The pharmacological properties of GABA receptor-coupled chloride channels using 36Cl-influx in cultured spinal cord neurons

Abstract

γ-Aminobutyric acid (GABA) synaptic pharmacology was studied in cultured mouse spinal cord using various biochemical techniques. The intact cultures were used to study GABA uptake, benzodiazepine (BZ) binding and GABA-stimulated 36Cl-influx, all under similar physiological conditions. The cultures appear to contain a population of GABAergic neurons, since there was evidence of neuronal active transport of [ 3H]GABA by these cells. There also was a significant population of neuronal BZ receptors based on the finding of clonazepam-displaceable [ 3H]flunitrazepam (FLU) binding. In addition, the presence of GABA receptors coupled to the BZ receptors was shown by the enhancement of FLU binding by GABA (10–100 μM). GABA A and glycine-coupled Cl − channels were also demonstrated using 36Cl-influx. GABA-induced 36Cl-influx was specific for GABA A agonists, since GABA B receptor agonist, baclofen and other excitatory neurotransmitters did not alter the 36Cl-influx. Both GABA ( K m = 9.1 μM ) and muscimol ( K m = 2.0 μM ) produced concentration-dependent increases in 36Cl-influx in the cultures. The GABA-stimulated Cl −-influx was blocked by the GABA A receptor antagonist (+)bicuculline ( IC 50 = 4.5 μM ) and by the Cl − channel antagonist picrotoxinin ( IC 50 = 25 μM ). These results indicate that spinal cord cultures are useful model for comparing the binding characteristics of the GABA receptor complex to the activation of the coupled Cl − channels using the 36Cl-influx assay.