The objective of this proposal is to develop a protocol to isolate synaptic vesicles (SVs) from frog saccular hair cells. Until a population can be successfully isolated, functional studies on SVs are severely limited. Isolated SV populations are essential for determining function and mechanism of neurotransmission, membrane synthesis, content and recycling, and enzymatic activity of SVs. In addition, the production of immunological probes for SV membrane proteins unique to hair cells requires isolated SVs. The frog saccule provides a good preliminary model for developing microisolation and purification techniques because of its relatively large size. However, the techniques to be perfected in this pilot project, and the information obtained about protein content of the SVs, should be applicable to mammalian models. In this pilot study we propose to isolate a SV-rich fraction from frog saccular hair cells by enzymatic cell separation followed by cell fractionation techniques. We will demonstrate the enrichment of vesicles in the isolated fraction by electron microscopy and immunoassay for a putative membrane protein marker, synaptophysin. We will also characterize the total protein content of the SV-rich fraction by one-dimensional SDS PAGE electrophoresis. The proposed project requires the use of microseparation techniques because of the minute quantities of tissue involved.
