Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons

The characterization of the Caenorhabditis elegans unc-47 gene recently all owed the identification of a mammalian gamma-amino butyric acid (GABA) tran sporter, presumed to be located in the synaptic vesicle membrane. In situ h ybridization data in rat brain suggested that it might also take up glycine and thus represent a general Vesicular Inhibitory Amino Acid Transporter ( VIAAT), In the present study, we have investigated the localization of VIAA T in neurons by using a polyclonal antibody raised against the hydrophilic N-terminal domain of the protein. Light microscopy and immunocytochemistry in primary cultures or tissue sections of the rat spinal cord revealed that VIAAT was localized in a subset (63-65%) of synaptophysin-immunoreactive t erminal boutons; among the VIAAT-positive terminals around motoneuronal som ata, 32.9% of them were also immunoreactive for GAD65, a marker of GABAergi c presynaptic endings. Labelling was also found apposed to clusters positiv e for the glycine receptor or for its associated protein gephyrin, At the u ltrastructural level, VIAAT immunoreactivity was restricted to presynaptic boutons exhibiting classical inhibitory features and, within the boutons, c oncentrated over synaptic vesicle clusters. Pre-embedding detection of VIAA T followed by post-embedding detection of GABA or glycine on serial section s of the spinal cord or cerebellar cortex indicated that VIAAT was present in glycine-, GABA- or GABA- and glycine-containing boutons, Taken together, these data further support the view of a common vesicular transporter for these two inhibitory transmitters, which would be responsible for their cos torage in the same synaptic vesicle and subsequent corelease at mixed GABA- and-glycine synapses.