Date of Award

5-2010

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Biological Science

Advisor/Committee Chair

John T. Schmidt

Abstract

Visual activity acts via NMDA (N-methyl D-aspartate) receptors in order to refine developing retinotectal maps by shaping retinal arbors. In zebrafish larvae, each arbor forms synapses by adding and deleting many trial and error branches. Branches with synapses in retinotopic sites are selectively stabilized and new branches are added next to existing synapses, while distant branches are deleted. The resulting arbor is compact and has a bushy appearance. When NMDARs are blocked, arbors become larger and dynamic rates increase almost twofold, suggesting that the release of a stabilizing signal has been prevented. A likely suspect for this retrograde stabilizing signal is arachidonic acid (AA), and this is for a few reasons. Firstly, Ca++ entering through NMDARs activates phospholipase A2 (PLA2) to release AA, and blocking PLA2 has the same effect as blocking NMDARs. Lastly, exogenous AA reverses the dynamic effects of NMDA blockers, such as MK801. Furthermore AA, released by DAG lipase, activates Protein Kinase C (PKC) which in turn phosphorylates GAP43 in order to stabilize F-actin. The polarity complex, consisting of Par3, Par6, and aPKC, has been found to play a central role in neuronal polarity. It is selectively localized to the growth cones of axons, and is required for specification of the axon (Banker 2008). Moreover, the polarity complex’s role in cell polarization is significant since it promotes the growth of actin filaments and microtubules, as enhanced axon outgrowth requires drastic rearrangements of actin filaments and microtubules within axonal growth cones (Yoshimura et al., 2006). As aforementioned, retinal axons have many trial and error branches, and it is hypothesized that each branch may reassemble the polarity complex before it grows out. A system for antisense suppression of either Par3 or Par6 in retinal ganglion cells was developed in order to test its effects on retinotectal arbors. After labeling zebrafish retinal ganglion axons with DiO on Day 2, images were taken the next day of iii the newly fluorescent arbors and analyzed as before treatment images. Then, the eyes of zebrafish larvae were injected with antisense oligonucleotides suppressing either Par3 or Par6 (100uM every 8 hours), and then reimaged after 24 hours. In some cases, the treatment was continued for another day and the arbor was imaged after two days of treatment. Overall, the width, and area of the treated arbors did not differ from the controls before or after treatment. However, the length of the Par6 arbors did show a slight increase in length. Additionally, treated arbors experienced less branching than the controls. On Day 3 (before treatment), the experimental and control arbors had similar numbers of branches (12.73 +/-1.11 vs. 11.83 branches +/-0.70, respectively). By Day 4, however, the control arbors had a mean value of 15.05 +/-1.36 branches, whereas arbors treated with Par3 antisense and Par6 antisense had mean values of 13.55 +/-1.63 and 10.06 +/-1.07 branches, respectively. These numbers indicate that arbors treated with the antisense oligos added fewer branches than the controls, and in some cases, added none at all. Suppression of Par3 and Par6 indeed had a differential impact on immature and mature arbors. Small arbors experienced less branching than the controls, but arbors beginning with more branches tended to maintain them and even added new ones. In contrast, the overall growth of the arbors was unaffected, indicating that the antisense suppression targets branch formation, and not the size of the arbors. Furthermore, these changes in growth patterns indicate the polarity complex might need to be activated and reassembled for branch formation as predicted.

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