Gr32a neurons contact OA neurons in the suboesophageal ganglion.

(A) Schematic depicting the SOG region targeted by Gr32a axons visualized in panel B. (B) Axons and presynaptic terminals of Gr32a-expressing neurons identified by immunofluorescence to CD8:GFP and the synaptotagmin:HA fusion protein in UAS-sytHA;;UAS-CD8:GFP/Gr32a-Gal4 progeny (green, anti-CD8, Invitrogen; red, anti-HA, Roche). Sensory neurons from the labellum project through the labial nerve (arrow), mouthpart neurons project through the pharyngeal/accessory nerve, and neurons from thoracic ganglia project via the cervical connective (arrowhead). Scale bar represents 30 µM (C–D) A subset of Tdc2-positive neurons located in the SOG in a schematic (C) and with GFP expression driven by the Tdc2-LexA line (Tdc2-LexA;lexAop-rCD4:GFP. Cell bodies are visible (D) with extensive arborizations apparent in a series of optical sections ventral to the cell bodies (Figure S1). (E) GRASP-mediated GFP reconstitution is observed between Gr32a neurons expressing CD4::spGFP1-10 and UAS-syt:HA (red, anti-HA, Roche) and OA neurons expressing CD4::spGFP11. GRASP reconstitution is detected by immunofluorescence using a rabbit monoclonal GFP antibody (Life Technologies). Regions in the SOG with only syt:HA expression are indicated (arrows) in addition to GFP-reconstitution contacts that show co-localization with syt-HA expression (arrowhead). Scale bar is 50 µM. (F–H) Optical sections of the same brain at higher magnification showing GRASP-mediated GFP reconstituted expression (H), syt:HA localization (G) and clear overlap or close association at synaptic-like puncta in the merged channel (F). Scale bar represents 20 µM. (See also Figure S2.) (I) Schematic representation of the GRASP reporter lines combined with the Gr32a-Gal4 and Tdc2-lexA driver lines.

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