Figure 1.
Morphological and immunostaining characterization of OLCs in rat primary cultures.
(A) Double immunostaining of cultured OPCs showing that A2B5-positive cells (red) were also immunopositive for anti-NG2 (green). (B) OPCs (NG2-positive, green) were negative for anti-GFAP (red). (C) There were NG2-positive cells (red) after differentiation in T3 contained medium for two days. Immature oligodendrocytes (IOs) were stained with O4 antibody (green), a marker of IOs. Some cells co-localized with O4 and NG2 (Asterisk) (D) Four days later, NG2-positive cells (red) still existed and mature oligodendrocytes (MOs) were stained with MBP antibody (green), a marker of MOs. Scar bar = 100 µm.
Figure 2.
RT-PCR analysis for primary cultured OLCs.
(A–D, left) The mRNA of GAPDH (243 bp), KCNQ2 (172 bp), KCNQ3 (121 bp), KCNQ4 (110 bp) and KCNQ5 (320 bp) was identified in OLCs and whole forebrain (positive control) by RT-PCR. (A, C, D, right) RNA from OLCs without reverse transcription was performed PCR procedure directly as negative control.
Figure 3.
Immunofluorescence localization of KV7.2-5/KCNQ2-5 subunits on the OLCs in rat primary cultures.
(A–D) Co-localization of immunostaining for KV7.2-5 subunits (green) and OPCs (NG2-positive, red) is displayed in the merged image. Higher magnification of the boxed area in A, B, C, D was shown in a1, a2; b1,b2; c1,c2; d1,d2. (E–H) The expression of KV7.2-5 subunits (green) on IOs (O4-positive, red). Higher magnification of the boxed area in E, F, G, H was shown in e1, e2; f1,f2; g1,g2; h1,h2. (I–L) The expression of KV7.2-5 subunits (green) on MOs (MBP-positive, red). Higher magnification of the boxed area in I, J, K, L was shown in i1, i2; j1,j2; k1,k2; l1,l2. Scar bar = 100 µm.
Figure 4.
Immunofluorescence localization of KV7.2-5/KCNQ2-5 on NG2-positive cells (green) of the rat brain slices.
(A–C) Co-localization of KV7.2, 3, 5 and OPCs is displayed in the merged image. Higher magnification of the boxed area in A, B and C was shown in a1–a3; b1–b3;c1–c3;d1–d3;e1–e3;f1–f3;g1–g3;h1–h3 and i1–i3. (D) KV7.4 was not detected on OPCs. j1–j3: higher magnification of the boxed area in D. The nuclei were stained with Hoechst (blue). (E) Schematic diagram of the brain coronal and the box represented A–D areas. Scar bar = 100 µm.
Figure 5.
KV7/KCNQ channel current (IK(Q)) in OPCs of rat primary cultures.
(A) IK(Q) was measured with whole cell patch clamp recording from OPCs. Left insert: Standard IM deactivation voltage protocol used to measure IK(Q). Hyperpolarizing voltage steps were given from a holding potential of −20 to −60 mV (in 10 mV decrements). Currents recorded are shown below; the dashed line represents the zero current level. Right: Current recorded in response to the voltage step to −40 mV. IK(Q) was measured as the inward relaxation current caused by deactivation of IK(Q) during the voltage step; i.e., the difference between the instantaneous current at the beginning and the steady-state current at the end of the voltage step (arrows). (B) Current–voltage relationship for IK(Q) (mean data from 17 OPCs) showing that IK(Q) amplitude was voltage dependent and was largest at −40 mV. (C) IK(Q) deactivation time constants were directly related to voltage (mean data from 17 OPCs). Correlation coefficient r = 0.93.
Figure 6.
Inhibition of IK(Q) by XE991 and TEA.
(A, B) Representative current traces, which were recorded before (Control) and after extracellular application of XE991 (10 µM, 30 µM) in OPCs. (C, D) Representative current traces, which were recorded before (Control) and after extracellular application of XE991 (10 µM, 30 µM) in differentiated oligodendrocytes. Insert: 1-s-long hyperpolarizing voltage step from a holding potential of −20 to −40 mV was given to monitor IK(Q). (E) Concentration–response curve showing the mean percentage inhibition of IK(Q) amplitude as a function of the log XE991 concentration for 54 OPCs. Smooth curve was fit with the Hill equation. IC50 value for XE991 inhibition determined from this pooled log concentration–response curve was 13.3 µM. n = 0.63, R2 = 0.99. (F) Concentration–response curve showing the mean percentage inhibition of IK(Q) amplitude as a function of the log TEA concentration for 20 OPCs. Smooth curve was fit with the Hill equation. IC50 value for TEA inhibition determined from this pooled log concentration–response curve was 0.84 mM. n = 0.7, R2 = 0.99.
Figure 7.
The inhibition of KV7/KCNQ channels promotes OPCs motility in vitro.
(A,B) Photomicrograph of OPCs transmigrated through the filter in the absence or presence of XE991 or TEA. Scar bar = 150 µm. (C, D) Quantitative assessment of migrated cells under different conditions. n(XE991) = 9; n(TEA) = 9. **P<0.01 versus control.
Table 1.
Primers for PCR.