'I 
Development and Function of the Synapse 
To study the functions of these proteins, we are 
using DNA transfection and antibody techniques. 
Wild-type and mutated forms of the proteins are 
being transfected into AtT-20 and PC 12 cells. In 
addition, some of the proteins have homologues 
in Drosophila. We have constructed vectors con- 
taining wild-type or mutated forms of the pro- 
teins driven by heat-shock promoters. These con- 
structs allow us to transfer the gene into flies and 
to turn on expression at any time during develop- 
ment and in the adult. 
Synapse Development 
Motor neurons in the spinal cord send axons to 
muscle fibers throughout the body. When axons 
contact muscle fibers, a highly ordered structure 
consisting of a presynaptic nerve terminal and a 
postsynaptic site develops. The presynaptic ter- 
minal comprises an active zone rich in synaptic 
vesicles containing neurotransmitter. The post- 
synaptic element is made up of a membrane rich 
in receptors for the neurotransmitter and an in- 
dentation in the membrane called the junctional 
fold. An extracellular matrix, or basal lamina, 
surrounds the muscle fiber, including the space 
between the nerve and muscle. 
One of the key events in the development of 
the neuromuscular junction is the redistribution 
of neurotransmitter receptors that occurs when 
nerve contacts muscle. Initially receptors for the 
neurotransmitter, in this case acetylcholine, are 
randomly distributed on the muscle fiber. When 
the nerve contacts muscle, neurotransmitter re- 
ceptors aggregate under the nerve terminal in an 
appropriate position to detect the chemicals re- 
leased during synaptic transmission. 
Agrin, a component of the extracellular matrix, 
causes acetylcholine receptors to cluster when 
added to muscle fibers growing in culture. We 
have isolated recombinant DNA clones encoding 
agrin molecules and, through an analysis of the 
nucleotide sequence, have defined the primary 
amino acid sequence of the molecule. When we 
compare the predicted agrin sequence with the 
proteins in the data bank, two types of similarities 
are revealed. The first is to a class of molecules 
that inhibit proteases, and the second to a protein 
motif called EGF (epidermal growth factor) re- 
peats. The gene is expressed in embryonic motor 
neurons at the time they are first contacting mus- 
cle fibers. We are currently attempting to express 
this gene in cell culture to study the roles of the 
agrin molecule in the formation of synapses. 
Since agrin is stably maintained in the synaptic 
basal lamina after nerve or muscle damage, it may 
also play a role in regeneration events. Under- 
standing the mechanisms of peripheral synapse 
regeneration may lead to procedures that could 
aid in central nervous system regeneration. 
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