Ch neurons in the Drosophila adult have been implicated as mechanosensory transducers for

acoustic signals ( Eberl, 1999), and also are presumed to be involved in larval propriosensation and mechanosensation ( Caldwell et al., 2003). To assess larval ch sensory neuron functions in a high throughput manner, we developed an assay for larval vibration sensation. Approximately 100 larvae were placed on a large agar-filled dish located above a loud speaker. We used the Multi-Worm Tracker (MWT) software (http://sourceforge.net) (Swierczek N., Giles A., Rankin C. and Kerr R., unpublished data) to automatically deliver vibration stimuli with the speaker while tracking the entire larval population on the dish. Prior to the onset of vibration larvae engage in normal foraging behavior, mostly crawling straight and occasionally MDV3100 making turns. We found that vibration induces a stopping response, followed by head turning ( Figures 7A and 7A′; Movie S1. Startle Response of a Single Wild-Type Drosophila Larvae to Vibration and Movie S2. Analyses of a Group

of Larvae following a Vibration Cabozantinib concentration Stimulus). Larval head turning in response to vibration is highly reproducible and readily quantifiable using the MWT software ( Figures 7B and 7E). This “startle” reaction to mechanical stimuli may allow the larva to sample its environment and change crawling direction following detection of potentially harmful stimuli. We found that atonal (ato1) mutant larvae, which lack ch neurons ( Jarman et al., 1993), do not exhibit a normal response to vibration. Upon stimulation, they show a small decrease in crawling speed (data not shown) with no head turning ( Figures 7C and

7E). We inhibited synaptic transmission in ch neurons by combining the iav-GAL4 with UAS-TNT (tetanus toxin) and found that iav-TNT larvae, which have inactivated ch neurons, do not show significant most increases in head turning in response to vibration as compared to control larvae that express GFP (iav-GFP) in ch neurons ( Figures S7A, S7B, and S7D). Therefore, ch neurons are a major class of larval sensory neurons involved in sensing vibration, and their proper synaptic input to the CNS is required for inducing normal head turning behavior in response to vibration. In Sema-2bC4 mutant larvae we also observed an abnormal response to vibration. Sema-2bC4 mutant larvae do reduce their speed significantly in response to vibration (data not shown), however they show no head turning ( Figures 7D and 7E), similar to the vibration responses observed in ato1 mutant larvae. These results suggest that defective larval vibration responses observed in the absence of Sema-2b result from ch neurons being unable to establish appropriate sensory afferent connectivity within the CNS ( Figure 6F).