Several neurodegenerative diseases have been tied to defects in presynaptic function, yet it has been difficult to precisely study these processes in living neurons. We hypothesize that several of mutations will specifically involve the processes of exocytosis and endocytosis. To study this, we propose to combine zebrafish genetic tools with evanescent field microscopy to study presynaptic proteins involved in the pathology of neurodegenerative diseases. As a first step in these studies, we propose here to generate and characterize, using evanescent field microscopy, four transgenic lines of zebrafish expressing fluorescent markers that will facilitate the measurement of presynaptic functions and use them to characterize the presynaptic properties of the zebrafish retinal bipolar cell terminal, a preparation well-suited for presynaptic studies.
1: To generate four lines of transgenic zebrafish for visualizing single synaptic vesicles and for studying rates and locations of exocytosis and endocytosis in isolated retinal bipolar cells using evanescent field microscopy. Specifically, we intend to generate lines of zebrafish that 1) express a pH sensitive variant of VAMP-GFP to be used as a reporter of net exocytosis and endocytosis; 2) express dynamin-GFP to be used as a dynamic marker of endocytosis; 3) express clathrin-GFP to be used as a dynamic marker of clathrin-mediated endocytosis; and 4) express a marker that can be used to visualize single vesicles. All of these lines will be expressed under control of a heat shock promoter.
2: To use transgenic zebrafish generated in Aim 1, to characterize the rates and locations of exocytosis and endocytosis in transgenic animals using evanescent field microscopy. These measurements will serve as a baseline for future work.
Evanescent field Microscopy: Synaptic terminals of retinal bipolar neurons isolated from zebrafish retina will be imaged using evanescent field fluorescence microscopy (EFM). EFM takes advantage of the sub-wavelength sized "evanescent field" of light created at the interface of two media during total internal reflection. In our case, the evanescent field is used to selectively excite the fluorophores nearest the portion of a synaptic terminal that adheres to a high refractive index coverslip. This method provides several advantages over other microscopy techniques. 1) The evanescent field only illuminates a thin region, which reduces interference from out-of-focus fluorescent objects. 2) The exponential decay in excitation light with distance allows one to monitor submicron scale movements of an object by tracking its intensity. 3) The high NA objective enables efficient light collection.
Retinal bipolar neurons as a model presynaptic system. A drawback to the EFM technique for studying presynaptic processes is that the "evanescent field" only penetrates a very short distance and thus can only be used to investigate cells that are closely adhered to a glass coverslip. Hence, in order to use this technique our studies are limited presynaptic preparations, which can be isolated from the post-synaptic cell and induced to adhere to glass. Zebrafish retinal bipolar cells are one such neuron, which can be readily isolated and adhered to a glass surface with its synaptic terminal intact. We intend to develop the zebrafish retinal bipolar neuron as a system for studying presynaptic mechanisms.
Transgenic zebrafish: Transgenic zebrafish are readily generated by injection of DNA into single cell zebrafish embryos, and generation times are short enough and clutch sizes large enough that stable lines of hundreds of fish can be generated within six to eight months. We intend to generate transgenics by using a promoter for the heat shock protein, Hsp70. Specifically, we intend to generate four transgenic zebrafish lines for our studies; one for studying net rates of exocytosis and endocytosis, two for visualizing endocytosis, and one for the visualizing single synaptic vesicles.