Spatial and temporal analysis of axonal transport in primary neuronal cultures from Drosophila larvae. Gary Iacobucci, Noura Abdel Rahman, Aida Andrades Valtueña, Shermali Gunawardena. Biological Sciences, State University of New York at Buffalo, Buffalo, NY.

   Efficient intracellular transport is essential for healthy cellular function and structural integrity. Problems in this pathway have recently been implicated in neuronal cell death and disease. To spatially and temporally determine how transport defects are initiated, we have developed a primary neuronal cell culture system from Drosophila larval brains. Immunohistochemical characterization indicates that these primary neurons are similar to larval neurons in vivo. The staining patterns of numerous synaptic markers mimic the patterns seen in fixed whole mount third instar larvae. We also visualize, live, the movement dynamics of several cargos/organelles. In day 1 and day 2 old cultures we observe robust bi-directional movement of six cargos/organelles. Using a MATLAB based single-particle tracker program we have analyzed the movement behavior of these cargos/organelles at each time point. Temporal analysis shows transport dynamics of these cargos change with time. Significant increases or decreases in segmental velocities observed at day 2 relative to day 1 negatively correlate to increases or decreases in pause frequency and/or duration. In contrast to WT larval brain cultures, neuronal cultures from motor protein reduction larval brains show reduced movement of cargos with increased numbers of stalled cargo and axonal blocks. Strikingly, we find that axonal blockages are not fixed, permanent blocks that impede transport as previously thought, but are instead dynamic. Under some motor reduction conditions, blocks resolve while under other conditions they do not. Over time, these neuronal cultures from mutant larval brains show defects in neuronal growth. Taken together, our results propose that non-resolving blocks may initiate deleterious pathways leading to death and degeneration while resolving blocks are benign.