Reduction of predator odor-induced anxiety in mice by the neurosteroid 3α-hydroxy-4-pregnen-20-one (3αHP)

The effects of the centrally produced allylic neurosteroid, 3α-hydroxy-4-pregnen-20-one (3αHP), on the responses of male mice to an aversive, anxiety-inducing, predator (cat) odor were examined in an odor preference test. Control untreated mice displayed an anxiogenic response to the cat odor, spending a minimal amount of time in a Y-maze in the vicinity of the cat odor. Intracerebroventricular (i.c.v.) administrations of 3αHP had an anxiolytic action, resulting in significant dose-related (0.01–1.0 μg) increases in the amount of time spent in the proximity of the cat odor. These anxiolytic effects of 3αHP were stereospecific, with the stereoisomer, 3β-hydroxy-4-pregnen-20-one (3βHP) having no significant effects on odor preferences. The analgesic, morphine, also had no significant effects on the response to cat odor indicating that the anxiolytic actions of 3αHP were unlikely to be related to any analgesic effects. The effects of 3αHP were significantly reduced by peripheral administrations of the GABA_A antagonists, bicuculline and picrotoxin, but were unaffected by either the benzodiazepine antagonist, Ro 15-1788, or the opiate antagonist, naloxone. These results indicate that the allylic neurosteroid 3αHP has anxiolytic actions involving interactions with the GABA_A receptor.     

Chronic ethanol administration alters the modulatory effect of 5α-pregnan-3α-ol-20-one on the binding characteristics of various radioligands of GABAA receptors

In this study, we investigated the modulatory effect of 5α-pregnan-3α-ol-20-one, a neurosteroid, on the binding characteristics of [ ]flunitrazepam (2 nM), [ ]muscimol (5 nM), and 4 nM [ ]t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5α-Pregnan-3α-ol-20-one potentiated the binding of [ ]flunitrazepam and [ ]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [ ]flunitrazepam as well as [ ]muscimol binding (E_max) in the ethanol-dependent rat cerebellum as compared to control group (p<0.025). Furthermore, 5α-pregnan-3α-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [ ]TBPS at lower concentrations (100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [ ]TBPS binding (−E_max) by 5α-pregnan-3α-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats (p<0.025). These observations suggest that the neurosteroid binding site associated with the γ-aminobutyric acid_A (GABA_A) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.     

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

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 GABA_A 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 GABA_A receptor.     

Synthesis, metabolism and levels of the neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), in rat pituitaries

The neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), is a metabolite of progesterone and a precursor of 3α-hydroxy-5α-pregnan-20-one (5αP3α; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA_A receptor complex, 3αHP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca²⁺-driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3αHP, and since there is the possibility of paracrine and/or autocrine regulation of FSH release, the capacity of pituitary cells to regulate levels (by synthesis, metabolism, and storage) of 3αHP was examined. Anterior pituitaries from random cycling female rats were incubated, either as fragments or as cultured cells, for 1, 4 or 8 h with ³H- or ¹⁴C-labeled progesterone. The steroid metabolites were identified by thin-layer chromatography, autoradiography, high pressure liquid chromatography (HPLC), derivatization and GC/MS. Pituitary cells actively converted progesterone to 3αHP along with 5αP3α, 5α-pregnane-3,20-dione, 20α-hydroxy-5α-pregnan-3-one, 3β-hydroxy-5α-pregnan-20-one, 5α-pregnane-3α(β), 20α-diols, 20α-hydroxy-4-pregnen-3-one, and 4-pregnene-3α(β), 20α-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3α-hydroxysteroid oxidoreductase (3α-HSO), 20α-HSO, 3β-HSO, and 5α-reductase. The activities of 5α-reductase and 3α-HSO were approximately equal and greatly exceeded those of the other enzymes. After 8 h of incubation with 100 ng progesterone per pituitary, about 20% of the progesterone was metabolized and 3.18 ng of 3αHP had been formed. The accumulation of 3αHP increased approximately linearly with the time of incubation. Metabolism studies using [1,2,6,7-³H]3αHP showed that pituitary cells convert about 29% and 8% of the 3αHP to progesterone and 5αP3α, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3αHP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3αHP were about 2–3-fold greater than those of progesterone in the same pituitaries. In older (80–100 days old) cycling rats, the levels of 3αHP were about 9.4 and 18.6 ng/pituitary at 13.00 h and 22.00 h, respectively, on the day of proestrus, while the concomitant circulating levels were 13.7 and 5.4 ng/ml. The results indicate a marked capacity of rat pituitary cells to synthesize the neuroactive and FSH regulating steroid, 3αHP, from progesterone, and in turn to metabolize 3αHP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3αHP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3αHP may be (in part, or in whole) via paracrine or autocrine mechanisms.     

Serotonergic agents modulate antidepressant-like effect of the neurosteroid 3α-hydroxy-5α-pregnan-20-one in mice

The present study demonstrated the antidepressant-like effect of neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α, 5α THP) in mouse forced swim test of depression and its modulation by different serotonergic agents. Pretreatment with the selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), the 5-HT releaser, fenfluramine (10 mg/kg, i.p.), the 5-HT_1A receptor agonist, 8-OH-DPAT (0.1 mg/kg, s.c.), the 5-HT_1B/1C receptor agonist, TFMPP (4 mg/kg, s.c.) and the 5-HT_2A/1C receptor agonist, DOI (2 mg/kg, s.c.) potentiated the antidepressant-like effect of 3α, 5α THP. At these doses the serotonergic agents per se did not modify the duration of immobility. However, fluoxetine (20 mg/kg, i.p.), fenfluramine (20 mg/kg, i.p.) or imipramine (5 or 20 mg/kg, i.p.) not only reduced immobility but also enhanced the antidepressant-like effect of 3α, 5α THP. Such a potentiating effect of the 5-HT_1A or the 5-HT_2A/1C receptor agonist was not antagonized by the sub-effective dose (0.1 mg/kg, s.c.) of their respective antagonists p-MPPI or ketanserin. Pretreatment with p-CPA (300×3 mg/kg, i.p.), a depleter of 5-HT neuronal store failed to block the influence of fluoxetine and fenfluramine on antidepressant-like effect of 3α, 5α THP. The accelerated effect of 3α, 5α THP in presence of serotonergic agents was antagonized by the GABA_A receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3α-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). These findings for the first time demonstrate that serotonergic agents potentiate the antidepressant-like action of 3α, 5α THP, by enhancing the GABAergic tone as a likely consequence of increased brain content of this neurosteroid.     

Cataleptic effect of neurosteroid 3α-hydroxy-5α-pregnan-20-one in mice: modulation by serotonergic agents

The neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) induced catalepsy in mice is modified by dopaminergic, adenosinergic and GABAergic agents. In light of serotonergic agents being implicated in antipsychotic-induced catalepsy and their ability to increase brain neurosteroid content, the present study was undertaken to investigate the effect of various 5-HT agents on catalepsy induced by 3α,5α-THP in mice. Pretreatment with selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), 5-HT releaser, fenfluramine (10 mg/kg, i.p.), 5-HT_1A receptor agonist, 8-OH-DPAT (0.3 mg/kg, s.c.), 5-HT_1B/1C receptor agonist, TFMPP (3 mg/kg, i.p.), 5-HT_2A/1C receptor agonist, DOI (1.5 mg/kg, s.c.) and 5-HT₃ agonist, 2-methylserotonin (5 mg/kg, i.p.) potentiated the catalepsy induced by exogenous administration of 3α,5α-THP. Furthermore, FGIN 1–27, an MDR agonist that increases endogenous content of 3α,5α-THP although per se failed to exhibit any cataleptic effect but enhanced the cataleptic response in combination with these serotonergic agents. The potentiating action of 5-HT_1A, 5-HT_2A/1C or 5-HT₃ receptor agonist on 3α,5α-THP induced catalepsy was not blocked by prior administration of sub-effective dose (1 mg/kg, s.c.) of their respective receptor antagonists pindolol, ritanserin or ondansetron or by pretreatment with serotonergic neurotoxin 5,7-DHT (100 μg/mouse, i.c.v.). However this effect of different serotonergic agents was antagonized by the GABA_A receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3α-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). The 5-HT agents enhance neurosteroid-induced catalepsy by increasing GABAergic tone, likely as a consequence of increased brain content of 3α,5α-THP.     

Evidence that 3α-hydroxy-5α-pregnan-20-one is a physiologically relevant modulator of gaba-ergic neurotransmission

3α-Hydroxy-5α-pregnan-20-one (HPO) is a progesterone metabolite which exhibits narcotic properties at high concentrations by interactions with the receptor for gamma-aminobutyric acid (GABA). The present investigation characterized low-dose effects of HPO on GABA_A receptor binding, by determining the allosteric properties of HPO on the in vitro binding of ³H-muscimol to membrane fractions from the cerebella of ovariectomized rats. A newly developed method for tissue preparation was used to wash out endogenous ligands interfering with the assay. HPO reduced the affinity of ³H-muscimol to GABA_A receptor sites by 52% and enhanced the number of accessible binding sites from 5.5±0.5 to 7.5±1.3 pmol/mg protein at subnanomolar (0.1 nM) HPO concentrations. The modulatory effects of HPO on GABA_A receptor binding provide evidence that this pregnane steroid might be a physiologically relevant modulator of GABAergic neurotransmission.     

GABA-ergic control of α-Melanocyte-Stimulating Hormone (α-MSH) release by frog neurointermediate lobe in vitro

Measurement of glutamate decarboxylase (GAD) activity in the intermediate lobe of the frog pituitary and brain showed that neurointermediate lobe extracts represented 12% of the GAD activity detected in the whole brain. No significant activity was measured in distal lobe extracts. Immunocytochemical studies revealed GAD-containing fibers among the parenchyma! cells of the pars intermedia. The localization of GAD-like material in the intermediate lobe of the frog pituitary suggested a possible role of γ-aminobutyric acid (GABA) in the regulation of melanotropic cell secretion. Administration of GAB A (10⁻⁶ to 10⁻⁴ M), to perifused neurointermediate lobes caused a brief stimulation of alpha-melanocyte stimulating hormone (α-MSH) release followed by an inhibition. Picrotoxin (10⁻⁴ M), a Cl⁻ channel blocker, abolished only the stimulatory effect of GAB A (10⁻⁴ M), whereas bicuculline (10⁻⁴ M), a specific antagonist of GABA_A receptors, totally inhibited the effects of GABA (both stimulatory and inhibitory phases). Bicuculline induced by itself a slight stimulation of α-MSH release, suggesting that GABA-ergic nerve fibers present in the intermediate lobe are functionally active in vitro. The GABA_A agonist muscimol (10⁻⁷ to 10⁻⁴ M) mimicked the biphasic effect of GABA on α-MSH release. Administration of baclofen, a specific GABA_BB agonist (10⁻⁷ to 10⁻⁴ M) induced a dose-dependent inhibition of α-MSH secretion. In contrast to GABA or muscimol, baclofen did not cause any stimulatory effect whatever the dose. Taken together these result suggested that GABA_A and GABa_b receptors were present on frog melanotrophs. Since bicuculline totally inhibited GABA effects (stimulation and inhibition) on α-MSH release, it appears however that the effect of GABA is mainly achieved through activation of G AB A_A receptors.     

Upregulation of GABAA-receptor α1- and α2-subunit mRNAs following ischemic cortical lesions in rats

Focal cortical lesions are associated with a functional downregulation of the GABAergic system in perilesional tissue lasting (at least) several weeks. The molecular mechanisms underlying this phenomenon are still poorly understood. Here we used RT-PCR to investigate whether mRNA-levels of two α-subunits of the GABA_A-receptor (α1- and α2-subunits) change following ischemic cortical lesioning. The results show that 7 days after lesion induction mRNA-levels for both the α1- and α2-subunits are increased threefold in perilesional tissue ipsilateral, but not contralateral to the lesion. Taken together with the results of a previous immunohistochemical study in which a moderate decrease of the α1-subunit-protein and no change for the α2-subunit [T. Neumann-Haefelin, J.F. Staiger, C. Redecker, K. Zilles, J.M. Fritschy, H. Mohler, O.W. Witte, Immunohistochemical evidence for dysregulation of the GABAergic system ipsilateral to photochemically induced cortical infarcts in rats. Neuroscience (Oxford) 87 (4) (1998) 871–879] was observed, this is interpreted as a partial block of translation in the perilesional tissue surrounding cortical ischemic lesions.     

Pregnanolone (3α-hydroxy-5α-pregnane-20-one), a progesterone metabolite, facilitates inhibition of synaptic transmission in the Schäffer collateral pathway of the guinea pig hippocampus in vitro

Pregnanolone (3α-hydroxy-5α-pregnane-20-one, a metabolite of progesterone) caused a significant depression of the amplitude of the population spike evoked in stratum pyramidale in CA1 of the guinea pig hippocampus in vitro. Local application of pregnanolone on the surface of the slice in stratum oriens depressed the population spike without effects on the presynaptic spike and the population excitatory postsynaptic potential simultaneously recorded in stratum radiatum. The depression was dose-dependent and was observed with a minimum latency of 10 s after application of a 0.5-nl droplet of 3.1 μM pregnanolone. The concentration at the recording site was computed to be 0.2 μM. The duration of the depression was 20–30 min. The depression was significantly reduced during perfusion of the slice bath with 100 μM picrotoxin in artificial cerebrospinal fluid. When pregnanolone was applied locally in stratum radiatum, the amplitudes of the presynaptic spike, the population excitatory postsynaptic potential and the population spike were depressed. The effects on the presynaptic spike and the population excitatory postsynaptic potential vanished with different time courses. It is concluded that the depression of the population spike was caused by GABA_A-mediated inhibition of the pyramidal neurones. The role of pregnanolone as a positive modulator of the GABA_A receptor and the effect of this modulation on the complex mechanisms underlying catamenial epilepsy are discussed.     

Inhibitory effect of 17β-estradiol in the parabrachial nucleus is mediated by GABA

In the present investigation, electrophysiological recordings of thalamic relay neurons were used to investigate the role of estrogen as a modulator of visceral afferent information through the PBN to forebrain structures. Experiments were done in anaesthetized (sodium thiobutabarbitol; 100 mg/kg) male and ovariectomized female rats supplemented for 7 days prior with either 17β-estradiol (OVX-E₂) or saline (OVX-S). A portion of the right cervical vagus was isolated for the electrical activation (0.8 Hz, 2 ms duration) of visceral afferents. The evoked single and multi-unit activity was recorded via a recording electrode in the ventrobasal thalamus. Exogenous microinjection of 17β-estradiol (0.1, 0.25 and 0.5 μM; 200 nl) into the parabrachial nucleus (PBN) produced a significant, dose-dependent attenuation in the magnitude of visceral afferent activation-evoked responses of neurons recorded in the thalamus in both male and OVX-E₂ groups. No effect on evoked thalamic activity was observed following injection of estrogen into the PBN of OVX-S animals. Co-injection of estrogen with the GABA_A receptor antagonist, bicuculine (0.1 μM; 200 nl) but not phaclofen (GABA_B; 0.1, 0.5 or 1 μM; 200 nl) resulted in an increase in the evoked thalamic response in males (55±11%) and OVX-E₂ female (68±15%) rats. These studies suggest that estrogen inhibits neurotransmission in the PBN via an interaction with the GABA_A receptor to modulate the flow of visceral information to the thalamus.     

GABAA α2 mRNA levels are decreased following induction of spontaneous epileptiform discharges in hippocampal-entorhinal cortical slices

Exposure of hippocampal slices to Mg²⁺ free media (0 Mg) has been shown to trigger full production of stimulus-induced seizure activity after restoration of physiological conditions [1]. In the present study employing hippocampal entorhinal cortical slices (HEC), spontaneous epileptiform discharges (SEDs) were induced using 0 Mg treatment following the return of the slices to physiological conditions. To evaluate the effect of sustained epileptiform activity on gene expression in this HEC slice preparation, changes in mRNA levels of the GABA_A α1 and α2 and β CaM Kinase II subunits were measured using in situ hybridization. HEC slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in the presence or absence of Mg²⁺ for 3 h, then placed in oxygenated ACSF containing Mg²⁺ for up to 3 h. Control slices were maintained in Mg²⁺ containing ACSF for up to 6 h. Recurrent SEDs were observed in 0 Mg pre-treated slices while no epileptiform discharges were seen in control slices. Following induction of SEDs by 0 Mg pre-treatment, a significant decrease in mRNA encoding GABA_A α2 was found in the CA1, CA2, CA3 and dentate gyrus (DG) regions of the hippocampus for up to 3 h after treatment. Levels of mRNA for GABA_A α1 and β CaM Kinase II were not affected. The results document a decrease in GABA_A α2 gene expression following the induction of SEDs in the HEC slice preparation and suggest that rapid changes in neuronal gene expression may contribute to long lasting excitability changes associated with the induction of epilepsy.     

Pyriform cortex β-waves: odor-specific sensitization following repeated olfactory stimulation

The first exposure to the odors of carbon tetrachloride, isopentenyl methyl sulfide, methyl ethyl ketone, 2-propylthietane, salicylaldehyde, toluene, 2,4,5-trimethyl thiazoline, or xylene elicits a weakly developed 20 Hz wave response (β-waves) in central olfactory structures in the rat. Repeated presentations of these odors produces a gradual enhancement or sensitization of olfactory β-waves over 5–10 trials given in 1–3 min. The odors of 2-aminoacetophenone and 2-hydroxyacetophenone produce sensitization after an average of 15–17 presentations. The sensitized β-wave response to the odors of 2-propylthietane and xylene persists for at least 5 days and probably much longer. Sensitization to one odor transfers partially or not at all to other novel odors even though repeated presentation of the new odor also produces sensitization. Since the initial negative response of the olfactory mucosa (presumably due to receptor depolarization) is not enhanced by repeated olfactory stimulation, it is presumed that the altered responsivity is due to synaptic changes in central olfactory structures such as the olfactory bulb or pyriform cortex. Finally, data are presented to show that behavioral antifeedant activity by an odorant does not invariably mean that the odorant has the ability to elicit an olfactory β-wave response.     

The testosterone metabolite and neurosteroid 3α-androstanediol may mediate the effects of testosterone on conditioned place preference

The abuse of androgens may be related to their ability to produce positive, hedonic interoceptive effects. Conditioned Place Preference (CPP) has been used in many experiments to examine hedonic effects of drugs. This review is focused on studies from our laboratory that utilized CPP to examine potential positive hedonic effects of testosterone (T), and its androgenic metabolite dihydrotestosterone (DHT), and its metabolite 3α-androstanediol (3α-diol). We hypothesized that administration of a high concentration of 3α-diol would produce a CPP, pharmacological concentrations of plasma androgens, and alter androgen receptors (AR) and the function of GABA_A/benzodiazepine receptor complexes (GBR). In our studies, we observed that systemic 3α-diol (1.0 mg/kg) prior to exposure to the non-preferred side of a CPP chamber significantly increased preference for the non-preferred side of the chamber compared to baseline preference and homecage controls. Furthermore, administration of T, DHT, or 3α-diol increased levels of these androgens, decreased ARs (decreased seminal vesicle weight and intrahypothalamic AR) and GBR function (decreased GABA-stimulated chloride influx in cortical synaptoneurosomes, and muscimol binding in the hippocampus compared to control groups). With systemic administration of 3α-diol that enhanced CPP, concentrations of 3α-diol were increased in the nucleus accumbens (NA). Central implants of T, DHT, or 3α-diol to the NA also produced a CPP compared to baseline preference and vehicle controls. These data indicate that systemic 3α-diol is more effective at enhancing CPP and increasing circulating 3α-diol levels than is T or DHT and that central administration of 3α-diol to the NA can condition a place preference. These findings indicate that 3α-diol produces positive hedonic effects and suggest that T’s variable effects on CPP may be due in part to T’s metabolism to 3α-diol.     

Influence of gender on chronic ethanol-induced alterations in GABAA receptors in rats

Ethanol dependence, arising from chronic ethanol exposure, is associated with neuroadaptations of GABA_A receptors, evidenced by alterations in various behaviors, receptor responsiveness and subunit gene expression. The present studies explored the effects of ethanol dependence in female rats for comparison with previous studies in our laboratory using male rats. We found that ethanol dependence resulted in differential effects on GABA_A receptor gene expression in female rat cerebral cortex compared to ethanol dependent male rats. Notably, chronic ethanol consumption did not change GABA_A receptor α1 subunit peptide levels in ethanol dependent female rat cortex, in contrast to previously observed decreases in α1 subunit expression in ethanol dependent male rat cortex. The effects of ethanol dependence on additional GABA_A receptor subunit peptide levels (α4, β2/3 and γ2) were similar, but not identical, between female and male rat cortex. When directly compared within the experiment, male and female rats had similar baseline bicuculline seizure thresholds and displayed a similar increase in seizure susceptibility during ethanol withdrawal. Ethanol withdrawn female rats were cross tolerant to the anticonvulsant effects of diazepam, similar to the findings in ethanol withdrawn male rats. Ethanol withdrawn female rats showed a dose-dependent enhancement of the anticonvulsant effect of the neuroactive steroid, THDOC (3α,21-dihydroxy-5α-pregnan-20-one) compared to control animals. This finding is similar to previous observations of increased sensitivity to the anticonvulsant effect of 3α,5α-THP (3α-hydroxy-5α-pregnan-20-one) in ethanol withdrawn male and female rats. In addition, low dose administration of THDOC elevated seizure thresholds in ethanol withdrawn female but not male rats, suggesting that ethanol withdrawn female rats were more responsive to the anticonvulsant effects of this neurosteroid than were ethanol withdrawn male rats. These findings show that gender impacts on adaptations in GABA_A receptors elicited by ethanol dependence. However, the physiological outcomes of the differential alterations are not clear. Taken together, these studies suggest that additional mechanisms, beyond effects on GABA_A receptor gene expression are involved in the mediation of ethanol dependence and withdrawal.     

Acute effects of γ-vinyl GABA (vigabatrin) on hippocampal GABAergic inhibition in vitro

The acute effects of γ-vinyl-GABA (GVG) on GABAergic inhibition were investigated in the hippocampal slice preparation using the paired-pulse test of inhibition during extracellular recordings. Superfusion of GVG (100–500 μM) for 60 min resulted in a concentration-dependent decrease in GABAergic inhibition. Slices superfused with higher concentrations of GVG (0.5–1 mM) were hyperexcitable as demonstrated by the appearance of multiple spikes. Binding studies showed that GVG (1 mM) had no effect on the binding of [³H]flunitrazepam or [³H]TBOB and displaced no more than 15% of specific [³H]GABA binding, which indicates that GVG-induced disinhibition is not mediated through an action at the GABA_A receptor complex. Consistent with this suggestion is the finding that GVG (500 μM) had little effect on the inhibition of the orthodromically evoked CA1 population spike produced by the GABA_A receptor agonist muscimol (10 μM), whereas this inhibition was considerably attenuated by the GABA_A receptor antagonist, bicuculline methiodide (5 μM). The results of this study suggest that the acute actions of GVG on the GABAergic neurotransmitter system are not involved in its anticonvulsant effect.     

Serotonin potentiates the response of neurons of the superficial laminae of the rat spinal dorsal horn to γ-aminobutyric acid

Employing the Nystatin-perforated whole-cell patch-clamp recording technique, the modulatory effects of serotonin (5-HT) on γ-aminobutyric acid (GABA)-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (laminae I and II) of the rat spinal dorsal horn. The results showed: (1) GABA acted on GABA_A receptors and elicited inward Cl⁻ currents (I_GABA) at a holding potential (V_H) of −40 mV; (2) 5-HT potentiated GABA-induced Cl⁻ current without affecting the reversal potential of I_GABA and the apparent affinity of GABA to its receptor; (3) α-methyl-5-HT, a selective agonist of 5-HT₂ receptor, mimicked the potentiation effect of 5-HT on I_GABA, whereas ketanserine, an antagonist of 5-HT₂ receptor, blocked the potentiation effect of 5-HT; (4) Chelerythrine, an inhibitor of protein kinase C, reduced the potentiation effect of 5-HT on I_GABA. The present results indicate: (1) The potentiation of 5-HT on I_GABA is mediated by 5-HT₂ receptor and through a protein kinase-dependent transduction pathway; (2) The interactions between 5-HT and GABA might play an important role in the modulation of nociceptive information transmission at spinal cord level.     

An investigation on the role of nitric oxide in the modulation of the binding characteristics of various radioligands of GABAA receptors by 5α-pregnan-3α-ol-20-one in the rat brain regions

Chronic administration of N^ω-nitro- -arginine methyl ester ( -NAME), an inhibitor of nitric oxide synthase, diminished the ability of 5α-pregnan-3α-ol-20-one, a neurosteroid, to potentiate the [³H]muscimol (5 nM) binding in the rat hippocampus but not in the cerebellum or cerebral cortex. This effect of NAME was stereospecific and susceptible to reversal by the pre-treatment of rats with -arginine. However, chronic administration of -NAME did not affect the modulation of the [³H]flunitrazepam (2 nM) or [³⁵S]TBPS (4 nM) binding by the neurosteroid in any of the brain regions investigated in this study. These results suggest that nitric oxide may be involved in some of the effects of neurosteroids in hippocampus.     

Dissociation of μ and δ opioid receptor-mediated reductions in evoked and spontaneous synaptic inhibition in the rat hippocampus in vitro

Modulation of γ-aminobutyric acid (GABA)-mediated inhibition, and glutamate-mediated excitation by highly selective μ and δ opioid agonists was studied using intracellular recordings of CA1 pyramidal neuron synaptic responses in superfused hippocampal slices. Equimolar concentrations of the selective μ agonist,

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(DAGO), or the δ selective agonist, [ -Pen², -Pen⁵]-enkephalin (DPDPE), reversibly increased the amplitudes of excitatory post-synaptic potentials (EPSPs), evoked by Schaffer collateral/commissural stimulation, without altering the input resistance or resting membrane potential of these CA1 pyramidal neurons. The increased EPSP amplitudes resulting from superfusion with the enkephalin analogs were qualitatively similar to those caused by the GABA_A receptor antagonist, bicuculline methiodide (BM1). Specific stimulation/recording protocols and micro-lesions of the slices were used to evoke relatively pure forms of recurrent and feed-forward GABA-mediated inhibitory post-synaptic potentials (IPSPs). The μ opioid agonist DAGO reduced both recurrent and feed-forward IPSPs, while the δ agonist DPDPE had no effect upon these responses. To test the hypothesis that the enhancement of pyramidal neuron EPSPs by δ (and μ) opioids was due to the reduction of an inhibitory potential that was coincident with the EPSP, DPDPE or the μ agonist, DAGO, were applied while recording monosynaptic IPSPs following the elimination of EPSPs by the glutamate receptor antagonists, -2-amino-5-phosphonovalerate (APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). The μ agonist, DAGO, reversibly reduced these pharmacologically isolated IPSPs, while the δ agonist, DPDPE, had no effect upon these responses. Despite the fact that the δ agonist, DPDPE, had no effect on recurrent, feed-forward or monosynaptic evoked IPSPs, this enkephalin did reversibly reduce the frequency of spontaneously occurring IPSPs, measured using whole-cell recordings with pipettes containing 65 mM KC1. The μ agonist, DAGO, and the GABA_A antagonist. BMI, similarly reduced spontaneous IPSP rates. We conclude from these data that μ and δ opioid receptor activation increases EPSPs via the reduction of a form of GABAergic inhibition that is difficult to characterize, and which may be distinct from conventional feed-forward and recurrent inhibition. Furthermore, δ opioids seem to reduce this form of GABAergic inhibition selectively, while μ opioids reduced this inhibition, and conventional feed-forward and recurrent IPSPs as well.     

Ontogeny of sexual difference in α-bungarotoxin binding capacity in the mouse amygdala

We have shown a sex difference in the nucleus amygdaloideus medialis posterior (NAMP) of the mouse with respect to the binding capacity for α-bungarotoxin (α-BGT) under various steroid-hormonal environments. The present study describes histochemically the postnatal development of α-BGT binding capacity in the NAMP of the intact male and female mouse, and characterizes biochemically the toxin binding component at different developmental stages. Light microscopic autoradiography using radio-iodinated α-BGT revealed characteristic patterns of development of α-BGT binding capacity in the NAMP of both sexes. On the day of birth, the autoradiographic grain density for [¹²⁵I]α-BGT binding sites was low in the NAMP and no sex difference was detected. During the next 4 days the grain density increased in each sex, but the density in the female increased to a lesser extent than in the male, resulting in a marked sex difference at 4 days after birth. The grain density in each sex was maximal at 7–10 days and then declined toward the adult level by 28 days of age. The density in the male exceeded that in the female throughout postnatal life except for the day of birth. The biochemical filtration assay on the tissue homogenates provided evidence suggesting that α-BGT binding sites in the posterior corticomedial amygdaloid region including the NAMP are sexually different in number, but not in the binding affinity, at an early postnatal age as well as in adulthood. These results indicate the importance of the early postnatal days for the sexual differentiation of α-BGT binding sites in the NAMP.