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Frank H. Kung
Biomedical Engineering Program
B.S. 2006, Cornell University
M.Eng. 2007, Cornell University
Thesis Advisor: Ellen Townes-Anderson, Ph.D.
Department of Neurology and Neurosciences
Thursday, May 8, 2014
2:00 PM., Cancer Center G-119
Photoreceptors, the light sensing cells of the retina, must make specific connections with second order retinal neurons for us to see. In retinal degenerative diseases and injury, photoreceptors show extensive synaptic plasticity and can form long (~50 Ám) sprouts and varicosities, swellings along the sprouts filled with synaptic vesicles, indicating a potential regenerative response by these cells to reform their connections. Understanding the mechanisms controlling this growth may be crucial to restoring function in degenerate or diseased retina.
We hypothesized that photoreceptor sprouting is receptive to spatiotemporal cues. To examine this hypothesis, we designed and fabricated microfluidic devices to pattern substrates and single cells. We patterned Sal-1, the antibody substrate for our retinal neurons, to the width of a single cell (10 Ám) to allow examination of individual interactions between photoreceptors and other retinal neurons. We then labeled retinal neurons from the left and right eye with dextran fluorescein and dextran tetramethyl rhodamine respectively, bisected the retina along either the dorsal-ventral or nasal-temporal axis, dissociated the retina halves, and plated the cell suspensions onto stripes of Sal-1. From interactions of labeled cells on these stripes, we discovered that rod photoreceptors are sensitive to spatial cues along the nasal-temporal axis of the retina and twice as likely to form contacts containing varicosities with cells from the opposite side of that axis. Thus, for transplantation of cells to repair the retina determining the spatial origin of the transplant and the host site for the implant may improve synaptic integration.
Next, we examined the effect of a specific axon guidance cue, Semaphorin3A, upon rod photoreceptor sprouting. We first determined that Sema3A binds to rod photoreceptors using a Sema3A linked to alkaline phosphatase. Then by using a microspritzer, we created gradients of Sema3A and exposed photoreceptors to those gradients over a 24 hr period. From this, we determined that rod photoreceptor sprouting is inhibited by Sema3A. Rod photoreceptors grew fewer processes in the presence of Sema3A, and those processes that did sprout tended to form away from high concentrations of Sema3A. In addition, rod photoreceptors reoriented in the presence of Sema3A indicating drastic cytoskeletal changes. Therefore Sema3A is inhibitory for rod photoreceptor sprouting. Thus while Sema3A is initially upregulated in retinal injury, a subsequent downregulation may allow sprouting to occur.
Finally, we devised a method of specifically stimulating rod photoreceptors and other retinal neurons in culture in order to examine the effect of activity on sprouting and synaptogenesis. With optical tweezers, we were able to maneuver individual neurons onto extracellular electrodes of a multiple electrode array (MEA), and measure rod cell calcium responses to a variety of types of stimulation. Precise placement of pre-identified cells onto an MEA provides a tool for future researchers to examine the effects of longterm stimulation on synaptic plasticity.
In conclusion, we demonstrated that sprouting in photoreceptors is stimulated and inhibited by specific cues and developed several methodologies to examine axon guidance in isolated retinal neurons.