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. 2017 Mar 6;8:14536. doi: 10.1038/ncomms14536

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Copyright © 2017, The Author(s)

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(a) Schematic of injection of AAV5-scrambled#1 (or #2) and AAV5-shRNA#1 (or #2) into opposing DG of rat, with time line for recovery and experiments. Slices were prepared 3 weeks after injection. (b) Tonic currents activated by 5 μM GABA in the presence of kynurenic acid (3 mM) of DG granule cells (DGGCs) in acute brain slices taken from hemispheres injected with either AAV5-shRNA#1 or AAV5-scrambled#1. GABA-mediated current shift and noise variance were lower in slices injected with AAV5-shRNA#1 compared with AAV5-scrambled#1 (current shift: n=10, 12; **P=0.0050; t-test; noise variance: n=9, 11; **P=0.025; t-test). (c) Tonic currents activated by 5 μM GABA in the presence of CGP54626 (5 μM) of DGGCs in acute brain slices taken from hemispheres injected with either AAV5-shRNA#1 or AAV5-scrambled#1. A lower GABA-mediated current shift and current variance in slices pre-treated with CGP54268 from AAV5-shRNA#1 compared with AAV5-scrambled#1 injected animals were observed (current shift: n=6, 7; *P=0.0352; t-test; noise variance: n=5, 7; *P=0.0215; t-test). (d) THIP (3 μM)-activated currents of DGGCs in the presence of kynurenic acid (3 mM). THIP current shift and current variance were lower in slices injected with AAV5-shRNA#1 compared with AAV5-scrambled#1 (current shift: n=9, 11; *P=0.0225; t-test; noise variance: n=10, 13; *P=0.0419; t-test). (e) THIP (3 μM)-activated currents from DGGCs in the presence of kynurenic acid (3 mM). A lower THIP current shift and current variance was also observed in slices injected with a second knockdown construct AAV5-shRNA#2 compared with AAV5-scrambled#2. Application of bicuculline (20 μM) demonstrates the GABA_AR-mediated specificity of the THIP current (current shift: n=9, 8; *P=0.0341; t-test; noise variance: n=10, 8; *P=0.0465; t-test). (f) Co-immunoprecipitation of GABA_BRs and δ but not γ2 subunits of GABA_ARs using brain extracts (full blot is shown in Supplementary Fig. 11) All histograms were presented as mean±s.e.m.