Imaging Protein and Synaptic Vesicle Dynamics in a Zebrafish Presynaptic Terminal
David Zenisek, Ph.D.
Yale University School of Medicine, New Haven, CT
David Mahoney Neuroimaging Program
June 2004, for 5 years
Imaging a Neurotransmitter at the Junction Between Brain Cells for Clues to Huntington’s Disease
Researchers will learn how disease-related proteins are recruited to brain cell “synapses,” the junctions that connect one brain cell to another, and how these proteins are released by one brain cell and taken up by another during synaptic transmission. This analysis may provide new insight into how the proteins involved in diseases such as Huntington’s exert their influence to impair cellular communication and memory.
Electrical impulses in the part of the neuron located prior to the synapse (called the “presynaptic” neuron) generate the release of neurotransmitters at the synaptic junction. The neurotransmitters are released when small structures (called “synaptic vesicles”) fuse with the nerve cell’s external membrane and the neurotransmitter exits the cell. This process is called exocytosis. The released neurotransmitter then is received by the synaptic vesicles within the adjoining nerve cell. This is called endocytosis. This transmission from one nerve cell to another at the synapse between the two is considered to be critical to learning and memory. Researchers believe that diseases that affect memory, such as Huntington’s disease, have defects in this process.
Yale investigators have found that they can look directly at a single synaptic vesicle near the nerve cell’s membrane, if they use evanescent field fluorescence microscopy (EFM) to visualize the retina of a living zebrafish. The researchers will fluorescently label the disease-related proteins and visualize them as they are recruited to sites of exocytosis and endocytosis in the zebrafish’s retina. If this technique is successful, the investigators then will seek funding from other sources to study in an animal model how the specific protein produced by the Huntington’s disease gene alters synaptic functions in brain cells.
Significance: This study of how disease-related neurotransmitter proteins are recruited to the brain cell synapse, released, and taken up by another brain cell may provide new insights into how certain diseases, such as Huntington’s disease, alter or disrupt this process. This could lead to new approaches to treatment or prevention.
Imaging Protein and Synaptic Vesicle Dynamics in a Zebrafish Presynaptic Terminal
We intend to establish the zebrafish bipolar cells as a system for studying presynaptic mechanisms using evanescent field microscopy. Zebrafish are an excellent vertebrate model for physiology and disease, conducive to both large-scale mutagenesis screens and the rapid and cheap generation of transgenic animals. Hence, several mutants have been generated that serve as human disease models and many more will be developed in the future.
We intend to take advantage of the large (~5 mm diameter) synaptic terminals of retinal bipolar neurons in these animals to image synaptic vesicle dynamics and to investigate the recruitment and interaction of presynaptic disease-related proteins to sites of exocytosis and endocytosis using evanescent field microscopy (EFM). To do this, we intend to first generate four lines of zebrafish with genetic fluorescent markers that will enable us to measure rates of exocytosis and endocytosis, determine the location of exocytosis and endocytosis, and image the movement of individual synaptic vesicles and clathrin coated vesicles. Expression of fluorescent proteins will be driven by a heat-shock promoter, allowing one to optimize expression for signal to noise and/or to target expression to specific cells or tissue types by selectively heating specific regions of the fish (Halloran et al., 2000; Sato and Yost, 2003). These fish will then serve as reference lines that can be crossed with mutant animals exhibiting known phenotypes or fluorescently tagged presynaptic proteins of a different color.
We intend to use the pilot data generated here to apply for other funding to use these techniques to investigate when and where huntingtin (protein product of the gene mutated in Huntington's disease), complexin (a protein involved in vesicle release that is down regulated in Huntington's disease, Schizophrenia and bipolar disorder), and selective huntingtin interacting proteins (e.g. Hip1, Hip12) act at the presynaptic terminal and to investigate whether the mutant form of the Huntington's disease protein, huntingtin, affects rates of exocytosis and/or endocytosis in presynaptic terminals. In addition, we believe these fish will be of general use to the neuroscience research community, and we will make these lines of fish available to other researchers, enabling them to study the effects of specific human disease related genes on presynaptic mechanisms.
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.
We have established a breeding colony of zebrafish that is specifically dedicated to this project; characterized the electrophysiological and exocytic properties of the zebrafish bipolar cell; set up techniques for recording from embryonic retinal slices; generated DNA constructs for transgenic fish; and generated three new lines of transgenic zebrafish expressing presynaptic proteins conjugated to fluorescent proteins that allow the monitoring of active zones and synaptic vesicles. In addition, we are currently screening offspring to find transgenic founders for two other lines.
Generation of transgenic lines: We have now generated three lines that are transgenic for fluorescent protein-labeled synaptic proteins that allow us to monitor vesicle trafficking and presynaptic active zones in both dissociated cells and retinal slices. In addition, fish injected with constructs for three other fluorescent lines are being raised and screened to look for germline transgenic animals.
Characterization of electrophysiological and exocytic properties of zebrafish bipolar terminals: We have continued to characterize the properties of exocytosis in wild-type zebrafish using whole-cell voltage clamp, including membrane capacitance measurements and evanescent field microscopy. Additionally, we have developed a retinal slice preparation that allows us to study synaptic transmission in young (5 to 20 day old) fish. Whole cell voltage clamp both in slice and in dissociated neurons reveal that bipolar cells have non-inactivating L-type calcium channels, which trigger neurotransmitter release. Results using evanescent field microscopy have shown that it is possible to visualize single synaptic vesicles, calcium entry sites near the membrane of bipolar cells and active zones in dissociated bipolar cells.
In addition to our work on dissociated cells, we have set up a slice preparation that allows us to study synaptic transmission in very young animals (4-10 days), which will allow us to study effects of transgenic manipulations without raising animals to adulthood. This, in combination with new techniques for expressing transgenes in a much larger proportion of injected cells, allows us to study transgenic fish within days of injection both optically and electrophysiologically. Experiments with 4 to 25 day old zebrafish slices indicate that slices can be made from the retinas of these young fish and that whole-cell voltage clamp recordings from a variety of cells can be made, including bipolar cells, amacrine cells and ganglion cells, making it feasible to record from pairs of neurons to assay synaptic transmission. We are currently using this preparation to characterize the developmental time course of the onset of calcium currents, synapses, and synaptic transmission in zebrafish retina, as well as to confirm that our fluorescent fish have normal synaptic properties.
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