Article Figures & Data
Figures
- Figure 1.
Phasic inhibition is altered in Cntnap2 KO mice in an age-dependent manner. A, Example traces of sIPSC activity recorded from layer 2/3 pyramidal cells in WT (upper traces) and KO (lower traces) mice at three to four weeks old (left) and six to eight weeks old (right). B, The frequency was lower in KO mice compared to WT at six to eight weeks. There was no effect of age on the frequency of sIPSCs, and no statistical difference between the three- to four-week-old KO and WT groups. C, The amplitude of sIPSCs was significantly increased during postnatal development, but there was no effect of genotype. D, Average sIPSC waveforms for Cntnap2 WT and KO mice in both the three- to four- and six- to eight-week age groups. E, There was no significant effect of genotype or age group on the sIPSC rise time. F, There was no significant effect of genotype or age group on the sIPSC decay time tau. Scale bars: 50 pA, 0.5 s (A); 10 pA, 100 ms (D); *p < 0.05. Error bars show SEM.
- Figure 2.
GABA-induced tonic inhibitory currents are reduced in Cntnap2 KO mice in an age-dependent fashion. A, Example traces showing tonic inhibitory currents induced by 5 µM GABA in six- to eight-week-old WT (left trace) and KO (right trace) animals. Peaks are capacitive transients in response to voltage steps used to monitor series resistance. B, C, In slices from three- to four-week-old animals, there was no effect of Cntnap2 KO on pyramidal cell tonic current (IGABA) amplitude (B), or normalized tonic current density (C). In slices from six- to eight-week-old animals, KO pyramidal cells exhibited reduced tonic current amplitude (B), and reduced tonic current density (C) compared to cells from WT mice. Scale bars: 100 pA, 50 s (A); *p < 0.05. Error bars show SEM.
- Figure 3.
Reduction of δ-subunit-mediated tonic currents in six- to eight-week-old Cntnap2 KO mice. A, Example traces from layer 2/3 pyramidal cells from six- to eight-week-old WT (top) and KO (bottom) mice showing the current induced by exposure to 10 µM THIP, an agonist of δ-subunit-containing GABAARs. Peaks are capacitive transients in response to voltage steps used to monitor series resistance. B, C, Cells from KO animals exhibited smaller THIP-induced tonic currents (ITHIP; B) and smaller normalized tonic current density (C). Scale bars: 100 pA, 50 s (A); *p < 0.05. Error bars show SEM.
- Figure 4.
GABABR-mediated slow inhibitory currents do not change in Cntnap2 KO mice. A, Example traces from layer 2/3 pyramidal cells from six- to eight-week-old WT (left) and KO (right) mice, showing the current induced by exposure to 100 µM R-Baclofen, an activator of GABABRs. Peaks are capacitive transients in response to voltage steps used to monitor series resistance. B, C, No effect of genotype was found on R-Baclofen-induced current (IBaclofen) amplitude (B), or normalized current density (C). Scale bars: 100 pA, 50 s (A). Error bars show SEM.
- Figure 5.
Intrinsic excitability in layer 2/3 pyramidal cells. A, Membrane excitability was tested by injection of a ramp current. An example trace is shown at top. No effect of genotype or age was found in the current threshold in ramp tests. B, The injection of step currents of increasing amplitude was used for the determination of membrane excitability. There was no significant difference in the function of firing rate versus input current between WT and KO mice in either age groups (3–4 or 6–8 weeks). C, Step current injection revealed no effect of genotype or age on the current threshold. D, Step current injection revealed no effect of genotype or age on the maximum firing rate. Scale bars: 20 mV, 250 pA, 1 s (A); 20 mV, 250 pA, 0.25 s (B). Error bars show SEM.
- Figure 6.
Application of THIP, the δ-subunit containing GABAAR agonist, reduces pyramidal cell excitability. A, Example traces of action potential firing induced by step-current injection before and after bath application of THIP. Step currents were applied to layer 2/3 pyramidal cells to probe action potential firing frequency before and after the application of 10 µM THIP. B, Graphical comparison of the input current versus firing rate relationship in three- to four-week-old WT and KO mice, before and after exposure to 10 µM THIP. We observed a main effect of drug treatment exposure on the input-output curve, but no effect of genotype and no treatment × genotype interaction. C, Graphical comparison of the input current versus firing rate relationship in six- to eight-week-old WT and KO mice, before and after exposure to 10 µM THIP. The increase in the current threshold of action potential induction was significantly higher in WT than in KO cells. D, Bath application of 10 µM THIP significantly increased the current threshold in both WT and KO mice at three to four weeks old. E, Bath application of 10 µM THIP significantly increased the current threshold of spike induction in both WT and KO mice at six to eight weeks old. F, Comparison of the change in current threshold of spike induction before and after THIP application in six- to eight-week-old Cntnap2 WT and KO littermates. G, Superfusion with 10 µM THIP significantly reduced the slope of current-firing curve in three- to four-week-old WT and KO mice. H, Superfusion with 10 µM THIP significantly reduced the slope of current-firing curve in six- to eight-week-old WT and KO mice. I, Comparison of the change in the initial firing-rate slope (Δ-slope) before and after THIP application in six- to eight-week-old Cntnap2 WT and KO littermates. The reduction in the slope was significantly lower in KO mice than in WT. Scale bars: 20 pA, 1 s (A); *p < 0.05. Error bars show SEM.
Tables
Description Data structure Type of test Statistical value a sIPSC frequency (Fig. 1B) Normal distribution t test t(29) = 0.5388, p = 0.5941 (3–4 weeks); t(29) = 2.116, p = 0.0431 (6–8 weeks) b sIPSC amplitude (Fig. 1C) Normal distribution t test; ANOVA t(29) = 0.5665, p = 0.5754 (3–4 weeks); t(29) = 0.4330, p = 0.6682 (6–8 weeks); age: F(1,58) = 20.27, p < 0.0001 c sIPSC rise time (Fig. 1E) Normal distribution ANOVA F(1,60) = 2.188, p = 0.1443 (WT vs KO) d sIPSC decay time (Fig. 1F) Normal distribution ANOVA F(1,60) = 0.7872, p = 0.3785 (WT vs KO) e IGABA in six to eight weeks (Fig. 2B) Non-normal distribution M–W test p = 0.0282 f Cm in IGABA measurement Normal distribution t test t(36) = 0.4044, p = 0.6883 g Normalized IGABA in six to eight weeks (Fig. 2C) Non-normal distribution M–W test p = 0.0058 h IGABA in three to four weeks (Fig. 2B) Normal distribution t test t(31) = 0.4277, p = 0.8637 i Normalized IGABA in three to four weeks (Fig. 2C) Non-normal distribution M–W test p = 0.5529 j Cm in ITHIP measurement Normal distribution t test t(45) = 0.1597, p = 0.8739 k ITHIP (Fig. 3B) Normal distribution t test t(45) = 2.788, p = 0.0077 l Normalized ITHIP (Fig. 3C) Normal distribution t test t(45) = 2.979, p = 0.0047 m Cm in IBaclofen measurement Normal distribution t test t(28) = 1.533, p = 0.1366 n IBaclofen (Fig. 4B) Normal distribution t test t(28) = 1.182, p = 0.247 o Normalized IBaclofen (Fig. 4C) Non-normal distribution M–W test p = 0.9834 p Ramp-current threshold (Fig. 5A) Normal distribution t test t(23) = 0.1924, p = 0.849 (3–4 weeks); t(22) = 1.064, p = 0.299 (6–8 weeks) q Current-firing curve (Fig. 5B) ANOVA F(1,23) = 0.0228, p = 0.881 (3–4 weeks); F(1,22) = 1.320, p = 0.263, (6–8 weeks) r Step-current threshold (Fig. 5C) Normal distribution t test t(23) = 0.2003, p = 0.843 (3–4 weeks); t(22) = 0.8177, p = 0.4223 (6–8 weeks) s Max. firing rate (Fig. 5D) Normal distribution t test t(23) = 0.69, p = 0.4971 (3–4 weeks); t(22) = 0.9258, p = 0.3646 (6–8 weeks) t Current-firing curve in three to four weeks (Fig. 6B) ANOVA F(1,24) = 28.45, p < 0.0001 (WT, before vs THIP); F(1,24) = 14.66, p = 0.0008 (KO, before vs THIP); F(1,24) = 1.199, p = 0.2844 (before, WT vs KO); F(1,24) = 0.0073, p = 0.9328 (THIP, WT vs KO) u Current-firing curve in six to eight weeks (Fig. 6C) ANOVA F(1,30) = 57.87, p < 0.0001 (WT, before vs THIP); F(1,32) = 30.66, p = 0.0008 (KO, before vs THIP); F(1,31) = 1.228, p = 0.2764 (before, WT vs KO); F(1,31) = 4.919, p = 0.0340 (THIP, WT vs KO) v Step-current threshold (Fig. 6D) Normal distribution
Non-normal distributionPaired t test
Wilcoxon testt(12) = 7.229, p < 0.0001 (WT); p = 0.0005(KO) w Step-current threshold (Fig. 6E) Non-normal distribution
Normal distributionWilcoxon test
Paired t testp < 0.0001 (WT);
t(16) = 6.154, p < 0.0001 (KO)x Δ current threshold (Fig. 6F) Non-normal distribution M–W test p = 0.0257 y Slope in three to four weeks (Fig. 6G) Non-normal distribution Wilcoxon test p = 0.0002 (WT)
p = 0.0002 (KO)z Slope in six to eight weeks (Fig. 6H) Normal distribution
Non-normal distributionPaired t test
Wilcoxon testt(15) = 5.761, p < 0.0001 (WT); p = 0.0001 (KO) aa Δ slope (Fig. 6I) Non-normal distribution M–W test p = 0.0063 - Table 2.
Membrane properties and spike features in pyramidal cells of layer 2/3 visual cortex
Three to four weeks Six to eight weeks WT KO Statistical value WT KO Statistical value Input resistance (MΩ) 91.51 ± 4.96 104.33 ± 13.07 t(23) = 1.125 p = 0.2721 75.49 ± 6.19 82.32 ± 5.57 t(22) = 0.8119 p = 0.4255 V_spike peak (mV) 46.41 ± 1.17 42.93 ± 2.65 t(23) = 1.407 p = 0.1727 48.54 ± 1.65 46.75 ± 1.34 t(22) = 0.8498 p = 0.4046 V_trough (mV) −47.87 ± 0.44 −49.13 ± 0.94 t(23) = 1.388 p = 0.1785 −48.08 ± 0.59 −48.46 ± 0.62 t(22) = 0.4247 p = 0.6752 Spike height (mV) 94.28 ± 1.41 92.06 ± 3.15 t(23) = 0.7528 p = 0.4592 96.63 ± 1.97 95.21 ± 1.62 p = 0.5036* Spike width (ms) 1.18 ± 0.04 1.22 ± 0.05 t(23) = 0.5595 p = 0.5812 1.05 ± 0.06 0.92 ± 0.03 t(22) = 2.069 p = 0.0505 V_threshold (mV) −37.25 ± 0.56 −38.67 ± 0.90 t(23) = 1.401 p = 0.1747 −38.20 ± 1.02 −39.14 ± 0.58 t(22) = 0.8592 p = 0.3995 Upstroke (mV/ms) 265.83 ± 8.82 250.42 ± 18.31 t(23) = 0.8626 p = 0.3973 349.53 ± 17.73 358.53 ± 12.56 p = 0.9431* Downstroke (mV/ms) −68.02 ± 2.31 −65.86 ± 3.44 t(23) = 0.5238 p = 0.6054 −79.90 ± 4.59 −91.96 ± 3.79 t(22) = 2.033 p = 0.0543 Upstroke/downstroke ratio −3.93 ± 0.11 −3.79 ± 0.18 t(23) = 0.7090 p = 0.4854 −4.45 ± 0.26 −3.93 ± 0.11 t(22) = 2.088p = 0.0486 RMP (mV) −68.40 ± 0.81 −67.01 ± 1.71 p = 0.6659* −70.27 ± 1.10 −69.72 ± 0.83 t(22) = 0.4048 p = 0.6895 First ISI (ms) 6.95 ± 0.26 7.86 ± 1.47 t(23) = 0.8638 p = 0.3966 6.41 ± 0.46 5.26 ± 0.58 p = 0.0220* Mean ISI (ms) 26.99 ± 0.76 28.63 ± 2.24 p = 0.7504* 29.41 ± 1.43 26.41 ± 1.44 t(22) = 1.436 p = 0.1650 Adaptation index 0.02 ± 0.001 0.02 ± 0.001 t(23) = 0.6344 p = 0.5321 0.02 ± 0.004 0.03 ± 0.002 p = 0.7879* Slope of current-firing curve 7.02 ± 0.23 6.41 ± 0.60 p = 0.0883* 6.35 ± 0.28 7.19 ± 0.47 p = 0.1583* Spike features were obtained from the first action potential evoked by the step current which was just above the threshold. ISIs were obtained from the response evoked by the step current which was 360 pA above the threshold. Two-tailed unpaired t test or M–W test (*) was used for statistical analyses. Bold text indicates statistical significance, p < 0.05.
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