Kv7 channels are upregulated during striatal neuron development and promote maturation of human iPSC-derived neurons

Figures

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  Fig. 1. Ontogeny of membrane potential and Kv7.2 and Kv7.3 channel protein expression in mouse striatal neurons isolated at embryonic stages E13, E15 and E17 and differentiated in vitro. a Exponential fits of membrane potential (Vm) changes in mouse striatal neurons isolated at embryonic stages E13 (black circles), E15 (grey triangles) and E17 (empty squares) and cultured for up to 15 days post-plate down (dpp). The dashed boxes represent the time windows for which the analyses in b have been derived. b Bar charts presenting means of Vm measured in mouse striatal neurons isolated at the embryonic stages indicated above each bar (E13 (black), E15 (grey) and E17 (white)) and assayed at 1 dpp (left), 7 dpp (centre) and 14 dpp (right). Number of observations shown in parentheses. Significant differences, determined by unpaired Student’s t test, are shown below the bars, where appropriate; ****p < 0.0001. c Kv7.2 (left panels, ×20 and ×100, as indicated) and Kv7.3 (right panels, ×20 and ×100, as indicated) protein expression by immunocytochemistry in E15 mouse striatal neurons during in vitro maturation at 1, 7 and 14 dpp

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  Fig. 2. Ontogeny of KCNQ2 and KCNQ3 mRNA expression in developing mouse striatum in vivo. a KCNQ2 mRNA expression during mouse striatal development in vivo. In a and b, mRNA levels were determined in laser-microdissected mouse striatal regions by qPCR and were normalised to the house-keeping genes (18S/β-actin) using the ΔΔCt method. By two-way ANOVA, there was a significant difference between KCNQ2 mRNA in mantle zone (MZ) and germinal zone (GZ), **p < 0.01, with a significant enrichment in MZ at E14.5, asterisk above the MZ bar, *p < 0.05. b KCNQ3 mRNA expression during mouse striatal development in vivo. By two-way ANOVA, there were significant regional differences, ***p < 0.001, with significant enrichments in the MZ at E14.5, E16.5 and E18.5, asterisks above the MZ bars, *p < 0.05, **p < 0.01. c KCNQ2 mRNA expression during mouse striatal development in vivo. For c and d, mRNA levels in laser-microdissected mouse striatal regions were determined by qPCR and were normalised to the house-keeping genes (18S/β-actin) using the ΔΔCt method. Data were analysed by one-way ANOVA with Tukey’s post hoc test, and showed that, compared to LGE, KCNQ2 mRNA levels in each zone did not…

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  Fig. 3. Ontogeny of KCNQ mRNA expression in developing mouse and human striatum. a KCNQ mRNA expression in mouse striatal neurons during development in vivo. mRNA levels were determined in mouse striatal neurons isolated at E13, E15 and E17 by qPCR and were normalised to the house-keeping genes (18S/β-actin) using the ΔΔCt method. By one-way ANOVA (Tukey’s post hoc), there were significant differences between KCNQ mRNA at E15 and E17 with its own control at E13, p < 0.05. b KCNQ mRNA expression in E13 mouse striatal neurons during in vitro differentiation and maturation. By one-way ANOVA (Tukey’s post hoc), there were significant differences of KCNQ2 and KCNQ3 mRNA between 1 and 7 dpp, p < 0.05. c KCNQ mRNA expression during human striatal development in vivo. mRNA levels in human striatal regions dissected from cryosections were determined by qPCR and were normalised to house-keeping genes (RPL13A and HSP90AB1) using the ΔΔCt method. By unpaired Student’s t test, there were significant differences between whole ganglionic eminence (WGE, 7–9 weeks post-conception) and adult striatum expression of KCNQ3, KCNQ4 and KCNQ5 mRNA, *p < 0.05

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  Fig. 4. Ontogeny of KCNQ mRNA and Kv7 channel protein expression during neuronal differentiation of CTR33Qn1 hiPSC-derived neurons. a Ontogeny of KCNQ2 (upper left), KCNQ3 (upper right), KCNQ4 (lower left) and KCNQ5 (lower right) mRNA expression during neuronal differentiation of CTR33Qn1 hiPSC-derived neurons from pre-patterned forebrain progenitors (16 dpp) to maturation (37 dpp). mRNA levels were determined by qPCR, were normalised to the house-keeping genes (B2M, RPL13A and HSP90AB1) using the 2−ΔΔCt method and expressed as a level relative to 16 dpp. Data were analyses by one-way AVOVA followed by Tukey’s post hoc test. Differences from 16 dpp are shown, **p < 0.01, ***p < 0.001. b Comparison of mean ∆Ct values of each KCNQ during CTR33Qn1 hiPSC neuronal differentiation. KCNQ2 and KCNQ3 values were significantly lower than KCNQ4 or KCNQ5, indicating that the former are expressed at a higher level than the latter. Two-way ANOVA, p < 0.001. c Immunocytochemistry showing expression of Kv7.2 (left) and Kv7.3 (right) protein in CTR33Qn1 hiPSC-derived neurons at 19 dpp and 31 dpp during neuronal differentiation, at ×20 and ×100, as indicated

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  Fig. 5. Spontaneous unit responses recorded from CTR33Qn1 hiPSC-derived neurons plated in 24-well multi-electrode arrays (MEAs). a Exemplars of 5-s snapshots of raw data taken from one electrode (position 24) within one MEA well. The first recording (left) is in the absence of drugs. The subsequent recordings (right) are in the presence of GABAA receptor-antagonist gabazine (5 μM), Kv7 channel opener retigabine (10 μM) and Kv7 channel selective blocker XE991 (10 μM). b Histograms of spike rate versus time for unit responses detected from individual MEA electrodes. Four consecutive 120-s recording epochs are shown from two electrodes, position 24 (top) and position 43 (bottom). The first epoch is in the absence of drugs. The inset shows 25 superimposed units detected during the first epoch. Drugs were applied immediately before the recording. Gabazine (5 μM) was applied initially to increase spontaneous activity of CTR33Qn1 hiPSC-derived neurons. Retigabine (10 μM) eliminated spiking and XE991 (10 μM) restored spiking to a rate similar to that observed before introduction of retigabine. c Bar charts presenting means of spike frequency ± SEM measured in CTR33Qn1 hiPSC-derived neurons…

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  Fig. 6. Forced expression of Kv7.2/7.3 channel subunits facilitates functional maturation of in CTR33Qn1 hiPSC-derived neurons. a Exemplar traces of current-clamp recordings (injected current = 0 pA) for CTR33Qn1 hiPSC-derived neurons exemplifying the model activity of no activity (Quiet), attempting activity (Attempting) or spontaneous activity (Spontaneous). b Bar graphs showing percentage of CTR33Qn1 hiPSC-derived neurons exhibiting no activity (Quiet), attempting activity (Attempting) or spontaneous activity (Spontaneous) (black bars, left y-axis) and their mean Vm (open bars, right y-axis) in sham-transfected (left panels) and Kv7.2/7.3 co-transfected CTR33Qn1 hiPSC-derived neurons (right panels) following 1, 2 and 3 weeks of in vitro neuronal differentiation

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  Fig. 7. Forced expression of Kv7.2/7.3 channels facilitates functional maturation of 34D6 hiPSC-derived neurons. a Pharmacological identification of Kv7.2/7.3 channels (retigabine-activated) in an exemplar (injected current = 0 pA), sham-transfected, spontaneously active 34D6 hiPSC-derived neuron following 2 weeks of in vitro neuronal differentiation. Vm was measured in current-clamp mode of the whole-cell patch-clamp configuration in the absence and presence of 10 μM retigabine (indicated above the main trace by a transparent box). Below the trace are extracted action potential events shown on a fester time base. b Pharmacological identification of Kv7.2/7.3 channels (retigabine-activated) in an exemplar (injected current = 0 pA), Kv7.2/7.3 co-transfected, spontaneously active 34D6 hiPSC-derived neuron following 2 weeks of in vitro neuronal differentiation. Recording and display parameters as in a. c Bar graphs showing percentage of 34D6 hiPSC-derived neurons exhibiting no activity (Quiet), attempting activity (Attempting) or spontaneous activity (Spontaneous) (black bars, left y-axis) and their mean Vm (open bars, right y-axis) in sham-transfected (left panel) and Kv7.2/7.3…

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  Fig. 8. Effect of forced expression of Kv7.2/7.3 channels on voltage-gated Na+ current activation and inactivation characteristics of CTR33Qn1 hiPSC-derived neurons. a Exemplar family of whole-cell currents (upper) during the activation/inactivation voltage-clamp step protocol (lower). Peak Na+ current activation and inactivation levels are shown by the single- and double-headed arrows, respectively. b Mean peak ± SEM Na+ current densities measured from the peak of the inward currents exemplified in a. Data from sham-transfected week 1 (n = 8), week 2 (n = 7) and week 3 (n = 9) are shown by black bars. Data from Kv7.2/7.3 co-transfection week 1 (n = 12), week 2 (n = 8) and week 3 (n = 10) are shown by grey bars. c–h Mean activation and inactivation curves of whole-cell Na+ currents recorded in CTR33Qn1-dervied neurons cultured in vitro for 1 (c, d), 2 (e, f) and 3 weeks (g, h) following sham transfection (c, e, g, black symbols and lines) or forced co-expression of Kv7.2/7.3 (d, f, h, grey symbols and lines). Activation curves are depicted by the squares and inactivation curves are shown by the circles. On each panel, also shown are individual Vm values (triangles) and mean Vm values…