Maria Cristina Mostallino

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 Vogels 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.

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

This study was undertaken to investigate the relationship between a reduction in brain GABAA receptor function and the cerebro‐cortical content of 3α‐hydroxy‐5α‐pregnan‐20 one (allopregnanolone, AP), a potent endogenous positive modulator of γ‐aminobutyric acid (GABA) action at GABAA receptors, with anticonflict and anticonvulsant effects in rodents. 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. 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. Pretreatment of handling‐habituated rats with the anxiolytic β‐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. In adrenalectomized and castrated rats foot shock or isoniazid failed to increase AP both in brain cortex and plasma. 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. 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.

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.

Ex vivo skin delivery of diclofenac by transcutol containing liposomes and suggested mechanism of vesicle–skin interaction

Recently, we described a novel family of liposomes, the Penetration Enhancer-containing Vesicles (PEVs), as carriers for enhanced (trans)dermal drug delivery. In this study, to go deeply into the potential of these new vesicles and suggest the possible mechanism of vesicle-skin interaction, we investigated transcutol containing PEVs as carriers for diclofenac, in the form of either acid or sodium salt. PEVs, prepared with soy phosphatidylcholine and aqueous solutions containing different concentrations of transcutol, were characterized by size distribution, zeta potential, incorporation efficiency, thermotropic behavior, and stability. (Trans)dermal diclofenac delivery from PEVs was investigated ex vivo through new born pig skin using conventional liposomes and a commercial gel as controls. The mode of action of the vesicles was also studied by performing a pre-treatment test and confocal laser scanning microscopy (CLSM) analyses. Results of the all skin permeation experiments showed an improved diclofenac (both acid and sodium salt) delivery to and through the skin when PEVs were used (especially in comparison with the commercial gel) thus suggesting intact PEVs penetration through the pig skin. Images of the qualitative CLSM analyses support this conclusion. Thus, this work shows the superior ability of the PEVs to enhance ex vivo drug transport of both hydrophilic and lipophilic diclofenac forms.

Molecular and functional adaptation of the GABAA receptor complex during pregnancy and after delivery in the rat brain

The abundance of γ‐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 γ2L 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 α5 subunit mRNA in the cerebral cortex, whereas no significant changes were apparent for α1, α2, α3, α4, β1, β2, β3 and γ2S subunit mRNAs. The amounts of γ2 and α1 proteins in the cerebral cortex were measured by immunoblot analysis; whereas the abundance of γ2 protein decreased during pregnancy, no change was detected in the amount of α1 protein. Evaluation for functional significance of the down‐regulated γ2 and α5 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.

Role of allopregnanolone in regulation of GABAA 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.

Changes in Expression and Function of Extrasynaptic GABAA Receptors in the Rat Hippocampus during Pregnancy and after Delivery

Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABAA-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABAA-Rs, and a tonic component mediated by extrasynaptic GABAA-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3α,5α-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the δ subunit of the GABAA-R and a concomitant decrease in that of the γ2 subunit in the hippocampus at P19. Expression of the α4 subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5α-reductase inhibitor finasteride prevented the changes in tonic current and in δ and γ2 subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABAA-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids.

Changes in GABAA receptor γ2 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.

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 γ‐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 μg) 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 3β‐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.

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.