Garry Wong

Desensitization of the NMDA receptor complex by glycinergic ligands in cerebellar granule cell cultures

Glutamate neurotoxicity was examined in cultured cerebellar granule neurons following both prolonged (20-24 h) and brief (45 min) exposure to compounds acting at strychnine-insensitive glycine receptors. Glutamate neurotoxicity was reduced in a concentration-dependent fashion by brief exposure to the glycine partial agonists 1-aminocyclopropanecarboxylic acid (ACPC) and (+-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) and the competitive antagonist, 7-chlorokynurenic acid (7-CK) with a rank order efficacy: 7-CK > HA-966 > ACPC. Neither D-cycloserine (D-CS) nor glycine affected neurotoxicity produced by maximum glutamate concentrations, while glycine but not D-CS augmented the effects of submaximum glutamate concentrations. Prolonged exposure of cultures to either full (glycine) or partial agonists (ACPC, D-CS, HA-966) abolished the neuroprotective effects of ACPC and significantly diminished the neuroprotective effects of HA-966. In contrast, the neuroprotective effects of 7-CK were only marginally reduced by prolonged exposure to glycinergic ligands, while the neuroprotection afforded by compounds acting at other loci on the NMDA receptor complex (e.g. 2-amino-5-phosphonopentanoate (APV) and dizocilpine (MK-801)) were unaltered. These effects may represent homologous desensitization of the NMDA receptor complex at its strychnine-insensitive glycine receptor induced by prolonged exposure to glycinergic agonists and partial agonists. Nonetheless, levels of the NMDA receptor subunit zeta 1 mRNA were unaffected by prolonged exposure to ACPC, indicating the apparent desensitization could involve a post-translational modification of the NMDA receptor complex.

Stereospecific transduction of behavioral effects via diazepam-insensitive GABAA receptors

Previous studies reported a positive correlation between ligand affinities at diazepam-insensitive GABAA receptors and substitution for the discriminative stimulus effects of the benzodiazepine receptor antagonist, flumazenil, in pigeons. In the present experiments, bretazenil and Ro 14-5974 (ethyl-(S)-11,12,13,13 a-tetrahydro-9-oxo-9H-imidazo[1,5-a]-pyrrolo-[2,1-c] [1,4]benzodiazepine-1-carboxylate) partially substituted for, and blocked the discriminative stimulus effects of midazolam, congruent with their actions at diazepam-sensitive GABAA receptors in vitro. In addition, bretazenil and Ro 14-5974, but not their R-enantiomers, had high affinity for diazepam-insensitive receptors and fully substituted for the discriminative stimulus effects of flumazenil. The R-enantiomers of these compounds had low affinity (Ki > 1 microM) for diazepam-sensitive and diazepam-insensitive receptors, and did not share discriminative stimulus effects with flumazenil or midazolam. Ro 19-0528 (7-chloro-3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dihydro-5-met hyl-6H- imidazo[1,5-a][1,4]benzodiazepin-6-one), a structurally related compound with full agonist actions at diazepam-sensitive GABAA receptors, had high diazepam-insensitive receptor affinity (Ki = 96 nM) and partially substituted for the discriminative stimulus effects of flumazenil. These results are consistent with stereospecific mediation of the discriminative stimulus effects of flumazenil through high affinity binding to diazepam-insensitive receptors in pigeons.

Diazepam enhancement of GABA-gated currents in binary and ternary GABAA receptors: relationship to benzodiazepine binding site density.

Although the predominant GABAA receptor isoform in the adult rodent central nervous system is a ternary complex composed of α1β2/3γ2-subunits, small populations of binary receptors lacking β-subunits (i.e., complexes containing αγ-subunits) have also been identified. When expressed in HEK 293 cells, recombinant GABAA receptors composed of either α1β2/3γ2- or α1γ2-subunits form benzodiazepine-responsive, GABA-gated chloride channels. The objective of this study was to compare the ability of a prototypic benzodiazepine (diazepam) to augment GABA-gated chloride currents in these binary and ternary receptor isoforms. The potency of GABA was characteristically increased by diazepam (1μM) in both receptor isoforms, but this increase was significantly greater (p<0.05) in receptors composed of α1β2γ2-subunits (approximately five-to sixfold) compared to α1γ2-subunits (∼2.2-fold). At GABA concentrations approximating its EC50 value (5 μM), the greater augmentation observed in ternary receptors was attributable to a higher efficacy of diazepam. Radioligand binding studies revealed that theBmax of [3H]flunitrazepam was increased ∼1.8- and 3.5-fold in cells expressing α1β2γ2- and α1β3γ2-subunits, respectively, compared to cells expressing α1γ2-subunits. A similar increase (∼3.8-fold) in theBmax of [3H]Ro 15-4513 was observed in HEK 293 cells transiently transfected with cDNAs encodign α6β3γ2- compared to α6γ2-subunits. TheKd values of these radioligands were not different in binary and ternary receptor isoforms. It is hypothesized that the greater efficacy of diazepam in α1β2γ2 compared to α1γ2 GABAA receptors results from the higher benzodiazepine binding site density produced by the formation of a ternary complex.

Localization and pharmacological characterization of pigeon diazepam-insensitive GABAA receptors

Transduction mechanisms associated with ligand binding at diazepam-insensitive subtypes of GABAA receptors remain largely unknown, but unique behavioral effects of ligands binding at these sites have been reported in pigeons. The present study further evaluated the pharmacological characteristics of diazepam-insensitive GABAA receptors in pigeon brain, using [3H]Ro 15-4513. Autoradiography detected diazepam-insensitive benzodiazepine sites on GABAA receptors in a number of brain regions, with the highest densities present in the olfactory bulb, hippocampus, thalamic nuclei and cerebellar granule cell layers, with densities of approximately 10-20% of total benzodiazepine receptor binding. Saturation analysis revealed significant densities (approximately 10% of total benzodiazepine receptor binding) of extracerebellar diazepam-insensitive benzodiazepine receptors in optic lobe, hippocampus, and brainstem compared to 27% in cerebellum. As reported for mammalian diazepam-sensitive benzodiazepine receptors, GABA (50 microM) generally increased the affinities of agonists and partial agonists, had little effect on the affinities of antagonists, and decreased the affinity of an inverse agonist for pigeon cerebellar diazepam-sensitive benzodiazepine receptors. GABA modulation of ligand binding to diazepam-insensitive benzodiazepine receptors was less than that observed for diazepam-sensitive sites, and no positive modulation was observed. These results demonstrate the presence of cerebellar and extracerebellar diazepam-insensitive benzodiazepine receptors in pigeon brain, with distribution patterns and pharmacology similar to those reported in mammals. The comparable central localization and pharmacological properties of drugs at diazepam-sensitive and -insensitive benzodiazepine receptors in pigeons and rats attests to the evolutionary conservation of GABAA systems.