Prevention of the stress-induced increase in the concentration of neuroactive steroids in rat brain by long-term administration of mirtazapine but not of fluoxetine

The effects of acute and chronic administration of fluoxetine on the basal and stress-induced increases in cerebrocortical and plasma concentrations of allopregnanolone (3alpha,5alpha-tetrahydroprogesterone; 3alpha,5alpha-TH PROG) and tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC) were compared with those of mirtazapine, an antidepressant that (unlike fluoxetine) is not a selective serotonin reuptake inhibitor. A single injection (20 mg/kg i.p.) of fluoxetine or mirtazapine resulted in significant increases in the cerebrocortical and plasma concentrations of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC. In contrast, long-term administration (10 mg/kg i.p., once daily for 2 weeks) of fluoxetine, but not that of mirtazapine, induced marked decreases in the cortical and plasma concentrations of these neuroactive steroids. Chronic treatment with fluoxetine, however, did not inhibit the increases in the cortical and plasma concentrations of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC induced by acute foot-shock stress. In contrast, chronic treatment with mirtazapine prevented or significantly reduced the stress-induced increases in neurosteroid concentrations in the cerebral cortex and plasma, respectively. These results show that mirtazapine, similar to fluoxetine, initially increases the cortical concentration of neuroactive steroids; however, chronic administration of this drug modulates the plasma and brain availability of these hormones in a manner distinct from that of fluoxetine.     

Anticipation and consumption of food each increase the concentration of neuroactive steroids in rat brain and plasma

Stressful stimuli and anxiogenic drugs increase the plasma and brain concentrations of neuroactive steroids. Moreover, in rats trained to consume their daily meal during a fixed period, the anticipation of food is associated with changes in the function of various neurotransmitter systems. We have now evaluated the effects of anticipation and consumption of food in such trained rats on the plasma and brain concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH DOC), two potent endogenous positive modulators of type A receptors for gamma-aminobutyric acid (GABA). The abundance of these neuroactive steroids was increased in both the cerebral cortex and plasma of the rats during both food anticipation and consumption. In contrast, the concentration of their precursor, progesterone, was increased in the brain only during food consumption, whereas it was increased in plasma only during food anticipation. Intraperitoneal administration of the selective agonist abecarnil (0.1 mg/kg) 40 min before food presentation prevented the increase in the brain levels of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC during food anticipation but not that associated with consumption. The change in emotional state associated with food anticipation may thus result in an increase in the plasma and brain levels of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC in a manner sensitive to the activation of GABA(A) receptor-mediated neurotransmission. A different mechanism, insensitive to activation of such transmission, may underlie the changes in the concentrations of these neuroactive steroids during food consumption.     

Gender impacts behavioral and neurochemical adaptations in ethanol-dependent rats

Previous investigations have found gender differences in the effects of chronic ethanol exposure on ethanol withdrawal behaviors as well as GABA(A) receptor gene expression. The present investigation extended these studies with additional behavioral and neurochemical measures of ethanol dependence and withdrawal. No significant gender differences in the elevated plus-maze assessment of ethanol withdrawal anxiety behaviors were found. However, the neuroactive steroid, 3alpha,5alpha-THP, increased exploratory behavior in ethanol withdrawn female, but not male, rats. GABA(A) receptor binding assays showed potent competition of [35S]TBPS binding by 3alpha,5alpha-THP. Control females displayed a decreased affinity for 3alpha,5alpha-THP compared to control males, as evidenced by a nearly 30% increase in the IC50 value. There was no significant effect of ethanol withdrawal on 3alpha,5alpha-THP modulation of [35S]TBPS binding. However, gender differences were observed in the effects of chronic ethanol exposure on GABA(A) receptor subunit peptide levels in the hypothalamus. Female rats had a significant increase in peptide levels for the alpha2 and alpha3 but not alpha4 subunit, whereas male rats displayed a significant increase in alpha4 and alpha3 but not alpha2 subunits compared to pair-fed control levels. Chronic ethanol-induced alterations in gene expression in the hypothalamus did not coincide with previous findings in the cerebral cortex. In particular, male rats showed an increase in alpha1 subunit peptide levels in the hypothalamus, whereas significant decreases in this subunit have been observed in the cerebral cortex. Both female and male rats showed significant increases in the alpha3 subunit in the hypothalamus but not the cerebral cortex. Taken together, these studies provide additional support for gender-selective effects of chronic ethanol-elicited adaptations at the molecular level.     

Quantification of ten neuroactive steroids in plasma in Withdrawal Seizure-Prone and -Resistant mice during chronic ethanol withdrawal

Rationale

The rapid membrane actions of neuroactive steroids, particularly via an enhancement of γ-aminobutyric acid_A receptors (GABA_ARs), participate in the regulation of central nervous system excitability. Prior evidence suggests an inverse relationship between endogenous GABAergic neuroactive steroid levels and behavioral changes in excitability during ethanol withdrawal.

Objectives

Previously, we found that ethanol withdrawal significantly decreased plasma allopregnanolone (ALLO) levels, a potent GABAergic neuroactive steroid, and decreased GABA_AR sensitivity to ALLO in Withdrawal Seizure-Prone (WSP) but not in Withdrawal Seizure-Resistant (WSR) mice. However, the effect of ethanol withdrawal on levels of other endogenous GABA_AR-active steroids is not known.

Methods

After validation of a gas chromatography-mass spectrometry method for the simultaneous quantification of ten neuroactive steroids, we analyzed plasma from control male WSP-1 and WSR-1 mice and during ethanol withdrawal.

Results

We quantified levels of nine neuroactive steroids in WSP-1 and WSR-1 plasma; levels of pregnanolone were not detectable. Basal levels of five neuroactive steroids were higher in WSR-1 versus WSP-1 mice. Ethanol withdrawal significantly suppressed five neuroactive steroids in WSP-1 and WSR-1 mice, including ALLO.

Conclusions

Due to lower basal levels of some GABA_AR-active steroids in WSP-1 mice, a withdrawal-induced decrease in WSP-1 mice may have a greater physiological consequence than a similar decrease in WSR-1 mice. Because WSP-1 mice also exhibit a reduction in GABA_AR sensitivity to neuroactive steroids during withdrawal, it is possible that the combined decrease in neuroactive steroids and GABA_AR sensitivity during ethanol withdrawal in WSP-1 mice represents a neurochemical substrate for severe ethanol withdrawal.     

Differential modulation of the gamma-aminobutyric acid type C receptor by neuroactive steroids.

Gamma-aminobutyric acid type C receptor channels (GABA(C)Rs) composed of rho subunits are pharmacologically distinct from GABA(A) receptor channels (GABA(A)Rs). This difference is illustrated by the insensitivity of homo-oligomeric rho(1) receptor channels to many known modulators of GABA(A)Rs, such as barbiturates and benzodiazepines. A number of endogenous metabolites of corticosterone and progesterone, known as neuroactive steroids, compose yet another class of compounds that can modulate GABA(A)Rs. Here, several neuroactive steroids are shown to also modulate the rho(1) receptor channel. 5alpha-Pregnane-3alpha,21-diol-20-one (allotetrahydrodeoxycorticosterone), 5alpha-pregnane-3alpha-ol-11, 20-dione (alphaxalone), and 5alpha-pregnane-3alpha-ol-20-one (allopregnanolone) potentiated the GABA-evoked currents from rho(1) receptor channels and concomitantly altered the deactivation kinetics by prolonging the decay time. In contrast, 5beta-pregnane-3alpha-ol-20-one (pregnanolone), 5beta-pregnane-3, 20-dione (5beta-dihydroprogesterone), and 5beta-pregnane-3alpha, 21-diol-20-one (tetrahydrodeoxycorticosterone), all potentiators of GABA(A)Rs, inhibited the GABA-elicited currents of the rho(1) receptor channel. In comparison to GABA(A)Rs, the modulation of rho(1) receptor channels by these neuroactive compounds occurred with relatively high concentrations of the neuroactive steroids and was more prominent in the presence of low concentrations of GABA, equivalent to fractions of the EC(50) value of the rho(1) receptor channel. Structural comparison of these six neuroactive steroids reveals that the key parameter in determining the mode of modulation for the rho(1) receptor channel is the position of the hydrogen atom bound to the fifth carbon, imposing a trans- or cis-configuration in the backbone structure. This is the first demonstration of isomeric compounds that can differentially modulate the activity of the rho(1) receptor channel.     

GABAergic and NPY-Y(1) network in the medial amygdala: a neuroanatomical basis for their functional interaction

We used Y(1)R/LacZ transgenic mice to investigate the interaction between NPY, GABA and Y(1) receptors in the amygdala. Immunolabeling of GABA and NPY positive neurons and histochemical staining of beta-galactosidase revealed NPY and GABA colocalization and close contacts of NPY-positive fibers with GABAergic neurons also expressing the Y(1)R/LacZ transgene.     

Chronic modulation of the GABA(A) receptor complex regulates Y1 receptor gene expression in the medial amygdala of transgenic mice

NPY exerts anxiolytic effects, which are mediated by activation of Y1 receptors in the amygdala. It has been shown that diazepam counteracts the anxiogenic effect of Y1 receptor antagonists, suggesting that NPYergic and GABAergic systems are coupled in the regulation of anxiety. We used a transgenic mouse model, expressing a mouse Y1 receptor-beta-galactosidase fusion gene (Y1R/LacZ), to study the effect of positive or negative modulators of GABA(A) receptors on Y1 receptor gene expression. Mice were treated for 14 days with diazepam (4 or 20 mg/kg), the anxiolytic beta-carboline-derivative abecarnil (0.3 or 6 mg/kg) and the anxiogenic beta-carboline FG7142 (20 mg/kg). Transgene expression was determined by quantitative analysis of beta-galactosidase histochemical staining in the medial amygdala and in the medial habenula as a control region. Chronic treatment with 20 mg/kg diazepam or 6 mg/kg abecarnil significantly increased, whereas FG 7142 decreased, transgene expression in the medial amygdala. A transient decrease in transgene expression was observed in the medial amygdala six hours after the acute treatment with 20 mg/kg FG 7142 but not with diazepam or abecarnil. No significant changes were observed in the medial habenula. These data suggest that modulation of GABA(A) receptor function may regulate Y1 receptor gene expression in medial amygdala.     

Epipregnanolone and a novel synthetic neuroactive steroid reduce alcohol self-administration in rats

This study was designed to compare the effects of several neuroactive steroids with varying patterns of modulation of gamma-aminobutyric acid (GABA)(A) and NMDA receptors on operant self-administration of ethanol or water. Once stable responding for 10% (w/v) ethanol was achieved, separate test sessions were conducted in which male Wistar rats were allowed to self-administer ethanol or water following pre-treatment with vehicle or one of the following neuroactive steroids: (3beta,5beta)-3-hydroxypregnan-20-one (epipregnanolone; 5, 10, 20 mg/kg; n=12), (3alpha,5beta)-20-oxo-pregnane-3-carboxylic acid (PCA; 10, 20, 30 mg/kg n=10), (3alpha,5beta)-3-hydroxypregnan-20-one hemisuccinate (pregnanolone hemisuccinate; 5, 10, 20 mg/kg; n=12) and (3alpha,5alpha)-3-hydroxyandrostan-17-one hemisuccinate (androsterone hemisuccinate; 5, 10, 20 mg/kg; n=11). The effect of the 3beta-epimer of PCA, (3beta,5beta)-20-oxo-pregnane-3-carboxylic acid (10, 20, 30 mg/kg; n=9), on ethanol self-administration was also examined. The compounds were administered using a Latin-square design 45 min prior to the weekly test sessions. The effects of the 30 mg/kg dose of the steroidal hemisuccinates on ethanol intake were also examined 5 min after administration of these drugs. Both epipregnanolone and PCA attenuated ethanol self-administration. However, neither of the hemisuccinate compounds significantly altered this behavior. The steroidal hemisuccinates (30 mg/kg; n=7) were also tested 5 min before behavior testing and had no effect on ethanol intake 5 min after administration. The 3beta-epimer of PCA also failed to alter ethanol intake. None of the test compounds altered water intake. In electrophysiological studies, the effects of PCA and androsterone hemisuccinate on evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A)-IPSCs) was examined in brain slices of the amygdala. PCA had a stimulatory effect at concentrations of 5 and 25 muM. Androsterone hemisuccinate had no agonist activity. The ability of epipregnanolone and PCA to alter ethanol intake appears to be related to different inhibitory actions of these compounds on either GABA(A) or NMDA receptors, respectively. Thus, dual modulation of these systems by selected neuroactive steroids may offer potential for modifying the reinforcing effects of alcohol.     

Dual potentiating and inhibitory actions of a benz[e]indene neurosteroid analog on recombinant alpha1beta2gamma2 GABAA receptors

Benz[e]indenes are tricyclic analogs of neuroactive steroids and can be modulators of GABA(A) receptor activity. We have examined the mechanisms of action of the benz[e]indene compound [3S-(3alpha,3aalpha,5abeta,7beta,9aalpha,9bbeta)]-dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]indene-3-carbonitrile (BI-2) using single-channel patch-clamp and whole-cell recordings from human embryonic kidney cells transfected with rat GABA(A) receptor alpha1, beta2, and gamma2L subunits. The data demonstrate that BI-2 is a positive modulator of GABA(A) receptor activity with a peak effect at 2 microM. The mechanism of modulation is similar but not identical to that of neuroactive steroids. Similar to steroids, BI-2 acts by prolonging the mean open time duration through an effect on the duration and prevalence of the longest open time component. However, in contrast to many steroids, BI-2 does not selectively reduce the channel closing rate. The potentiating action of BI-2 seems to be mediated through interactions with the classic neuroactive steroid binding site. Mutation to the membrane-spanning region in the alpha1 subunit Q242W and the double mutation alpha1N408A/Y411F, previously shown to abolish potentiation by neurosteroids, also diminish potentiation by BI-2. At higher concentrations (>5 microM), BI-2 inhibits receptor function by enhancing the apparent rate of desensitization. From single-channel recordings, we estimate that the entry rate into the inhibited or blocked state, k(+B), is 0.50 microM(-1) s(-1). Based on the kinetic mechanism of action, and the finding that this effect is blocked by the alpha1V256S mutation, we propose that BI-2 acts through an inhibitory site first postulated for the inhibitory neurosteroid pregnenolone sulfate.     

Chronic intermittent ethanol (CIE) administration in rats decreases levels of neurosteroids in hippocampus, accompanied by altered behavioral responses to neurosteroids and memory function

The administration of ethanol on a chronic intermittent regimen (CIE) involving multiple withdrawal episodes is a model for ethanol dependence. After CIE, rats exhibited reduced seizure threshold, increased anxiety, tolerance to GABAergic sedative-hypnotic drugs, and changes in GABA(A) receptor function and subunit composition in hippocampus. Previous studies have shown that acute and chronic ethanol may induce changes in the levels of the neurosteroid 3alpha-hydroxysteroid-5alpha-pregnan-20-one (3alpha, 5alpha-THP) in the brain. Therefore, the current study analyses the correlation between chronic intermittent ethanol effects on the level of 3alpha, 5alpha-THP in hippocampus of CIE rats and the behavioral changes in sensitivity to neurosteroids. After CIE, the levels for 3alpha, 5alpha-THP were significantly reduced in hippocampus of rats. The mRNA levels for the enzymes 5alpha-reductase and 3alpha-HSD in hippocampus were also reduced. In vivo, (in contrast to a tolerance to the hypnotic effect of steroids), CIE rats showed increased sensitivity to the anticonvulsant and to the anxiolytic effect of the steroid alphaxalone. Perhaps, this is a response to lowered levels of endogenous neuroactive steroids. CIE rats also showed impairment of hippocampus-dependent memory function. These results suggest that changes in neurosteroids level and in vivo sensitivity to these compounds are involved in the development of ethanol dependence in the CIE model.     

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions

BACKGROUND AND PURPOSE: Neuroactive steroids are potent modulators of GABA(A) receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABA(A) receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABA(A) receptor, such as (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP, allopregnanolone). EXPERIMENTAL APPROACH: To assess the versatility of cyclodextrins as steroid modulators, we investigated interactions between gamma-cyclodextrin and neuroactive steroids of different structural classes. KEY RESULTS: Both a bioassay based on electrophysiological assessment of GABA(A) receptor function and optical measurements of cellular accumulation of a fluorescent steroid analogue suggest that gamma-cyclodextrin sequesters steroids rather than directly influencing GABA(A) receptor function. Neither a 5beta-reduced A/B ring fusion nor a sulphate group at carbon 3 affected the presumed inclusion complex formation between steroid and gamma-cyclodextrin. Apparent dissociation constants for interactions between natural steroids and gamma-cyclodexrin ranged from 10-60 microM. Although gamma-cyclodextrin accommodates a range of natural and synthetic steroids, C(11) substitutions reduced inclusion complex formation. Using gamma-cyclodextrin to remove steroid not directly bound to GABA(A) receptors, we found that cellular retention of receptor-unbound steroid rate limits potentiation by 3alpha- hydroxysteroids but not inhibition by sulphated steroids. CONCLUSIONS AND IMPLICATIONS: We conclude that gamma-cyclodextrins can be useful, albeit non-specific, tools for terminating the actions of multiple classes of naturally occurring neuroactive steroids.     

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.     

Opposite effects of short- versus long-term administration of fluoxetine on the concentrations of neuroactive steroids in rat plasma and brain

RATIONALE: Recent preclinical and clinical studies have shown that selective serotonin re-uptake inhibitors modulate neurosteroid synthesis in an opposite manner. OBJECTIVES: The action of long-term administration of fluoxetine was investigated on the peripheral and central concentrations of 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-TH PROG) and 3alpha,5alpha-tetrahydrodeoxycorticosterone (of 3alpha,5alpha-TH DOC), progesterone, and pregnenolone in rats. We also investigated the effect of chronic treatment with fluoxetine on the foot-shock stress-induced increase in the plasma and brain concentrations of these steroids. METHODS: Fluoxetine was administered acutely (20 mg/kg) or chronically (10 mg/kg, once daily for 15 days). Steroids were extracted from plasma and brain, separated and purified by means of high-performance liquid chromatography, and quantified by means of radioimmunoassay. RESULTS: A single dose of fluoxetine (20 mg/kg, i.p.) induced in 20 min significant increases in the cerebral cortical and plasma concentrations of 3alpha,5alpha-TH PROG (+96% and +13%, respectively), 3alpha,5alpha-TH DOC (+129 and +31%, respectively), progesterone (+111 and +58%, respectively), and pregnenolone (+151 and +59%, respectively). In addition, the plasma concentration of corticosterone was also significantly increased (+24%) after acute administration of fluoxetine. In contrast, long-term administration of fluoxetine reduced the basal concentrations of these various steroids (ranging from -22 to -43%), measured 48 h after the last drug injection, in both brain and plasma. A challenge injection of fluoxetine (20 mg/kg, i.p.), however, was still able to increase the concentrations of steroids in both the brain and plasma of rats chronically treated with this drug. Acute foot-shock stress increased the cortical and plasma concentrations of steroids in rats chronically treated with fluoxetine to extents similar to those apparent in control rats. CONCLUSIONS: A repetitive increase in the brain concentrations of neuroactive steroids may contribute to the therapeutic action of fluoxetine.     

Effects of voluntary ethanol consumption on emotional state and stress responsiveness in socially isolated rats

Social isolation of rats immediately after weaning is thought to represent an animal model of anxiety-like disorders. This mildly stressful condition reduces the cerebrocortical and plasma concentrations of 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) as well as increases the sensitivity of rats to the effects of acute ethanol administration on the concentrations of this neuroactive steroid. We further investigated the effects of voluntary consumption of ethanol at concentrations increasing from 2.5 to 10% over 4 weeks of isolation. Isolated rats showed a reduced ethanol preference compared with group-housed animals. Ethanol consumption did not affect the isolation-induced down-regulation of BDNF or Arc, but it attenuated the increase in the cerebrocortical concentration of 3α,5α-TH PROG induced by foot-shock stress in both isolated and group-housed animals as well as increased the percentage of number of entries made by socially isolated rats into the open arms in the elevated plus-maze test. Ethanol consumption did not affect expression of the α₄ subunit of the GABA_A receptor in the hippocampus of group-housed or isolated rats, whereas it up-regulated the δ subunit throughout the hippocampus under both conditions. The results suggest that low consumption of ethanol may ameliorate some negative effects of social isolation on stress sensitivity and behavior.     

Neurosteroid analogues. 14. Alternative ring system scaffolds: GABA modulatory and anesthetic actions of cyclopenta[b]phenanthrenes and cyclopenta[b]anthracenes

Although the structural features of binding sites for neuroactive steroids on gamma-aminobutryic acid type A (GABA A) receptors are still largely unknown, structure-activity studies have established a pharmacophore for potent enhancement of GABA A receptor function by neuroactive steroids. This pharmacophore emphasizes the importance of the position and stereochemistry of hydrogen-bonding groups on the steroid. However, the importance of the steroid ring system in mediating hydrophobic interactions with the GABA A receptor is unclear. We have taken the cyclopenta[ b]phenanthrene (tetracyclic compounds with a nonlinear ring system different from that of steroids) and cyclopenta[ b]anthracene (tetracyclic molecules with a linear 6-6-6-5 carbocyclic ring system) ring systems and properly substituted them to satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids. We have found these cyclopenta[ b]phenanthrene and cyclopenta[ b]anthracene analogues to have potent activity at the GABA A receptor, rivaling that of the most potent steroid modulators. Single-channel analysis of electrophysiological data indicates that similarly substituted analogues in the different ring systems affect the kinetic components of macroscopic currents in different ways. Mutations to the hydrogen bonding amino acids at the putative steroid binding site (alpha1Q241L mutation and alpha1N407A/Y410F double mutation) produce similar effects on macroscopic current amplitude by the different ring system analogues suggesting that the different kinetic effects are explained by the precise interactions of each analogue with the same binding site(s).     

Mechanisms of potentiation of the mammalian GABAA receptor by the marine cembranoid eupalmerin acetate

BACKGROUND AND PURPOSE: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABA(A) receptor. EXPERIMENTAL APPROACH: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. KEY RESULTS: Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat alpha1beta2gamma2L GABA(A) receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues alpha1Q241 and alpha1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABA(A) receptors activated by very high concentrations (1-10 microM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex. CONCLUSIONS AND IMPLICATIONS: We conclude that EPA either interacts with the putative neurosteroid binding site on the GABA(A) receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABA(A) receptor modulators.     

Auto-modulation of neuroactive steroids on GABA A receptors: a novel pharmacological effect

GABA(A) receptor function is modulated by various important drugs including neuroactive steroids that act on allosteric modulatory sites and can directly activate GABA(A) receptor channels at high concentrations. We used whole cell patch-clamp recordings and rapid applications of the neuroactive steroid alphaxalone to investigate repetitive steroid effects. Alphaxalone potentiation of submaximal GABA-evoked currents was enhanced significantly by repetitive coapplications at all investigated recombinant isoforms (alpha1beta3delta, alpha1beta3gamma2L, alpha6beta3delta, alpha6beta3gamma2L) and at GABA(A) receptors of differentiated human NT2 neurons. A similar increase of current amplitudes was induced by repetitive applications of a high steroid concentration without GABA. We refer to these reversible effects as auto-modulation because repeated interactions of steroids enhanced their own pharmacological impact at the receptor sites in a time and concentration dependent manner without affecting GABA controls. Pronounced auto-modulatory actions were also measured using the neurosteroid 5alpha-THDOC in contrast to indiplon, THIP, and pentobarbital indicating a steroid specificity. Protein kinase A inhibition significantly reduced alphaxalone auto-modulation at alpha1beta3gamma2L, alpha6beta3gamma2L, and alpha6beta3delta subtypes while it enhanced potentiation at alpha1beta3delta isoforms suggesting a crucial influence of receptor subunit composition and phosphorylation for steroid actions. Especially at extrasynaptic GABA(A) receptor sites containing the delta subunit steroid auto-modulation may have a critical role in enhancing potentiation of GABA-induced currents.     

Gamma-hydroxybutyric acid and diazepam antagonize a rapid increase in GABA(A) receptors alpha(4) subunit mRNA abundance induced by ethanol withdrawal in cerebellar granule cells

Both benzodiazepines and gamma-hydroxybutyric acid (GHB) are used to treat alcohol withdrawal syndrome. The molecular basis for this therapeutic efficacy was investigated with primary cultures of rat cerebellar granule cells. Long-term exposure of these cells to ethanol (100 mM, 5 days) reduced the abundance of mRNAs encoding the gamma(2)L and gamma(2)S subunits of the GABA type A receptor (-32 and -23%, respectively) but failed to affect that of alpha(1), alpha(4), or alpha(6) subunit mRNAs. Subsequent ethanol withdrawal resulted in decreases in the amounts of alpha(1) (-29%), alpha(6) (-27%), gamma(2)L (-64%), and gamma(2)S (-76%),subunit mRNAs that were maximal after 6 to 12 h. In contrast, 3 h after ethanol withdrawal, the abundance of the alpha(4) subunit mRNA was increased by 46%. Ethanol withdrawal did not affect neuronal morphology but reduced cellular metabolic activity. The increase in alpha(4) subunit was confirmed by functional studies showing a positive action of flumazenil in patch clamp recordings of GABA-stimulated currents after ethanol withdrawal. Diazepam (10 microM) or GHB (100 mM) prevented the increase in the amount of the alpha(4) subunit mRNA, the metabolic impairment, and the positive action of flumazenil induced by ethanol withdrawal but failed to restore the expression of the alpha(1) and gamma(2) subunits. The antagonism by GHB seems not to be mediated by a direct action at GABA(A)R because GHB failed to potentiate the effects of GABA or diazepam on Cl(-) currents mediated by GABA type A receptor.     

Neurosteroids, GABAA receptors, and ethanol dependence

Rationale Changes in the expression of type A receptors for γ-aminobutyric acid (GABA) represent one of the mechanisms implicated in the development of tolerance to and dependence on ethanol. The impact of such changes on the function and pharmacological sensitivity of GABA_A receptors (GABA_ARs) has remained unclear, however. Certain behavioral and electrophysiological actions of ethanol are mediated by an increase in the concentration of neuroactive steroids in the brain that results from stimulation of the hypothalamic–pituitary–adrenal (HPA) axis. Such steroids include potent modulators of GABA_AR function.Objectives We have investigated the effect of ethanol exposure and withdrawal on subunit expression and receptor function evaluated by subunit selective compounds, as well as the effects of short-term exposure to ethanol on both neurosteroid synthesis and GABA_AR function, in isolated neurons and brain tissue.Results Chronic treatment with and subsequent withdrawal from ethanol alter the expression of genes for specific GABA_AR subunits in cultured rat neurons, and these changes are associated with alterations in receptor function and pharmacological sensitivity to neurosteroids, zaleplon, and flumazenil. Acute ethanol exposure increases the amount of 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) in hippocampal slices by a local action independent of the activity of the HPA axis. This effect of ethanol was associated with an increased amplitude of GABA_AR-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurons in such slices.Conclusions Chronic ethanol exposure elicits changes in the subunit composition of GABA_ARs, which, in turn, likely contribute to changes in receptor function associated with the altered pharmacological and behavioral sensitivity characteristic of ethanol tolerance and dependence. Ethanol may also modulate GABA_AR function by increasing the de novo synthesis of neurosteroids in the brain in a manner independent of the HPA axis. This latter mechanism may play an important role in the central effects of ethanol.     

The effects of acute and chronic ethanol administration and its withdrawal on gamma-aminobutyric acid receptor binding in rat brain.

1 The effects of acute and chronic ethanol administration, and withdrawal on the binding of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), was investigated in rat brain. 2 Acute ethanol (2 to 4 g/kg i.p. 30 min before removal of brain) produced an increase in the binding capacity of the low affinity GABA receptor binding site. 3 Following chronic ethanol administration (1 to 21 days), the GABA receptor binding characteristics were not altered. These results suggest a possible adaptation of GABA receptors to the continuous presence of ethanol at the GABA synapse. 4 During ethanol withdrawal, the affinity of the low affinity GABA receptor binding site was significantly lower than pair-fed controls at 8 and 16 h withdrawal. 5 These results suggest that GABA receptor sensitivity may play a role in some of the neuropharmacological effects of ethanol and in its withdrawal symptoms.