Worm Breeder's Gazette 14(5): 40 (February 1, 1997)

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.

A genetic screen for mutations affecting D neuron synapse formation and synaptic plasticity

Mei Zhen, Yishi Jin

329 Sinsheimer Laboratories, Dept. of Biology, University of California, Santa Cruz, CA 95064 (e-mail: zhen@darwin.ucsc.edu)

     Long-term synaptic plasticity is widely observed in diverse
organisms from insects to mammals.  It often involves two aspects of
concurrent changes in the neurons: the structural change and the change
in synaptic strength.  In C. elegans, one example of such neuronal
plasticity is the synaptic rewiring of the DD motor neurons. 

     DD motor neurons are GABAergic inhibitory neurons born during
embryogenesis.  In L1 larval stage, DDs receive synaptic inputs from DA
and DB motor neurons on the dorsal side and innervate ventral body wall
muscles.  However in older larvae and adults, DD neurons receive inputs
from VA and VB neurons on the ventral side and form NMJs on the dorsal
side.  The original DD neuron connectivity is replaced by another class
of GABAergic neurons, VDs, which are generated by postembryonic cell
division (l).  Little is known about either the detailed process of the
synaptic rewiring of DD neurons or the molecular mechanism underlying
such a synaptic polarity switch.

     We are interested in elucidating the molecular mechanisms
regulating the remodeling of DD synapses.  We are using a synapse
specific GFP marker to visualize the synaptic rewiring (2), and to
perform a genetic screen for genes regulating the structural and
synaptic changes of DD neurons during the rewiring.  The GFP marker,
Punc-25-VAMP-GFP, is specifically expressed in the pre-synaptic zones of
DD and VD neurons (together called type D neurons).  VAMP-GFP chimeric
protein is localized to the presynaptic zone of neurons (3).  The
promoter of the unc-25 gene, which encodes GABA biosynthetic enzyme
glutamic acid decarboxylase, is used to specifically activate the
expression of VAMP-GFP in DD neurons in L1 stage and DD&VD neurons in L2
to adult stages.  In this screen we are recovering mutant animals
showing any abnormal GFP expression patterns in the D neurons so that a
wide spectrum of genes involved in the synapse formation of the D
neurons can be isolated.  But the further characterization will be
focused on those disrupting the DD neuron synaptic rewiring (most likely
to be the ones with little or abnormal GFP expression in the dorsal cord
from L2 stage onward).

     So far we have screened 8,000 haploid genomes using EMS as the
mutagen.  New alleles of known genes required for axonal growth and
synaptic formation, such as unc-5, unc-6, unc-11, unc-30, unc-33,
unc-51, unc-76, and unc-104 have been isolated.  The new allele (ul9) of
unc-104 gene, which encodes the kinesin heavy chain and is essential
for vesicle transport, is particularly interesting since the mutant
animals show very little abnormality in movement.  The GFP expression
pattern in ju19 indicates that this mutant is able to make nearly normal
synapses in the ventral cord, but fail to make synapses in the
dorsal code.  This suggests that ju19 may be defective in long-distance
vesicle transport. More than 100 lethal/sterile mutants, most of which
are defective in postembryonic cell division (thus fail to generate VD
neurons) were also isolated from the screen.  Intriguingly we have
isolated a number of viable mutants with severely disrupted GFP
expression pattern in either the DD or VD neurons (or in both), yet only
subtle abnormality in locomotion.  Because of the subtle visible
phenotype of these mutants, they might represent new genes which are
essential for the normal D neuron synaptic formation, but fail to be
uncovered by previous unc screens.  We are in the process of
characterizing and mapping these mutations.

Ref: 1) White JG, Albertson DG and Anness M. Nature 271: 764-766, 1978
     2) Jin YS and Horvitz HR. 1995 Worm Meeting Abstract: 291.
     3) Nonet ML WBG 13(5): 40, 1995