GABA Modulators

Abstract: The GABA-modulating and GABA-mimetic activities of the monoterpenoid thymol were explored on human GABAA and Drosophila melanogaster homomeric RDLac GABA receptors expressed in Xenopus laevis oocytes, voltage-clamped at -60 mV. The site of action of thymol was also investigated. Thymol, 1-100 microm, resulted in a dose-dependent potentiation of the EC20 GABA response in oocytes injected with either alpha1beta3gamma2s GABAA subunit cDNAs or the RDLac subunit RNA. At 100 microm thymol, current amplitudes in response to GABA were 416+/-72 and 715+/-85% of controls, respectively. On both receptors, thymol, 100 microm, elicited small currents in the absence of GABA. The EC50 for GABA at alpha1beta3gamma2s GABAA receptors was reduced by 50 microm thymol from 15+/-3 to 4+/-1 microm, and the Hill slope changed from 1.35+/-0.14 to 1.04+/-0.16; there was little effect on the maximum GABA response. Thymol (1-100 microm) potentiation of responses to EC20 GABA for alpha1beta1gamma2s, alpha6beta3gamma2s and alpha1beta3gamma2s human GABAA receptors was almost identical, arguing against actions at benzodiazepine or loreclezole sites. Neither flumazenil, 3-hydroxymethyl-beta-carboline (3-HMC), nor 5alpha-pregnane-3alpha, 20alpha-diol (5alpha-pregnanediol) affected thymol potentiation of the GABA response at alpha1beta3gamma2s receptors, providing evidence against actions at the benzodiazepine/beta-carboline or steroid sites. Thymol stimulated the agonist actions of pentobarbital and propofol on alpha1beta3gamma2s receptors, consistent with a mode of action distinct from that of either compound. These data suggest that thymol potentiates GABAA receptors through a previously unidentified binding site.

Abstract: 1. In this study in vitro and in vivo approaches were combined in order to investigate if the anti-epileptic mechanism(s) of action of levetiracetam (LEV; Keppra) may involve modulation of inhibitory neurotransmission. 2. GABA- and glycine-gated currents were studied in vitro using whole-cell patch-clamp techniques applied on cultured cerebellar granule, hippocampal and spinal neurons. Protection against clonic convulsions was assessed in vivo in sound-susceptible mice. The effect of LEV was compared with reference anti-epileptic drugs (AEDs): carbamazepine, phenytoin, valproate, clonazepam, phenobarbital and ethosuximide. 3. LEV contrasted the reference AEDs by an absence of any direct effect on glycine-gated currents. At high concentrations, beyond therapeutic relevance, it induced a small reduction in the peak amplitude and a prolongation of the decay phase of GABA-gated currents. A similar action on GABA-elicited currents was observed with the reference AEDs, except ethosuximide. 4. These minor direct effects contrasted with a potent ability of LEV (EC(50)=1 - 10 microM) to reverse the inhibitory effects of the negative allosteric modulators zinc and beta-carbolines on both GABA(A) and glycine receptor-mediated responses. 5. Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of beta-carbolines on GABA-gated currents. Blockade of zinc inhibition of GABA responses was observed with clonazepam and ethosuximide. Phenytoin was the only AED together with LEV that inhibited the antagonism of zinc on glycine-gated currents and only clonazepam and phenobarbital inhibited the action of DMCM. 6. LEV (17 mg kg(-1)) produced a potent suppression of sound-induced clonic convulsions in mice. This protective effect was significantly abolished by co-administration of the beta-carboline FG 7142, from a dose of 5 mg kg(-1). In contrast, the benzodiazepine receptor antagonist flumazenil (up to 10 mg kg(-1)) was without any effect on the protection afforded by LEV. 7. The results of the present study suggest that a novel ability to oppose the action of negative modulators on the two main inhibitory ionotropic receptors may be of relevance for the anti-epileptic mechanism(s) of action of LEV.

Abstract: Previous studies have shown that the glutamate/glutamine (Glu/Gln) neurotransmitter cycle and neuronal glucose oxidation are proportional (1:1), with increasing neuronal activity above isoelectricity. GABA, a product of Glu metabolism, is synthesized from astroglial Gln and contributes to total Glu/Gln neurotransmitter cycling, although the fraction contributed by GABA is unknown. In the present study, we used 13C NMR spectroscopy together with i.v. infusions of [1,6-13C2]glucose and [2-13C]acetate to separately determine rates of Glu/Gln and GABA/Gln cycling and their respective tricarboxylic acid cycles in the rat cortex under conditions of halothane anesthesia and pentobarbital-induced isoelectricity. Under 1% halothane anesthesia, GABA/Gln cycle flux comprised 23% of total (Glu plus GABA) neurotransmitter cycling and 18% of total neuronal tricarboxylic acid cycle flux. In isoelectric cortex, glucose oxidation was reduced >3-fold in glutamatergic and GABAergic neurons, and neurotransmitter cycling was below detection. Hence, in both cell types, the primary energetic costs are associated with neurotransmission, which increase together as cortical activity is increased. The contribution of GABAergic neurons and inhibition to cortical energy metabolism has broad implications for the interpretation of functional imaging signals.

Abstract: There has been a resurgence of interest in synthetic and plant-derived flavonoids as modulators of γ-amino butyric acid-A (GABAA) receptor function influencing inhibition mediated by the major inhibitory neurotransmitter GABA in the brain. Areas of interest include (i) flavonoids that show subtype selectivity in recombinant receptor studies in vitro consistent with their behavioural effects in vivo, (ii) flumazenil-insensitive modulation of GABAA receptor function by flavonoids, (iii) the ability of some flavonoids to act as second-order modulators of first-order modulation by benzodiazepines and (iv) the identification of the different sites of action of flavonoids on GABAA receptor complexes. An emerging area of interest is the activation of GABAA receptors by flavonoids in the absence of GABA. The relatively rigid shape of flavonoids means that they are useful scaffolds for the design of new therapeutic agents. Like steroids, flavonoids have wide-ranging effects on numerous biological targets. The challenge is to understand the structural determinants of flavonoid effects on particular targets and to develop agents specific for these targets.