Molecular Characterization of Invertebrate Neural Regeneration
Prof. Hadley Horch
Bowdoin College Department of Neuroscience
Prior research (Hoy et al. 1985, Schildberger et a.l 1986) has shown that the cricket auditory system exhibits a great deal of plasticity when responding to neural damage. Cricket auditory neurons will sprout new dendritic processes which seek and form new synapses with undamaged afferent neurons. This regeneration is quite contrary to the normative process of dendritic atrophy or cell death following deafferentation. The plasticity of neurons is controlled by several conserved molecular cues and receptors. The proteins slit and robo, a ligand-receptor pair, have been identified in several organisms and are known to regulate axon and dendrite behavior at established midlines. Axons in robo mutants will repeatedly cross a midline, suggesting the receptor is involved in cellular effects that repel growing axons from the midline.
We propose that slit and robo are responsible for the dendritic sprouting observed at the midline of the cricket auditory system. To test the hypothesis we have designed primers to identify Slit and Robo genes in the cricket species Gryllus bimaculatus. The primers were designed using the CODEHOP degenerate algorithm based on protein sequences identified in Drosophila, C. elegans, Apis mellifera and Danio rerio. Thus far several sets of primers have been used in an attempt to identify the gene in genomic DNA. Recent primers have been producing diffuse bands of the predicted size, but PCR parameters are being modified to achieve better amplification specificity.
Future work includes isolating mRNA from Gryllus nervous tissue for use in gene amplification. This would avoid the possibility of intron presence in genomic DNA. Also, once the genes have been identified, in situ hybridization will be performed to quantify the expression patterns of slit and robo to better understand their roles in dendritic sprouting.