Inhibitory neurotransmission in rat spinal cord: co-localization of glycine- and GABAA-receptors at GABAergic synaptic contacts demonstrated by triple immunofluorescence staining

Abstract

Synaptic inhibition in rat spinal cord is mediated by the amino acids gamma-aminobutyric acid (GABA) and glycine. Most spinal cord neurons respond to both neurotransmitters, suggesting co-expression of GABAA- and strychnine-sensitive glycine-receptors in individual cells. While the distribution of glycine-receptors has been extensively characterized, much less is known about the cellular localization of GABAA-receptors in spinal cord neurons. In the present study, the distribution of GABAA-receptors was analyzed immunohistochemically with a subunit-specific antiserum recognizing the alpha 1-subunit. Their co-localization with glycine-receptors and their apposition to GABAergic axon terminals were assessed by confocal laser microscopy in sections processed for double- and triple-immunofluorescence staining, using a monoclonal antibody against the 93 kDa glycine-receptor-associated protein, gephyrin, and an antiserum to glutamic acid decarboxylase. Staining for the GABAA-receptor alpha 1-subunit decorated the soma and dendrites of numerous neurons in laminae III-VIII and X of the spinal cord, revealing their morphology in clear detail. By contrast, laminae II and IX contained little immunoreactivity for these GABAA-receptors. Double-immunofluorescence staining showed that most GABAA-receptor-positive cells in layers III-VIII and X also exhibited a prominent glycine-receptor immunoreactivity. Both types of receptors had very similar distribution patterns in the cell membrane and were frequently co-localized in sites apposed to GABAergic axon terminals. These results indicate that GABAA- and glycine-receptors may co-exist within single postsynaptic densities, suggesting a possible synergism in the action of GABA and glycine in spinal cord neurons.