Effect of RNAi-mediated knockdown of cac on gene expression in motor neurons of Drosophila melanogaster third instar larva

Entry of calcium into the terminal bouton of a neuron is critical step in action potential-dependent neurotransmitter release. At the Drosophila larval neuromuscular junction, the conversion of the presynaptic motor neuron's electrical activity into a chemical signal is mediated by the voltage-gated calcium channel Cacophony (Cac). While RNAi knockdown of cac dramatically reduces the probability of evoked neurotransmitter release at the NMJ, locomotion remains largely unaffected (Hoagland et al., 2025). Here we investigate the mechanism of this compensation by sequencing the transcriptomes of Cac RNAi motor neurons. We find decreased expression of K+ channels as seen with the Rbp or unc-13 knockdown models of presynaptic dysfunction. Overall design: We assessed the consequences of impaired neurotransmission by cac knockdown in motor neurons using the Gal4-UAS system, under control of the motor neuron-specific promoter OK6, to drive expression of both the dsRNA construct against Cac as well as nuclear mCherry as a marker. We used the empty attP2 insertion site as a negative control for the effect of the RNAi knockdown. RNA-seq libraries were prepared from motor neurons of each genotype after selecting mCherry-positive cells from dissected and dissociated brain tissue by FACS.