatment also enhanced pargyline-induced 5-HT accumulation, suggesting increased 5-HT activity, in the medial preoptic nucleus and interstitial nucleus of the stria terminalis, but reduced 5-HT accumulation, suggesting decreased 5-HT activity, in the ventromedial nucleus. 5-HT activity was unaffected after EB alone, either in the morning or afternoon, or by the administration of naloxone to EB-primed rats. Specific depletion of 5-HT in the medial preoptic/stria terminalis area, achieved by microinjection of the neurotoxic indoleamine 5,7-dihydroxytryptamine resulted in a blockade of the LH surge induced by EB plus P. The present results suggest that central 5-HT neurons innervating the preoptic area are involved in the LH surge induced by progesterone, but not in the increases in LH occurring after treatment with estradiol alone or after blockade of opiate receptors.     

Partial characterization of a neurotransmitter pathway regulating the in vivo release of prolactin.

Nicotinic cholinergic, opiate and serotonergic agonists as well as dopaminergic antagonists induce the release of pituitary prolactin. The purposes of the present studies were to determine if nicotine, morphine and the serotonin1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) utilize a common synaptic pathway to release prolactin and, if so, to establish the serial order of the receptors involved. We also sought to determine whether the pathway under investigation leads to the secretion of prolactin via a mechanism involving dopamine, the prolactin inhibitory factor. Male rats with indwelling jugular catheters were pretreated with saline, mecamylamine, naltrexone, methysergide or bromocriptine. In the saline-treated animals, administration of nicotine, morphine, 8-OH-DPAT and haloperidol resulted in significant increases in plasma prolactin levels. Mecamylamine pretreatment prevented the prolactin response to nicotine only. Naltrexone blocked the stimulation of prolactin release by morphine and by nicotine. Methysergide inhibited the effects of 8-OH-DPAT, morphine and nicotine but not haloperidol. Bromocriptine blocked the prolactin secretion induced by haloperidol as well as by each of the above agonists. Also, in dual-immunocytochemically stained sections, tyrosine hydroxylase-immunoreactive cells and serotonin-immunoreactive processes were detected in close anatomical proximity in the dorsomedial arcuate nucleus. These data indicate that nicotine, morphine and 8-OH-DPAT act to release prolactin via a common synaptic pathway expressing nicotinic cholinergic, opiate, and 5-HT1A receptors at synapses arranged serially in that functional order. Furthermore, the data indicate that the in vivo secretion of prolactin via this pathway may ultimately occur through the inhibition of dopamine release.     

In vitro effect of leptin on LH release by anterior pituitary glands from female rats at the time of spontaneous and steroid-induced LH surge.

OBJECTIVE: The purpose of this work was to study the direct effect of leptin on LH release by anterior pituitary glands from female rats at the time of spontaneous and steroid-induced LH surge. METHODS: LH responsiveness to leptin by pituitaries from rats killed in the afternoon (1500 h) at different stages of the 4-day estrous cycle (diestrus-1: D1; diestrus-2: D2; proestrus; estrus), ovariectomized (OVX; 15 days post-castration) and ovariectomized steroid-primed (OVX-E(2)/Pg; pretreated with 5 microg estradiol and 1 mg progesterone), was evaluated in vitro. Hemi-adenohypophyses were incubated in the presence of synthetic murine leptin for 3 h. RESULTS: Addition of increasing concentrations of leptin (0.1-100 nmol/l) to the incubation medium of proestrus pituitaries produced a dose-related stimulation of LH release; the maximal increase to 315% of control was obtained with 10 nmol/l leptin. Leptin (10 nmol/l) enhanced LH release at all days of the estrous cycle, the greatest response occurring in proestrus (318%) and the lowest at D1 (123%). In order to evaluate the role of nitric oxide (NO) in the action of leptin on LH release, glands from proestrus rats were incubated in the presence of 10 nmol/l leptin with or without 0.3 mmol/l N(G)-monomethyl-l-arginine (NMMA), a competitive inhibitor of NO synthase (NOS). NMMA completely suppressed the stimulation of LH release induced by leptin. Leptin also stimulated LH release by pituitaries from OVX rats, and treatment with steroid hormones led to a marked increase in the response (OVX: 162% compared with OVX-E(2)/Pg: 263%; P<0.05). For comparative analysis, a similar experimental procedure was carried out using GnRH (10 nmol/l). Leptin acts at the pituitary level in a similar manner as GnRH, although with significantly lower potency. CONCLUSIONS: These results confirm and extend previous reports regarding a direct action of leptin at the pituitary level, stimulating LH release by anterior pituitaries from female rats at the time of spontaneous and steroid-induced LH surge. In the female rat pituitary this leptin action is controlled by gonadal steroids and mediated by NO.     

Acute activation of the adrenocorticotropin-adrenal axis: effect on gonadotropin and prolactin secretion in the female rat.

The purpose of this study was to examine the effect of a single injection of ACTH-(1-39) on gonadotropin secretion in the estrogen-primed intact and ovariectomized female rat and to determine if the effect of ACTH was mediated through the release of adrenal steroids. Administration of 100 micrograms ACTH-(1-39/rat caused a significant elevation in serum LH and FSH levels 6 h after injection. This stimulatory action of ACTH was abolished by adrenalectomy in both intact and ovariectomized rats, suggesting that the effect was mediated through the adrenal. A requirement for estrogen priming was demonstrated by a lack of ACTH effect in nonestrogen-primed intact rats. The specificity of ACTH-(1-39) was demonstrated by the absence of effect on FSH and LH secretion by alpha MSH and ACTH-(4-10). Fifteen and 30 min after ACTH stimulation, the adrenal secreted significantly elevated amounts of both progesterone and corticosterone. RU486, which has both antiglucocorticoid and antiprogestin activity, effectively blocked the action of ACTH on gonadotropin secretion. Of the two steroids secreted by the adrenal after ACTH administration, progesterone, but not corticosterone, was able to induce a gonadotropin surge. Since estrogen priming is important for progesterone receptor development, the stimulatory action of ACTH appears to be mediated through progesterone acting through the progesterone receptor. These studies demonstrate that ACTH treatment in the estrogen-primed rat can stimulate LH and FSH secretion through adrenal progesterone secretion.     

Central orexin A has site-specific effects on luteinizing hormone release in female rats

Orexin A stimulates GnRH release from hypothalamic explants in vitro. The sites of action of orexin A in the regulation of LH release have been investigated in vivo in ovariectomized rats that were given vehicle or estradiol benzoate (EB), with or without an injection of progesterone 48 h later. Orexin A was administered intrahypothalamically under Saffan anesthesia, 50 h after the EB or vehicle; its effects on plasma LH levels were monitored in sequential blood samples. Orexin A (1.0 microg/side) injected into the rostral preoptic area (rPOA) at the level of the organum vasculosum of the lamina terminalis had a stimulatory effect on LH release in EB-treated ovariectomized rats. When orexin A was injected into the medial POA (mPOA) or the arcuate/median eminence, it had an inhibitory effect on the LH surge that occurs in ovariectomized rats primed with EB plus progesterone. Orexin A injected into the mPOA also reduced LH levels in ovariectomized rats untreated with ovarian steroids. Both the stimulatory and inhibitory effects of orexin A were antagonized by SB334867A, a selective orexin 1 receptor antagonist. Furthermore, when given alone into the rPOA, this antagonist attenuated the LH surge induced by EB plus progesterone. Thus, orexin appears to have a dual effect on LH release, being stimulatory in the rPOA and inhibitory in the mPOA or arcuate/median eminence. Both effects may be mediated, at least in part, by the orexin 1 receptor. Double label immunohistochemistry revealed close appositions between orexin A immunoreactive varicosities and a small proportion of GnRH cell bodies in the rPOA. It is suggested that the stimulatory effect of orexin A on LH release may involve direct actions on GnRH neurons.     

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.     

Evidence that norepinephrine and epinephrine systems mediate the stimulatory effects of ovarian hormones on luteinizing hormone and luteinizing hormone-releasing hormone

Results from previous investigations have suggested an important role for central epinephrine (EPI) systems in mediating the stimulatory effects of ovarian hormones on LH release in ovariectomized female rats. The purpose of these experiments was 1) to test whether selective inhibition of EPI synthesis blocks the sequential accumulation and decline of LHRH concentrations in the median eminence that precedes the ovarian hormone-induced LH surge and 2) to test whether the stimulatory ovarian hormone regimen enhances the activity of EPI systems in the hypothalamus. Ovariectomized rats were treated with estradiol, followed 2 days later by progesterone. Animals were treated before progesterone administration with saline, one of the EPI synthesis inhibitors [SK&F 64139 (2,3-dichloro-tetrahydroisoquinoline HCl) or LY 78335 (dichloro-alpha-methylbenzylamine)], or the dopamine-beta-hydroxylase inhibitor FLA-63 (bis-4-methyl-1-homopiperazinyl thiocarbonyl disulfide), which inhibits NE and EPI synthesis. The catecholamine synthesis inhibitors blocked or delayed the afternoon LH surge. FLA-63 completely prevented the accumulation of LHRH in the median eminence that preceded the rise in LH release. However, selective EPI synthesis inhibition with SK&F 64139 only partially prevented this increase in LHRH. A second EPI synthesis inhibitor, LY 78335, delayed both the LH surge and the rise in LHRH. In a second experiment, the administration of estradiol and progesterone to ovariectomized rats increased the alpha-methyltyrosine-induced depletion of hypothalamic EPI, suggesting increased activity in this system during the LH surge. Further experiments localized this effect to the medial basal hypothalamus. The depletion of both NE and EPI after synthesis inhibition was also enhanced during an earlier period, approximating the time of LHRH accumulation. These results suggest that the ovarian hormones activate both NE and EPI systems to stimulate the early afternoon rise of LHRH in the median eminence and to induce the subsequent LH surge.     

Dorsal raphe lesion alters the estrous cycle and the preovulatory gonadotropin release

The purpose of this study was to examine the role that the dorsal raphe (DR)-median eminence (ME) serotonergic projection may have in the proestrous gonadotropin and prolactin (PRL) release. DR electrolytic lesions were performed in cycling rats during the first day of diestrus. The effect of DR lesions after 48-72 h of survival (short-term lesioned animals) or after 35-40 days of survival (long-term lesioned animals) on estrous cyclicity, preovulatory gonadotropin and PRL releasing pattern, ovulation and serotonin (5-HT) content of the ME were studied. Following DR lesions the estrous cycle became irregular, remaining in the diestrus phase for several days. Preovulatory gonadotropin release in short-term lesioned animals was increased; on the contrary, in long-term lesioned rats a delay in the surge of these two hormones and a decrease in LH secretion were detected. Long-term lesioned animals also showed a diminished secretion of PRL. The number of ova did not differ between control and lesioned animals. DR lesions in both short- and long-term lesioned rats reduced 5-HT levels in the ME by about 50% and nullified the normal 5-HT decline during the afternoon of proestrus. Our results suggest that the DR exerts a stimulatory influence on the preovulatory gonadotropin release by means of its 5-HT projection to the ME. As the pattern of hormonal secretion in lesioned animals remains similar to that of controls, it may be suggested that this pathway acts as a fine modulator of the mechanisms involved in the regulation of LH and FSH release in cycling rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of 5-HT1A receptor subtype in the paraventricular nuclei of the hypothalamus induces CRH and ACTH release in the rat.

Previous studies have shown that activation of the 5-HT1A receptor subtype enhances rat plasma ACTH concentration. Such receptors have been suggested to be located on CRH neuronal cell bodies in the paraventricular nuclei of the hypothalamus (PVN). In this report, microinjection of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a selective 5-HT1A agonist, into the PVN increased rat plasma ACTH concentration in a dose-related manner. Similar responses were observed when two other 5-HT1A agonists, busipirone and gepirone, were used. (+/-)-Pindolol, known to have 5-HT1A antagonist properties, blocked the effect induced by an optimal dose of 8-OH-DPAT after injection into the PVN. This same dose of 8-OH-DPAT also induced a decrease of hypothalamic CRH concentration, which was completely antagonized as well by pretreatment injection of (+/-)-pindolol into the PVN. A significant inverse correlation was found between hypothalamic CRH and plasma ACTH levels. These results confirm that elevation of the plasma ACTH concentration induced by 5-HT1A receptor subtype activation is mediated by the release of CRH from the paraventricular nuclei of the hypothalamus in rats, but do not exclude other mechanisms.     

Evidence that stimulation of two modalities of pituitary luteinizing hormone release in ovarian steroid-primed ovariectomized rats may involve neuropeptide Y Y1 and Y4 receptors.

A large body of evidence indicates that neuropeptide Y (NPY) is involved in stimulation of basal and cyclic release of hypothalamic LHRH and pituitary LH. To identify the NPY receptor subtypes that mediate the excitatory effects of NPY in these two modalities of LH release, we studied the effects of 1229U91, a selective Y1 receptor antagonist and Y4 receptor agonist, in two experimental paradigms that reproduce the two modalities of LH secretion in steroid-primed ovariectomized (OVX) rats. Rats were ovariectomized and implanted with a permanent cannula into the lateral cerebroventricle. In the first experiment, rats received estradiol benzoate (EB, 30 microg/rat) on day 5, followed 2 days later with progesterone (2 mg/rat) at 1000 h to induce an afternoon LH surge. 1229U91 (30 microg/3 microl) or vehicle (control) was injected intracerebroventricularly into these rats either once at 1300 h or twice (15 microg/injection) at 1100 and 1200 h. Blood samples were collected before progesterone injection at 1000 h and at hourly intervals from 1300 -1800 h via an intrajugular cannula implanted on the previous day. In control rats, serum LH levels rose significantly at 1400 h, and these high levels were maintained until 1700 h. After two injections of 1229U91, LH levels displayed a tendency to rise at 1300-1400 h, as in controls, but thereafter, decreased rapidly below the control range. In the second experiment, the acute effect of 1229U91 on LH release was evaluated in OVX rats pretreated with EB alone. Saline alone or saline containing 1, 3, 10, or 30 microg 1229U91 was injected intracerebroventricularly at 1000 h, and the effects on LH release were analyzed at 10, 20, 30, and 60 min. 1229U91 elicited a dose-dependent stimulation of LH release, with maximal response (950% of basal levels) occurring at 10 min after the 30-microg dose; elevated levels were maintained for 1 h. Because 1229U91 is a potent Y4 agonist with some affinity for Y5 receptors, these results raised the possibility that activation of Y4/Y5 receptors by 1229U91 may augment LH release. Therefore, we examined the effects of icv administration of rat pancreatic polypeptide, a Y4-selective agonist, and [D-Trp32]-NPY, a Y5 agonist on LH release in EB-primed rats. Rat pancreatic polypeptide (0.5-2 microg/rat) stimulated LH release in a dose-related manner, and peak levels (280% of basal levels) were seen at 10-20 min; the response evoked by a higher dose (10 microg) was smaller than that induced by 0.5 or 2 microg. [D-Trp32]-NPY was relatively less effective, because only the highest (10-microg) dose elicited a modest stimulation (244% of basal levels). These results demonstrate that 1229U91, a Y1 antagonist and Y4 agonist, evokes two types of responses; it suppresses the protracted ovarian steroid-induced LH surge, and acutely, it also stimulates LH. These results imply that normally two different types of NPY receptors may mediate the stimulation of LH release. Because 1229U91 is a Y1 receptor antagonist, inhibition of the steroid-induced LH surge by 1229U91 suggests that Y1 receptors may mediate the cyclic release of LH. On the other hand, acute stimulation of LH by 1229U91 implies that the Y4 agonist-like activity of 1229U91 may mediate the basal release of LH and that either NPY or a yet-to-be-identified endogenous Y4 receptor agonist may activate Y4 receptors in the hypothalamus to stimulate LH release.     

Serotonin agonists cause parallel activation of the sympathoadrenomedullary system and the hypothalamo-pituitary-adrenocortical axis in conscious rats

The effects of three potent serotonin (5-HT) agonists with different structures and 5-HT receptor binding profiles on sympathoadrenomedullary and hypothalamo-pituitary-adrenocortical axis functions were assessed in conscious, freely moving male Sprague-Dawley rats. The 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5-HT1C agonist, m-chlorophenylpiperazine (m-CPP), and the 5-HT2/5-HT1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)2-amino-propane (DOI), all produced marked dose-dependent increases in plasma epinephrine and ACTH concentrations. Both epinephrine and ACTH responses peaked at 10 min and showed strong positive correlations across all drugs and doses studied. Corticosterone increases showed a saturable response pattern and were close to the maximum level with a relatively small (approximately 2.5-fold) increase in plasma ACTH concentrations. Norepinephrine levels showed small dose-dependent increases after 8-OH-DPAT and m-CPP and decreases after DOI treatment. These results suggest that both the sympathoadrenomedullary system and the hypothalamo-pituitary-adrenocortical axis can be activated via 5-HT1 and 5-HT2 receptors and that these two systems may have common or similar regulatory mechanisms triggered by these stimuli.     

Role of endogenous opioid peptides in mediating progesterone-induced disruption of the activation and transmission stages of the GnRH surge induction process.

How progesterone blocks the E2-induced GnRH surge in females is not known. In this study we assessed whether the endogenous opioid peptides (EOPs) that mediate progesterone negative feedback on pulsatile GnRH secretion also mediate the blockade of the GnRH surge. We treated ovariectomized ewes with physiological levels of E2 and progesterone to stimulate and block the GnRH surge, respectively, using LH secretion as an index of GnRH release. A pilot study confirmed that blocking opioidergic neurotransmission with the opioid receptor antagonist, naloxone (NAL; 1 mg/kg.h, i.v.), could prevent the suppression of pulsatile LH secretion by progesterone in our model. By contrast, antagonizing EOP receptors with NAL did not restore LH surges in ewes in which the E2-induced GnRH surge was blocked by progesterone treatment during the E2-dependent activation stage (Exp 1) of the GnRH surge induction process. However, in ewes treated with progesterone during the E2-independent transmission stage (Exp 2), NAL partially restored blocked LH surges, as indicated by increased fluctuations in LH that, in some cases, resembled LH surges. We conclude, therefore, that the EOPs that mediate progesterone negative feedback on pulsatile GnRH secretion are not involved in blockade of activation of the E2-induced GnRH surge by progesterone, but do appear to be part of the mechanism by which progesterone disrupts the transmission stage.     

Opioidergic modulation of the oestradiol-induced LH surge in the rat: roles of ovarian steroids

Sex steroids convey information on the status of the reproductive system, which the brain is able to integrate to promote ovulation, in the form of the LH surge. The present studies examined the influence of alterations in central opioidergic tone to initiate the LH surge, and the roles of oestradiol and progesterone to effect changes in opioidergic tone, by antagonizing this activity using either naloxone or nalmefene (N-cyclopropylmethyl-6-desoxy-6-methylene-noroxy-morphone), a long-acting mu- and kappa-opiate antagonist. The timing and amplitude of the LH surge was examined in (1) cyclic rats in pro-oestrus and (2) ovariectomized rats with varying doses of oestradiol supplementation. Plasma was obtained hourly through an indwelling intra-atrial catheter between 13.00 and 19.00 h, and later assayed for LH and oestradiol concentrations by radioimmunoassay. Rats treated with either nalmefene or progesterone on pro-oestrus demonstrated similar advances in the time of initiation of the LH surge by 1-2 h compared with control rats. The effects of nalmefene and progesterone were evident within 2 and 3-5 h of their administration respectively. Conversely, rats treated with progesterone on dioestrus demonstrated low pro-oestrous oestradiol levels and abolition of the pro-oestrous LH surge, but continuous naloxone infusion restored the pro-oestrous LH surge, with raised oestradiol concentrations. In ovariectomized rats without oestradiol supplentation, nalmefene alone was able to increase basal LH levels, but unable to facilitate a spontaneous rise in LH amplitude indicative of an LH surge. Supplementation with low doses of oestradiol was itself ineffective in facilitating a spontaneous rise in LH concentration, but nalmefene co-administration significantly potentiated the ability of low doses of oestradiol to induce augmented LH secretion, in addition to advancing the timing of the spontaneous LH rise. Similarly, progesterone co-administration to ovariectomized, oestradiol-primed rats significantly advanced and augmented LH hypersecretion. The results of these experiments are consistent with the concept that central opioidergic systems normally restrain the initiation of the LH surge, and that blocking opiate receptors removes this inhibition. They advance the hypothesis that oestradiol, the essential signal for LH surge induction, has, as one consequence of its action, the time-specific inhibition of hypothalamic opiodergic tone in the afternoon, which would otherwise restrain the LH surge-generating mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)