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. 2002 Dec 9;159(5):747–752. doi: 10.1083/jcb.200209011

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Copyright © 2002, The Rockefeller University Press

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Figure 1. — Exogenous BDNF and NT-4 down-regulate KCC2 mRNA and protein expression in organotypic hippocampal slices. (A) A representative RT-PCR experiment (out of four similar ones) showing the down-regulatory effect of BDNF and NT-4 on KCC2 mRNA expression. The effects of BDNF and NT-4 were inhibited by 200 ng/ml TrkB-Fc. The amplified light neurofilament (NF-L) fragment indicates an equal amount of mRNA in the reactions. Note the increase in KCC2 mRNA brought about by exposure to TrkB-Fc only. (B) Western blot analysis showing the dose-dependent effect of BDNF. A representative blot (left) and the average normalized optical densities displayed as a percentage of control (right, mean ± SD, n = 5). (C) Representative Western blot (top) showing that KCC2 protein levels are down-regulated by BDNF or NT-4. The Trk inhibitor K252a and TrkB-Fc receptor body inhibited this effect. Note the significant increase in KCC2 protein level after treatment with TrkB-FC or K252a only. The bottom panel shows the average normalized optical densities displayed as percentage of control (mean ± SD, n = 24). (D) Inhibition of network activity with TTX or with glutamate blockers (CNQX and AP5) does not inhibit the down-regulation of KCC2 by BDNF in organotypic slices. The top panel shows a representative Western blot, and the bottom one the normalized optical densities (mean ± SD, n = 7; ***, P < 0.001; **, P < 0.05 as compared with control using the t test).