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. 2021 Nov 17;7(47):eabh2399. doi: 10.1126/sciadv.abh2399

Fig. 3. Characterization of an infralimbic mGluR2 knockdown in CamKII-iCre rats.

Fig. 3.

(A) Dual luciferase assay of control AAV and mGluR2 knockdown AAV reveals a significant down-regulation of luciferase activity. (B) Schematic representation of the AAV construct. The short hairpin–mediated RNA (shRNA) coding sequence (in red) was split in the middle and inserted in opposite directions into the lox71 and lox66 flanked cassette. The reporter gene eYFP was also inverted and inserted opposite of the EF1α promoter. After Cre recombination, shRNA expression is driven by U6 promoter and eYFP by EF1α. (C) Schematic representation of AAV injection into the infralimbic and projection to the NAc. mGluR2 mRNA was detected via RNAscope, protein levels by Western blot using NAc shell tissue punches (see Fig. 3F). (D) Injection placements of Cre-inducible knockdown AAV and control AAV are represented by black circles. (E) Left: Representative image of eYFP and mGluR2 fluorescent in situ hybridization of CamKII-Cre mGluR2 knockdown AAV rats. Arrow indicates a single cell for mGluR2. Triangle indicate double-positive cells expressing eYFP and mGluR2. Scale bar, 10 μm. Right: Quantification of knockdown efficiency of mGluR2 on mRNA level using fluorescent in situ hybridization in CamkII-Cre rats. (F) Left: Representative Western blot for mGluR2/ß-actin from NAc shell tissue of CamKII-Cre knockdown AAV (KD) or control AAV (Ctrl) injection. M, marker. Right: Signal intensity of mGluR2 protein in NAc shell of CamKII-Cre knockdown AAV (black bar) or control (white bar). *P < 0.05 and **P < 0.01. (G) LY379268-stimulated G protein coupling (GPC) was decreased in the NAc shell following infralimbic mGluR2 knockdown (n = 4 per group; shUNC BL: 291 ± 20 nCi/g; shRNA BL: 279 ± 9 nCi/g; means ± SEM). (H) Normalized EPSP amplitude (means ± standard deviation) before (BL), during a 5-min application of 100-nM mGluR2 agonist LY379268, and during prolonged washout. Representative voltage traces of control and knockdown are shown (“1” labels BL traces; “2” labels traces after 45 min of LY379268 washout).