Treatment with gut bifidobacteria improves hippocampal plasticity and cognitive behavior in adult healthy rats.

At the present time, gut microbiota inspires great interest in the field of neuroscience as a function of its role in normal physiology and involvement in brain function. This aspect suggests a specific gut-brain pathway, mainly modulated by gut microbiota activity. Among the multiple actions controlled by microbiota at the brain level, neuronal plasticity and cognitive function represent two of the most interesting aspects of this cross-talk communication. We address the possible action of two-months implementation of gut Bifidobacteria using a mixture of three different strains (B-MIX) on hippocampal plasticity and related cognitive behavior in adult healthy Sprague Dawley rats. B-MIX treatment increases the hippocampal BDNF with a parallel gain in dendritic spines' density of hippocampal CA1 pyramidal neurons. Electrophysiological experiments revealed a significant increment of HFS-induced LTP formation on the CA1 hippocampal region in B-MIX treated rats. All these effects are accompanied by a better cognitive performance observed in B-MIX treated animals with no impairments in locomotion activity. Therefore, in adult rats, the treatment with different strains of bifidobacteria is able to markedly enhance neuronal plasticity and the CNS function influencing cognitive behavior, an effect that may suggest a potential therapeutic treatment in brain diseases associated with cognitive functions.

Social enrichment reverses the isolation-induced deficits of neuronal plasticity in the hippocampus of male rats.

Environmental enrichment is known to improve brain plasticity and protect synaptic function from negative insults. In the present study we used the exposure to social enrichment to ameliorate the negative effect observed in post weaning isolated male rats in which neurotrophic factors, neurogenesis, neuronal dendritic trees and spines were altered markedly in the hippocampus. After the 4 weeks of post-weaning social isolation followed by 4 weeks of reunion, different neuronal growth markers as well as neuronal morphology were evaluated using different experimental approaches. Social enrichment restored the reduction of BDNF, NGF and Arc gene expression in the whole hippocampus of social isolated rats. This effect was paralleled by an increase in density and morphology of dendritic spines, as well as in neuronal tree arborisation in granule cells of the dentate gyrus. These changes were associated with a marked increase in neuronal proliferation and neurogenesis in the same hippocampal subregion that were reduced by social isolation stress. These results further suggest that the exposure to social enrichment, by abolishing the negative effect of social isolation stress on hippocampal plasticity, may improve neuronal resilience with a beneficial effect on cognitive function.

Maternal separation attenuates the effect of adolescent social isolation on HPA axis responsiveness in adult rats.

Adverse early life experiences that occur during childhood and adolescence can have negative impacts on behavior later in life. The main goal of our work was to assess how the association between stressful experiences during neonatal and adolescent periods may influence stress responsiveness and brain plasticity in adult rats. Stressful experiences included maternal separation and social isolation at weaning. Three hours of separation from the pups (3-14 PND) significantly increased frequencies of maternal arched-back nursing and licking-grooming across the first two weeks postpartum. Separation also induced a long-lasting increase in dams blood levels of corticosterone. Maternal separation did not modify brain and plasma allopregnanolone and corticosterone levels in adult offspring, but they demonstrate partial recovery from the reduction induced by social isolation during adolescence. Moreover, the enhancement of corticosterone and allopregnanolone levels induced by foot shock stress in socially isolated animals that were subjected to maternal separation was markedly reduced with respect to that observed in animals that were just socially isolated. All experimental groups showed a significant reduction of BDNF and Arc protein expression in the hippocampus. However, the reduction of BDNF observed in animals that were maternally separated and subjected to social isolation was less significantly pronounced than in animals that were just socially isolated. The results sustained the mismatch hypothesis stating that aversive experiences early in life trigger adaptive processes, thereby rendering an individual to be better adapted to aversive challenges later in life.

Penetration enhancer-containing vesicles: composition dependence of structural features and skin penetration ability.

In this work, we focused on how composition and preparation method of vesicles might affect their morphological features and delivery performances. Penetration Enhancer-containing Vesicles, PEVs, vesicles containing a water miscible penetration enhancer (Transcutol® P; 10%, 20%, 30% v/v) and encapsulating diclofenac sodium, were formulated and compared with conventional liposomes. A cheap and unpurified commercial mixture of phospholipids, fatty acids, and triglycerides (Phospholipon® 50) was used, and the effects of this heterogeneous composition (along with the presence or absence of transcutol and the production method) on vesicle morphology, size, surface charge, drug loading, and stability were investigated. The variations in vesicle structure, bilayer thickness, and number of lamellae were assessed by TEM and Small and Wide Angle X-ray Scattering, which also proved the liquid state of the vesicular bilayer. Further, vesicles were evaluated for ex vivo (trans)dermal delivery, and their mode of action was studied performing a pre-treatment test and confocal laser scanning microscopy analyses. Results showed the formation of multi- and unilamellar vesicles that provided improved diclofenac delivery to pig skin, influenced by vesicle lipid composition and structure. Images of the qualitative CLSM analyses support the conclusion that PEVs enhance drug transport by penetrating intact the stratum corneum, thanks to a synergic effect of vesicles and penetration enhancer.

Social isolation stress and neuroactive steroids.

Social isolation of rats immediately after weaning is associated to a reduction in the cerebrocortical and plasma concentrations of progesterone and its metabolites 3alpha,5alpha-TH PROG and 3alpha,5alpha-THDOC. Although we found that the basal plasma concentration of adrenocorticotropic hormone in isolated rats was slightly decreased compared with that in group-housed animals no other significant changes were found in the steroidogenic machinery (peripheral benzodiazepine receptors, steroidogenic regulatory protein (StAR)). However, the functional response of the hypothalamic-pituitary-adrenal axis HPA axis to an acute stressful stimulus (foot shock), or to an acute injection of ethanol or isoniazid is markedly increased in isolated rats. Behavioral studies have also indicated that the ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect of isolation is prevented by treatment with the 5alpha-reductase inhibitor finasteride. Social isolation modified the effects of ethanol on the amounts of StAR mRNA and protein in the brain suggesting an alteration in the mechanism of cholesterol transport in mitochondria. Moreover, the amounts of the alpha4 and delta subunits of the GABA(A) receptor in the hippocampus were increased in isolated rats, and these effects were accompanied by an increase in GABA(A) receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. Ethanol also increased the amplitude of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents (mIPSC) recorded from CA1 pyramidal neurons with a greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed, an effect inhibited by finasteride.

Changes in GABAA receptor gamma 2 subunit gene expression induced by long-term administration of oral contraceptives in rats.

The effects of oral contraceptives (OCs) on neurosteroid concentrations were evaluated in female rats and women. In rats, ethynylestradiol and levonorgestrel (0.030 and 0.125 mg, respectively, subcutaneously) administered daily for 6 weeks reduced the concentrations of pregnenolone (-41%) progesterone (-74%) and allopregnanolone (-79%) in the cerebral cortex; the plasma concentrations of these steroids were also reduced but by smaller extents. OC administration for 3 months also reduced the serum concentrations of pregnenolone, progesterone and allopregnanolone in women. Chronic administration of OCs in rats increased the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor gamma 2L and gamma 2S subunit mRNAs and the relative protein in the cerebral cortex, while the amounts of various alpha and beta subunit mRNAs were unaffected. Ovariectomy did not modify the effect of OCs administration on the concentrations of neurosteroids in the rat cerebral cortex (but not in the plasma) as well as on the GABA(A) receptor gene expression, suggesting a direct effect of OCs in brain. Finally, rats treated with OCs exhibited an anxiety-like behavior in the elevated plus-maze test. These results indicate that long-term treatment with OCs induced a persistent reduction in the concentrations of pregnenolone, progesterone and its GABA(A) receptor-active metabolite, allopregnanolone, both in rats and women. In rats this effect was associated with a plastic adaptation of GABA(A) receptor gene expression in the rat cerebral cortex.

Role of allopregnanolone in regulation of GABA(A) receptor plasticity during long-term exposure to and withdrawal from progesterone.

Here we summarize recent data from our laboratory pertaining to the effects of fluctuations in the brain concentrations of the progesterone (PROG) metabolite allopregnanolone (3alpha,5alpha-TH PROG) on the expression and function of gamma-aminobutyric acid type A (GABA(A)) receptors. The effects of long-term exposure to progesterone and of its sudden withdrawal on the activity of GABA(A) receptors and on the abundance of receptor subunit mRNAs were examined in cultured rat cerebellar granule cells and cortical neurons. The effects of a persistent reduction in the brain concentration of 3alpha,5alpha-TH PROG on GABA(A) receptor function and gene expression were examined in vivo in rats subjected to long-term administration of oral contraceptives. Our results demonstrate that long-lasting changes in the exposure of GABA(A) receptors to this PROG metabolite induce marked effects on receptor structure and function. These effects of 3alpha,5alpha-TH PROG appear to be mediated through modulation of GABA(A) receptor signaling mechanisms that control the expression of specific receptor subunit genes. Furthermore, the specific outcomes of such signaling appear to differ among neurons derived from different regions of the brain. Neuroactive steroids such as 3alpha,5alpha-TH PROG might thus exert differential actions on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological effects induced by these compounds.

Antagonism by pivagabine of stress-induced changes in GABAA receptor function and corticotropin-releasing factor concentrations in rat brain.

Pivagabine [4-(2.2-dimethyl-l-oxopropylamino) butanoic acid] (PVG) is a hydrophobic 4-aminobutyric acid derivative with neuromodulatory activity. The effects of subchronic treatment with PVG on stress-induced changes both on brain concentrations of corticotropin-releasing factor (CRF) and neurosteroids and on the function of the gamma-aminobutyric acid type A (GABAA) receptor complex were investigated in male rats. Subchronic treatment with PVG (100-200 mg/kg, i.p.) resulted in a dose-dependent inhibition of the foot shock-induced increase in the binding of t-[35S]butylbicyclophosphorothionate to unwashed membranes prepared from the cerebral cortex of rats killed immediately after stress; PVG treatment alone had no effect on this parameter. This antagonistic action of PVG was also shown in adrenalectomized-orchietomized rats. Foot-shock stress decreased by 74% and increased by 125% the CRF concentration in the hypothalamus and cerebral cortex, respectively. PVG prevented these effects of stress on CRF concentration in both brain regions; this drug per se reduced hypothalamic CRF concentration by 52% but had no effect in the cortex. Moreover, intracerebroventricular injection of CRF, like stress, induced a dose-dependent increase of [35S]TBPS binding to cerebral cortical membranes: an effect not prevented by subchronic treatment of PVG. Finally, PVG did not antagonize the stress-induced increases in the concentrations of neuroactive steroids in brain or plasma. These results suggest that the marked antistress action of PVG is mediated by antagonizing the effects of stress on GABA(A) receptor function and CRF concentrations in the brain, but not by altering the stress-induced increase in neurosteroid concentrations.

Increased abundance of GABAA receptor subunit mRNAs in the brain of Long-Evans Cinnamon rats, an animal model of Wilson's disease.

The abundance of mRNAs encoding various subunits of the gamma-aminobutyric acid type A (GABAA) receptor was examined in different regions of the brain of Long-Evans Cinnamon (LEC) rats, an animal model of Wilson's disease (WD). The measurements were performed at two different stages of disease: at 9 weeks of age, when no symptoms are evident, and at 15 weeks of age, when 90% of the animals develop jaundice. The amounts of the gamma2L and gamma2S subunit mRNAs in the striatum, cerebellum, and cerebral cortex of LEC rats at 9 weeks of age were increased (+25 to +35%) compared with those in LE rats of the same age; these differences were no longer apparent in 15-week old animals. The amount of alpha1 subunit mRNA was also significantly increased (+30%) in the cerebellum of LEC rats at 9 weeks of age; although a smaller increase (+20%) was still evident at 15 weeks of age, this was not statistically significant. The amount of beta2 subunit mRNA was increased in the cerebellum (+32%) and hippocampus (+21%) of LEC rats at 9 weeks of age, but no differences with LE rats were apparent at 15 weeks. The onset of isoniazid-induced seizures in LEC rats at 9 weeks of age was significantly delayed compared with that in LE rats. These results demonstrate abnormal expression of GABAA receptor subunit genes in the brain of LEC rats, and they suggest that this altered expression is associated with an increase in GABAergic tone.

Role of brain allopregnanolone in the plasticity of gamma-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery.

The relation between changes in brain and plasma concentrations of neurosteroids and the function and structure of gamma-aminobutyric acid type A (GABAA) receptors in the brain during pregnancy and after delivery was investigated in rats. In contrast with plasma, where all steroids increased in parallel, the kinetics of changes in the cerebrocortical concentrations of progesterone, allopregnanolone (AP), and allotetrahydrodeoxycorticosterone (THDOC) diverged during pregnancy. Progesterone was already maximally increased between days 10 and 15, whereas AP and allotetrahydrodeoxycorticosterone peaked around day 19. The stimulatory effect of muscimol on 36Cl- uptake by cerebrocortical membrane vesicles was decreased on days 15 and 19 of pregnancy and increased 2 days after delivery. Moreover, the expression in cerebral cortex and hippocampus of the mRNA encoding for gamma2L GABAA receptor subunit decreased during pregnancy and had returned to control values 2 days after delivery. Also alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, and gamma2S mRNAs were measured and failed to change during pregnancy. Subchronic administration of finasteride, a 5alpha-reductase inhibitor, to pregnant rats reduced the concentrations of AP more in brain than in plasma as well as prevented the decreases in both the stimulatory effect of muscimol on 36Cl- uptake and the decrease of gamma2L mRNA observed during pregnancy. These results indicate that the plasticity of GABAA receptors during pregnancy and after delivery is functionally related to fluctuations in endogenous brain concentrations of AP whose rate of synthesis/metabolism appears to differ in the brain, compared with plasma, in pregnant rats.

Molecular and functional adaptation of the GABA(A) receptor complex during pregnancy and after delivery in the rat brain.

The abundance of gamma-aminobutyric acid receptor type A (GABAA receptor) subunit mRNAs and polypeptides as well as muscimol-stimulated 36Cl- uptake were measured in rat cerebral cortex or hippocampus at various times during pregnancy and after delivery. RNase protection assays revealed that the amount of the gamma2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, and then returned to control values around the time of delivery. A similar pattern was observed for the alpha5 subunit mRNA in the cerebral cortex, whereas no significant changes were apparent for alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3 and gamma2S subunit mRNAs. The amounts of gamma2 and alpha1 proteins in the cerebral cortex were measured by immunoblot analysis; whereas the abundance of gamma2 protein decreased during pregnancy, no change was detected in the amount of alpha1 protein. Evaluation for functional significance of the down-regulated gamma2 and alpha5 subunit was made by determining the GABAA receptor function assessed by measurement of muscimol-stimulated 36Cl- uptake in cerebral cortical membrane vesicles. Muscimol-induced 36Cl- uptake was markedly reduced during of pregnancy compared with rats in oestrus. At this same time, the potentiating effects of diazepam and allopregnanolone on muscimol stimulation of 36Cl- uptake also were reduced. In contrast, the effects of muscimol, allopregnanolone and diazepam were significantly increased, relative to animals in oestrus, after delivery.

Modulation of gamma-aminobutyric acid (GABA) receptors and the feeding response by neurosteroids in Hydra vulgaris.

Gamma-Aminobutyric acid (GABA) receptors are present in membrane preparations from Hydra vulgaris, one of the most primitive organisms with a nervous system. These receptors are sensitive to muscimol and benzodiazepines and appear to be important in the regulation of the feeding response. The effects of neurosteroids, general anaesthetics, and GABA antagonists on GABA(A) receptors in membranes prepared from Hydra and on the feeding response have now been investigated. The neurosteroids tetrahydroprogesterone and tetrahydrodeoxycorticosterone increased [3H]GABA binding to hydra membranes with nanomolar potency (EC50, 141+/-11 and 623+/-36 nM, respectively) and high efficacy (maximal increase 79+/-6.5 and 62+/-4%, respectively), whereas the 3beta-hydroxy epimer of tetrahydroprogesterone was ineffective. The benzodiazepine receptor ligands diazepam (100 microM), clonazepam (100 microM) and abecarnil (30 microM) enhanced [3H]GABA binding to Hydra membranes by 22, 20 and 24%, respectively; effects abolished by the specific benzodiazepine antagonist flumazenil (100 microM). On the contrary, the peripheral benzodiazepine receptor ligand 4'chlorodiazepam failed to affect [3H]GABA binding to Hydra membranes. The general anaesthetics propofol and alphaxalone similarly increased (+38% and +30% respectively) [3H]GABA binding. Moreover, [3H]GABA binding to Hydra membranes was completely inhibited by the GABA(A) receptor antagonist SR 95531, whereas bicuculline was without effect. The modulation of GABA(A) receptors in vitro by these various drugs correlated with their effects on the glutathione-induced feeding response in the living animals. Tetrahydroprogesterone and tetrahydrodeoxy-corticosterone (1 to 10 microM) prolonged, in a dose-dependent manner, the duration of mouth opening induced by 10 microM glutathione, with maximal effects of +33 and +29%, respectively, apparent at 10 microM neurosteroid. Alphaxalone (10 microM) similarly increased (+33%) the effect of glutathione. The effects of steroids on the feeding response were inhibited by SR 95531 in a dose-dependent manner; t-butylbyclophosphorothyonate (1 microM), a specific Cl- channel blocker, which per se, like picrotoxin but not bicuculline, shortened the duration of the response, also counteracted the steroids effects at 1 microM. These results suggest that the modulation of GABA(A) receptors by steroids is an ancient characteristic of the animal kingdom and that the pharmacological properties of these receptors have been highly conserved through evolution.

The effects of inhibitors of GABAergic transmission and stress on brain and plasma allopregnanolone concentrations.

1. This study was undertaken to investigate the relationship between a reduction in brain GABAA receptor function and the cerebro-cortical content of 3 alpha-hydroxy-5 alpha-pregnan-20 one (allopregnanolone, AP), a potent endogenous positive modulator of 7-aminobutyric acid (GABA) action at GABAA receptors, with anticonflict and anticonvulsant effects in rodents. 2. An acute depletion of the cerebral content of GABA or an attenuation of GABAA receptor-mediated transmission by systemic injections of isoniazid (375 mg kg-1, s.c.) or FG 7142 (15 mg kg-1, i.p.) induced a transient increase in the cerebro-cortical and plasma concentrations of AP in handling-habituated (not stressed) rats. 3. Two stress paradigms, handling in naive rats and mild foot shock in handling-habituated rats, that reduce central GABAergic tone mimicked the effects of isoniazid and FG 7142 on cortical AP content; foot shock in handling-habituated rats, but not handling in naive animals, also increased plasma AP. Isoniazid, FG 7142, and foot shock also each increased the concentrations of the AP precursors, pregnenolone and progesterone, in both brain and plasma of handling-habituated rats, whereas handling in naive rats increased the concentrations of these steroids only in brain. 4. Pretreatment of handling-habituated rats with the anxiolytic beta-carboline derivative abecarnil, a positive allosteric modulator of GABAA receptors, which per se failed to affect the AP concentration in brain or plasma, prevented the increase in brain and plasma AP induced by foot shock or isoniazid. 5. In adrenalectomized and castrated rats foot shock or isoniazid failed to increase AP both in brain cortex and plasma. 6. These observations indicate that inhibition of GABAergic transmission, induced by foot shock or pharmacological manipulations, results in an increase in the concentrations of AP in brain and plasma, possibly via a modulation of hypothalamic-pituitary-adrenal (HPA) axis. 7. Given that AP enhances GABAA receptor function with high efficacy and potency, an increase in brain AP concentration may be important in the fine tuning of the GABA-mediated inhibitory transmission in the central nervous system.

Functional correlation between allopregnanolone and [35S]-TBPS binding in the brain of rats exposed to isoniazid, pentylenetetrazol or stress.

1. The relation between changes in the cerebral cortical concentration of allopregnanolone and gamma-aminobutyric acid (GABA) type A receptor function after intracerebroventricular injection of this neurosteroid was investigated in male rats. 2. Intracerebroventricular administration of allopregnanolone (1.25 to 15 micrograms) produced a maximal increase (100 fold at the highest dose) in cortical allopregnanolone concentration within 5 min; the concentration remained significantly increased at 15 and 30 min, before returning to control values by 60 min. 3. The same treatment induced a rapid and dose-dependent decrease in the binding of t-[35S]-butylbicyclophosphorothionate ([35S]-TBPS) to cerebral cortical membranes measured ex vivo, an effect mimicked by the benzodiazepine midazolam but not by the 3 beta-hydroxyepimer of allopregnanolone. The time course of changes in [35S]-TBPS binding paralleled that of brain allopregnanolone concentration. 4. In a dose-dependent manner, allopregnanolone both delayed the onset of convulsions and inhibited the increase in [35S]-TBPS binding to cortical membranes induced by isoniazid. The potency of allopregnanolone in inhibiting [35S]-TBPS binding in isoniazid-treated rats was approximately four times that in control animals. 5. The ability of allopregnanolone to decrease [35S]-TBPS binding in isoniazid-treated rats also correlated with its anticonvulsant activity against pentylenetetrazol-induced seizures as well as its inhibitory effect on the increase in [35S]-TBPS binding induced by foot shock. 6. The results indicate that the in vivo administration of allopregnanolone enhances the function of GABAA receptors in rat cerebral cortex and antagonizes the inhibitory action of stress and drugs that reduce GABAergic transmission.

Stress-induced increase in brain neuroactive steroids: antagonism by abecarnil.

Acute foot shock stress elicits a selective and time-dependent increase of neuroactive steroid (pregnenolone, progesterone, allotetrahydrodeoxycorticosterone) concentrations in rat brain cortex, accompanied by a marked increase of plasma corticosterone. The brain cortical neuroactive steroid levels peaked between 10 and 30 min poststress and returned to control values by 2 h. Abecarnil (0.3 mg/kg), i.p.), a beta-carboline derivative with anxiolytic properties, completely antagonized the effect of foot shock on brain cortical neuroactive steroids. A single administration of the anxiogenic beta-carboline FG 7142 (15 mg/kg, i.p.), in contrast, mimicked the effect of foot shock. These data support the hypothesis for the existence of a functional relationship between brain neuroactive steroid concentrations and GABAA receptor function/emotional state of the animal.

Chronic ethanol intoxication enhances [3H]CCPA binding and does not reduce A1 adenosine receptor function in rat cerebellum.

The effects of acute and chronic treatment with ethanol on the function of A1 adenosine receptor in the rat cerebellar cortex were investigated. Acute administration of ethanol (0.5-5 g/kg) had no effect on the binding of the A1-receptor agonist [3H]2-chloro-N6-cyclopentyladenosine ([3H]CCPA) or that the antagonist [3H]8-cyclopentyl-1-3-dipropylxanthine ([3H]DPCPX) in rat cerebellar cortical membranes. Rats were rendered ethanol dependent by repeated forced oral administration of ethanol (12-18 g/kg per day) for 6 days. [3H]CCPA binding was increased by 23% in cerebellar cortical membranes prepared from rats killed 3 h after ethanol withdrawal compared with saline-treated animals. The increase in [3H]CCPA binding was still apparent 12-24 h after the last ethanol administration, but was no longer detectable 3-6 days after ethanol withdrawal. In contrast, the binding of [3H]DPCPX was not modified in the cerebellar cortex of rats killed at various times after ethanol withdrawal. The acute administration of CCPA [0.25-1 mg/kg, intraperitoneally (IP)] suppressed the tremors and audiogenic seizures apparent 24 h after ethanol withdrawal. Moreover, repeated coadministration of CCPA (0.5 mg/kg, IP, four times daily) and ethanol did not prevent the generation of audiogenic seizures during withdrawal but completely prevented mortality. Finally, CCPA antagonized with similar potencies and efficacies the isoniazid-induced convulsions observed in control and ethanol-withdrawn rats. These results indicate that long-term treatment with intoxicating doses of ethanol enhances [3H]CCPA binding but does not reduce the anticonvulsant efficacy of CCPA or the function of A1 adenosine receptors.

Time-dependent changes in rat brain neuroactive steroid concentrations and GABAA receptor function after acute stress.

The time courses of changes in rat brain neuroactive steroid concentrations and gamma-aminobutyric acid type A (GABAA) receptor function elicited by acute stress were investigated in animals exposed to CO2 for 1 min, a treatment known to induce stress in rats and panic attacks in humans. Inhalation of CO2 induced increases in cerebral cortical steroid concentrations, the time dependence of which varied with the steroid examined. Thus, progesterone and deoxycorticosterone showed maximal increases (10- and 4-fold, respectively) 10 min after CO2 inhalation and had returned to basal values by 30 and 60 min, respectively. In contrast, pregnenolone and 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) concentrations showed maximal increases (+174 and + 200%, respectively) at 30 min, were still higher than control at 60 min and returned to control values 120 min after stress. Inhalation of CO2 also resulted in increases in plasma steroid concentrations, most of which peaked at 30 min and had returned to control values by 60 min. A parallel analysis of the stress-induced changes in GABAA receptor function, assessed either biochemically by t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to cerebral cortical membranes or behaviorally by the punished responding score in Vogel's test, showed that the effects of CO2 inhalation on both parameters were maximal (+51 and -40%, respectively) after 10 min; the behavioral reaction returned to normal after 60 min, whereas [35S]TBPS binding had returned to control values 120 min after stress. The results show that: (a) the maximal increase in the brain concentrations of allopregnanolone, a potent and efficacious positive modulator of GABAA receptors, occurred at a time (30 min) when both conflict behavior and [35S]TBPS binding begun to decrease, and (b) both allopregnanolone concentrations and [35S]TBPS binding had returned to control values 120 min after CO2 inhalation. The data are thus consistent with a physiological role of neuroactive steroids in restoring GABAergic tone after stress.

Isoniazid-induced inhibition of GABAergic transmission enhances neurosteroid content in the rat brain.

Isoniazid (375 mg/kg, s.c.), a drug that decreases GABAA receptor-mediated transmission, elicited a time-dependent increase of neuroactive steroid (pregnenolone, progesterone and allotetrahydrodeoxycorticosterone) concentrations in rat brain and plasma. This treatment also time-dependently increased the plasma concentration of corticosterone. Brain and plasma neuroactive steroid levels peaked between 40 and 120 min after isoniazid administration, respectively, and returned to control values by 5 hr. Acute foot shock stress mimicked the effect of isoniazid by increasing in a time-dependent manner the same neuroactive steroids both in brain and plasma. Abecarnil (0.3 mg/kg, i.p.), a beta-carboline derivative with anxiolytic properties, antagonized the effect of both isoniazid and foot shock on brain and plasma neuroactive steroids and on plasma corticosterone level. These data indicate that an inhibition of central GABAergic transmission enhances the concentrations of THDOC and its precursors pregnenolone and progesterone in the rat brain and plasma as well as the plasma levels of corticosterone. This finding suggests that GABA exerts a tonic inhibitory action on the mechanisms involved in the regulation of the synthesis and release of these neuroactive steroids in the central nervous system and plasma.

Biochemical and functional identification of GABA receptors in Hydra vulgaris.

GABA binding sites labelled in vitro with [3H]GABA are present in crude membrane preparations from Hydra vulgaris. [3H]GABA binding is specific (70%), saturable and it is completely displaced by the GABA mimetic muscimol but not by bicuculline or baclofen. Scatchard analysis of saturation data indicates the presence of only one population of binding sites with a Bmax of 4, 75 pmol/mg of protein and a KD of 76 nM. In the living animal GABA and benzodiazepines increase the duration of mouth opening during the glutathione-induced feeding response. Bicuculline prevents the GABA-induced increase of the feeding response. Diazepam activity is enhanced by simultaneous GABA administration and it is suppressed by the specific antagonist flumazenil. In contrast, no [3H]-flunitrazepam binding is detected in membrane preparations. We conclude that a population of GABA receptors is present in Hydra tissues, where they are involved in the modulation of response to chemical stimulation.