The role of GABA in the regulation of the dopamine/tyrosine hydroxylase-containing neurons of the rat retina

Dopamine (DA) and gamma-aminobutyric acid (GABA) are putative neurotransmitters in two separate populations of amacrine neurons in the mammalian retina. Pharmacological studies have been conducted to determine if GABA neurons regulate the neuronal activity of the neurons that secrete DA. Tyrosine hydroxylase (TH) activity, a biochemical indicator of changes in activity of DA/TH-containing neurons, was low in dark-adapted retinas and high in light-exposed retinas. Muscimol (a GABA receptor agonist) produced a dose-related, biphasic effect on the light-evoked activation of TH, when the drug was injected into the vitreous (intravitreal injection) of dark-adapted rats. At low doses, (35 and 60 pmol) muscimol enhanced the light-evoked activation of TH, but at higher doses (greater than or equal to 120 pmol) it inhibited the light-evoked increase in enzyme activity. Muscimol had no significant effect on the TH activity of dark-adapted retinas. GABA antagonists, bicuculline and picrotoxin, produced effects on TH activity that were dependent on both dose and light-exposure. At low doses (0.4-0.5 nmol), bicuculline and picrotoxin both inhibited the light-evoked activation of TH, but had no effect on TH activity of the dark-adapted retinas. At a higher dose (2.0 nmol), both antagonists increased TH activity in the dark-adapted retina and attenuated the further activation of the enzyme by light. Rat retinas were dissociated into suspensions of viable cells in order to investigate the direct effects of muscimol and picrotoxin on the DA/TH-containing cells. The process of dissociating dark-adapted retinas resulted in an apparent activation of TH. Incubation of the cells with muscimol resulted in a decrease of TH activity in a concentration-dependent manner. Picrotoxin antagonized the inhibitory effect of muscimol, but had no effect when incubated alone. The biphasic effects of GABA agonists and antagonists in vivo suggest that a certain subpopulation of GABA neurons are involved in the activation of the DA/TH-containing neurons by photic stimulation, while another subpopulation of GABA neurons produce a tonic inhibition of the DA/TH-containing neurons in darkness. The experiments with retinal cell suspensions indicate that the tonic inhibition is probably mediated by synapses of GABA neurons directly onto the DA/TH-containing cells.     

Time-dependent effects of estradiol and progesterone on the number of striatal dopaminergic D2-receptors

Recent evidence has shown that sexual steroids are able to modify the activity of the dopaminergic nigrostriatal pathway. Most of this evidence has been obtained from the individual effects of these hormones, but there is less information about possible interrelationships between both. In order to further explore this question, ovariectomized adult rats were submitted to estradiol (E2) or vehicle injections during 3 days and, at the third day, were also submitted to a single injection of progesterone (P) or vehicle at 4, 10, 24 and 32 h before decapitation. Additionally, the effect of injections of 2-hydroxyestradiol (2OH-E2), which has been involved as local mediator in the effects of E2, was also examined. The two striata of each animal were removed and used for determination of number and affinity of dopamine D2-receptors, using [3H]spiroperidol as ligand. Administration of E2 produced a significant reduction in the number of striatal dopaminergic receptors 10 h after the last steroid injection, which was followed by an increase at 24 h. Administration of P briefly decreased the number of dopaminergic receptors at 4 h after the steroid injection. This effect was not observed in animals pretreated with E2, in which administration of P produced an apparent increase 24 h after the steroid treatment. On the other hand, the 2-hydroxylated derivative of E2 does not seem to mediate in the stimulatory action of this estrogen, since it was unable to increase the number of dopaminergic receptors by itself or priming the action of P. The affinity of dopaminergic receptors for [3H]spiroperidol was not significantly altered after all the steroid treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypothalamic tyrosine hydroxylase activity and plasma gonadotropin and prolactin levels in ovariectomized-steroid treated rats

Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase (TH) activity were evaluated in ovariectomized (OVX) estradiol benzoate (EB) or progesterone (P) treated rats. Single injection of 10 μg or daily injection of 5 μg EB/rat for 7 days significantly lowered gonadotropin levels in OVX animals and elevated PRL levels. Single injection of 2 mg or daily injection of 200 μg P/rat for 7 days increased gonadotropin and PRL levels. Hypothalamic TH activity was significantly elevated by estradiol. Single injection of 2 mg P suppressed TH activity in contrast to the elevation in enzyme activity following chronic treatment. These results indicate that hypothalamic noradrenergic as well as dopaminergic neurons participate in the stimulatory or inhibitory feedback effects of ovarian hormones on gonadotropin and PRL secretion.     

Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat

We have previously shown that the density of dendritic spines on hippocampal CA1 pyramidal cells is dependent on circulating estradiol and progesterone and fluctuates naturally during the 5 day estrous cycle in the adult rat. To date, however, no detailed characterization of the roles that these hormones play in regulation of spine density has been made. In order to determine the time courses and extent of the effects of estradiol and progesterone on dendritic spine density, we have analyzed the density of dendritic spines on the lateral branches of the apical dendritic tree of Golgi-impregnated CA1 hippocampal pyramidal cells in several experiments. In summary, our findings included the following: (1) Following ovariectomy, circulating estradiol is undetectable within 24 hours; however, spine density decreases gradually over a 6 day period. (2) Spine density does not decrease any further up to 40 days following ovariectomy. (3) Treatment with estradiol alone can reverse the ovariectomy-induced decrease in spine density. (4) Spine density begins to increase within 24 hours following estradiol benzoate injection in an ovariectomized animal, peaks at 2 and 3 days, then gradually decreases over the next 7 day period. (5) Although free estradiol is metabolized more rapidly than estradiol benzoate, there is no difference in the rate of decrease in spine density following injection of either form. (6) Progesterone has a biphasic effect on spine density in that progesterone treatment following estradiol initially increases spine density for a period of 2 to 6 hours but then results in a much sharper decrease than is observed following estradiol alone. By 18 hours following progesterone treatment, spine density is decreased nearly to 6 day ovariectomy values. (7) Treatment of intact rats with the progesterone receptor antagonist, RU 486, during the proestrus phase of the estrous cycle inhibits the proestrus to estrus drop in spine density. These findings account for both the gradual increase and rapid decrease in spine density which we have previously observed during the estrous cycle and indicate that progesterone in particular may be an important factor in the regulation of rapid morphologic changes which occur naturally in the adult brain. © 1993 Wiley-Liss, Inc.     

Inhibitory Effect of Progesterone on Occupied Estrogen Receptors of Anterior Pituitary and Uterus in Adult Rats

The acute effects of a single progesterone injection on estradiol receptor (E₂R) binding in the anterior pituitary and uterus were examined in the mature ovariectomized rat. Adult ovariectomized female rats receiving estradiol were injected with various doses of progesterone followed 1 h later by a final injection of 2 μg of estradiol. All animals were sacrificed 1 h after estradiol and the E₂R binding in the nuclei and cytosol was determined. In the anterior pituitary, progesterone decreased total nuclear E₂R in a biphasic inhibitory pattern, with maximal effects at 0.8 and 4 mg/kg body wt doses of progesterone and a smaller decrease with the 2 mg/kg body wt dose. This progesterone-induced decrease of nuclear E₂R was due to a decrease in the occupied form of nuclear E₂R. The unoccupied nuclear E₂R and cytosol E₂R binding were not altered by progesterone administration. In the uterus, progesterone caused a dose-dependent decrease in total nuclear E₂R binding. This effect also occurred in the occupied form of E₂R. The uterine unoccupied nuclear E₂R and cytosol E₂R were not altered by progesterone administration. These studies emphasize differences in the tissue specificity of progesterone action on occupied nuclear E₂R which are presumably responsible for the mediation of estradiol action, and provide a biochemical basis for our previous observations of multiphasic effect of progesterone on gonadotropin secretion.     

Estradiol facilitates kainic acid-induced, but not flurothyl-induced, behavioral seizure activity in adult female rats

PURPOSE: This study was designed to determine whether previously demonstrated increases in hippocampal axospinous synapse density and NMDA receptor function induced by estradiol are paralleled by increased susceptibility to limbic (kainic acid induced) or generalized (flurothyl induced) behavioral seizures. METHODS: Kainic acid was injected systemically to ovariectomized adult female rats treated with either estradiol or oil vehicle. The latencies to each of five stages of seizure-related behaviors (staring, wet-dog shakes, head waving and chewing, forelimb clonus, rearing, and falling) were recorded for each animal. Flurothyl was administered by inhalation to ovariectomized adult female rats treated with estradiol alone, estradiol followed by short-term progesterone, or oil vehicle. The latencies to each of three stages of seizure-related behaviors (first myoclonic jerk, forelimb clonus, wild running and bouncing) were recorded for each animal. RESULTS: Estradiol treatment decreased the latency to seizure-related behaviors induced by kainic acid, but neither estradiol alone nor estradiol followed by progesterone had any effect on flurothyl-induced seizure-related behaviors. CONCLUSIONS: The same estradiol treatment paradigm known to induce structural and functional changes in the excitatory circuitry of the hippocampus facilitates the progression of kainic acid-induced seizures, which are known to involve the hippocampus, but has no effect on flurothyl-induced seizures. The lack of an effect of estradiol alone or estradiol followed by progesterone on flurothyl-induced seizures indicates that estradiol's effects on seizure susceptibility do not result from increased neuronal excitability throughout the brain, but rather involve action within the limbic system. The data suggest that structural and functional changes in hippocampal circuitry induced by estradiol may contribute to increased susceptibility to limbic seizure activity.     

Estradiol decreases the orexigenic effect of neuropeptide Y, but not agouti-related protein, in ovariectomized rats

Available data suggest that estradiol exerts an inhibitory effect on food intake by modulating the actions of multiple gut- and brain-derived peptides implicated in the control of food intake. For example, recent studies have shown that estradiol decreases the orexigenic effects of ghrelin and melanin-concentrating hormone. In the present study, we examined estradiol's ability to decrease the actions of two additional orexigenic peptides, neuropeptide Y (NPY) and agouti-related protein (AgRP). Food intake was monitored following lateral ventricular infusions of 5 microg NPY, 10 microg AgRP, or saline vehicle in ovariectomized rats treated with either 1 microg estradiol or sesame oil vehicle. NPY increased food intake for 2h in both oil- and estradiol-treated ovariectomized rats. During this interval, the orexigenic effect of NPY was significantly greater in oil-treated rats, relative to estradiol-treated rats. In contrast to the short-term action of NPY, a single injection of AgRP increased food intake for 3 days in oil- and estradiol-treated rats. Meal pattern analysis revealed that the orexigenic effect of AgRP is mediated by an increase in meal size, not meal number. Unlike that observed following NPY treatment, estradiol failed to modulate the magnitude by which AgRP increased food intake and meal size. We conclude that a physiological regimen of estradiol treatment decreases the orexigenic effect of NPY, but not AgRP, in ovariectomized rats.     

δ9-Tetrahydrocannabinol affects mesolimbic dopaminergic activity in the female rat brain: interactions with estrogens

Summary In this work, we studied the possible estrogenic modulation of the effects of ⁹-tetrahydrocannabinol (THC) on mesolimbic dopaminergic activity, by examining the effects of an acute dose of this cannabinoid: (i) during the estrous cycle; (ii) after ovariectomy, chronic estrogen-replacement and tamoxifen (TMX)-induced blockade of estrogenic receptors; and (iii) combined with a single and physiological injection of estradiol to ovariectomized rats. THC significantly decreased the density of D1 dopaminergic receptors and non-significantly increased the L-3,4-dihydroxyphenylacetic acid (DOPAC) content in the limbic forebrain of ovariectomized rats chronically replaced with estrogens. The decrease in D1 receptors was also produced by TMX, whereas the coadministration of both THC and TMX did not lead to a major decrease. In addition to the trend of THC increasing DOPAC content, this cannabinoid was also able to increase the ratio between DOPAC and dopamine, although this last effect only occurred after coadministration of THC and TMX, which had been ineffective administered individually. All these effects were not seen when THC was administered to normal cycling rats during each phase of estrous cycle and to ovariectomized rats without chronic estrogen replacement or only submitted to a single and acute dose of estradiol. This observation might be related to the fact that the density of limbic cannabinoid receptors increased in chronic estrogen-replaced ovariectomized ratsversus normal cycling, ovariectomized or acutely estrogen-treated ovariectomized rats. Interestingly, THC administration in ovariectomized rats was followed by a slight, although significant, increase in tyrosine hydroxylase activity, which was also observed after coadministration of THC with a short-time and acute dose of estradiol. In summary, THC stimulated the presynaptic activity of mesolimbic dopaminergic neurons, but accompanied by a decrease in their postsynaptic sensitivity. These effects did not appear in normal cycling rats being only evident after ovariectomy and chronic estrogen replacement, which might be related to changes in binding characteristics of cannabinoid receptors in this area. Moreover, some of them appeared after TMX-induced blockade of estrogenic cytosolic receptors, which likely suggests the existence of a certain estrogenic modulation of the actions of THC on mesolimbic neurons. On the contrary, coadministration of THC with a single and shortly tested dose of estradiol was always ineffective in modifying THC effects.Presented in abstract form to the Third IBRO World Congress of Neuroscience, Montreal (Canada), August 4–9, 1991     

Proestrus levels of estradiol during transient global cerebral ischemia improves the histological outcome of the hippocampal CA1 region: perfusion-dependent and-independent mechanisms.

We conducted this study to determine whether high physiological levels of estradiol (proestrus) could protect the hippocampal CA1 neurons following transient global ischemia. Ovariectomized or ovary-intact female rats were subjected to 20 min of ischemia and allowed to survive for 96 h. Estradiol was administered subcutaneously in a group of ovariectomized rats 24 h before ischemia induction. Ending serum estrogen levels were correlated to cerebral blood flow (CBF), histologic assessment and immunofluorescent caspase-3 active peptide (C-3AP) positive cell count. Estradiol administration significantly improved CBF in the hippocampus (compared with intact or ovariectomized rats) but not in the parietal cortex. No significant differences in CBF between intact or ovariectomized rats were noted. Estradiol administration maintained serum levels of the steroid in estradiol-treated rats-about 10 times that of intact animals and more than 20 times that of ovariectomized animals. Morphologically, live cell counts in estradiol-treated rats were significantly higher than in intact or ovariectomized rats. Live cell counts were also significantly higher in intact than ovariectomized rats. C-3AP positive cell counts were much higher in ovariectomized rats than in intact and estradiol-treated rats. In conclusion, proestrus levels of 17beta-estradiol protect hippocampal CA1 neurons against transient global ischemia, through mechanisms that appear to involve improvement of perfusion and inhibition of caspase-3 activity.     

Somatostatin augments the spread of limbic seizures from the hippocampus

The role of the neuropeptide somatostatin in limbic seizures was studied using electrical stimulation of the hippocampus in kindled rats. Cysteamine, an agent which selectively and reversibly depletes brain somatostatin stores, had a biphasic action. An early proconvulsant effect was seen within a few hours, consisting of prolonged electrographic seizures in the hippocampus and more severe behavioral convulsions. A later anticonvulsant effect, maximal at 1 to 2 days and dissipating within a week, was manifested by less intense behavioral convulsions without change in the duration of electrical seizure activity. Both effects were dose-dependent. No change in afterdischarge thresholds was detected at any time after the administration of cysteamine. Intraventricular administration of somatostatin to animals with behavioral seizures attenuated by cysteamine treatment restored the responses to precysteamine levels. We conclude that somatostatin facilitates the spread of seizures over limbic circuits from a region of focal seizure initiation.     

Progesterone decreases the concentration of hypothalamic and anterior pituitary estrogen receptors in ovariectomized rats

In a study of cellular mechanisms of progesterone's antiestrogenic action on behavior and neuroendocrine responses, we investigated the influence of progesterone on the concentration of estrogen receptors in the hypothalamus-preoptic area (HP), anterior pituitary gland (AP), and uterus of chronically estradiol-treated ovariectomized rats. Ovariectomized (OVX) rats were implanted s.c. with 15 mm silastic capsules of estradiol. One week later, they were injected with progesterone or oil vehicle and killed 6 h or 24 h later. Confirming previous reports, progesterone caused a decrease in the concentration of uterine cytosol and nuclear estrogen receptors at both times. Less consistent results were obtained in HP and AP; a decrease in the concentration of HP cytosol estrogen receptors was detected at 6 h, as was a small decrease in the concentration of HP nuclear estrogen receptors at 24 h. More consistent results were seen when a low priming dose of estradiol was used. Although progesterone was without effect on the concentration of nuclear estrogen receptors in HP and AP at 6 h, cytosol receptor levels were depressed by 25% in HP and 14% in AP. At 24 h after progesterone injection, nuclear estrogen receptor levels were decreased in all tissues, while cytosol estrogen receptor levels remained depressed. A study of the time course of progesterone's suppression of cytosol estrogen receptor concentration revealed that the effect is transient, occurring by 6 h after progesterone injection, but returning to baseline by 48 h after injection. Scatchard analysis confirmed that the decreased concentration of cytosol binding in HP was due to a decrease in the concentration of binding sites. As with nearly all of progesterone's neuroendocrine effects, the suppression of estrogen receptor levels requires estrogen priming. HP and AP cytosol from progesterone-treated rats did not seem to contain an estrogen receptor-regulatory factor as do uterine cell nuclei; loss of binding sites at 37 degrees C was no faster in cytosol from progesterone-treated rats. These results demonstrate that, under some conditions, progesterone decreases HP and AP estrogen receptor concentrations. Unlike progesterone's action in the uterus, the primary effect in the brain and pituitary gland seems to be on the cytosol receptor.     

Progesterone decrease the concentration of hypothalamic and anterior pituitary estrogen receptors in ovariectomized rats

In a study of cellular mechanisms of progesterone's antiestrogenic action on behavior and neuroendocrine responses, we investigated the influence of progesterone on the concentration of estrogen receptors in the hypothalamus-preoptic area (HP), anterior pituitary gland (AP), and uterus of chronically estradiol-treated ovariectomized rats. Ovariectomized (OVX) rats were implanted s.c. with 15 mm silastic capsules of estradiol. One week later, they were injected with progesterone or oil vehicle and killed 6 h or 24 h later. Confirming previous reports, progesterone caused a decrease in the concentration of uterine cytosol and nuclear estrogen receptors at both times. Less consistent results were obtained in HP and AP; a decrease in the concentration of HP cytosol estrogen receptors was detected at 6 h, as was a small decrease in the concentration of HP nuclear estrogen receptors at 24 h.More consistent results were seen when a low priming dose of estradiol was used. Although progesterone was without effect on the concentration of nuclear estrogen receptors in HP and AP at 6 h, cytosol receptor levels were depressed by 25% in HP and 14% in AP. At 24 h after progesterone injection, nuclear estrogen receptor levels were decreased in all tissues, while cytosol estrogen receptor levels remained depressed. A study of the time course of progesterone's suppression of cytosol estrogen receptor concentration revealed that the effect is transient, occuring by 6 h after progesterone injection, but returning to baseline by 48 h after injection. Scatchard analysis confirmed that the decreased concentration of cytosol binding in HP was due to a decrease in the concentration of binding sites. As with nearly all of progesterone's neuroendocrine effects, the suppression of estrogen receptor levels requires estrogen priming. HP and AP cytosol from progesterone-treated rats did not seem to contain an estrogen receptor-regulatory factor as do uterine cell nuclei; loss of binding sites at 37 °C was no faster in cytosol from progesterone-treated rats.These results demonstrate that, under some conditions, progesterone decreases HP and AP estrogen receptor concentrations. Unlike progesterone's action in the uterus, the primary effect in the brain and pituitary gland seems to be on the cytosol receptor.     

The ovarian hormones and absence epilepsy: a long-term EEG study and pharmacological effects in a genetic absence epilepsy model

In the first experiment, the relationship between the phase of the estrous cycle and the number of spontaneously occurring spike-wave discharges was investigated in WAG/Rij rats, a model for generalized absence epilepsy. The electroencephalogram (EEG) was continuously recorded for 96 h in eight rats chronically equipped with cortical EEG electrodes. A circadian pattern emerged for the number of spike-wave discharges: a nadir during the first hours of the light period, and an acrophase during the first hours of the dark period. This daily maximum was increased at proestrus day compared with the other days of the cycle, when the plasma level of progesterone is enhanced specifically at these hours of this day. This suggests that progesterone enhances spike-wave discharges. There was no difference in the first few hours of the light period in the number of spike-wave discharges between proestrus and the three other days, suggesting that estradiol has no effect on spike-wave discharges. In the second study, the effects of the systemic administration of progesterone and 17 beta-estradiol on spike-wave discharges and spontaneous behavior were investigated. It was shown that progesterone (20 and 30 mg/kg) but not estradiol (0.17-1.5 mg/kg) increased the number and total duration of spike-wave discharges. On the other hand, injection of RU 38486 (10 and 30 mg/kg), an antagonist of intracellular progesterone receptors, had no effect on spike-wave discharges and did not block the stimulatory effect of progesterone. The antagonist of 17 beta-estradiol tamoxifen (1 and 3 mg/kg) did not evoke alterations in the number or duration of spike-wave discharges. Our results indicate that progesterone aggravates spike-wave discharges, but is not mediated through intracellular receptors. Since progesterone is rapidly metabolized in the brain to the positive modulator of GABA(A) receptor allopregnanolone, which increases spike-wave discharges in WAG/Rij rats, it is possible that the epileptiformic effects of progesterone are mediated through this metabolite.     

The possible role of 2-hydroxyestradiol in the development of estrogen-induced striatal dopamine receptor hypersensitivity

In the present study, we have confirmed the existence of a biphasic response in striatal dopamine receptor sensitivity following the administration of estradiol benzoate (EB). This biphasic response consists of a hyposensitive phase 24 h after the last injection of EB, followed by a hypersensitive phase 72 h after the last injection of EB. In contrast to this, the administration of 2-hydroxyestradiol (2-OHE2), a catechol metabolite of estrogen, resulted in a striatal dopamine receptor hypersensitivity at both 24 and 72 h after the last injection of 2-OHE2. Studies on the in vivo metabolism of [3H]estradiol to its [3H]catechol metabolites indicated that the administration of piperonyl butoxide (PBO; a microsomal enzyme inhibitor) significantly decreased the level of [3H]catechol metabolites of [3H]estradiol in the striatum and in the medial basal hypothalamus. In addition, PBO administration resulted in about a 7-fold decrease in the ability of estradiol to induce a striatal dopamine receptor hypersensitivity. These data indicate that the biphasic response in striatal dopamine receptor sensitivity following estrogen, may be mediated by separate molecular mechanisms. The association of the hypersensitive phase with pharmacological doses and/or treatment paradigms, the development of a similar hypersensitivity following the administration of the 2-OHE2 metabolite of estrogen and the attenuation of the estrogen-induced striatal dopamine receptor hypersensitivity in PBO pretreated animals all suggest that this striatal dopamine receptor hypersensitivity may be mediated, at least in part, by the catecholestrogens.     

Progesterone-induced decrease of pineal protein synthesis in rats. Possible participation in estrous-related changes of Pineal function

Summary Pineal protein synthesis in female rats, estimated from the incorporation of labeled amino acids into proteinsin vitro, exhibited significant changes as a function of the stage of the estrous cycle. These changes were restricted to the proestrous and estrous days; pineal protein synthesis attained its maximum on the morning of proestrus declining abruptly by 53% during the evening, at the time of the expected gonadotrophin and prolactin release. Pineal serotonin-N-acetyltransferase activity increased by 10 to 15 times during night-time on every day of cycle; no appreciable modification of its daily rhythm was detected along the estrous cycle. Spayed rats treated for 2 days with progesterone showed a dose-dependent decrease of amino acid incorporation into pineal proteins, regardless of whether estradiol was simultaneously administered or not. Pineal protein synthesis in spayed rats administered with estradiol for 2 days and killed at 11 a.m. and 5 p.m. on the third day, did not show differences as a function of time of sacrifice. When progesterone was injected on the morning of the third day a significant decline in protein synthesis was observed at 5 p.m. Only in the latter group serum LH levels showed significantly greater values at 5 p.m. Pineal serotonin content of estradiol-treated rats increased significantly at evening, an effect which was obliterated by the administration of progesterone; progesterone alone did not affect pineal serotonin content. Radioactivity uptake by pineal glands incubated with labeled progesterone did not show changes along the estrous cycle. These data argue in favour of the involvement of progesterone in the changes of pineal protein synthesis observed during the critical period for gonadotrophin and prolactin release.     

Preproenkephalin mRNA is Regulated by an Interaction Between Steroid Hormones and Nociceptive Stimulation

The expression of preproenkephalin (PPE) mRNA has previously been shown to be regulated by steroid hormones in the ventromedial nucleus of the hypothalamus (VMH) and to be regulated by noxious stimuli in the dorsal horn of the spinal cord (DH). The present in situ hybridization study in ovariectomized rats showed that PPE mRNA expression in both the VMH and the lumbar DH, responds to the interaction between a noxious peripheral stimulus and ovarian steroid hormones. In the VMH, either estradiol or estradiol+progesterone increased the mean PPE mRNA content per cell by 100% compared with vehicle-treated rats. Unilateral hindpaw injection of 5% formalin, as compared to saline, significantly increased mean PPE mRNA content per VMH cell in rats treated with vehicle or estradiol but not those treated with estradiol+progesterone. Regression analysis for mean PPE mRNA content per VMH cell as a function of intensity of hindpaw inflammation showed a significant positive correlation coefficient after vehicle and estradiol treatment (P<0.02) but a strong trend towards a negative correlation coefficient after estradiol+progesterone treatment (P<0.06). ANOVA for homogeneity of regression coefficients showed a significant difference across hormone groups (P<0.01).     

Ovarian Steroid Regulation of Pulsatile Luteinizing Hormone Release During Early Gestation in the Rat

The object of this study was to examine ovarian regulation of pulsatile luteinizing hormone (LH) secretion during early gestation. This was done primarily by analyzing pulsatile LH release in rats that were either sham ovariectomized (OVX) on Day 7 of pregnancy, implanted with empty Silastic capsules, and bled on Day 8, or OVX on Day 7, immediately implanted with Silastic capsules producing plasma levels of estradiol and/or progesterone characteristic of Day 7 to 8 of pregnancy, and bled on Day 8. In addition, the role of progesterone in regulating pulsatile LH secretion was also examined by administration of the progesterone receptor antagonist, RU486, on Day 7 and examining pulsatile LH release on Day 8 of pregnancy. OVX caused a marked increase in LH pulse amplitude and frequency within 24 h. Replacement with physiological plasma levels of estradiol or progesterone alone had no suppressive effect on this OVX-induced increase in pulsatile LH secretion. Restoration of physiological plasma levels of both estradiol and progesterone returned LH pulse amplitude to values seen in sham OVX controls, and prevented the OVX-induced increase in LH pulse frequency. The group mean LH pulse frequency tended to be less in estradiol + progesterone-treated rats than in sham OVX controls, but this difference was not statistically significant. RU486 blocked uterine progesterone receptors as evidenced by endometrial hemorrhaging. In agreement with the OVX + steroid replacement data, RU486 administration also resulted in increases in LH pulse amplitude and frequency. These data demonstrate that the frequency and amplitude of LH pulses on Day 8 of gestation are held in check by negative feedback signals coming from the ovary. Neither steroid alone exerts any suppressive influence over pulsatile LH secretion during early gestation, but both steroids acting together exert a prominent negative feedback regulation on the pulsatile LH release process.     

Abbreviation of the period of sexual behavior in female guinea pigs by the progesterone antagonist RU 486

In previous studies we have tested the hypothesis that the termination of the period of sexual behavior in female guinea pigs results from the loss of progestin receptors from hypothalamic cell nuclei. We have shown that hormonal manipulations that delay heat termination also delay loss of hypothalamic nuclear progestin receptors. In order to determine if accelerated nuclear receptor loss results in abbreviation of the period of sexual behavior, we tested the effect of 17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propyl)-estra-4,9-diene-3-one (RU 486), a progesterone antagonist, on heat termination. Ovariectomized guinea pigs were treated with estradiol benzoate. Forty hours later, they received progesterone followed 4 h later by injection of RU 486 or vehicle. RU 486 injected 4 h after progesterone caused heat abbreviation. We have found that RU 486 administration to estradiol-treated guinea pigs causes accumulation of progestin receptors in cell nuclear extract. Because this accumulation can be detected only when assay conditions are used that promote exchange of RU 486 progestin receptor complexes (15 degrees C incubation rather than 0 degree C); our routine assay conditions (at 0 degree C) can be used to measure primarily receptors that are occupied by progesterone. In order to confirm that RU 486 decreased progesterone-occupied nuclear progestin receptor levels when injected 4 h after progesterone, animals treated as in the behavioral experiment were killed 6 or 10 h after progesterone injection (2 or 6 h after RU 486), and nuclear progestin receptor levels were measured. RU 486 treatment resulted in lowered nuclear concentrations of hypothalamic progestin receptors at both times. These results support our hypothesis that the termination of the period of sexual receptivity in female guinea pigs is the result of loss of progestin receptors from hypothalamic cell nuclei.     

Cyclic estradiol replacement attenuates stress-induced c-Fos expression in the PVN of ovariectomized rats

Estradiol modulates stress reactions in female rats. Several studies showed anxiolytic effects of estradiol in behavioral tests, but the underlying mechanisms are still unclear. The aim of the current study was to explore how estradiol-treated rats respond to acute and chronic stress compared to ovariectomized rats. Ovariectomized rats received vehicle or 17beta-estradiol injections (10 microg/250 g) once every 4 days, which induced alternating high and low plasma 17beta-estradiol levels. Stress was presented by daily exposure to an adverse environment in which the animals received five footshocks for either 3 or 22 days. Under control conditions no differences were observed, but as soon as stress was applied, reactions of ovariectomized and estradiol-treated rats diverged. Both acute and chronic stress increased the c-Fos protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Cyclic estradiol treatment reduced this stress-induced activation of the PVN, an effect that seems to be dependent on the plasma estradiol levels. No differences in stress-induced corticosterone responses were revealed between the treatment groups. An increase in the number of ERbeta-expressing cells in the PVN of ovariectomized and estradiol-treated rats during chronic stress implied increased ERbeta-mediated mechanisms during these conditions. The dampening effect of estradiol on the excessive stress-induced activity in the PVN may be beneficial for the animal in its response to chronic recurrent stress by reducing the output of the PVN.     

Evidence that nitric oxide-glutamate cascade modulates spinal antinociceptive effect of morphine: a behavioural and microdialysis study in rats

We evaluated the ability of spinally administered nitric oxide (NO) synthase inhibitor to modulate antinociceptive action of intrathecal (i.t.) morphine in rats by measuring the early and late phases of flinching and licking/biting in the formalin test. To determine the contribution of spinal NO and glutamate, we measured the release of NO metabolites (nitrite/nitrate) and glutamate from the spinal cord in rats, using a microdialysis probe placed in the lumbar space. The i.t. administration of NG-nitro L-arginine methyl ester (L-NAME) produced a dose-dependent reduction in the number of flinches during the late phase, whereas there were no significant alterations in the late phase licking/biting, and early phase flinching and licking/biting. Spinal administration of morphine at low doses produced a significant antinociceptive activity in the early and late phases of the flinching behaviour, whereas higher doses of morphine were required to obtain a significant effect in the licking/biting behaviour during both phases. Combination of L-NAME with morphine resulted in an enhanced reduction in the early and late phase flinching. Enhanced antinociceptive activity was observed in the late phase licking/biting by i.t. combined administration of L-NAME (400 nmol) and morphine (1.25 nmol). In the present study, we have confirmed our prior results that injection of formalin (5.0%) into the plantar surface of the paw evoked a biphasic spinal release of nitrite/nitrate and a transient release of glutamate. Formalin-evoked release of nitrite/nitrate and glutamate was also reduced markedly by i.t. combined administration of L-NAME and morphine. These behavioural and biochemical results suggest that i.t. administered L-NAME may enhance morphine-induced antinociception through an increased inhibition of nitrite/nitrate and glutamate releases evoked by formalin injection at the spinal cord level.