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Journal of Neuroscience, Vol 16, 2179-2190, Copyright © 1996 by Society for Neuroscience
Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles
ML Gilmor, NR Nash, A Roghani, RH Edwards, H Yi, SM Hersch and AI Levey
Graduate Program in Neuroscience, Emory University, Atlanta, Georgia 30322, USA.
A cholinergic locus has recently been identified consisting of a unique
mammalian genomic arrangement containing the genes for choline
acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter
(VAChT). Although transcripts for ChAT and VAChT protein have been
localized in cholinergic neurons, little is known about the encoded VAChT
protein. Here we describe production of highly specific rabbit polyclonal
antibodies, generated using a VAChT C- terminus/glutathione-S-transferase
fusion protein, and immunological characterization of the native VAChT
protein. These antibodies specifically recognized full-length recombinant
VAChT expressed in transfected HeLa cells by Western blotting, with the
prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single
VAChT- immunoreactive band of approximately 70 kDa was predominant in known
areas of cholinergic innervation, including striatum, cortex,
hippocampus,and amygdala. Light microscopic immunocytochemistry revealed
reaction product in cholinergic cell groups but not in noncholinergic
areas. More significantly, immunoreactivity was also concentrated in axonal
fibers in many regions known to receive prominent cholinergic innervation,
such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic
nuclei, and brainstem regions. Electron microscopy using immunoperoxidase
revealed that VAChT was localized in axon terminals, and using more precise
immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya,
the immunogold particles were localized to the cytoplasmic face of the
Golgi complex. These findings confirm that VAChT protein is expressed
uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at
least for the C terminus, topologically oriented as predicted by models.
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