Presynaptic membrane of inhibitory crayfish axon terminals is stained by antibodies raised against mammalian GABA(A) receptor subunits alpha3 and beta(2/3)

The opener muscle of the dactyl of the walking leg of crayfish is innervated by one excitatory axon releasing glutamate and one inhibitory axon releasing GABA. Functional GABA(A) receptors are present postsynaptically on the muscle and presynaptically on terminals and release boutons of the excitatory axon, whereas presynaptic GABA(A) autoreceptors have not been reported on terminals or release boutons of the inhibitory axon. Using antibodies raised against mammalian GABA(A) receptor subunits alpha3 and beta(2/3), we obtained highly specific staining of the presynaptic membrane of the inhibitory bouton and of the postsynaptic membrane of the muscle. Using pre- and postembedding techniques, staining was localized to only presynaptic and postsynaptic membranes of synaptic active zones. We also found extrasynaptic receptor subunit immunoreactivity near (up to 100 nm) to the active zones. Staining with antibodies for the alpha3 and beta(2/3) subunits showed colocalization of particles of the two subunits. We suggest that presynaptic inhibitory boutons of the crayfish possess GABA(A)-like autoreceptors composed of at least the alpha3 and beta(2/3) subunits.