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Increasing evidence suggests that interleukin-1beta (IL-1beta) regulates luteinizing hormone (LH) release primarily through modulation of the gonadotropin-releasing hormone (GnRH) neuronal activity. This study was undertaken to elucidate the effect of IL-1beta on GnRH as well as GnRH receptor (GnRHR) gene expression in the preoptic area. IL-1beta (100 ng/rat) or saline was administered into the lateral ventricle of castrated rats. RNA samples were isolated from micropunches of the preoptic area and mediobasal hypothalamus from individual brain slices and GnRH mRNA levels in the preoptic area and GnRHR mRNA levels in the mediobasal hypothalamus were determined by competitive reverse transcription-polymerase chain reaction (RT-PCR) protocols. Serum LH concentrations were decreased from 1 h to 3 h after IL-1beta treatment, but rebounded at 5 h, while serum concentrations of follicle-stimulating hormone (FSH) and prolactin were not altered. There were no significant changes in GnRH mRNA levels from the micropunched preoptic area, while GnRHR mRNA levels from the preoptic area and mediobasal hypothalamus micropunch samples, but not in the anterior pituitary, showed a pattern similar to the serum LH profile following i.c.v. administration of IL-1beta. We then examined the effect of IL-1beta on the translational efficiency of the GnRH mRNA. After the separation and fractionation of polyribosome-associated cytoplasmic RNA from the hypothalamic fragments containing the preoptic area-anterior hypothalamic area of control (saline-treated) and IL-1beta-treated group 3 h after administration, GnRH transcript levels were examined from the each fraction. IL-1beta decreased the translational efficiency of the transcribed GnRH mRNA. These results clearly demonstrate that central administration of IL-1beta suppresses the translational activity of GnRH mRNA. Moreover, GnRHR may play an important role in the modulation of GnRH neuronal activity through GnRHR-expressing neurones (or glia) in the hypothalamus.

There is increasing evidence that in the rat prior to and during the preovulatory LH surge, release rates of GABA in the preoptic area (POA) are decreased while no such changes occurred in the mediobasal hypothalamus (MBH). In addition, GnRH release appears to be facilitated by an increased preoptic excitation of glutamate (GLU). To investigate whether such changes of secretory activity of intrahypothalamic GABA or GLU neurons are associated with altered gene expression of biosynthetic enzymes or transporter proteins characteristic for either neuronal system, we determined mRNA levels of the two forms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD65 and GAD67), the glutamate-synthesizing enzyme glutaminase (GLS), the GABA transporter type 1 (GAT-1) and the glutamate-aspartate transporter type 1 (GLAST). Competitive RT-PCRs using mutant cRNAs as internal standards were conducted with mRNA extracted from microdissected tissue of POA and MBH from diestrous, proestrous, and estrous rats. Proestrous animals were subgrouped according to their endocrine status as follows: 'prior to', on the 'ascending' or on the 'descending' limb of the LH peak, and 'after the LH surge (post)'. During the preovulatory LH surge, mRNA concentrations of GAD67 and GAT-1 in the POA were significantly increased compared to those observed on diestrous (2.8-fold for GAD67 and 2.5-fold for GAT-1, p < 0.01), while in the MBH the amount of both mRNAs remained constant. The expression levels of GAD65, GLS and GLAST were without any changes in the POA as well as in the MBH. These findings support the hypothesis that in rats induction of the preovulatory LH surge is controlled at the level of GnRH perikarya, and suggest that altered activities of intrapreoptic GABA neurons at both transcriptional and secretory levels are pivotal for the preovulatory activation of GnRH neurons., (Copyright 2000 S. Karger AG, Basel.)

Alterations in the immune system are often accompanied by reproductive disorders. The bacterial endotoxin lipopolysaccharide (LPS) has central inflammatory effects, and activates the release of cytokines (immune system mediatory factors) in the hypothalamus, where the LHRH neurons are located. Therefore, these studies were designed to investigate a possible effect of the LPS and LHRH secretion and the possible interaction with excitatory and inhibitory amino acids. Animals were decapitated at 09.00 h, and the preoptic mediobasal hypothalamic area (PO/MBH) dissected and superfused. Superfusate fractions were collected at 15-min intervals. After a stabilization superfusion period of 60 min four fractions were collected; LPS (100 ng/ml) was then added to the superfusion medium for the duration of 1 h. This was followed by a washout period of 60 min. The PO/MBH fragments were then subjected to a 56 mM K+ stimulus. Control PO/MBH fragments were continuously superfused with Earle's balanced salt solution. LHRH release was significantly (p < 0.05) reduced during and following exposure to LPS. At this time GABA and taurine concentrations increased in the superfusion medium, while glutamate decreased significantly compared with the control group. These observations indicate that LPS inhibits LHRH and glutamate release, but stimulates taurine and GABA secretion. These effects may be explained by the stimulation of cytokines of neuronal and/or glial source which may interact with the excitatory and inhibitory amino acids.

Inhibitory and excitatory amino acid neurotransmitters have been suggested to participate in the feedback actions of estradiol (E2) on LH secretion. In the rat estrogen-receptive neurons have been demonstrated in the preoptic/anterior hypothalamic area (POA) and mediobasal hypothalamus/median eminence (MBH) and many of these neurons utilize gamma-aminobutyric acid (GABA) as neurotransmitter. The actions of excitatory amino acids (EAA) differ in ovariectomized (ovx) and ovx E2-substituted rats indicating that EAAs also participate in the positive feedback action of E2 on LH release. However, little information is available as to whether in vivo these transmitters exert their effects in the POA, where most of the GnRH perikarya are located, or in the MBH, i.e. at the nerve terminals. Therefore we conducted push pull cannula perfusions to compare the release rates of GABA, aspartate (ASP) and glutamate (GLU) in the MBH and POA. A subcutaneous implant of a silastic tube containing E2 resulted in LH surges in the afternoon of all treated animals. Prior to and during this LH surge the MBH release rates of neither GABA nor ASP nor GLU were significantly altered. In contrast, a conspicuous drop in preoptic GABA release occurred prior to and during the time of estrogen-induced LH surges and this was accompanied by enhanced preoptic secretion of ASP and GLU. In conclusion, we present the first data about amino acid release in the MBH during the E2-induced LH surge. Since only in the POA the LH surge is associated with changes in amino acid release, it appears that both inhibitory and excitatory amino acids act at the level of the GnRH cell bodies and/or dendrites and not on GnRH nerve terminals to mediate the feedback mechanism of E2 on LH release.

gamma-Aminobutyric acid (GABA) exerts an inhibitory action on gonadotropin-releasing hormone (GnRH) release from the hypothalamus. In vivo, this inhibitory action appears to be mediated via the GABAA receptor since in ovariectomized (ovx) rats and sheep direct application of muscimol (MUS), a GABAA agonist, into the preoptic area (POA), the site were the GnRH cell bodies are located, caused an immediate reduction of LH release. This effect may be the result of an inhibition of GnRH release but also GnRH biosynthesis may be affected. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) we now addressed the question, whether an acute inhibition of the GnRH pulse generator in ovx rats by GABA involves reduction of GnRH biosynthesis as determined by GnRH mRNA levels in micropunches of the POA. To activate either the GABAA or GABAB receptor, we injected intraventricularly (icv) MUS or baclofen (BAC). Intracerebroventricular injection of 10 nmol MUS caused a rapid and lasting inhibition of LH release from about 7.5 ng/ml (pretreatment value) to approximately 1.5 ng/ml. Neither application of BAC or saline (control injections) affected LH secretion. Two hours after icv injections, rats were decapitated and GnRH mRNA levels were determined. MUS induced a pronounced decrease of GnRH levels in the POA (control rats: 2.26 pg GnRH mRNA; MUS-treated rats: 0.85 pg, n = 10/group). BAC was without any effect on GnRH mRNA levels. Thus, we confirm the inhibitory action of GABA on LH release in vivo which is exerted via the A-subtype of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

In rats, beta-endorphin (beta-END) and gamma-aminobutyric acid (GABA) suppress LH secretion by hypothalamic mechanisms involving the preoptic area (POA). Systemic injection of naloxone (NAL) increases LH secretion in male rats, an effect which can be prevented by coadministration of GABA agonists. Application of NAL into the POA of ovariectomized (ovx), progesterone substituted sheep modulates preoptic GABA release. These findings have been interpreted such that the endogenous opioids act via the preoptic GABAergic system to regulate LH release. To evaluate this hypothesis we implanted ovx rats with push pull cannula into the POA and measured GABA secretion prior to and during the preoptic application of either NAL or beta-END. Blood samples were collected to assess the effects of the drugs on LH secretion. In addition, ovx rats were substituted with estradiol (E2) to induce a negative feedback effect on LH release. Intrapreoptic application of beta-END caused a rapid decline of LH release in ovx rats which was completely reversible after termination of beta-END perfusion. Though LH levels were clearly suppressed, no change of GABA release in the POA was observed. During preoptic NAL perfusion both LH secretion and GABA release remained unaffected. Likewise, during beta-END perfusion into the POA of E2 treated rats neither LH nor GABA secretion changed. In contrast, NAL perfusion rapidly increased LH release but again this action of the opioidergic drug was not accompanied by alterations of GABA release. We conclude from these data: 1) Intrapreoptically applied beta-END inhibits LH release only in the absence of steroids. In turn, blockade of opioid receptors is effective only in the presence of steroids. Both findings indicate that in the POA opioidergic activity is low in ovx rats, but high during negative feedback of E2. 2) No changes of GABA secretion were observed during manipulations of the opioidergic tonus in the POA suggesting that both beta-END and GABA do not interact to regulate LH release. Thus, beta-END may directly inhibit the activity of GnRH neurons located in the POA or acts via a neurotransmitter other than GABA.

Though the pivotal importance of pulsatile gonadotrophin releasing hormone (GnRH) release from the hypothalamus for sufficient luteinizing hormone (LH) secretion is well recognized, the mechanisms causing GnRH pulses are still largely unknown. In the rat, the GnRH neurons are concentrated in the preoptic area, which is a bilateral structure of the anterior hypothalamus. This distribution into two distinct brain areas raises the question of whether the GnRH neurons in both preoptic areas are simultaneously or alternatively active and whether an interaction between both areas is required to generate GnRH pulses. We addressed these questions by unilateral manipulations of the activity of GnRH neurons using either electrical lesion or local infusion of gamma-aminobutyric acid (GABA), a neurotransmitter known to inhibit LH release via a hypothalamic action. Acute lesion of one preoptic area caused complete cessation of pulsatile LH release which was restored after 48 h. An acute inhibition of LH release was also observed after unilateral preoptic application of GABA. It is concluded that an interaction of both preoptic areas is mandatory for GnRH pulse generation. However, we suggest that the pacemaker function is not intrinsic to GnRH nor GABA neurons but the cross-talk between these neurons within and between both preoptic areas results in episodic GnRH release.

A competitive polymerase chain reaction (PCR) for quantitating gonadotropin-releasing hormone (GnRH) mRNA level in a single micropunch of the rat preoptic area (POA) is described. The POA (600 microns in depth) was micropunched from frozen rat brain slices and used for mRNA isolation using Dynabeads-oligo(dT) magnetic separation technique. The target RNA combined with a synthetic, deletion mutant GnRH cRNA as an internal standard, is co-reverse transcribed, and their cDNAs are subsequently co-amplified by Taq DNA polymerase in the same tube in which the same GnRH primers are used. This PCR protocol is sensitive enough to detect GnRH mRNA level in a single POA micropunch derived from an individual rat. There is a linear increase of the amount of GnRH PCR products as a function of input RNA and of the number of PCR cycles. Addition of mutant GnRH cRNA as an internal standard allows us to quantitate GnRH mRNA level in biological samples and to compensate variations of PCR reaction between samples. Following preoptic treatment with 5'-ADMP, which depletes selectively norepinephrine (NE), GnRH mRNA level was significantly reduced. This simple, yet highly sensitive PCR method appears to be a valuable tool for the study of the cellular and molecular regulation of GnRH gene expression in a variety of experimental models.

In order to evaluate the participation of amino acid neurotransmitters (AANT) in pubertal development, we measured the preoptic release rates of glutamate (Glu), aspartate (Asp), homocysteic acid (HCA), gamma-aminobutyric acid (GABA), glycine (Gly), and taurine (Tau) in female rats aged 26, 35 and 42 days. Push-pull cannulae were implanted in the preoptic area (POA) after reinforcing the os calvarium by cementing a slightly curved 2-mm thick polyethylene plaque. Four days later, 120-min perfusion experiments (0-90 min: artificial CSF; 90-120 min: CSF containing 50 mM KCl) were performed. Amino acid concentrations were measured in 15-min fractions after derivatization with phenylisothiocyanate, using reverse-phase high-performance liquid chromatography and UV detection. Rats aged 42 days showed significantly (p < 0.01) higher preoptic Asp release rates, as compared to 26-day-old rats; Glu output increased significantly (p < 0.05) in animals aged 35 and 42 days, as compared to 26-day-old rats. No significant changes in HCA release were observed. Preoptic release rates of GABA, Gly and Tau were significantly higher in 26-day-old animals as compared to 35-day-old (GABA, p < 0.05) and 35- and 42-day-old rats (Gly, Tau, p < 0.01). Basal release of Asp, Glu, HCA, Tau and GABA, but not of Gly, was significantly stimulated (p < 0.05) by KCl in all age groups. It is suggested that in peripubertal female rats, increased preoptic Asp and Glu release rates and decreased GABA and Tau output might enhance luteinizing hormone-releasing hormone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The preoptic/anterior hypothalamic area (PO/AH) contains the majority of LHRH neurons of which the function is regulated by a variety of neurotransmitters and peptides. In this area, numerous estrogen-receptive neurons utilize gammaaminobutyric acid (GABA) as neurotransmitter and these neurons communicate directly with LHRH neurons. Angiotensin II (AII) and atrial natriuretic peptide (ANP) are known to be involved in the regulation of LH secretion. The site of action of these peptides and the mechanisms by which they influence LHRH neurons, are largely unknown. Therefore the effects of intrapreoptic application of AII and ANP on serum LH levels of ovariectomized (ovx) and of ovx estrogen-primed rats were investigated. The peptides were applied into the PO/AH by means of push-pull cannula and in the effluent fractions GABA was measured. In the ovx estrogen-primed rat, prominent LH and prolactin surges were observed. At the time of increased LH levels preoptic GABA release was significantly reduced. At this time application of AII or ANP into the PO/AH was without effect on either LH or prolactin levels in the serum or on preoptic GABA release rates. In ovx, not steroid-primed rats intrapreoptic AII application suppressed serum LH levels significantly and this treatment had a slight stimulatory effect on preoptic GABA release rates. This effect of AII could be antagonized by prior preoptic treatment with saralasin, a specific AII receptor blocking peptide. Preoptic treatment with ANP resulted in a slight increase in serum LH levels which was accompanied by a slight, but significant reduction of preoptic GABA release rates.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the role of amino acid neurotransmitters in the regulation of LH secretion in ovariectomized (ovx) rats with or without estrogen substitution, we measured the release rates of gamma-aminobutyric acid (GABA), taurine, glycine, aspartate, glutamate, homocysteic acid, and also of the neurally inactive amino acids serine and glutamine in push-pull perfusate samples of the preoptic/anterior hypothalamic area (PO/AH) collected at 30-min intervals. To achieve this we had to develop a highly sensitive assay utilizing phenylisothiocyanate prederivatization which was followed by HPLC chromatography. In confirmation of our earlier results we observed again a conspicuous drop of preoptic GABA release prior to and during the time of estrogen-induced LH surge. In addition, the release rates of the excitatory amino acid neurotransmitters aspartate and glutamate in the PO/AH increased during this time. Interestingly, also secretion of taurine and glycine was increased during the LH surge, whereas preoptic release rates of serine and glutamine and of homocysteic acid, the putative endogenous ligand of the so-called N-methyl-D-aspartate receptor, remained unchanged. No such changes of amino acid neurotransmitters release rates were observed in ovx rats. This finding underlines that the changes of amino acid secretion in ovx estrogen-primed rats are likely due to the influence of the steroid rather than due to a diurnal rhythm. We conclude that GnRH neurons are under a tonic inhibitory tone exerted by GABA which is relieved during the time of the estrogen-induced LH surge. During this time, aspartate and glutamate may have additional stimulatory effects on GnRH neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine (NE) in the preoptic/anterior hypothalamic area (PO/AH) is known to be involved in the regulation of luteinizing hormone (LH) secretion. The effects of selective and complete depletion of NE in the PO/AH of ovariectomized (ovx) rats on LH secretion were studied. PO/AH concentrations of NE were reduced by 90% within 6 h and were undetectable (more than 98% depletion) 52 h after bilateral stereotaxic microinjections of 50 micrograms of 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine (5-ADMP). LH levels in the blood were significantly reduced within 60 min after NE depletion but remained low only for several hours. Despite continuously low preoptic NE concentrations episodic LH secretion reoccurred within 4-6 h such that normal blood LH levels were present 6 and 52 h after selective NE depletion. While the alpha 1-adrenoreceptor antagonist prazosin was inhibitory to LH secretion in control rats the drug was totally ineffective in the NE depleted animals. NE may be inhibitory to LH secretion via a beta-adrenergic receptor mechanism. It was therefore also tested whether 5-ADMP causes a massive NE release which might be inhibitory to LH secretion. Propranolol (PROP), a beta-adrenoreceptor blocking drug, was given 30 min prior to preoptic injection of 5-ADMP. Blockade of beta-receptors did not prevent the transient inhibition of LH release. These results indicate that under physiologic conditions the GnRH pulse generator functions only properly when NE is present in the PO/AH and that the stimulatory effect of NE is mediated via an alpha 1-adrenoreceptor.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

The push-pull cannula technique was used to examine the endogenous release of GABA from the medial preoptic area (MPO) of unanesthetized rats. In diestrous females the mean resting release of GABA was 27.1 +/- 2.0 pmol/min. GABA release was significantly elevated by increasing the potassium concentration in the perfusion solution to 50 mM, whereas it was dramatically inhibited by mercaptoproprionic acid (1.0 mM), a glutamic acid decarboxylase inhibitor. A comparison between diestrous females and chronically castrated animals indicated that endogenous GABA release in OVX animals was only 60-70% of that in diestrous animals. A model for the presynaptic inhibition of NE by estrogen receptive GABAergic neurons in the MPO is proposed.

The noradrenergic innervation of the medial preoptic area (MPO) and the hypothalamus is provided by mesencephalic neurons via the ventral noradrenergic tract. Fibers of these neurons emerge through the MPO. Bilateral microinjections of 6-OHDA into the ventral noradrenergic bundle (VNB) depletes large parts of the diencephalon of norepinephrine (NE) and dopamine (DA). Since the total hypothalamic DA content is of intrahypothalamic origin, 6-OHDA injection into the VNB does not reduce hypothalamic DA content. Similarly microinjections of 6-OHDA into the MPO reduces hypothalamic and preoptic NE content without altering NE concentrations in other diencephalic structures. Microinjections of 6-OHDA and of the carrier solution of 6-OHDA into the VNB or into the MPO of female rats with regular estrous cycles results in a slight disturbance of the cyclic activity for few days. Within 1--4 days normal cyclic activity is resumed. Preovulatory LH release is substantially reduced 8--12 days after injection of 6-OHDA into the VNB or into the MPO. On the basis of these and previous results it is concluded that the availability of NE in the MPO is an important factor in determining the hight of the preovulatory LH surge.

Single cell activity from preoptic neurons was extracellularly recorded in normal female rats and the effects of electrical stimulation of the mediocortical amygdala (AMY), the N. med. raphes (MES) and the mediobasal hypothalamus (MBH) was tested. 1. One type of preoptic neurons (55%) reacted with primary excitation to a single electrical stimulus of AMY, MES or MBH, which was usually followed by a period of postexcitatory inhibition. Another type of neurons (29%) were first inhibited after stimulation of AMY, MES or MBH and then showed a period of postinhibitory excitation. The discharge rate of a third type was not affected by electrical stimulation of these structures (16%). If a neuron was affected by a stimulus in a given area it predictably reacted in the same fashion to stimulation of the other areas. 2. The postexcitatory inhibition of the majority of the primarily excited neurons was strong enough to prevent the stimulus response to a second stimulus. Occasional recordings from two neighboured cells simultaneously indicate that they can be inhibitory to each other. 3. By pulse train stimulation of the AMY or MES with varying frequencies it could be demonstrated that low frequencies (10 Hz) had a facilitatory action whereas higher frequencies (100 Hz) were inhibitory to preoptic discharge rates. 4. No obvious alteration of neuronal properties we found neither in rats treated intraventricularly with 5,6-dihydroxytryptamine, which strongly reduces central nervous system serotonin content, nor in 6-hydroxydopamine treated rats, which had low central nervous system catecholamine levels.

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.

High serum prolactin levels in ovariectomized rats were induced by transplantation of additional pituitaries under the kidney capsule. Such high prolactin levels reduced serum LH but not FSH levels 3 days after pituitary transplantion. LH and FSH values were at a control levels at day 15 and above these levels at day 24, although prolactin values were still high. Dopamine (DA) and norepinephrine (NE) turnover in the anterior part of the mediobasal hypothalamus (AMBH) and in the medial preoptic area (MPO) was measured by following the decrease in NE or DA content after synthesis blockage with a-methyl-p-tyrosine. The content was measured using a radioenzymatic assay. DA turnover in the AMBH was significantly increased 3, 15 and 24 days after induction of hyperprolactinemia. Although NE concentration in the AMBH was also increased at these times an increased NE turnover was statistically significant only at day 24. DA as well as NE turnover rates in the MPO were reduced by day 3 after pituitary transplantation and at control values at day 15 and 24. It is concluded that the increased DA turnover in the AMBH depresses pituitary LH release probably by inhibiting hypothalamic LH-RH secretion. This inhibition is counteracted by the somewhat slower increase of NE turnover resulting in normalization of LH levels. The increased gonadotropin levels after long-lasting hyperprolactinemia may be due to desensitization of the DA receptor, which was reported earlier. The reduced NE turnover in the MPO 3 days after induction of hyperprolactinemia may be an additional factor in reducing pituitary LH release acting at the level of LH-RH-producing perikarya.

To study the interactions of catecholestrogens with the catecholamine system we estimated the catecholamine concentrations and turnover rates in the anterior part of the mediobasal hypothalamus (AMBH) and medial preoptic area (MPO) following 2-hydroxyestradiol-17 beta (2-OHE2) or 2-hydroxyestrone (2-HOE1) treatment in castrated male and female rats. Serum concentrations of LH and prolactin were also measured. The turnover rates of catecholamines were calculated by monitoring the catecholamine loss 1 h after blocking the catecholamine synthesis with alpha-methyl-p-tyrosine. Dopamine, epinephrine and norepinephrine concentrations were measured by a radioenzymatic assay. In males, 2-OHE2 (50 micrograms/kg) and 2-OHE1 (50 micrograms/kg) resulted in decreased serum LH values (p less than 0.05) 4 and 5 h after treatment. None of these 2-hydroxylated estrogens were able to alter serum prolactin levels significantly. There was a decline in epinephrine and norepinephrine concentrations in the AMBH. The greatest change in catecholamine turnover rates in response to catecholestrogen treatment also occurred in the AMBH. 2-OHE2 and 2-OHE1 reduced turnover rates of dopamine, norepinephrine and epinephrine in the AMBH. Only the dopamine turnover rate was affected in the MPO, where it increased following 2-OHE2 treatment. In females, only 2-OHE2 (50 micrograms/kg) was effective in decreasing serum LH (p less than 0.05) and increasing prolactin (p less than 0.01) levels. Dopamine and epinephrine concentrations as well as their turnover rates declined in the AMBH after treatment with catecholestrogens. The concentration and turnover rate of epinephrine also decreased in the MPO. There was no significant change in norepinephrine concentration or turnover rate. It is suggested that 2-hydroxyestrogens are possibly involved in mechanisms which are inhibitory to LH secretion and stimulatory to prolactin release. These actions appear to be partly mediated by catecholamines.

The effects of intraventricular injections of the highly specific gamma-amino-butyric acid (GABA) agonist muscimol (5 nmol/animal) on blood LH and prolactin levels were measured in ovariectomized (ovx) and in ovx estrogen-progesterone (OEP) primed rats. While the drug stimulated pituitary prolactin release in both experimental groups, pituitary LH release was significantly inhibited in the ovx animals. Muscimol was without any effect on LH levels in ovx-OEP primed rats. Bilateral implantation of tubes containing a muscimol-mannitol mixture into the medial preoptic/anterior hypothalamic (MPO/AH) area abolished pulsatile LH release whereas blood prolactin values were elevated. The intraventricular injection of GABA (8 mumol) also reduced LH and increased prolactin levels in the blood. Measurements of catecholamine turnover rates in the MPO/AH and in the mediobasal hypothalamus (MBH) yielded reduced preoptic but unchanged hypothalamic norepinephrine (NE) and stimulated hypothalamic dopamine (DA) turnover. In view of the well known stimulatory involvement of the NE system in the mechanism of pulsatile LH release and the inhibitory effect of GABA and its agonist muscimol on pulsatile LH release, it is suggested that GABA inhibits NE release in the MPO/AH by the mechanism of presynaptic inhibition. The observation that muscimol is unable to suppress LH release in vox OEP-primed rats may indicate that those estrogen receptive neurons in the MPO/AH which mediate the negative feedback action of the steroid may use GABA as neurotransmitter and that they are the neurons which inhibit NE release. The inhibitory effect of locally implanted muscimol into the MPO/AH also supports this hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Estrogen target neurons are numerous in the medial preoptic/anterior hypothalamic area (MPO/AH) of the female rat brain, and they are thought to play a crucial role in reproductive functions. This brain region is also known to contain high concentrations of the inhibitory transmitter gamma-aminobutyric acid (GABA) and of its synthesizing enzyme glutamate decarboxylase (GAD). Since it is known that GABA is involved in the regulation of gonadotropin release from the pituitary gland it has been proposed that estrogen feedback may be mediated by this transmitter. Here we show, by a combined method of estrogen autoradiography and GAD immunocytochemistry, that estrogen-receptive neurons of GABAergic nature exist in the MPO/AH.