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. 2015 May 26;112(24):E3131–E3140. doi: 10.1073/pnas.1506488112

Fig. 5.

Fig. 5.

PSD-95 controls both the maturational unsilencing and the stabilization of the matured state of synapses onto pyramidal neurons. (A) Knockdown efficiency of AAV-sh95 tested in dissociated mouse neuronal cultures by immunoblotting. PSD-95 protein levels were reduced by ∼90% in AAV-sh95 transduced cultures, whereas AAV-GFP had no effect on PSD-95 protein levels. Mortalin was probed as a loading control. (B) Sample image of a patched AAV-sh95–transduced V1 pyramidal neuron with differential interference contrast imaging (Left) and fluorescence imaging (Right). (Scale bar, 10 µm.) (C) Sample traces of EPSCs were recorded at Vh = −60 mV (downward deflection) and Vh = +40 mV (upward deflection) from nontransduced control (black), AAV-sh95–transduced neuron (blue) and AAV-GFP transduced neuron (green). (Scale bar, 20 ms and 50 pA.) (D) Summary graph of AAV-sh95 transduced neurons at P0, recorded on P30–33 and P60–70 [Fag(1,40) = 7.71, P < 0.01; Fgt(1,40) = 20.78, P < 0.001; control P30–33, n/m = 8/3 vs. sh95 P30–33, n/m = 8/3, P > 0.05; control P60–70, n/m = 17/6; sh95 P60–70, n/m = 11/3; sh95 P30–33 vs. sh95 P60–70, P > 0.05]. AAV-GFP transduced neurons on P0, recorded on P60–70 (GFP P60–70, n/m = 19/5 vs. control P60–70, P = 0.99). (E and F) Sample traces of EPSCs with minimal stimulation. AAV-sh95 was injected on P0 into the visual cortex of WT mice and nontransduced control (E) and AAV-sh95 transduced pyramidal neurons (F) recorded on ∼P30. (G) Summary graph of WT mice, injected with AAV-sh95 on P0 and sh95-transduced and control neurons analyzed on P30–33. (H and I) Sample traces of EPSCs with minimal stimulation. AAV-sh95 was injected on P40 into V1 and control (H) and AAV-sh95–transduced pyramidal neurons (I) recorded on P60–70. (J) Summary graph of control or transduced pyramidal neurons. AAV-sh95 was injected on P40 into V1 and analyzed on P60–70. (K) AMPA/NMDA receptor EPSC ratios of AAV transduced neurons at P60, recorded at P80–90. (G, J, and K) Number of cells/mice in the foot of the bar. *P < 0.05; **P < 0.01; ***P < 0.001. Values in Table S2.