Abstract
We describe a protocol to image the complex axonal branching structure of identified mechanosensory neurons in Drosophila, combined with a behavioral assay to evaluate the functional output of the neuron. The stimulation of identified mechanosensory neurons in live animals produces a stereotyped grooming reflex. The mechanosensory axonal arbor within the CNS is subsequently labeled with a lipophilic fluorescent dye and imaged by fluorescence microscopy. The behavioral output can therefore be correlated to the axonal morphology of the stimulated neuron in the same animal. Combining this protocol with genetic analysis provides a powerful tool for identifying the roles of molecules involved in different aspects of hard-wired neural circuit formation underlying an innate behavior. From behavioral analysis to axonal imaging, the protocol takes 4 d.
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Acknowledgements
We thank M. Ghiasi, A. Hibbert, V. Stoudenikina, S. Neufeld and F. Emran for assistance with experiments, and we thank B. Douba for graphic arts assistance in the fly drawings. This work was supported by a Canadian Institutes of Health Research grant (no. 119610 to B.E.C.) and a Canadian Institutes of Health Research Graduate Scholarship (no. 223158 to I.K.).
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I.K. and V.C. performed the experiments and collected the data. I.K., V.C. and B.E.C. wrote the manuscript.
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Kays, I., Cvetkovska, V. & Chen, B. Structural and functional analysis of single neurons to correlate synaptic connectivity with grooming behavior. Nat Protoc 9, 1–10 (2014). https://doi.org/10.1038/nprot.2013.157
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DOI: https://doi.org/10.1038/nprot.2013.157
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