Daniel Bitran

Anxiolytic effects of 3α-hydroxy-5α[β]-pregnan-20-one: endogenous metabolites of progesterone that are active at the GABAA receptor

Abstract The effects of intracerebroventricular administration of reduced metabolites of progesterone on locomotor activity and on exploration in the elevated plus-maze were assessed in adult female rats. Allopregnanolone (3α-hydroxy-5α-pregnan-20-one; 1.25, 5.0, and 10 μm) and pregnanolone (3α-hydroxy-5β-pregnan-20-one; 2.5, 5.0, and 10 μg) elicited anxiolytic effects and, at the highest dose tested, allopregnanolone resulted in sedation. In contrast, the 3β-hydroxy-epimer of allopregnanolone was without effect in either behavioral paradigm. The anxiolytic response to pregnanolone was blocked by picrotoxin (0.75 mg/kg, i.p.), a dose that by itself did not affect behavior in the plus-maze. These data suggest that the anxiolytic effect of 3α-hydroxy metabolites of progesterone is mediated by brain GABAA receptors in a stereospecific manner, and are in good agreement with the well-documented in vitro effects of these steroids as potent modulators of the GABAA receptor.

Pharmacological analysis of male rat sexual behavior

Pharmacological influences on male rat sexual behavior are reviewed in an attempt to identify neurotransmitters and their respective receptor types that regulate various factors comprising the behavioral pattern. Evidence is presented that: (1) serotonergic influence is generally inhibitory to sexual behavior, although two receptor subtypes may lower ejaculation threshold; (2) dopaminergic agonists facilitate several aspects of copulatory behavior and ex copula genital responses; (3) noradrenergic activity appears to increase sexual arousal; (4) cholinergic agonists facilitate ejaculation, or in some cases, delay or prevent initiation of copulation; (5) GABA agonists inhibit sexual responses both in and ex copula; (6) opiate agonists appear to inhibit copulation and penile reflexes, although antagonists have mixed effects; (7) ACTH and MSH peptides promote copulatory behavior and genital responses; (8) oxytocin facilitates ex copula penile responses, but may contribute to postejaculatory refractoriness; and (9) long-term exposure to prolactin inhibits sexual behavior and penile responses. Although some progress has been made in identifying neurotransmitter-receptor effects on behavioral components, copulatory behavior is complex and no drug has been found to affect only a single component. Furthermore, drug specificity is only relative.

Activation of estrogen receptor-beta regulates hippocampal synaptic plasticity and improves memory.

Estrogens have long been implicated in influencing cognitive processes, yet the molecular mechanisms underlying these effects and the roles of the estrogen receptors alpha (ERα) and beta (ERβ) remain unclear. Using pharmacological, biochemical and behavioral techniques, we demonstrate that the effects of estrogen on hippocampal synaptic plasticity and memory are mediated through ERβ. Selective ERβ agonists increased key synaptic proteins in vivo, including PSD-95, synaptophysin and the AMPA-receptor subunit GluR1. These effects were absent in ERβ knockout mice. In hippocampal slices, ERβ activation enhanced long-term potentiation, an effect that was absent in slices from ERβ knockout mice. ERβ activation induced morphological changes in hippocampal neurons in vivo, including increased dendritic branching and increased density of mushroom-type spines. An ERβ agonist, but not an ERα agonist, also improved performance in hippocampus-dependent memory tasks. Our data suggest that activation of ERβ can regulate hippocampal synaptic plasticity and improve hippocampus-dependent cognition.

Anxiolytic Effect of Progesterone is Mediated by the Neurosteroid Allopregnanolone at Brain GABAA Receptors

Previous studies from this laboratory have shown that progesterone (PROG) treatment in ovariectomized rats produces an anti‐anxiety response similar to that observed after the administration of prototypical anxiolytic benzodiazepine (BDZ) compounds. The PROG‐induced anxiolytic response was highly correlated with an increased level of 3α‐hydroxy‐5α‐pregnan‐20‐one (allopregnanolone) in the blood and brain, and was also associated with a facilitation of GABA‐stimulated chloride ion (Cl−) influx in cortical synaptoneurosomes. This correlative evidence suggested that the anxiolytic effect of PROG was a result of its in vivo reduction to the neuroactive steroid, allopregnanolone. In this report, a series of studies was conducted to determine the mechanism(s) by which PROG alters behavior in animal models of anxiety. In the first experiment, ovariectomized rats were injected with PROG (1 mg/0.2 ml, SC) 4 h prior to a test in the elevated plus‐maze. Some animals also received an injection of picrotoxin (0.75 mg/kg, IP), a GABAA receptor‐gated Cl− channel antagonist, whereas other animals were pretreated with RU 38486 (5 mg/0.2 ml, SC), a progestin receptor antagonist. PROG elicited anxiolytic behavior in the plus‐maze, an effect that was blocked by picrotoxin administration. Pretreatment with RU 38486 was not effective in altering PROG‐induced anxiolytic behavior in the plus‐maze. In a second experiment, the effect of PROG on behavior in the plus‐maze was determined in the presence of N,N‐diethyl‐4‐methyl‐3‐oxo‐4‐aza‐5α‐androstane‐17β‐carboxamide (4‐MA; 10 mg/0.2 ml, SC), a 5α‐reductase inhibitor. The enzyme inhibitor was potent in preventing the anxiolytic effect observed in the plus‐maze after PROG administration. In the defensive burying paradigm, PROG treatment also produced anxiolysis by reducing the duration of burying behavior, and this effect was prevented by 4‐MA pretreatment, but not by RU 38486 administration. After the completion of the behavioral assays, analysis of blood allopregnanolone levels revealed a marked increase in PROG‐treated females. The PROG‐induced elevation in circulating allopregnanolone was blocked by pretreatment with 4‐MA. In cortical synaptoneurosomes, the sensitivity (inverse of the EC50) and the maximal response (Emax) in GABA‐stimulated 36Cl− uptake were increased in PROG‐treated females. The potentiation of PROG on both of these neurochemical measures was not observed in animals pretreated with 4‐MA. Together, these studies provide evidence that the anxiolytic effect of PROG is not associated with an intracellular steroid receptor that initiates genomic‐mediated responses. The evidence is consistent with a nongenomic mechanism whereby PROG is metabolized to allopregnanolone, a neuroactive steroid that potentiates GABAA receptor‐mediated responses.

Anxiolytic effect of progesterone is associated with increases in cortical alloprenanolone and GABAA receptor function

The effects of a SC injection of progesterone (0, 1, or 4 mg) on locomotor behavior and exploration of an elevated plus-maze were examined in ovariectomized rats. At the completion of the behavioral tests, blood serum and cerebral cortical level of the 3 alpha-hydroxy ring-A metabolite of progesterone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone), was also assessed. GABA-stimulated 36Cl- influx was studied in cortical synaptoneurosomes from a subgroup of ovariectomized females treated with vehicle or 4 mg progesterone. Whereas progesterone treatment did not affect ambulation in a novel arena, significant anxiolytic behavior was detected in the plus-maze 4 h after administration of 1 or 4 mg progesterone. A dose-dependent increase in allopregnanolone level was found in serum and cortical homogenates. Studies of GABA-stimulated Cl- influx demonstrated that progesterone treatment increased the sensitivity of cortical synaptoneurosomes to GABA (i.e., decreased the EC50) and increased the maximal efficacy with which GABA stimulated Cl- transport (i.e., increased the Emax). Together, these data support the hypothesis that the psychotropic effects observed after progesterone administration are due to the bioconversion of progesterone to allopregnanolone, which subsequently augments GABAA receptor-mediated function.

Dopaminergic control of male sex behavior in rats: Effects of an intracerebrally-infused agonist

Systemically-administered dopaminergic drugs have been found to facilitate sexual behavior of men and male rats. The present experiments investigated the localization within the brain of dopaminergic effects on copulation of male rats. Apomorphine, a dopamine agonist, was microinfused into the medial preoptic area, caudate-putamen, nucleus accumbens, lateral septum and lateral ventricle. The lowest dose of apomorphine (0.2 microgram) infused into the ventricle reduced the number of ejaculations, slowed the rate of intromitting and decreased the percentage of mounts on which the male gained vaginal intromission. The higher two doses (0.5 and 2.0 micrograms) infused into the medial preoptic area and, in some cases, the ventricle, increased the number of ejaculations and the percentage of mounts with vaginal intromission, increased the rate of intromitting and decreased the latency to ejaculate and the postejaculatory interval before resuming copulation. Infusions into the caudate-putamen and lateral septum were without effect. Those into nucleus accumbens produced only a slight dose-related decrease in latency to begin copulating. The copulatory impairments associated with infusions of the lowest dose into the ventricle may have resulted from stimulation of autoreceptors, or from preferential stimulation by low doses of an undetermined area. The facilitative effects of the two higher doses into the medial preoptic area and lateral ventricle may have been due to stimulation of dopaminergic postsynaptic receptors.

Treatment with an Anabolic-Androgenic Steroid Affects Anxiety-Related Behavior and Alters the Sensitivity of Cortical GABAA Receptors in the Rat

The putative psychotropic effect of the anabolic-androgenic steroid, testosterone propionate (TP), was determined in intact adult male rats after 1 or 2 weeks of continued exposure via subcutaneously implanted capsules. Behavior was assessed in a novel open-field arena and in the elevated plus-maze. In addition, gamma-aminobutyric acid (GABA)-stimulated 36chloride (Cl-) influx was determined in cerebral cortical synaptoneurosomes as a function of TP exposure. The weight of the prostate gland was taken and blood serum level of total testosterone (T) was assayed. One week of TP exposure (approximately 3.5-5.0 mg/kg per day) resulted in anxiolytic behavior, as evidenced by an increase in the exploration of the open arms of the elevated plus-maze. The behavioral effect in the elevated plus-maze was not observed in animals exposed to TP for a 2-week period. Ambulation scores in the novel open field did not change as a function of TP exposure. Blood T levels were increased 7-fold by 1 week of exposure, and increased 10-fold in animals with implants for a 2-week period. After 1 week of TP exposure, the concentration of GABA that elicited 50% of the maximal Cl- influx in cortical synaptoneurosomes (i.e., EC50) was significantly decreased; this effect was not seen in animals exposed to TP for 2 weeks. The maximal efficacy of the GABAA receptor-gated Cl- influx was not affected after 1 or 2 weeks of TP treatment. Thus, 1 week of treatment with TP resulted in anxiolytic behavior that was accompanied by an increase in the sensitivity of cortical GABAA receptors. However, the behavioral and neurochemical changes were no longer present after 2 weeks of TP exposure. These results are discussed in terms of the agonist effects of reduced androgen metabolites at the GABAA receptor and the possible development of tolerance to these effects.

Anxiolytic effects of the neuroactive steroid pregnanolone (3α-OH-5β-pregnan-20-one) after microinjection in the dorsal hippocampus and lateral septum

The anxiolytic effects of the neuroactive steroid, 3α-OH-5β-pregnan-20-one (pregnanolone), were determined after injection into the dorsal hippocampus or lateral septum in adult male rats. An increase in the proportion of time spent on the open arms of the elevated plus-maze was found after 2.5 and 5 μg of pregnanolone in the hippocampus, but not in the lateral septum. Intrahippocampal injection of 2.5 μg of the 3β-epimer of pregnanolone did not affect behavior in the plus-maze; a higher dose of 5 μg produced an anxiogenic effect. In the shock-probe burying test latency to burying behavior was increased by intrahippocampal or intraseptal injection of 2.5 and 5 μg of pregnanolone; the duration of burying behavior was decreased by 0.5, 2.5 and 5 μg of pregnanolone injection in the dorsal hippocampus or lateral septum. The number of contacts with the shock probe was not affected by any dose of pregnanolone in either intracranial site of injection. The anxiolytic effects of intrahippocampal or intraseptal injection of pregnanolone were blocked by intracranial pretreatment with 20 ng of picrotoxin, but not by microinjection of 5 μg of flumazenil or 200 ng of PK 11195. Thus, inhibition of the hippocampus, mediated by the pregnanolones action at the GABAA receptor, produces a general anxiolytic effect. However, similar inhibition in the lateral septum attenuates active avoidance of anxiogenic stimuli (i.e., decreased burying behavior), but not passive avoidance of aversive stimuli (i.e., exploration of open arms of the plus-maze and number of shocks in the probe burying test).

Ovarian endocrine status modulates the anxiolytic potency of diazepam and the efficacy of !g-aminobutyric acid-benzodiazepine receptor-mediated chloride ion transport.

The effect of ovarian steroid hormones on the behavioral and neurochemical sensitivity of the gamma-aminobutyric acid (GABA)-benzodiazepine (BZD) receptor chloride ion channel complex was studied. Locomotor activity and behavior in the elevated plus maze were examined in female rats of various ovarian states as was the efficacy and potency of GABA-stimulated chloride uptake in cortical synaptoneurosomes from proestrous and ovariectomized rats. A significant increase in the exploration of the open arms of the plus maze was observed in lactating females, in relation to diestrous, proestrous, ovariectomized, and pregnant females. The anxiolytic effect of diazepam (DZ) was decreased in ovariectomized females, in relation to proestrus females. Although 1.0 mg/kg DZ in proestrous females resulted in significant anxiolytic activity, this dose was ineffective in ovariectomized females but was reinstated by injection of estradiol benzoate and progesterone. A reduced efficacy of GABA-stimulated chloride ion transport in cortical synaptoneurosomes from ovariectomized females, in relation to that from proestrous females, was observed. Furthermore, the facilitative effect of DZ on the potency of GABA-stimulated chloride ion influx that was observed in cortical synaptoneurosomes from proestrous females was absent in synaptoneurosomes from ovariectomized females. These results are discussed in terms of the effect of ovarian steroids and reduced metabolites on GABA-BZD receptor-mediated functions.

Microinjection of cis-flupenthixol, a dopamine antagonist, into the medial preoptic area impairs sexual behavior of male rats

Systemically administered dopamine agonists have been shown to facilitate copulation in male rats. Microinjection of the dopamine agonist apomorphine into the medial preoptic area has also been reported to facilitate sexual behavior. The present experiments investigated the effects of medial preoptic microinjections of the dopamine antagonist cis-flupenthixol on male rat copulatory behavior. Fewer males initiated copulation and fewer ejaculated following flupenthixol administration. Those males that did ejaculate following flupenthixol injections had fewer ejaculations and longer interintromission intervals. Flupenthixol also antagonized the facilitative effects of apomorphine injections into the medial preoptic area. Flupenthixol and apomorphine produced only minor alterations in noncopulatory behaviors. The results suggest that dopamine receptors within the medial preoptic area are important in the regulation of masculine sexual behavior in the rat.