(A) Representative images of larval muscle 4 NMJs at segment A4 stained for Brp (yellow), GluRIIC (magenta) and anti-Hrp (cyan, only shown in inset) in control (+;UAS-trol-RNAi.2/+;+) and trol RNAi knockdown in the indicated cell types (UAS-trol-RNAi.2 (one copy) driven by elavC155, elavC155 and mef2-Gal4, repo-Gal4, ppl-Gal4, Lsp2-Gal4, Hml-Gal4, and c564-Gal4 (one copy)). The merged image is shown below with location of the insets highlighting single boutons. Inset scale bar is 2 μm. (B) Quantification of Hrp+/GluRIIC +positive Ib bouton number at muscle 4 NMJs in segment A4 in controls and following cell-type specific trol RNAi knockdown. Each knockdown was analyzed in separate experiments with both UAS only and Gal4 only controls. Significance was calculated for each experimental comparison, but a single control that represents the average bouton number of every experiment is plotted for ease of visualization. Quantification of trol knockdown with tubulin-Gal4 at A4 (from Figure 2—figure supplement 1) is included for comparison. Unlike pan-cellular RNAi, cell-type specific RNAi does not induce synaptic retraction. Quantification of bouton number: neuron UAS only control: 23.25±1.399, 12 NMJs from 6 larvae; neuron Gal4 only control: 23.75±1.226, 12 NMJs from 6 larvae; elavC155 >UAS-trol-RNAi.2: 21.17±1.359, 12 NMJs from 6 larvae; p=0.4424 compared to UAS control; p=0.2994 compared to Gal4 control; neuron and muscle UAS only control: 21.21±1.130, 14 NMJs from 7 larvae; neuron and muscle Gal4 only control: 23.43±1.189, 14 NMJs from 7 larvae; elavC155, mef2 >UAS-trol-RNAi.2: 26.44±1.248, 16 NMJs from 8 larvae; p=0.0066 (<0.01) compared to UAS control; p=0.1437 compared to Gal4 control; glia UAS only control: 26.83±1.375, 12 NMJs from 6 larvae; glia Gal4 only control: 23.79±1.407, 14 NMJs from 7 larvae; repo >UAS-trol-RNAi.2: 20.25±1.643, 12 NMJs from 6 larvae; p=0.0078 (<0.01) compared to UAS control; p=0.1666 compared to Gal4 control; fat body (ppl) UAS only control: 25.57±1.312, 14 NMJs from 7 larvae; fat body (ppl) Gal4 only control: 26.21±0.7644, 14 NMJs from 7 larvae; ppl >UAS-trol-RNAi.2: 19.75±1.216, 16 NMJs from 8 larvae; p<0.01 compared to UAS and Gal4 controls (0.0014 compared to UAS; 0.0004 compared to Gal4); fat body 2 (Lsp2) UAS only control: 19.08±1.412, 13 NMJs from 7 larvae; fat body 2 (Lsp2) Gal4 only control: 25.07±0.7593, 14 NMJs from 7 larvae; Lsp2 >UAS-trol-RNAi.2: 23.64±2.053, 14 NMJs from 7 larvae; p=0.0731 compared to UAS control; p=0.7249 compared to Gal4 control; hemocyte UAS only control: 18.93±0.8285, 14 NMJs from 7 larvae; hemocyte Gal4 only control: 26±1.441, 14 NMJs from 7 larvae; Hml >UAS-trol-RNAi.2: 25.19±0.9841, 16 NMJs from 8 larvae; p=0.0005 (<0.001) compared to UAS control; p=0.8242 compared to Gal4 control; hemocyte and fat body UAS only control: 23.14±1.181, 14 NMJs from 7 larvae; hemocyte and fat body Gal4 only control: 24.25±0.8972, 12 NMJs from 6 larvae; c564 >UAS-trol-RNAi.2: 23.44±0.9999, 16 NMJs from 8 larvae; p=0.9705 compared to UAS control; p=0.8149 compared to Gal4 control. (C) Representative images of muscle 4 NMJs stained for Perlecan and Hrp in control (TrolGFP,UAS-trol-RNAi.1) or trol RNAi knockdown by elavC155, elavC155 and mef2-Gal4, or repo-Gal4 (one copy of UAS and Gal4 constructs). (D–G) Line scanning profiles of Perlecan (D,F) or Hrp (E,G) mean fluorescent intensity through axons (D,E) or synaptic boutons (F,G) at muscle 4 in segment A4. Measurements are color-coded as indicated: control (green), trol RNAi in neurons (magenta), neurons and muscles (blue), or glia (light blue). No reduction in Perlecan around nerves or on the muscle surface surrounding boutons was observed compared with tubulin-Gal4 knockdown (Figure 1F–I, replicated here (‘All cells’) for comparison).