Figure 5. BDNF signaling was critical for activity-induced presynaptic changes at GABAergic synapses. A, BMI-induced upregulation of BDNF mRNA (10.70 ± 1.78-fold, p < 0.001) was blocked by coapplication of TTX (TTX: 0.40 ± 0.09-fold, p > 0.05 vs Ctrl; TTX + BMI: 0.97 ± 0.17, p > 0.05 vs TTX). B, K252a blocked BMI-induced increase in mIPSC frequency (Ctrl: 1.68 ± 0.23 Hz; BMI: 2.67 ± 0.24 Hz, p < 0.01 vs Ctrl; K252a: 1.45 ± 0.16 Hz, p > 0.05 vs Ctrl; BMI + K252a: 1.68 ± 0.22 Hz, p > 0.05 vs K252a); TrkB-Fc also blocked BMI-induced increase in mIPSC frequency (IgG-Fc: 1.16 ± 0.12 Hz; IgG-Fc + BMI: 2.95 ± 0.37 Hz, p < 0.001 vs IgG-Fc; TrkB-Fc: 1.53 ± 0.23 Hz, p > 0.05 vs IgG-Fc; TrkB-Fc + BMI, 1.78 ± 0.30 Hz, p > 0.05 vs TrkB-Fc). C, Four hour BDNF treatment did not affect mIPSC amplitude, but significantly increased its frequency (Ctrl: 1.19 ± 0.17 Hz, BDNF: 1.93 ± 0.21 Hz, p < 0.01). D, BMI increased the mIPSC amplitude of both neurons transfected with scrambled RNAi (ScrRNAi: 15.52 ± 0.58 pA; ScrRNAi + BMI: 20.67 ± 0.91 pA, p < 0.001) and those transfected with BDNF RNAi (BDNF RNAi: 14.75 ± 0.83 pA, p > 0.05 vs ScrRNAi; BDNF RNAi + BMI, 18.02 ± 1.01 pA, p < 0.05 vs BDNF RNAi). E, Corresponding cumulative distributions: ScrRNAi versus ScrRNAi + BMI, p < 0.001; BDNF RNAi versus BDNF RNAi + BMI, p < 0.01. F, BMI increased the mIPSC frequency of neurons transfected with ScrRNAi (ScrRNAi: 0.38 ± 0.05 Hz; ScrRNAi + BMI: 0.87 ± 0.14 Hz, p < 0.05), an effect blocked by BDNF RNAi (BDNF RNAi: 0.55 ± 0.10 Hz; BDNF RNAi + BMI: 0.64 ± 0.13 Hz, p > 0.05 vs BDNF RNAi). G, Corresponding cumulative distributions: ScrRNAi versus ScrRNAi + BMI, p < 0.001; BDNF RNAi versus BDNF RNAi + BMI, p = 0.41. H–J, Representative images and quantifications of the average density and integrated intensity of synaptic vGAT in neurons transfected with ScrRNAi or BDNF RNAi, in Ctrl or BMI conditions. Scale bar is 5 μm. I, BMI treatment increased the density of synaptically localized vGAT puncta (1.21 ± 0.07, p < 0.05), an effect blocked by BDNF RNAi (BDNF RNAi: 0.91 ± 0.05, p > 0.05 vs ScrRNAi; BDNF RNAi + BMI: 0.94 ± 0.06, p > 0.05 vs BDNF RNAi). J, BMI treatment did not affect the integrated intensity of synaptic vGAT in neurons transfected with ScrRNAi (ScrRNAi + BMI 1.02 ± 0.04, p > 0.05) or BDNF RNAi (BDNF RNAi: 0.82 ± 0.04, p < 0.01 vs ScrRNAi; BDNF RNAi + BMI: 0.92 ± 0.04, p > 0.05 vs ScrRNAi, p > 0.05 vs BDNF RNAi). K–N, Postsynaptic TrkB-T1 did not block BMI-induced increase in mIPSC amplitude or frequency. K, An illustration showing that recording was made from a low-density transfected neuron surrounded by many untransfected neurons. L, BMI increased the mIPSC amplitude of both neurons transfected with GFP (Ctrl: 12.66 ± 1.00 pA; Ctrl + BMI: 17.11 ± 0.92 pA, p < 0.01) and those transfected with TrkB-T1 (TrkB-T1: 13.16 ± 0.86 pA, p > 0.05 vs Ctrl; TrkB-T1 + BMI, 17.06 ± 1.04 pA, p < 0.05 vs TrkB-T1). M, BMI increased the mIPSC frequency of both neurons transfected with GFP (Ctrl: 1.02 ± 0.14 Hz; Ctrl + BMI: 1.82 ± 0.17 Hz, p < 0.01), and those transfected with TrkB-T1 (TrkB-T1: 0.99 ± 0.13 Hz, p > 0.05 vs Ctrl; TrkB-T1 + BMI: 1.86 ± 0.20 Hz, p < 0.01 vs TrkB-T1). N, Cumulative distributions of mIPSC frequency: Ctrl versus BMI, p < 0.001; TrkB-T1 versus TrkB-T1 + BMI, p < 0.001. O–R, Interfering with presynaptic BDNF signaling with high-efficiency TrkB-T1 transfection significantly blocked the BMI-induced increase of mIPSC frequency. O, An illustration showing that recording was made from an untransfected neuron surrounded by several transfected neurons. P, BMI increased the mIPSC amplitude of both untransfected neurons neighboring GFP-expressing neurons (GFP neighbor: 25.62 ± 1.57 pA; GFP neighbor + BMI: 37.98 ± 3.34 pA, p < 0.01), and untransfected neurons neighboring TrkB-T1-expressing neurons (TrkB-T1 neighbor: 25.95 ± 1.75 pA, p > 0.05 vs GFP neighbor; TrkB-T1 neighbor + BMI: 36.36 ± 2.59 pA, p < 0.05 vs TrkB-T1 neighbor). Q, BMI-induced increase in mIPSC frequency (GFP neighbor: 0.83 ± 0.18 Hz; GFP neighbor + BMI: 2.33 ± 0.39 Hz, p < 0.01) was significantly blocked by expression of TrkB-T1 in neighboring neurons (TrkB-T1 neighbor: 1.16 ± 0.25 Hz, p > 0.05 vs GFP neighbor; TrkB-T1 neighbor + BMI: 1.48 ± 0.28 Hz, p > 0.05 vs TrkB-T1 neighbor). R, Corresponding cumulative distributions: GFP neighbor versus GFP neighbor + BMI, p < 0.001; TrkB-T1 neighbor versus TrkB-T1 neighbor + BMI, p = 0.10. *p < 0.05, **p < 0.01, ***p < 0.001.