Worm Breeder's Gazette 13(5): 40 (February 1, 1995)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Visualization of Presynaptic Terminals Using GFP.

Michael L. Nonet

Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110

A protein fusion between the synaptic vesicle associated
membrane protein VAMP and GFP localizes relatively specifically
to synaptic vesicles permitting the visualization of
synaptic terminals in live animals. VAMP (also called
synaptobrevin and formally dubbed snb-l ) is a member of
a family of proteins that have been implicated in vesicular
docking and fusion. VAMP is a small protein of 120 amino
acids with its C-terminus anchored into the synaptic vesicle
membrane (fig lA). Antibodies directed against C. elegans
VAMP co-localize with another synaptic vesicle associated
protein, synaptotagmin. I chose to fuse GFP to the C-terminus
of VAMP because R. Kelly's lab (UCSF) had demonstrated
that sequences sufficient to localize VAMP to synaptic
vesicles were present in the cytoplasmic domain of VAMP.
The construct inserts the GFP coding sequence in frame
just prior to the termination codon in a snb-l genomic clone.
The resulting fusion protein would be predicted to be oriented
in the synaptic vesicle membrane as illustrated in figure
When the VAMP-GFP construct is present in nematodes as
an integrated array, called jsIsl, synaptic rich regions
of the nervous system can be visualized directly using
epi-fluorescence. The ventral cord, dorsal cord, nerve
ring, and portions of the pharyngeal nervous system fluoresce
brightly. In most animals commissures are not visible,
nor are the dendritic portions of the amphid bundles. The
cell bodies of some
neurons are detectable. In addition, discrete patches
of fluorescence in sub-lateral bundles and the SAB processes
are readily visible in all animals. Finally, in young adults
patches are also visible in the proper location in the vulval
region to represent the terminals of HSN and
VC neuron synapses with vulval muscles. Unfortunately,
in jsIsl adults GFP-derived fluorescence appears in the
spermatheca and proceeds to spread through the central
part of gonad in older adults making the visualization
of these patches difficult.
While it is unlikely that all of the neuronal VAMP-GFP fusion
product is localized to synaptic vesicles, several lines
of experiment suggest that most of the fusion product is
vesicularly localized. First, in unc-104 (el265); jsIsl
animals fluorescence is concentrated in neuronal cell
bodies and absent from the dorsal cord. This suggest that
most axonal membrane does not contain significant amounts
of VAMP-GFP. Second, in most jslsl; unc 5 (e53) animals
variable sized patches of fluorescence are observed in
sub-lateral and lateral positions. These patches are
likely to be the ectopic neuromuscular synapses which
Hedgecock, Culloti and Hall (1990) observed in unc-5 (e53)
animals. Third, in strains bearing extrachromosomal
arrays expressing the VAMP-GFP fusion under control of
the mec-7 touch cell promoter, fluorescence in the nerve
ring was limited to several patches ventrally and laterally.
These patches were in similar positions as the synaptic
connections described for the mec-7 expressing neurons
ALMR, ALML and AVM in White et al's 'the mind of a worm'. Finally,
in collaboration with Yishi Jin in Bob Horvitz's lab VAMP-GFP
was placed under the unc-25 (glutamic acid decarboxylase)
promoter. These VAMP-GFP constructs yielded a patchy
fluorescent pattern in the ventral and dorsal cord and
some nerve ring staining. The numbers of patches in the
cords are consistent with the number of GABAergic inhibitory
neuromuscular synapses formed by GABAergic neurons (personal
communication, E. Jorgensen). Taken together these data
provide strong evidence that the VAMP-GFP fusion localizes
to synaptic vesicles when expressed in C. elegans. I am
presently using VAMP-GFP fusions to screen for mutants
with synaptogenesis defects. My work with VAMP and VAMP-GFP
was initiated while I was a postdoctoral fellow in Dr. Barbara
Meyer's laboratory.