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. 2021 Dec 20;298(2):101508. doi: 10.1016/j.jbc.2021.101508

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© 2021 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

shRNA-mediated NCLX knockdown does not grossly affect synaptic activity–dependent gene transcription. QRT–PCR analysis of activity-induced genes Arc (A), Atf3 (B), Bdnf (C), cFos (D), and Npas4 (E), as well as Nclx (F) for primary hippocampal cultures infected with either rAAV-shCTRL or rAAV-shNCLX and stimulated (or not) with the GABA_AR antagonist, bicuculline (Bic; 50 μM) for 1 h or 24 h to evoke action potential bursting (n = 4–5 independent cultures; mixed-effects model two-way ANOVAs followed by Tukey's multiple comparisons tests; A, Arc: main effect of treatment F_(2,8) = 28.39, p = 0.0002, shCTRL control versus 1 h Bic q_(5,5) = 8.871, p = 0.0058, shCTRL control versus 24 h Bic q_(5,4) = 0.7924, p = 0.9904, shNCLX control versus 1 h Bic q_(5,5) = 9.742, p = 0.0036, shNCLX control versus 24 h Bic q_(5,4) = 0.3635, p = 0.9998; main effect of shRNA F_(1,4) = 0.3612, p = 0.5802, control shCTRL versus shNCLX q_(5,5) = 0.1479, p > 0.9999, 1 h Bic shCTRL versus shNCLX q_(5,5) = 2.427, p = 0.5637, 24 h Bic shCTRL versus shNCLX q_(4,4) = 0.9323, p = 0.9806. B, Atf3: main effect of treatment F_(2,8) = 32.38, p = 0.0001, shCTRL control versus 1 h Bic q_(5,5) = 7.302, p = 0.0011, shCTRL control versus 24 h Bic q_(5,4) = 0.9848, p = 0.0854, shNCLX control versus 1 h Bic q_(5,5) = 8.907, p = 0.0057, shNCLX control versus 24 h Bic q_(5,4) = 2.498, p = 0.5424; main effect of shRNA F_(1,4) = 67.98, p = 0.0012, control shCTRL versus shNCLX q_(5,5) = 3.171, p = 0.3385, 1 h Bic shCTRL versus shNCLX q_(5,5) = 9.589, p = 0.0039, 24 h Bic shCTRL versus shNCLX q_(4,4) = 7.389, p = 0.0145. C, Bdnf: main effect of treatment F_(2,8) = 12.26, p = 0.0037, shCTRL control versus 1 h Bic q_(5,5) = 1.983, p = 0.7269, shCTRL control versus 24 h Bic q_(5,4) = 7.969, p = 0.0100, shNCLX control versus 1 h Bic q_(5,5) = 2.318, p = 0.6061, shNCLX control versus 24 h Bic q_(5,4) = 5.661, p = 0.0488; main effect of shRNA F_(1,4) = 12.77, p = 0.0233, control shCTRL versus shNCLX q_(5,5) = 0.2197, p > 0.9999, 1 h Bic shCTRL versus shNCLX q_(5,5) = 1.769, p = 0.8005, 24 h Bic shCTRL versus shNCLX q_(4,4) = 9.928, p = 0.0032. D, cFos: main effect of treatment F_(2,8) = 23.48, p = 0.0004, shCTRL control versus 1 h Bic q_(5,5) = 8.607, p = 0.0068, shCTRL control versus 24 h Bic q_(5,4) = 1.320, p = 0.9237, shNCLX control versus 1 h Bic q_(5,5) = 9.268, p = 0.0069, shNCLX control versus 24 h Bic q_(5,4) = 0.7911, p = 0.9285; main effect of shRNA F_(1,4) = 0.1792, p = 0.6938, control shCTRL versus shNCLX q_(5,5) = 0.1527, p > 0.9999, 1 h Bic shCTRL versus shNCLX q_(5,5) = 4.251, p = 0.1456, 24 h Bic shCTRL versus shNCLX q_(4,4) = 2.973, p = 0.3916. E, Npas4: main effect of treatment F_(2,8) = 25.40, p = 0.0003, shCTRL control versus 1 h Bic q_(5,5) = 9.196, p = 0.0048, shCTRL control versus 24 h Bic q_(5,4) = 0.7618, p = 0.9920, shNCLX control versus 1 h Bic q_(5,5) = 8.980, p = 0.0055, shNCLX control versus 24 h Bic q_(5,4) = 0.4546, p = 0.9993; main effect of shRNA F_(1,4) = 0.8438, p = 0.4103, control shCTRL versus shNCLX q_(5,5) = 0.2204, p > 0.9999, 1 h Bic shCTRL versus shNCLX q_(5,5) = 0.9754, p = 0.9766, 24 h Bic shCTRL versus shNCLX q_(4,4) = 1.419, p = 0.9012. F, Nclx: main effect of treatment F_(2,8) = 5.121, p = 0.0.0370, main effect of shRNA F_(1,4) = 251.2, p < 0.0001, shCTRL control versus 1 h Bic q_(5,5) = 1.065, p = 0.9664, shCTRL control versus 24 h Bic q_(5,4) = 5.678, p = 0.0482, shNCLX control versus 1 h Bic q_(5,5) = 0.1500, p > 0.9999, shNCLX control versus 24 h Bic q_(5,4) = 1.402, p = 0.9054, control shCTRL versus shNCLX q_(5,5) = 17.82, p = 0.0001, 1 h Bic shCTRL versus shNCLX q_(5,5) = 16.80, p = 0.0002, 24 h Bic shCTRL versus shNCLX q_(4,4) = 11.44, p = 0.0015). ns, ∗p < 0.05, ∗p < 0.01, and ∗∗∗p < 0.001. Bar graphs show the mean + SEM. Arc, activity regulated cytoskeletal-associated protein; Atf3, activating transcription factor 3; Bdnf, brain derived neurotrophic factor; Bic, bicuculline; cFos, FBJ osteosarcoma oncogene; GABA_AR, gamma-aminobutyric acid (GABA) A receptor; NCLX, solute carrier family 8 sodium/calcium/lithium exchanger, member B1; Npas4, neuronal PAS domain protein 4; ns, not significant; qRT–PCR, quantitative reverse transcription polymerase chain reaction; rAAV, recombinant adeno-associated viral vector.