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. Author manuscript; available in PMC: 2016 Sep 9.
Published in final edited form as: Nature. 2016 Mar 9;531(7594):371–375. doi: 10.1038/nature17187

Extended Data Figure 6. Igf1 knockdown in VIP neurons affects inhibitory inputs onto VIP neurons but not onto neighboring neurons.

Extended Data Figure 6

a) AAV shRNA constructs. shRNA cassettes against Igf1 or a control gene (Luc) were cloned downstream of the U6 promoter into an AAV vector that drives Cre-dependent expression of EGFP.

b) qPCR validation of the efficacy of Igf1 shRNA constructs. Cultured cortical neurons were infected with lentiviral constructs either expressing no shRNA (Vector only), a control shRNA (against Luc) or shRNAs against Igf1. Four days post infection the cultures were quieted overnight with TTX and AP-5 and then harvested either prior to or after being depolarized with 55 mM KCl for 6 hours; RNA was then isolated and qPCR was performed. Data are normalized to the maximal value in each replicate and represent the mean and standard error of three biological replicates.

c) Bar graph showing normalized soma size of P21 visual cortex VIP neurons infected with control shRNA or shRNA targeting Igf1 (shRNA Control: n = 103, shRNA Igf1: n = 174, p =0.41, Mann Whitney U-Test).

d) Bar graphs showing the levels of IGF-1 in the serum of P20 mice that were injected intracortically with AAVs driving the expression of control shRNA (black) or Igf1 shRNA. Data represent the mean and SEM of the serum IGF-1 levels of 4 mice per group.

e–l) Bar graphs and cumulative distribution plots showing mIPSC amplitudes and frequency/interevent interval upon early widespread knockdown of Igf1 in VIP (e,f), SST (g,h), PV (i,j) and excitatory (k,l) neurons after injection of AAVs into P3 cortices of the respective Cre-mice. VIP neurons (identified by EGFP-positive cells in Vip-Cre mice): control shRNA and Igf1 shRNA (Amplitude p = 0.96, Frequency p = 0.04). SST neurons (identified by EGFP-positive cells in Sst-Cre mice): control and Igf1 shRNA (Amplitude p = 0.89, Frequency p = 0.55). PV neurons (identified by EGFP-positive cells in Pv-Cre mice): control and Igf1 shRNA (Amplitude p = 0.084, Frequency p = 0.93). Pyramidal neurons (identified by morphology): control and Igf1 shRNA (Amplitude p = 0.84, Frequency p = 0.15). (All p values are derived from Mann Whitney U-Tests; numbers inside bars indicate the number of cells recorded)