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We have identified four neurons (VC4, VC5, HSNL, HSNR) in Caenorhabditis elegans adult hermaphrodites that express both the vesicular acetylcholine transporter and the vesicular monoamine transporter. All four of these cells are motor neurons that innervate the egg-laying muscles of the vulva. In addition, they all express choline acetyltransferase, the synthetic enzyme for acetylcholine. The distributions of the vesicular acetylcholine transporter and the vesicular monoamine transporter are not identical within the individual cells. In mutants deficient for either of these transporters, there is no apparent compensatory change in the expression of the remaining transporter. This is the first report of neurons that express two different vesicular neurotransmitter transporters in vivo.

The nematode Caenorhabditis elegans has a number of advantages for the analysis of synaptic molecules. These include a simple nervous system in which all cells are identified and synaptic connectivity is known and reproducible, a large collection of mutants and powerful methods of genetic analysis, simple methods for the generation and analysis of transgenic animals, and a number of relatively simple quantifiable behaviors. Studies in C. elegans have made major contributions to our understanding of vesicular transmitter transporters. Two of the four classes of vesicular transporters so far identified (VAChT and VGAT) were first described and cloned in C. elegans; in both cases, the genes were first identified and cloned by means of mutations causing a suggestive phenotype (1, 2). The phenotypes of eat-4 mutants and the cell biology of the EAT-4 protein were critical in the identification of this protein as the vesicular glutamate transporter (3, 4). In addition, the unusual gene structure associated with the cholinergic locus was first described in C. elegans (5). The biochemical properties of the nematode transporters are surprisingly similar to their vertebrate counterparts, and they can be assayed under similar conditions using the same types of mammalian cells (6, 7). In addition, mild and severe mutants (including knockouts) are available for each of the four C. elegans vesicular transporters, which has permitted a careful evaluation of the role(s) of vesicular transport in transmitter-specific behaviors. Accordingly, it seems appropriate at this time to present the current status of the field. In this review, we will first discuss the properties of C. elegans vesicular transporters and transporter mutants, and then explore some of the lessons and insights C. elegans research has provided to the field of vesicular transport.

Mutations in the unc-17 gene of the nematode Caenorhabditis elegans produce deficits in neuromuscular function. This gene was cloned and complementary DNAs were sequenced. On the basis of sequence similarity to mammalian vesicular transporters of biogenic amines and of localization to synaptic vesicles of cholinergic neurons in C. elegans, unc-17 likely encodes the vesicular transporter of acetylcholine. Mutations that eliminated all unc-17 gene function were lethal, suggesting that the acetylcholine transporter is essential. Molecular analysis of unc-17 mutations will allow the correlation of specific parts of the gene (and the protein) with observed functional defects. The mutants will also be useful for the isolation of extragenic suppressors, which could identify genes encoding proteins that interact with UNC-17.