ShakB RNAi prevents NB coupling of JONs with GF.

<p>In these animals, <i>peb-GAL4</i> was used to drive expression of CD8::GFP, and also Dcr-2. (A, B) Diagrammatic views of the brain, showing the pair of GF neurons (magenta) in relation to the antennal afferent projection (green and cyan). (A) Oblique side and (B) dorsal views, arrows indicate anterior (A). The asterisk in B indicates the approximate site of GF axon impalement for dye injection. (C) Enlarged dorsal view of the GF dendrites, showing the anterior large dendrite (den) inserted within a cylinder of JO-A axons, to which they are NB-coupled (and hence appear cyan), and also the lateral (lat) and medial (med) dendrites. The olfactory glomeruli, also labeled by <i>peb-GAL4</i>, are indicated (olf). (D) Dorsal view of a complete confocal stack from a control animal, in which <i>peb-GAL4</i> drives expression of CD8::GFP (green) and Dcr-2 in JON axons (and other sensory neurons—for clarity, olfactory axons are shown as partially transparent). Neurobiotin (NB: blue) and Lucifer Yellow (LY: red) are injected into the GF (making it appear magenta). NB alone transfers across gap junctions into the green JO-A axons (to appear cyan). Both LY and NB transfer into the commissural interneurons (GCI). (E) Projection from 3–5 horizontal sections through the antennal nerve of a control animal, showing peb-driven GFP and a subset of axons containing NB. (F) Projection of 3 confocal sections taken from the anterior end of the GF dendrite shown in D, showing its insertion within a ring of several GFP-expressing JO-A afferents (A), to which it is NB coupled. (G) Higher magnification view of the NB-coupled A afferents. In the right panel, the green channel is omitted for clarity. (H-J) Animal in which <i>peb-GAL4</i> drives CD8::GFP, Dcr2 and <i>shakB-RNAi</i>. (H). Section through the nerve, showing absence of NB coupling in the axons. (I) Low- and (J) high-power views of the A group of JON axons surrounding the GF dendrite, showing the absence of NB coupling. For full genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152211#pone.0152211.s004" target="_blank">S1 Table</a>. Scale bar in D: 20 μm in D, E, F, H, I; Scale bar in G: 10 μm in G and J.