Homeostatic regulation of extracellular signal-regulated kinase 1/2 activity and axonal Kv7.3 expression by prolonged blockade of hippocampal neuronal activity

FIGURE 1. Prolonged activity blockade transiently reduces ERK1/2 activity before decreasing TrkB activity. Cultured hippocampal neurons (DIV 10) were treated for 24 h, 36 h, and 48 h with TTX (1 μM) to block their activity or its vehicle control (0.1% H2O), and subjected to immunoblot analysis for pTrkB (pTrkATyr674/675/pTrkBTyr706/707) and TrkB (A,B) or pERK1/2 (pERK1Thr202/Tyr204/pERK2Thr185/Tyr187) and ERK1/2 (C,D). GAPDH served as a loading control. (A,B) TTX treatment for 48 h decreased pTrkB level. (A) Representative immunoblots. (B) Quantification of pTrkB and total TrkB immunodensities. The immunodensity ratios (pTrkB/GAPDH and TrkB/GAPDH) were normalized to vehicle control. Number of culture dishes used: pTrkB (24 h: H2O = 6, TTX = 6; 36 h: H2O = 6, TTX = 6; 48 h: H2O = 13, TTX = 13), TrkB (24 h: H2O = 6, TTX = 6; 36 h: H2O = 6, TTX = 6; 48 h: H2O = 17, TTX = 20). (C,D) TTX treatment for 36 h decreased pERK1/2 level. (C) Representative immunoblots. (D) Quantification of pERK1/2 and total ERK1/2 immunodensities. The immunodensity ratios (pERK1/2/GAPDH and ERK1/2/GAPDH) were normalized to vehicle control. Number of culture dishes used: pERK1/2 (24 h: H2O = 6, TTX = 6; 36 h: H2O = 6,…

FIGURE 2. Prolonged pharmacological inhibition of ERK1/2 decreased mRNA and protein expression of Kv7.3. (A,B) Cultured hippocampal neurons (DIV 10) were treated for 48 h with vehicle control (0.1% v/v DMSO) or 20 μM U0126. Immunoblot analysis was performed with antibodies for pERK1/2 (pERK1Thr202/Tyr204/pERK2Thr185/Tyr187), ERK1/2, and GAPDH (n = 6 per treatment). (A) Representative immunoblots. (B) Quantification of pERK1/2 and total ERK1/2 immunodensities normalized to vehicle control. (C) Both TTX and U0126 treatment for 48 h decreased BDNF and KCNQ3 expression. Cultured hippocampal neurons (DIV 10) treated for 48 h with 1 μM TTX or its vehicle control (0.1% H2O), and 20 μM U0126 or its vehicle control (0.1% DMSO). QPCR was performed using the cDNA which was prepared from 2 μg of total RNA isolated from cultured neurons and validated primers for Kcna1, Kcna4, Kcnq3, Bdnf, Camk4, and Gapdh (n = 5 per treatment). Data were analyzed using the comparative threshold cycle (Ct) method and Gapdh internal control gene. Following normalization to Gapdh cDNA levels (which is reflected in the ΔCt values), the relative mRNA quantification (RQ) of the fold change for each condition compared to…

FIGURE 3. Prolonged activity blockade decreases Kv7.3 and ankyrin-G expression at the AIS. Cultured hippocampal neurons (DIV 11–12) were treated for 48 h with 1 μM TTX or its vehicle control (0.1% H2O) and subjected to immunocytochemistry with antibodies for the AIS marker ankyrin-G, somatodendritic marker MAP2, or Kv7.3. (A) Representative images of ankyrin-G and MAP2. (B) Representative images of ankyrin-G and Kv7.3. (C) Quantification of the background-subtracted raw integrated fluorescent intensity of ankyrin-G-positive segment in the axon, which was normalized to vehicle control, and the length of the ankyrin-G-positive segment. (D) Quantification of the background-subtracted raw integrated fluorescent intensity of Kv7.3 within the ankyrin-G-positive segment, which was normalized to vehicle control. Data shown represent the mean ± SEM with individual data points (H2O = 62 neurons from 46 images, TTX = 61 neurons from 43 images). Images were collected from 2 independent experiments. The Student’s t-test was used (*p < 0.05). Scale bar = 20 μm.

FIGURE 4. Prolonged pharmacological inhibition of ERK1/2 decreases Kv7.3 and ankyrin-G expression at the AIS and the AIS length. Cultured hippocampal cultured neurons (DIV 11–12) were treated for 48 h with 20 μM U0126 or its vehicle control (0.1% DMSO) and subjected to immunocytochemistry with antibodies for the AIS marker ankyrin-G, somatodendritic marker MAP2, or Kv7.3. (A) Representative images of ankyrin-G and MAP2. (B) Representative images of ankyrin-G and Kv7.3. (C) Quantification of the background-subtracted raw integrated fluorescent intensity of ankyrin-G-positive segment in the axon, which was normalized to vehicle control, and the length of the ankyrin-G-positive segment. (D) Quantification of the background-subtracted raw integrated fluorescent intensity of Kv7.3 within the ankyrin-G-positive segment, which was normalized to vehicle control. Data shown represent the mean ± SEM with individual data points (DMSO = 132 neurons from 69 images, U0126 = 126 neurons from 53 images). Images were collected from 2 independent experiments. The Student’s t-test was used (***p < 0.005). Scale bar = 20 μm.