Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats.

The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17ß-estradiol (free E2). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats.

Estrogen receptors regulate the estrous behavior induced by progestins, peptides, and prostaglandin E2.

The role of classical estrogen receptors (ERs) in priming female reproductive behavior has been studied previously; however, the participation of this receptor during activation of estrous behavior has not been extensively studied. The purpose of this work was to test the possibility that the facilitation of lordosis behavior in estrogen-primed rats by progesterone (P) and its 5α- and 5ß-reduced metabolites, gonadotropin-releasing hormone (GnRH), leptin, prostaglandin E2 (PGE2) and vagino-cervical stimulation (VCS) involves interactions with classical ERs by using the selective ER modulator, tamoxifen. To further assess the role of ERs, we also explored the effects of the pure ER antagonist, ICI182780 (ICI), on estrous behavior induced by P and GnRH. Ovariectomized, estrogen-primed rats (5µg estradiol benzoate 40h earlier) were injected intraventricularly with the above-mentioned compounds, or they received VCS. All compounds and VCS effectively facilitated estrous behavior when tested at 60, 120 or 240min after infusion or application of VCS. Intraventricular infusion of tamoxifen (5µg), 30min before, significantly attenuated estrous behaviors induced in estradiol-primed rats by P, most of its 5α- and 5ß-reduced metabolites, GnRH, and PGE2, but not by VCS. Although there was a trend for reduction, tamoxifen did not significantly decrease lordosis in females treated with 5ß-pregnan-3,20-dione. ICI also inhibited lordosis behavior induced by P and GnRH at some testing intervals. These results suggest that activation of classical ERs participates in the triggering effects on estrous behavior induced by agents with different chemical structures that do not bind directly to ERs.

Chronic estradiol treatment increases CA1 cell survival but does not improve visual or spatial recognition memory after global ischemia in middle-aged female rats.

Transient global ischemia induces selective, delayed neuronal death in the hippocampal CA1 and cognitive deficits. Physiological levels of 17beta-estradiol ameliorate ischemia-induced neuronal death and cognitive impairments in young animals. In view of concerns regarding hormone therapy in postmenopausal women, we investigated whether chronic estradiol treatment initiated 14 days prior to ischemia attenuates ischemia-induced CA1 cell loss and impairments in visual and spatial memory, in ovariohysterectomized (OVX), middle-aged (9-11 months) female rats. To determine whether the duration of hormone withdrawal affects the efficacy of estradiol treatment, hormone treatment was initiated immediately (0 week), 1 week, or 8 weeks after OVX. Age-matched, OVX and gonadally intact females were studied at each OVX interval. Ischemia was induced 1 week after animals were pretested on a variety of behavioral tasks. Global ischemia produced significant neuronal loss in the CA1 and impaired performance on visual and spatial recognition. Chronic estradiol modestly but significantly increased the number of surviving CA1 neurons in animals at all OVX durations. However, in contrast with previous results in young females, estradiol did not preserve visual or spatial memory performance in middle-aged females. All animals displayed normal locomotion, spontaneous alternation and social preference, indicating the absence of global behavioral impairments. Therefore, the neuroprotective effects of estradiol are different in middle-aged than in young rats. These findings highlight the importance of using older animals in studies assessing potential treatments for focal and global ischemia.

Regulation of soluble guanylyl cyclase activity by oestradiol and progesterone in the hypothalamus but not hippocampus of female rats.

Oestradiol and progesterone act in the hypothalamus to coordinate the timing of lordosis and ovulation in female rats in part through regulation of nitric oxide (NO) and cyclic guanosine monophosphate (cyclic GMP) signalling pathways. Soluble guanylyl cyclase is an enzyme that produces cyclic GMP when stimulated by NO and plays a crucial role in the display of lordosis behaviour. We examined the effects of oestradiol and progesterone on the stimulation of cyclic GMP synthesis by NO-dependent and independent activators of soluble guanylyl cyclase in preoptic-hypothalamic and hippocampal slices. Ovariectomised Sprague-Dawley rats were injected with oestradiol (2 microg oestradiol benzoate, s.c.) or vehicle for 2 days. Progesterone (500 microg, s.c.) or vehicle was injected 44 h after the first dose of oestradiol. Rats were killed 48 h after the first oestradiol or vehicle injection, and hypothalamus and hippocampus were obtained. NO-dependent activation of soluble guanylyl cyclase was induced by NO donors, sodium nitroprusside or diethylamine NONOate; NO-independent activation of soluble guanylyl cyclase was induced with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole and 5'-cyclopropyl-2-[1-2fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyridine-4-ylamine. The NO-dependent activators of soluble guanylyl cyclase produced a concentration-dependent increase in cyclic GMP accumulation and induced significantly greater cyclic GMP accumulation in preoptic-hypothalamic slices from animals treated with oestradiol and progesterone than in slices from rats injected with vehicle, oestradiol or progesterone alone. Hormones did not modify soluble guanylyl cyclase activation by NO-independent stimulators or influence NO content in preoptic-hypothalamic slices. Oestradiol and progesterone did not affect activation of soluble guanylyl cyclase in hippocampal slices by any pharmacological agent, indicating a strong regional selectivity for the hormone effect. Thus, oestradiol and progesterone, administered in vivo, enhance the ability of NO to activate soluble guanylyl cyclase in brain areas modulating female reproductive function without an effect on production of NO itself.

Ovarian hormone dependence of alpha(1)-adrenoceptor activation of the nitric oxide-cGMP pathway: relevance for hormonal facilitation of lordosis behavior.

The ovarian hormones estradiol (E(2)) and progesterone (P) facilitate rat lordosis behavior in part by regulating the expression of and signal transduction by adrenoceptors in the hypothalamus (HYP) and preoptic area (POA). The major adrenoceptor subtype mediating E(2) and P facilitation of lordosis is the alpha(1)-adrenoceptor. In the present studies, we tested the hypotheses that (1) alpha(1)-adrenoceptors in the HYP enhance lordosis responses by activating the nitric oxide (NO)-cGMP signaling pathway, and (2) coupling of alpha(1)-adrenoceptors to this signal transduction pathway is hormone-dependent. Basal levels of cGMP were significantly higher in HYP and POA slices from animals treated with E(2) and P when compared with slices from ovariectomized controls or females treated with only E(2) or P. When slices of HYP and POA from ovariectomized female rats were incubated with norepinephrine or the selective alpha(1)-adrenoceptor agonist phenylephrine, cGMP accumulation was observed only if slices had been derived from females treated with both E(2) and P before experimentation. Moreover, alpha(1)-adrenoceptor stimulation of cGMP synthesis was blocked by an inhibitor of NO synthase, confirming that these receptors act by NO-mediated stimulation of soluble guanylyl cyclase. Behavioral studies demonstrated further that the cell-permeable cGMP analog 8-bromoadenosine-cGMP reverses the inhibitory effects of the alpha(1)-adrenoceptor antagonist prazosin on lordosis behavior in E(2)- and P-treated female rats. Thus, the NO-cGMP pathway mediates the facilitatory effects of alpha(1)-adrenoceptors on lordosis behavior in female rats, and previous exposure of the HYP and POA to both E(2) and P are required to link alpha(1)-adrenoceptors to this pathway.

1-(o-chlorophenyl)-1-(p-chlorophenyl)2,2,2-trichloroethane: a probe for studying estrogen and progestin receptor mediation of female sexual behavior and neuroendocrine responses.

A number of chlorinated insecticides have been shown to interact with estrogen receptors and to mimic estrogen action in peripheral reproductive tissues (e.g. vagina and uterus). The present study was designed to assess whether the dichlorodiphenyltrichloroethane (DDT) isomer o,p'-DDT has estrogenic or antiestrogenic activity in neural estrogen target tissues. Single sc injections of up to 500 mg/kg o,p'-DDT had no effect on female sexual behavior (lordosis). However, more prolonged treatments induced high levels of lordosis behavior, inhibited compensatory ovarian hypertrophy, and reduced body weight gain in ovariectomized adult female rats. Since o,p'-DDT was able to mimic the action of estrogen on these three neuroendocrine responses, further experiments were performed to determine whether the compound could be used as a tool to investigate the role of hypothalamic steroid receptors in estrogen stimulation of reproductive behavior. It was found that single injections of 500 mg/kg o,p'-DDT (which did not induce sexual receptivity) interacted with cytosol estrogen receptors in both the hypothalamus and pituitary. However, depletion of cytosol receptors by o,p'-DDT was very slow and incomplete; maximal depletion was only 51% and did not occur until 8 h postinjection. It was also observed that neither behaviorally effective nor ineffective injections of o,p'-DDT were able to induce progestin receptor synthesis in the hypothalamus or pituitary. Thus, it appears that the inability of the compound to promote lordosis behavior after a single injection probably results from inadequate receptor interaction in hypothalamic cell nuclei, but that failure to induce neural progestin receptor synthesis is not the critical factor. These data also suggest that the study of o,p'-DDT action in the brain may provide useful information regarding mechanisms of steroid mediation of neuroendocrine responses.

Antagonism of female sexual behavior with intracerebral implants of antiprogestin RU 38486: correlation with binding to neural progestin receptors.

The steroidal antiprogestin 17 beta-hydroxyl-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propynl)estra-4,9-dien-3-one (RU 38486) was administered systemically or was implanted into the ventromedial hypothalamus and other brain regions (habenula, preoptic area, interpeduncular region, in order to determine whether the compound could antagonize progesterone (P) activation of estrous responsiveness and whether the compound would exert its behavioral effects at the presumed site of P action and/or at other neural sites implicated in the regulation of female sexual behavior. RU 38486 (5 mg) administered sc 1 h before 200 micrograms P inhibited P facilitation of lordosis behavior in estrogen-primed rats. Intracerebral application of RU 38486 to the ventromedial hypothalamus reduced lordosis responses in 14 of the 25 animals tested. Similar implants in the habenula also inhibited lordosis in 5 of the 14 animals tested. Antiprogestin implants in the interpeduncular region and preoptic area were virtually without effect (1 of 7 inhibited in each group). Interactions of RU 38486 with steroid binding sites in the hypothalamus-preoptic area (HPOA) were also assessed. RU 38486 appeared to be a competitive inhibitor of progestin ([3H] R5020) binding in HPOA cytosols. Scatchard analysis of [3H]RU 38486 binding showed that when unlabeled P was used as the competitor to assess nonspecific binding, the antiprogestin bound with high affinity [dissociation constant Kd = 8.4 nm] to brain cytosols. In addition, the number of [3H]RU 38486 binding sites in HPOA cytosol increased by approximately 50% in estrogen-primed female rats. Competition studies indicated that unlabeled RU 38486 was the most effective competitor for [3H]RU 38486 binding but that P and R5020 were nearly as effective. Corticosterone, hydrocortisone, deoxycorticosterone, and triamcinolone also competed for [3H]RU 38486 binding but were somewhat less effective than the progestins. Testosterone and estradiol did not displace [3H]RU 38486 except at very high molar excesses. Thus RU 38486 appears to bind with highest affinity to HPOA progestin receptors, but it also binds to glucocorticoid receptors. These data are consistent with the interpretation that inhibition of estrous responsiveness by RU 38486 is associated with the antagonist's interference with brain progestin binding.

Insulin-like growth factor-1 regulation of alpha(1)-adrenergic receptor signaling is estradiol dependent in the preoptic area and hypothalamus of female rats.

Recently, we demonstrated that estradiol (E(2)) modulates cross-talk between protein tyrosine kinases and norepinephrine (NE) receptor signaling in the hypothalamus (HYP) and preoptic area (POA), brain areas that govern female reproductive function. We are now investigating the identity of protein tyrosine kinase(s) that modify NE receptor signaling in the HYP and POA. Incubation of POA and HYP slices with insulin-like growth factor I (IGF-I), which signals via a receptor (IGF-IR) with endogenous tyrosine kinase activity, enhances NE-stimulated cAMP accumulation only in tissue derived from ovariectomized, E(2)-primed animals. JB-1, an antagonist for IGF-IR, prevents the IGF-I enhancement of NE-stimulated cAMP accumulation in both POA and HYP slices. IGF-I enhances NE-stimulated cAMP accumulation via modulation of alpha(1)-adrenoceptor potentiation of adenylyl cyclase. Binding studies in membranes demonstrate that ovariectomized, E(2)-primed animals show a significant increase in the density of [(125)I]IGF-I-binding sites in both POA and HYP compared with ovariectomized control animals. Neither the IC(50) for [(125)I]IGF-I displacement by IGF-I nor the levels of IGF-I binding proteins in serum or brain tissue are affected by E(2). RIA results showed that E(2) does not modify serum or brain IGF-I levels. These results indicate that E(2) regulation of NE receptor function in the POA and HYP involves increased expression of IGF-IR, and that after E(2) treatment, IGF-IR activation augments alpha(1)-adrenoceptor signaling.

Estradiol elevates protein kinase C catalytic activity in the preoptic area of female rats.

Estrogen acts in the brain to regulate female reproductive physiology and behavior, and protein kinase C (PKC) is estrogen-regulated in many estrogen-responsive tissues. We examined whether estrogen regulates PKC in the hypothalamus (HYP) and preoptic area (POA), brain regions which mediate estrogenic control of female reproductive function. PKC activity in tissue from hormone-treated and control female rats was measured, in the presence of phorbol ester and calcium, by quantifying 32p incorporation into a substrate peptide. PKC catalytic activity increased significantly in POA tissue extracts from estradiol-treated, ovariectomized (OVX) female rats but not in HYP or cortical extracts. Phorbol ester potentiation of cAMP accumulation also was examined to determine whether the ability of PKC to potentiate adenylyl cyclase activity was affected by estrogen. PKC stimulation potentiated forskolin-induced cAMP accumulation to a greater degree in POA, but not HYP, slices from estrogen-treated OVX female rats. PKC enzyme levels were examined using phorbol-12,13-dibutyrate binding assays and immunoblots. Estrogen treatment did not change phorbol ester binding affinity or the density of binding sites in the POA or HYP. Immunoblots for the alpha, beta, and gamma PKC isoforms combined, or the gamma PKC isoform alone, did not detect differences between hormone-treated and control OVX female rats. Therefore, estrogen treatment increased PKC catalytic activity in the POA of OVX female rats but not in the HYP. However, the increased PKC catalytic activity was not correlated with detectable changes in the level of the alpha, beta, or gamma PKC isoforms or in the density of phorbol ester binding sites.

Progesterone attenuation of alpha 1-adrenergic receptor stimulation of phosphoinositol hydrolysis in hypothalamus of estrogen-primed female rats.

These experiments examined whether the previously observed abolition by progesterone (P) of alpha 1-adrenergic potentiation of adenylyl cyclase activity in brain slices of estrogen-primed female rats is attributable to a reduced capacity of alpha 1-adrenoceptors to stimulate phosphoinositol hydrolysis. In preoptic area and hypothalamic slices from ovariectomized (OVX) female rats, both norepinephrine (NE) and the alpha 1-adrenergic agonist phenylephrine (PHE) were robust stimulators of inositol phosphate (IP) formation. The NE response was completely blocked by the alpha 1-adrenergic antagonist prazosin. PHE-induced IP formation in tissue from OVX females exposed for 48 h to estrogen alone was comparable to that in OVX controls. In hypothalamic tissue from OVX rats given estrogen plus P, NE and PHE activation of phosphoinositol hydrolysis was attenuated. Both chlorethylclonidine, an irreversible antagonist of alpha 1b-adrenoceptors, and 5-methylurapidil, an alpha 1a-selective antagonist, reduced NE-induced IP formation regardless of the hormonal condition of the animals. Analysis of PHE competition for [3H]prazosin binding indicated that agonist binding was of high affinity (Ki, 16-30 microM) and was unaffected by hormonal status. Therefore, P abolition of alpha 1-adrenergic augmentation of adenylyl cyclase is correlated with reduced phosphoinositol hydrolysis, but not with changes in alpha 1-adrenoceptor agonist binding affinity. Moreover, both alpha 1a- and alpha 1b-adrenoceptors appear to work together to stimulate this second messenger system in the female rat hypothalamus.

Estrogen uncouples beta-adrenergic receptor from the stimulatory guanine nucleotide-binding protein in female rat hypothalamus.

The responsiveness of adenylyl cyclase to beta-adrenergic receptor stimulation was investigated in membranes prepared from hypothalamus-preoptic area and cortex of ovariectomized female rats injected with oil vehicle or estradiol benzoate 24 or 48 h before death. Membranes from the hypothalamus-preoptic area of ovariectomized animals displayed a concentration-dependent stimulation of adenylyl cyclase when incubated with the beta-adrenergic receptor agonist, isoproterenol (10(-7)-10(-5) M). This response was suppressed in membranes from estrogen-treated animals. The effect of estrogen was observed 48 h, but not 24 h, after hormone administration. In addition, estrogen had no measurable effect on hypothalamic adenylyl cyclase activation by either GTP (10(-8)-10(-5) M) or forskolin (10(-8)-10(-6) M), on beta-adrenergic receptor density, or on antagonist binding affinity measured with the beta-adrenergic antagonist [125I]iodocyanopindolol. Analysis of isoproterenol displacement of iodocyanopindolol binding revealed that estrogen reduced agonist binding affinity in hypothalamus-preoptic area membranes. In membranes from ovariectomized controls, high affinity agonist binding to the beta-adrenergic receptor was apparent and was abolished by guanine nucleotides. However, membranes from estradiol-treated rats demonstrated only low affinity agonist binding that was unaffected by guanine nucleotides. Estradiol did not detectably alter concentrations of either cholera or pertussis toxin substrates in hypothalamus-preoptic area membranes. These data indicate that estrogen promotes a stable time-dependent desensitization of beta-adrenergic receptor activation of adenylyl cyclase in hypothalamus and preoptic area by uncoupling the receptor from the guanine nucleotide-binding protein, G8.

Dilute estradiol implants and progestin receptor induction in the ventromedial nucleus of the hypothalamus: correlation with receptive behavior in female rats.

The ventromedial nucleus of the hypothalamus (VMN) is thought to constitute an essential neural substrate for hormonal induction of female sexual behavior. In previous work, implants of dilute (0.4%) estradiol (E2) have been found to prime progesterone-facilitated female sexual behavior in rats only when they are within or very close to the VMN. In other studies, induction of cytosolic progestin receptors (PRc) in the hypothalamus has been correlated with systemic E2 priming of receptive behavior. We combined dilute E2 implants with a Palkovits punch microassay for PRc in the an effort to examine the relationship between estrous behavior and estrogen-induced PRc in the VMN of individual female rats. Ovariectomized rats were given bilateral guide cannulae aimed at the VMN and were primed for 3 days with 1) blank implants plus oil sc, 2) blank implants plus 0.5 micrograms E2 benzoate sc, 3) implants of 0.4% E2 in cholesterol, or 4) implants of 2.0% E2. On day 4 all rats received progesterone (P) sc and were tested for receptive behavior. The following week all subjects received the same estrogen-priming treatment but were killed on day 4 without P treatment. The VMN was punch dissected and assayed for PRc. High levels of lordosis were seen in the E2 benzoate-treated and 2.0% E2-implanted animals, while little behavior was seen in the oil-treated animals. Among 0.4% E2-implanted animals, higher levels of lordosis were observed in subjects with implant placements in the rostral than in the caudal VMN; however, lordosis quotients were not correlated with PRc induction measured in the entire VMN of individual animals. In general, our findings suggest that local estrogenic stimulation of target cells in the VMN is capable of both priming the estrous response and induction of P receptors. Whether these two effects are causally related awaits future study.

Estradiol plus progesterone promote glutamate-induced release of gamma-aminobutyric acid from preoptic area synaptosomes.

Treatment of ovariectomized rats with both estradiol and progesterone in vivo resulted in a marked enhancement of glutamate-induced release of newly synthesized [3H]gamma-aminobutyric acid (GABA) from synaptosomes of the preoptic area in vitro. With this treatment, as little as 0.01 nM glutamate, in vitro, enhanced release of GABA. In contrast, glutamate, in vitro, did not stimulate release of GABA from synaptosomes, obtained from rats treated with either estradiol or progesterone alone and only large concentrations of glutamate (1.0 and 10 mM) caused a modest release of GABA from synaptosomes from ovariectomized, vehicle-treated rats. Also, treatment with estradiol plus progesterone did not alter glutamate-induced release or exchange of [3H]glutamate. Glutamate-induced release of GABA was calcium-independent and attenuated by the putative chloride channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-DL-disulfonic acid. Thus, glutamate-induced, steroid-enhanced release of GABA may occur through a chloride-dependent carrier rather than by exocytosis. In addition to enhancement by glutamate, release of GABA was also enhanced by D-aspartate, an agent that is transported by the neuronal glutamate carrier. It is postulated that enhancement of glutamate-induced release of GABA, by estradiol plus progesterone in the preoptic area, represents one process by which these steroids modulate reproductive function in female rats.

Characterization of glutamate efflux from preoptic area synaptosomes.

Treatment of ovariectomized rats in vivo with ovarian steroids has been found to influence the efflux of glutamate and gamma-aminobutyric acid from preoptic area synaptosomes incubated in vitro. Since these studies indicated a possible role of the glutamate carrier in steroid-modulated release of amino acids, the present studies examined the characteristics of efflux of glutamate and of the carrier system for glutamate in synaptosomes of the preoptic area derived from ovariectomized hormone-treated rats. The efflux of [3H]glutamate from preoptic area synaptosomes, was induced by glutamate and by the glutamate carrier agonist, D-aspartate; the putative glutamate carrier antagonist dihydrokainate failed to block this efflux. Dihydrokainate inhibited the uptake of glutamate but it was less effective than D-aspartate. The excitatory amino acid receptor agonists, N-methyl-D-aspartate and kainate were without effect while quisqualate modestly stimulated the efflux of [3H]glutamate. Efflux of [3H]glutamate, induced by glutamate itself or by D-aspartate was not blocked by the excitatory amino acid receptor antagonists, D-2-amino-5-phosphonovaleric acid, 6,7-dinitroquinoxaline-2,3-dione or kynurenate. Glutamate-induced efflux of [3H]glutamate did not require external Ca2+. Glutamate altered neither the basal nor the potassium-induced increases in the intrasynaptosomal concentration of Ca2+ as measured by the fura-2 method. Glutamate-induced efflux of [3H]glutamate was blocked by the putative chloride channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. It is concluded that the glutamate-induced efflux of [3H]glutamate in synaptosomes of the preoptic area is a carrier-mediated process that does not require activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Gender-related differences exist in cortical [3H]nisoxetine binding and are not affected by prenatal morphine exposure.

The present study was designed to test the hypothesis that prenatal morphine, which differentially affects hypothalamic norepinephrine content and turnover in male and female rats, has sexually dimorphic effects on the density of hypothalamic norepinephrine uptake sites in adult offspring. The binding characteristics of norepinephrine transporters were examined in the hypothalamus, preoptic area and frontal cortex of adult male and female rats exposed to morphine (5-10 mg/kg, twice daily) or saline on gestation days 11-18. There was a gender-related difference in the density of norepinephrine uptake sites measured by [3H]nisoxetine binding in the frontal cortex of saline controls, with control males having significantly fewer binding sites than control females. Prenatal morphine administration did not reverse or eliminate this difference. Additionally, prenatal morphine exposure had no effects on either the binding capacity or the affinity of norepinephrine uptake sites in the hypothalamus, preoptic area or frontal cortex of adult progeny. Thus, alterations in hypothalamic norepinephrine content and turnover following prenatal morphine exposure are not reflected in alterations in norepinephrine uptake sites. However, recent immunocytochemical work in our laboratory correlated reductions in hypothalamic norepinephrine content and turnover rate with reductions in tyrosine hydroxylase and dopamine-beta-hydroxylase fiber density in the hypothalamus of morphine-exposed female rats. Therefore, the present results may suggest that compensatory mechanisms increase the density of norepinephrine uptake sites in hypothalamic terminal fields of morphine-exposed females.

Estradiol reduction of alpha 2-adrenoceptor binding in female rat cortex is correlated with decreases in alpha 2A/D-adrenoceptor messenger RNA.

We developed a quantitative RNAse protection assay for alpha 2A/D-adrenoceptor messenger RNA in rats to test the hypothesis that decreases in cortical alpha 2-adrenoceptor binding observed following estrogen treatment of ovariectomized rats correlate with reduced levels of messenger RNA encoding the predominant alpha 2-adrenoceptor subtype expressed in cortex. Estradiol treatment for 48 h reduced cortical alpha 2A/D-adrenoceptor messenger RNA by approximately 50% when compared to ovariectomized, oil-treated control animals. Estradiol down-regulation of alpha 2A/D-adrenoceptor messenger RNA was accompanied by a significant decrease in cortical alpha 2-adrenoceptor density, as measured by 3H-RX821002 binding. Estrogen treatment did not alter alpha 2A/D-adrenoceptor messenger RNA or alpha 2-adrenoceptor binding in female rat hypothalamus-preoptic area. This study provides the first evidence that estradiol regulates expression of postsynaptic alpha 2-adrenoceptors in female rat frontal cortex, suggesting a possible molecular substrate for hormonal modulation of cognitive function.

Estrogen regulation of noradrenergic signaling in the hypothalamus.

Hypothalamic circuits utilizing the monoamine neurotransmitter norepinephrine (NE) may be key elements upon which the ovarian steroids estradiol (E2) and progesterone (P) act to regulate female reproductive behavior. Recent studies have focused on the modulation of hypothalamic NE release by E2 and P treatments that facilitate sexual behavior. Brain microdialysis studies suggest that oxytocin, a neuropeptide known to enhance lordosis when infused into the ventromedial hypothalamus (VMH) of E2 + P-primed females, modulates NE release in the VMH. Systemic administration of oxytocin reliably enhances extracellular NE levels in the VMH of animals primed with moderate doses of both E2 and P. Thus, ovarian steroids may facilitate female sexual behavior in part by promoting oxytocin-induced NE release in the VMH. Studies examining the release of 3H-NE from superfused hypothalamic slices indicate that estrogen treatment also facilitates NE neurotransmission by attenuating alpha 2-adrenergic receptor-mediated inhibition of NE release. Hypothalamic alpha 2-adrenergic receptors are not downregulated by estrogen, suggesting that brain adrenoceptor function can be modulated by E2 independent of changes in receptor density. A model is proposed wherein E2 and P enhance hypothalamic NE release, leading to increased excitability of VMH neuronal activity and the expression of lordosis behavior.

Behavioral effects of progestin in the brain.

In this article we review research on the role of progestins in the regulation of estrous responsiveness in female rats. Estrous responsiveness normally results from a synergistic action of estradiol (E2) and progesterone (P). E2 primes the system but normally does not result in estrous behavior. The full expression of estrous responsiveness results from the action of P on the E2-primed system. It has been demonstrated with implants of dilute E2 (1 part E2: 250 parts cholesterol) that the site of E2 priming is the ventromedial nucleus of the hypothalamus (VMN). In females primed with systemically administered E2, P also acts on the VMN to facilitate full estrous responsiveness. It has been shown in addition that estrous responsiveness results from sequential application of E2 and P to the VMN but not to other areas of the brain. The VMN is also the site at which P produces sequential inhibition of estrous responsiveness. The time course of P action in facilitating full estrous responsiveness is about two hours, regardless of whether the hormone is administered intracerebrally or intravenously. The duration of estrous responsiveness is directly correlated with the length of time P is in contact with brain tissue. Experiments with the protein synthesis inhibitor anisomycin are consistent with the view that P acts in the VMN by way of a protein synthetic mechanism to facilitate estrous behavior; however, other mechanisms must be considered as alternatives. Finally, we address the question of whether estrogenic priming depends upon induction of progestin receptors in the VMN. Results indicate that estrogenic priming of estrous responsiveness may occur without concomitant induction of progestin receptors.

Progesterone depression of norepinephrine-stimulated cAMP accumulation in hypothalamic slices.

The present experiments examined the effects of progesterone on adrenergic receptor coupling to adenylate cyclase in hypothalamic and preoptic area slices by monitoring norepinephrine (NE)-stimulated increases in cAMP accumulation. Progesterone treatment of estrogen-primed rats decreased NE-induced slice cAMP accumulation. The reduced cAMP response was estrogen-dependent since it was not demonstrable in slices from rats exposed to progesterone without prior estrogen priming. Neither generalized increases in phosphodiesterase activity nor decreases in the catalytic activity of adenylate cyclase could account for the reduced ability of NE to stimulate cAMP accumulation in hypothalamic slices. Moreover, the cAMP response to two other activators of adenylate cyclase, adenosine and vasoactive intestinal peptide, was not decreased in slices from rats treated with estrogen plus progesterone. Selective adrenergic agonists and antagonists were employed to determine which adrenergic receptors mediate cAMP accumulation in progesterone-exposed slices. Slice cAMP levels were elevated by the beta receptor agonist isoproterenol but not by alpha 1 (phenylephrine) or alpha 2 (clonidine) agonists. However, clonidine potentiated the effect of isoproterenol on slice cAMP formation whereas phenylephrine did not. Likewise, NE-stimulated cAMP accumulation was completely antagonized only by a combination of both beta (propranolol) and alpha 2 (yohimbine) antagonists. The data suggest that in slices from estrogen plus progesterone-treated rats, alpha 2 receptors contribute significantly to NE stimulation of cAMP accumulation. The overall depression of the cAMP response to NE in progesterone-exposed slices may involve a decrease of alpha 1 receptor facilitation of cAMP synthesis.

Hormonal Activation of Female Sexual Behavior is Accompanied by Hypothalamic Norepinephrine Release.

Abstract The present study employed the intracranial microdialysis technique to measure norepinephrine release in the ventrolateral dendritic fields of the ventromedial hypothalamus of freely-moving animals before and during ovarian steroid (estradiol and progesterone) activation of female sexual behavior (lordosis). One day after implantation of a dialysis probe, animals were injected with 3 mug of estradiol benzoate followed 44 h later by 200 mug of progesterone. Introduction of a male rat 4 h after progesterone treatment was correlated with dramatic increases in extracellular norepinephrine levels measured in dialysates of the ventrolateral ventromedial hypothalamus of female rats which displayed high levels of lordosis behavior. In contrast, female rats given the same steroid treatment but which did not show lordosis responses did not have elevated norepinephrine levels in their dialysates. Moreover, animals that received an estrogen antagonist concurrently with the estrogen treatment had neither an increase in ventromedial hypothalamic levels of norepinephrine during behavior testing nor did they display lordosis. These results indicate a close relationship among ovarian steroids, noradrenergic transmission in the ventromedial hypothalamus, and the expression of female sexual behavior.