EXPRESSION OF THE PUTATIVE VESICULAR ACETYLCHOLINE TRANSPORTER IN RAT-BRAIN AND LOCALIZATION IN CHOLINERGIC SYNAPTIC VESICLES

A cholinergic locus has recently been identified consisting of a uniqu e mammalian genomic arrangement containing the genes for choline acety ltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been lo calized in cholinergic neurons, little is known about the encoded VACh T protein. Here we describe production of highly specific rabbit polyc lonal antibodies, generated using a VAChT C-terminus/glutathione-S-tra nsferase fusion protein, and immunological characterization of the nat ive VAChT protein. These antibodies specifically recognized full-lengt h recombinant VAChT expressed in transfected HeLa cells by Western blo tting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT-immunoreactive band of similar to 70 kDa w as predominant in known areas of cholinergic innervation, including st riatum, cortex, hippocampus, and amygdala. Light microscopic immunocyt ochemistry revealed reaction product in cholinergic cell groups but no t in noncholinergic areas. More significantly, immunoreactivity was al so concentrated in axonal fibers in many regions known to receive prom inent cholinergic innervation, such as cerebral cortex, hippocampus, a mygdala, striatum, several thalamic nuclei, and brainstem regions. Ele ctron microscopy using immunoperoxidase revealed that VAChT was locali zed in axon terminals, and using more precise immunogold techniques, t o synaptic vesicles. In VAChT-positive perikarya, the immunogold parti cles were localized to the cytoplasmic face of the Golgi complex. Thes e findings confirm that VAChT protein is expressed uniquely in choline rgic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.