Fig. 3: MDN-responsive neurons identified by functional imaging.

a Schematic of the functional imaging and data analysis pipeline. b Maximum intensity projections of imaged VNC volumes during photostimulation, using MDN-1-GAL4 to drive Chrimson88 in MDNs. Cyan represents activated voxels; red represents inhibited voxels. See also Supplementary Movie 3. We recorded from at least 5 independent samples for each genotype and obtained similar results across the samples. c Top, pipeline for discovering cell types that are functionally connected to MDN. Bottom, an example showing how an MDN-responsive cell type is identified. The three images are, from left to right, a maximum intensity projection of activated voxels in the VNC of a fly in which Chrimson88 expression is driven by MDN-1-GAL4 and GCaMP6s expression is driven by 28F07-LexA, a multi-color flip-out (MFCO) image from the corresponding 28F07-GAL4 driver, and a single cell segmented from the MCFO image that matches the morphology of the MDN-responsive neurons. d Twenty-four MF interneuron types, whereby segmentally repeated cells are classified as the same cell type. Maximum intensity projections of segmented and registered images are shown on the left; averaged responses to MDN activation on the right (mean ± s.e.m. of n ≥ 5 samples; red shading indicates the 20 s red-light stimulus; see also Supplementary Fig. 2). Cell types labeled in red were also identified in the trans-Tango experiments (see Supplementary Fig. 1 for pairwise comparisons). e Eight MF ascending neuron types and their calcium responses to MDN activation. f The single MF motor neuron type and its calcium response to MDN activation. Each cell type in d-f is given a provisional name beginning with “MF” (“MDN functional imaging”).