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Structural and functional analysis of single neurons to correlate synaptic connectivity with grooming behavior

Nature Protocols volume 9, pages 1–10 (2014)Cite this article

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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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Figure 1: A single mechanosensory neuron innervates a single bristle and elaborates a stereotypical axonal branching pattern within the CNS.
Figure 2: The behavioral output and structure of a single mechanosensory neuron can be measured in single flies.
Figure 3: Flowchart of the experimental procedure, starting from behavioral assay to imaging axonal morphology, performed in 4 d.
Figure 4: Each of the 12 pairs of macrochaetae mechanosensory neurons has a characteristic axonal arbor that increases in complexity from anterior to posterior macrochaeta location.
Figure 5: Different Gal4 drivers can be used to express dsRNA solely within specific mechanosensory neurons.
Figure 6: Specific Gal4 drivers for the presynaptic mechanosensory neuron can be combined with a behavioral assay to examine the functional output of the neuron after experimental manipulations.

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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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Author notes

  1. Ibrahim Kays and Vedrana Cvetkovska: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, Québec, Canada.,

    Ibrahim Kays, Vedrana Cvetkovska & Brian E Chen

  2. Department of Medicine, McGill University, Montréal, Québec, Canada.,

    Brian E Chen

  3. Department of Neurology and Neurosurgery, McGill University, Montréal, Québec, Canada.,

    Brian E Chen

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  1. Ibrahim Kays
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Contributions

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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Correspondence to Brian E Chen.

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The authors declare no competing financial interests.

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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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