Recombinant GABA Research Articles

The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood-brain barrier and exhibits anticonvulsive effects.

The functional characterization of hispidulin (4',5,7-trihydroxy-6-methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. After chemical synthesis, hispidulin was investigated at recombinant GABA(A)/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nm and higher stimulated the GABA-induced chloride currents at tested receptor subtypes (alpha(1-3,5,6)beta(2)gamma(2)S) indicating positive allosteric properties. Maximal stimulation at alpha(1)beta(2)gamma(2)S was observed with 10 microm hispidulin. In contrast to diazepam, hispidulin modulated the alpha(6)beta(2)gamma(2)S-GABA(A) receptor subtype. When fed to seizure-prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg(-1) body weight (BW) per day) and diazepam (2 mg kg(-1) BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). Permeability across the blood-brain barrier for the chemically synthesized, (14)C-labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (K(in)) of 1.14 ml min(-1) g(-1), measurements approached the values obtained with highly penetrating compounds such as diazepam. Experiments with Caco-2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 microm, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites.

Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors.

Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater potentiation than loreclezole (90 +/- 14% and 109 +/- 14% at beta3 and beta2, respectively, compared with 62 +/- 6% and 56 +/- 3%), whereas niflumic acid exhibited the lowest efficacy. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity. This study identifies and characterizes a variety of allosteric modulators for which betasubunits are an important determinant of efficacy and potency.

GABA A receptor α1 and α6 subunits mediate cell surface anchoring in cultured cells

The clustering and immobility of γ-aminobutyric acid type A receptors (GABA ARs) at discrete and functionally significant domains on the nerve cell surface is an important determinant in the integration of synaptic inputs. To investigate the role that different GABA AR α subunit isoforms play in determining receptor mobility, α xβ3γ2s subunits (where x=α subunit isoforms 1–6) were co-transfected into COS 7 and human embryonic kidney (HEK) 293 cells and the surface mobility of these recombinant complexes was measured by fluorescence photobleach recovery (FPR). In addition, the lateral mobility of endogenous GABA ARs in cerebellar granule (CG) cells was measured. We show that the α1 and α6 subunits immobilize recombinant GABA AR in transfected cells. This is consistent with the immobility of native receptors in CG cells, which express α1 and α6.

Inhibition by miltirone of up-regulation of GABA A receptor α 4 subunit mRNA by ethanol withdrawal in hippocampal neurons

Miltirone, a tanshinone isolated from the root of Salvia miltiorrhiza, has been characterized as a low-affinity ligand for central benzodiazepine receptors. We have now shown that this compound bound with low affinity (micromolar range) to central benzodiazepine recognition sites but did not interact with peripheral benzodiazepine receptors. It failed to potentiate Cl − currents induced by γ-aminobutyric acid (GABA) both in Xenopus oocytes expressing recombinant human GABA A receptors and in cultured rat hippocampal pyramidal cells, but it inhibited the ability of diazepam to potentiate the effect of GABA in these systems. Miltirone (1–10 μM) also partially inhibited the increase in the abundance of the mRNA for the α 4 subunit of the GABA A receptor induced by ethanol withdrawal in cultured hippocampal neurons. These results suggest that miltirone might ameliorate the symptoms associated with discontinuation of long-term administration of ethanol or of other positive modulators of the GABA A receptor.

Activation of single heteromeric GABAA receptor ion channels by full and partial agonists

The linkage between agonist binding and the activation of a GABA(A) receptor ion channel is yet to be resolved. This aspect was examined on human recombinant alpha1beta2gamma2S GABA(A) receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), isonipecotic acid, piperidine-4-sulphonic acid (P4S), imidazole-4-acetic acid (IAA), 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) and 5-(4-piperidyl)-3-isoxazolol (4-PIOL). Whole-cell concentration-response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25-27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5-fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, alpha, ranged from 200 to 600 s(-1). The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, beta, and the total dissociation rates, k(-1), for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E approximately 7-9) compared to the weak partial agonists ( approximately 0.4-0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABA(A) receptor activation does not occur in a switch-like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel.

Conductance of Recombinant GABA Channels Is Increased in Cells Co-expressing GABAA A Receptor-associated Protein

High conductance gamma-aminobutyric acid type A (GABA(A)) channels (>40 picosiemens (pS)) have been reported in some studies on GABA(A) channels in situ but not in others, whereas recombinant GABA(A) channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA(A) channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA(A) channels. GABARAP, a protein associated with native GABA(A) channels, has been reported to cause clustering of GABA(A) receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing alpha(1), beta(1), and gamma(2S) subunits of human GABA(A) receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA(A) receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.

Palmitoylation regulates the clustering and cell surface stability of GABA A receptors

GABA A receptors are the major mediators of fast synaptic inhibition in the brain. These receptors are ionotropic, hetero-pentameric, ligand-gated ion channels, which are predominantly composed of α, β, and γ2 subunits. Here, we reveal that the γ2 subunit of neuronal and recombinant GABA A receptors is palmitoylated. We further establish that palymitoylation of the γ2 subunit occurs on multiple cysteine residues within the major intracellular domain of this receptor subunit. In cultured hippocampal neurons, inhibitors of protein palymitoylation reduced the synaptic clustering of GABA A receptors and steady-state cell surface receptor number. These effects are likely to be mediated by direct palmitoylation of the γ2 subunit, as mutation of palmitoylation sites within this protein reduces GABA A receptor clustering. Taken together, these results suggest that palmitoylation of GABA A receptors plays an essential role in regulating the clustering of these receptors at synaptic sites.

Flumazenil-independent positive modulation of γ-aminobutyric acid action by 6-methylflavone at human recombinant α 1β 2γ 2L and α 1β 2 GABA A receptors

In view of the ability of flavones to displace radiolabelled benzodiazepines from brain tissue and the interesting behavioural profile of these compounds, the present study investigated the activity of 6-methylflavone at ionotropic γ-aminobutyric acid (GABA) receptors expressed in Xenopus laevis oocytes. 6-Methylflavone (1–100 μM) was found to be a positive allosteric modulator at α 1β 2γ 2L and α 1β 2 GABA A receptors with no significant difference between the enhancement seen at either receptor subtype. At 100 μM, 6-methylflavone enhanced the response to 5 μM GABA by 183±20% at α 1β 2γ 2L GABA A receptors. The methyl substituent was important since the parent flavone was significantly weaker as a positive modulator (103±24% enhancement of 5 μM GABA by 100 μM flavone). This enhancement is not mediated via high-affinity benzodiazepine sites as it was not inhibited by the classical benzodiazepine antagonist flumazenil under conditions where flumazenil inhibits the potentiation of the GABA response to diazepam. 6-Methylflavone (60 μM) did not significantly affect the GABA dose–response curve at ρ 1 GABA C receptors. 6-Methylflavone acts as a positive modulator of recombinant GABA A receptors at sites independent of flumazenil-sensitive benzodiazepine sites.

Topiramate modulates recombinant GABA(A) receptors by a nonbenzodiazepine mechanism

Purpose: The study investigates the mechanism of action of topiramate at recombinant GABA(A) receptor subtypes.

Selected amino acids, dipeptides and arylalkylamine derivatives do not act as allosteric modulators at GABA B receptors

Based on recent reports describing enhancing actions of arylalkylamines (fendiline [ N-(3,3-diphenylpropyl)-α-methylbenzylamine] and prenylamine [ N-(3,3-diphenylpropyl)-α-methylphenethylamine]), amino acids ( l-phenylalanine, l-leucine and l-isoleucine), and dipeptides ( l-Phe-Phe and l-Phe-Leu) on baclofen-induced responses in cortical slices, we have examined whether these compounds might act as positive allosteric modulators at GABA B receptors. Unlike the previously described allosteric GABA B receptor modulator CGP7930 (2,6-Di- tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), these compounds did not enhance GABA B receptor-mediated guanosine 5′- O-(3-thiotriphosphate) [GTP(γ) 35S] binding in native or recombinant cell membrane preparations. Similarly, in a competition binding assay using the antagonist radioligand [ 3H]CGP62349, CGP7930, but not the other compounds, enhanced the affinities of γ-aminobutyric acid (GABA) for native GABA B receptors from rat brain cortex. Finally, in a cellular assay (Ca 2+ signaling in a recombinant cell line), CGP7930 was again the only compound found to enhance the GABA response. It is concluded that the arylalkylamines, amino acids and dipeptides tested do not act as allosteric modulators at native and recombinant GABA B receptors.

Prolonged exposure to γ-aminobutyric acid up-regulates stably expressed recombinant α1β2γ2s GABA A receptors

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABA A receptors following their prolonged exposure to drugs. Exposure (48 and/or 96 h) of human embryonic kidney (HEK 293) cells stably expressing recombinant α1β2γ2s GABA A receptors for γ-aminobutyric (GABA, 1 mM) and muscimol (100 μM), but not for diazepam (1 μM), enhanced the maximum number ( B max) of [ 3H]flunitrazepam binding sites without affecting their affinity ( K d). The GABA-induced enhancement in B max was reduced by the GABA receptor antagonist, bicuculline (100 μM), and by cycloheximide (10 μl/ml), a protein synthesis inhibitor. GABA (100 μM) enhanced the affinity of [ 3H]flunitrazepam binding to vehicle- and GABA-pretreated, but not to diazepam-pretreated, HEK 293 cells. The results suggest that chronic GABA treatment up-regulates stably expressed GABA A receptors, presumably by stimulating their synthesis. Unlike chronic diazepam, which produced functional uncoupling of GABA and benzodiazepine binding sites, chronic GABA failed to produce this effect.

Recombinant α1β2γ2 GABA A receptors expressed in HEK293 and in QT6 cells show different kinetics

Neuronal γ-aminobutyric acid (GABA) A receptors are extremely heterogeneous and therefore GABAergic currents represent responses of unknown mixture of receptor subtypes. Expression of recombinant receptors in foreign cells allows to investigate a defined receptor subtype but its properties can be altered due to, e.g., differences in the endogenous modulators. In the present study the α1β2γ2 receptors were expressed in HEK293 and QT-6 cells and current responses to ultrafast GABA applications were recorded. Rise time and rapid deactivation component were faster in responses recorded from QT-6 cells. Moreover, in QT-6 cells desensitization was faster and more profound. Recovery in the paired pulse experiments was faster in HEK293 cells. In conclusion, we provide evidence that recombinant receptors may show functional differences when expressed in different cells.

GABA A receptor modulation by the novel intravenous general anaesthetic E-6375

E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABA A receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABA A (α 1β 2γ 2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABA A receptors. The GABA enhancing action of E-6375 on recombinant GABA A receptors was unaffected by the subtype of the α isoform (i.e. α x β 2γ 2L; x=1–3) within the receptor, but was increased by the omission of the γ 2L subunit. Receptors incorporating β 2, or β 3, subunits were more sensitive to modulation by E-6375 than those containing the β 1 subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the β-subunit. The various in vivo actions of general anaesthetics may be mediated by GABA A receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABA A receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile.

The modulatory effects of the anxiolytic etifoxine on GABA A receptors are mediated by the β subunit

The anxiolytic compound etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4 H-3,1-benzoxazine hydrochloride) potentiates GABA A receptor function in cultured neurons (Neuropharmacology 39 (2000) 1523). However, the molecular mechanisms underlying these effects are not known. In this study, we have determined the influence of GABA A receptor subunit composition on the effects of etifoxine, using recombinant murine GABA A receptors expressed in Xenopus oocytes. Basal chloride currents mediated by homomeric β receptors were reduced by micromolar concentrations of etifoxine, showing that β subunits possess a binding site for this modulator. In oocytes expressing α 1β x GABA A receptors ( x=1, 2 or 3), etifoxine evoked a chloride current in the absence of GABA and enhanced GABA (EC10)-activated currents, in a dose-dependent manner. Potentiating effects were also observed with α 2β x , β x γ 2s or α 1β x γ 2s combinations. The extent of potentiation was clearly β-subunit-dependent, being more pronounced at receptors containing a β 2 or a β 3 subunit than at receptors incorporating a β 1 subunit. The mutation of Asn 289 in the channel domain of β 2 to a serine (the homologous residue in β 1) did not significantly depress the effects of etifoxine at α 1β 2 receptors. This specific pattern of inhibition/potentiation was compared with that of other known modulators of GABA A receptor function like benzodiazepines, neurosteroids, barbiturates or loreclezole.

Evidence for the involvement of GABA A receptor blockade in convulsions induced by cephalosporins

There is accumulating evidence that most β-lactam antibiotics (i.e., cephalosporins and penicillins) have some degree of convulsive activity, both in laboratory animals as well as in clinical settings. The proposed mechanism is suppression of inhibitory postsynaptic responses, mainly mediated by γ-amino butyric acid (GABA) A-receptors (GABA A-R). However, comprehensive studies on the convulsive activities of various β-lactam antibiotics in vivo and in vitro have not been performed. We have therefore examined the convulsive activities of seven different cephalosporins using both in vivo and in vitro models: intracerebroventricular (ICV) administration in mouse; [ 3H]muscimol binding assay (BA) in mouse brain synaptosome; and inhibition of recombinant mouse α1β2γ2s GABA A-Rs in Xenopus oocyte (GR). The rank orders of convulsive activities in mouse (cefazolin>cefoselis>cefotiam>cefpirome>cefepime>ceftazidime>cefozopran) correlated with those of inhibitory potencies on [ 3H]muscimol binding and GABA-induced currents of GABA A-R in vitro, with correlation coefficients of ICV:GR, ICV:BA and BA:GR of 0.882, 0.821 and 0.832, respectively. In contrast, none of the antibiotics had affinities for N-methyl- d-aspartate (NMDA) receptors nor facilitatory actions on NMDA receptor-mediated current in oocytes. These results clearly demonstrate that the mechanism of cephalosporin-induced convulsions is mediated predominantly through the inhibition of GABA A-R function and not through NMDA receptor modulation.

The in vitro and in vivo enantioselectivity of etomidate implicates the GABA A receptor in general anaesthesia

General anaesthetics exhibiting enantioselectivity afford valuable tools to assess the fundamental mechanisms underlying anaesthesia. Here, we characterised the actions of the R-(+)- and S-(−)-enantiomers of etomidate. In mice and tadpoles, R-(+)-etomidate was more potent (~10-fold) than S-(−)-etomidate in producing loss of the righting reflex. In electrophysiological and radioligand binding assays, the enantiomers of etomidate positively regulated GABA A receptor function at anaesthetic concentrations and with an enantioselectivity paralleling their in vivo activity. GABA-evoked currents mediated by human recombinant GABA A receptors were potentiated by either R-(+)- or S-(−)-etomidate in a manner dependent upon receptor subunit composition. A direct, GABA-mimetic, effect was similarly subunit dependent. Modulation of GABA receptor activity was selective; R-(+)-etomidate inhibited nicotinic acetylcholine, or 5-hydroxytryptamine 3 receptor subtypes only at supra-clinical concentrations and ionotropic glutamate receptor isoforms were essentially unaffected. Acting upon reticulothalamic neurones in rat brain slices, R-(+)-etomidate prolonged the duration of miniature IPSCs and modestly enhanced their peak amplitude. S-(−)-etomidate exerted qualitatively similar, but weaker, actions. In a model of locomotor activity, fictive swimming in Xenopus laevis tadpoles, R-(+)- but not S-(−)-etomidate exerted a depressant influence via enhancement of GABAergic neurotransmission. Collectively, these observations strongly implicate the GABA A receptor as a molecular target relevant to the anaesthetic action of etomidate.

SL651498, a GABAA receptor agonist with subtype-selective efficacy, as a potential treatment for generalized anxiety disorder and muscle spasms.

SL651498 (6-fluoro-9-methyl-2-phenyl-4-(pyrrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one) was identified as a drug development candidate from a research program designed to discover subtype-selective GABA(A) receptor agonists for the treatment of generalized anxiety disorder and muscle spasms. The drug displays high affinity for rat native GABA(A) receptors containing alpha(1) (K(i) = 6.8 nM) and alpha(2) (K(i) = 12.3 nM) subunits, and weaker affinity for alpha5-containing GABA(A) receptors (K(i) = 117 nM). Studies on recombinant rat GABA(A) receptors confirm these findings and indicate intermediate affinity for the alpha(3)beta(2)gamma(2) subtype. SL651498 behaves as a full agonist at recombinant rat GABA(A) receptors containing alpha(2) and alpha(3) subunits, and as a partial agonist at recombinant GABA(A) receptors expressing alpha(1) and alpha(5) subunits. SL651498 produced anxiolytic-like and skeletal muscle relaxant effects qualitatively similar to those of benzodiazepines (BZs) [minimal effective dose (MED): 1 to 10 mg/kg, i.p. and 3 to 10 mg/kg, p.o.]. However, unlike these latter drugs, SL651498 induced muscle weakness, ataxia or sedation at doses much higher than those having anxiolytic-like activity (MED: 30 to 100 mg/kg, i.p. or p.o.). Moreover, in contrast to BZs, SL651498 did not produce tolerance to its anticonvulsant activity or physical dependence. It was much less active than BZs in potentiating the depressant effects of ethanol or impairing cognitive processes in rodents. The differential profile of SL651498 as compared to BZs may be related to its selective efficacy at the alpha(2)- and alpha(3)-containing GABA(A) receptors. This suggests that selectively targeting GABA(A) receptor subtypes can lead to drugs with increased clinical specificity. SL651498 represents a promising alternative to agents currently used for the treatment of anxiety disorders and muscle spasms without the major side effects seen with classical BZs.

Intact synaptic GABAergic inhibition and altered neurosteroid modulation of thalamic relay neurons in mice lacking delta subunit.

Robust GABA-mediated inhibitory postsynaptic currents (IPSCs) in neurons of the thalamic relay (TC) nuclei are important in sustaining oscillatory activity within thalamic and thalamocortical circuits. The biophysical properties and pharmacological sensitivities of these IPSCs both depend on the subunit combination of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors. Recombinant GABA(A) receptors containing the delta subunit (heavily expressed in TC nuclei) have been shown to exhibit slowed desensitization rates and high affinity for GABA in heterologous expression systems. We tested whether the GABA(A)-mediated synaptic inhibition in TC neurons would be affected by loss of the delta subunit. Spontaneous and evoked IPSCs were recorded from neurons in the ventral basal complex (VB) of the thalamus from brain slices of wild-type (delta(+/+)) and homozygous delta subunit deficient mice (delta(-/-)). Spontaneous IPSCs (sIPSCs) from delta(-/-) mice had no significant differences in amplitude, duration, or frequency compared with their delta(+/+) counterparts. However, baseline noise (63% of control) and the relative contribution of the slow component to overall decay (79% of control) were significantly lower in delta(-/-) VB recordings. Evoked IPSCs (eIPSCs) in delta(-/-) neurons showed no difference in peak amplitude, but had an accelerated slow decay component (40- vs. 55-ms time constant). We further tested whether neurosteroid modulation of GABA(A) receptors was dependent on the presence of the delta subunit, as previously reported in recombinant systems. Pregnenolone sulfate (PS) significantly reduced eIPSC peak amplitude (-30%) and increased duration in delta(-/-), but not in delta(+/+) mice. sIPSCs were not affected in any neurons, delta(-/-) or delta(+/+). In contrast, 3-alpha,5-alpha-tetrahydrodeoxycorticosterone (THDOC) increased the durations of eIPSCs and sIPSCs in both delta(-/-) and delta(+/+) VB neurons. Our findings show that although the delta subunit confers a striking PS insensitivity to eIPSCs in VB neurons, it plays only a minor role in the synaptic inhibition of VB neurons. This suggests delta subunit containing GABA(A) receptors may be functionally limited to an extrasynaptic locus in VB neurons.

Alpidem analogues containing a GABA or glycine moiety as new anticonvulsant agents

Alpidem analogues containing a GABA ( 1– 3) or glycine ( 4– 6) moiety were synthesized and their interaction with the GABA/benzodiazepine receptor complex at central (CBR) and peripheral (PBR) level was evaluated. In particular, their ability to modulate the specific binding of [ 3H]-GABA to washed membrane preparations from the rat cerebral cortex, as well as their effects on human recombinant GABA A receptors in Xenopus laevis oocytes, were assessed. Results from these in vitro assays showed that the most active compounds were 1 and 4. Intraperitoneal administration of compound 1 at a dose of 150 mg/kg significantly antagonized pentylenetetrazole-induced seizures in rats and the protective effects were evident for 90 min. However, compound 4 failed to interact with strychnine-sensitive Gly-binding sites. Consistent with these binding results, intraperitoneal administration of compound 4 at 150 mg/kg showed no effect against convulsions induced by strychnine, except for a prolonged time of the latency of convulsions. The results obtained suggest that compound 1 possesses interesting anticonvulsant activity and deserves further investigation as a novel lipophilic GABA derivative.

Low doses of ethanol and a neuroactive steroid positively interact to modulate rat GABA(A) receptor function.

Fast inhibitory responses in the central nervous system are mediated by the GABA(A) receptor. The activation and function of the GABA(A) receptor can be modulated by a variety of compounds including benzodiazepines, barbiturates and neuroactive steroids. Modulation of the GABA(A) receptor function by ethanol has been observed in some but not all studies. We have studied the effect of ethanol at concentrations corresponding to light intoxication on the function of the recombinant GABA(A) receptor containing alpha1beta2gamma2 subunits. The experiments were performed both in the absence and presence of low, subthreshold concentrations of a neuroactive steroid. The results demonstrate that, in the presence of the steroid, 0.05 % (9 mM) ethanol potentiates the GABA(A) receptor function by increasing the channel mean open duration. No effect was observed on the channel closed time durations. The data suggest that ethanol influences channel closing with no effect on the affinity of the receptor for GABA or the channel opening rate constant.