Molecular analysis of presynaptic differentiation

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Molecular and Cellular Pharmacology

First Committee Member

John L. Bixby - Committee Chair


The formation of synaptic connections between neurons and their target cells is a key event in the development of the nervous system. The major goal of this work is to understand the molecular basis of presynaptic differentiation. One approach to pursue this goal was to identify molecules specific for "innervatable" muscle with the potential to induce presynaptic differentiation. Using a differential immunization method, we obtained 4 monoclonal antibodies that stain embryonic (innervatable) but not adult (non-innervatable) muscle sections, with the exception that 1 also stains adult neuromuscular junctions. All antigens exhibit temporal and tissue distributions that are consistent with roles in muscle innervation and that are different from those of any reported muscle proteins. These results suggest that these antigens may be novel molecules that are important in muscle innervation. Another strategy to pursue our goal was to examine the regulation of genes encoding presynaptic proteins during synaptogenesis. For this approach, we cloned a full length chicken synaptotagmin I cDNA and a fragment of synaptotagmin II cDNA. We examined the expression of synaptotagmin I and synaptophysin II proteins and mRNAs in chicken forebrain. All 3 vesicle protein mRNAs showed a major upregulation roughly coincident with the time of target contact. In contrast, their protein expression showed a relatively steady rise throughout embryonic development. To study the relationship between presynaptic protein gene regulation and target contact more precisely, we examined the temporal expression of synaptophysin IIa, IIb, synaptotagmin I, II and choline acetyltransferase (ChAT) mRNAs in chick ciliary ganglia (CG), where target contact occurs during a well-defined two-day period. We found a 2,5-3 fold increase in the expression of synaptotagmin I, synaptophysin IIa and IIb mRNAs, well correlated with target contact. In addition, we saw an abundance switch in both synaptotagmin and synaptophysin II isoforms during the period of synaptic maturation, and the major upregulation (4-5 fold) of ChAT mRNA expression also occurred around this time. Therefore, although the major upregulation of vesicle protein mRNA expression follows target contact, the regulation of synaptic vesicle protein genes is complicated, and might also be linked to the maturation of synapses.


Biology, Neuroscience

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