828B Poster - 13. Neural development and physiology
Friday April 08, 2:00 PM - 4:00 PM

Authors:
Grace Woods ¹; Karli Corey ¹; Sonya Lee ¹; Eve Lowenstein ²; Isa Maxwell ¹; Asia Wooten ³; Allie Osgood ⁴; Luke Rotello ⁵; Daniela Mendoza Ortiz ⁶; Norma Velazquez Ulloa ¹

Affiliations:
1) Department of Biology, Lewis & Clark College, Portland, OR; 2) PMCB Graduate Program, OHSU, Portland, OR; 3) Medical School Program, OHSU, Portland, OR; 4) National Human Genome Research Institute, NIH, Bethesda, MD; 5) Biochemistry and Molecular Biology Program, Lewis & Clark College, Portland, OR; 6) University of Turku, Turku, Finland

Keywords:
f. neuromuscular junction; c. synaptogenesis

Thrombospondin (TSP) is an extracellular matrix glycoprotein that plays a role in synaptogenesis at glutamatergic synapses in the mammalian brain. While there are 5 TSP genes in humans, there is a single homologous gene in Drosophila melanogaster (D-TSP) and there is conservation in the protein domains involved in TSP’s function in synaptogenesis. It has not been investigated if D-TSP plays a role in synaptogenesis in D. melanogaster. Here we determined if D-TSP modulates synaptogenesis and locomotor behavior in the D. melanogaster third instar larval NMJ. We hypothesized that D-TSP would be necessary for normal NMJ formation and locomotor behavior. We used the GAL4-UAS system to knock down D-TSP in neurons with two different RNAi lines and quantified features of the NMJ structure and locomotor behavior in larvae with normal or decreased D-TSP expression. Both RNAi experiments showed no significant survival differences between the parental lines and the cross. For the anatomical analysis, we took images of NMJs from muscle 4 of segments A3-4 in female third instar larvae from both RNAi crosses. We manually counted the number of boutons, number of branches, number of branch points, and number of islands of each NMJ, and generated a complexity index comprising the number of branches, number of branch points, and number of islands. Our preliminary results from experiments with one of the RNAi lines suggest an increase in NMJ complexity when D-TSP is knocked down in neurons. We also calculated the polygon area for each NMJ, and found no differences in the D-TSP knockdowns. We are currently normalizing our morphological data by muscle area, increasing the sample size of NMJs for the first RNAi line, and completing our anatomical analysis of the second RNAi line. To investigate locomotor behavior, we took videos of male and female larvae moving in a gridded arena, and analyzed the larval trajectories for distance, velocity, curvature, and zones (a measure of how far larvae move from their point of origin). We found that TSP knockdown larvae from the second RNAi line exhibited a significantly higher distance travelled compared to control larvae. We are in the process of validating the knockdowns of D-TSP by RT-qPCR. In the future, we will analyze additional behaviors, such as head-turning and body contractions.