Gr32a neurons contact OA neurons in the suboesophageal ganglion.

<p>(<b>A</b>) Schematic depicting the SOG region targeted by Gr32a axons visualized in panel B. (<b>B</b>) Axons and presynaptic terminals of Gr32a-expressing neurons identified by immunofluorescence to CD8:GFP and the synaptotagmin:HA fusion protein in <i>UAS-sytHA;;UAS-CD8:GFP/Gr32a-Gal4</i> 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 (<b>C–D</b>) A subset of Tdc2-positive neurons located in the SOG in a schematic (C) and with GFP expression driven by the Tdc2-LexA line (<i>Tdc2-LexA;lexAop-rCD4:GFP</i>. Cell bodies are visible (D) with extensive arborizations apparent in a series of optical sections ventral to the cell bodies (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004356#pgen.1004356.s001" target="_blank">Figure S1</a>). (<b>E</b>) GRASP-mediated GFP reconstitution is observed between Gr32a neurons expressing CD4::spGFP1-10 and <i>UAS-syt:HA</i> (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. (<b>F–H</b>) 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004356#pgen.1004356.s002" target="_blank">Figure S2</a>.) (<b>I</b>) Schematic representation of the GRASP reporter lines combined with the <i>Gr32a-Gal4</i> and <i>Tdc2-lexA</i> driver lines.</p>

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