Dissecting the role of presynaptic GABAA receptors in nerve terminal function

Fast synaptic inhibition in the mammalian brain is mediated principally by GABAA receptors, a large and diverse family of Cl- permeable ion channels. Emerging evidence points to the presynaptic localisation of these receptors and their role in regulating intraterminal Ca2+ levels and neurotransmitter release in some areas of the CNS. We used purified nerve terminals (synaptosomes) to establish the presence of functional presynaptic GABAA receptors in the rat neocortex. As a biochemical read-out of presynaptic GABAA receptor activity, we measured Ca2+-dependent changes in the phosphorylation state of synapsin I. A dose-dependent decrease in synapsin I phosphorylation was detected in response to bicuculline, picrotoxin and GABase, while muscimol and isoguvacine caused a dose-dependent increase in synapsin I phosphorylation. Immunohistochemical analysis revealed this phospho-form of synapsin I to be localised to glutamatergic nerve terminals. In functional studies, GABAA receptor activation by muscimol caused a dose-dependent inhibition of glutamate release, which was abolished by picrotoxin. In order to investigate the mechanism(s) by which presynaptic GABAA receptor activity leads to inhibition of glutamate release, we measured release in the presence of the NKCC1 antagonist bumetanide, which abolished the muscimol-induced inhibition of glutamate release. When we subsequently investigated the role of voltage-gated Ca2+ channels on muscimol-dependent inhibition of glutamate release, we found that muscimol inhibited glutamate release in the presence of conotoxin GVIA, agatoxin IVA and NiCl2. However, the inhibition of release by muscimol was abolished by SNX-482 and nifedipine. Muscimol- dependent decrease in release was also abolished in the presence of W7. Our results indicate that the activation of presynaptic GABAA receptors in the rat neocortex is coupled to depolarisation of the nerve terminal membrane, leading to inhibition of glutamate release via Ca2+-dependent Ca2+ channel inhibition linked to L- and R-type VDCCs