Figure 3. Spontaneous Firing of Cholinergic Interneurons Decreased via Elevated Expression of GABA_A α2 Receptors in Chat-Mecp2^-/y Mice.

(A, B) Example traces of spontaneous spiking (A) or averaged frequency (B) measured in a cell-attached configuration from NAc cholinergic interneurons. P-values were calculated by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test. F (2, 41)=13.93, p<0.0001. n = 18 neurons from three mice (Chat^Cre), 13 neurons from three mice (Mecp2^flox/y), 13 neurons from five mice (Chat-Mecp2^-/y). (C, D) Example traces of spontaneous spiking (C) or averaged frequency (D) measured in a whole-cell configuration from NAc cholinergic interneurons. P-values were calculated by one-way ANOVA with Bonferroni’s multiple comparisons test. F (2, 34)=5.336, p=0.0096. n = 16 neurons from three mice (Chat^Cre), 13 neurons from four mice (Mecp2^flox/y), 8 neurons from five mice (Chat-Mecp2^-/y). (E, F) Example traces of spontaneous spiking (E) or averaged frequency (F) measured in a whole-cell configuration from NAc neurons infected by AAV-GFP or AAV/Mecp2 for indicated genotype. Data are means ± SEM. P-values were calculated by one-way ANOVA with Tukey’s multiple comparisons test. F (2, 27)=6.201, p=0.0061. n = 9 neurons from three mice (Chat^Cre-AAV/GFP), 9 neurons from three mice (Chat-Mecp2^-/y-AAV/GFP), 12 neurons from three mice (Chat-Mecp2^-/y-AAV/Mecp2). (G, H) Example mIPSC traces (G) measured in whole-cell configuration from NAc cholinergic interneurons. (H) Cumulative distribution of mIPSC amplitude of NAc cholinergic interneurons. P-values were calculated by one-way ANOVA with Bonferroni's multiple comparisons test. F (2, 39)=15.46, p<0.0001. n = 12 neurons from four mice (Chat^Cre), 14 neurons from four mice (Mecp2^flox/y), 16 neurons from four mice (Chat-Mecp2^-/y). (I) Confocal images showing NAc sections stained for PV, ChAT, GABA_A α2 receptor, and DAPI. Bar graph showing percentage of ChAT or PV-positive cells co-expressing GABA_A α2 receptor in wild-type (WT) mice. n = 8 sections from two mice. (J) Immunoblotting of GABA_A α2 receptor in NAc extracts prepared from Chat^Cre and Chat-Mecp2^-/y mice. Each lane was loaded with 40 μg of protein, with GAPDH as loading control, and normalized to Chat^Cre levels. P-values were calculated by two-tailed unpaired t-test. t = 2.153, df = 28, p=0.0401. n = 15 mice per group. (K) Representative images of NAc slices from Chat^Cre and ChAT-Mecp2^-/y mice. Statistically integrated immunofluorescence data normalized to Chat^Cre levels. P-values were calculated by two-tailed unpaired t-test. t = 2.986, df = 14, p=0.0098. n = 7–9 sections from two mice per group. (L, M) Example traces (L) and statistical results (M) of spontaneous spiking recorded from cholinergic interneurons in NAc of different groups. P-values were calculated by two-tailed unpaired t-test. t = 3.977, df = 25, p=0.0005. n = 14 neurons from four mice for ACSF, n = 13 neurons from four mice for L-838,417. L-838,417: an α2-subunit-selecive selective positive allosteric modulator of GABA_A receptors. Data are means ± SEM. *p<0.05, **p<0.01, ***p<0.001, NS means no significance.

Figure 3—figure supplement 1. Chat-Mecp2^-/y Mice Exhibited No Statistical Difference in Intrinsic Properties in NAc Cholinergic Interneurons, Related to Figure 3.

(A–D) Quantitative analysis of resting membrane potential (RMP), membrane time constant (Tau), input resistance (Rin), and membrane capacitance (Cm) of cholinergic interneurons from NAc slices. P-values were calculated by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test. F (2, 32)=0.005866, p=0.9942 for RMP. F (2, 32)=0.3762, p=0.6894 for Tau. F (2, 32)=0.3748, p=0.6904 for Cm. F (2, 32)=1.809, p=0.1801 for Rin. n = 13 neurons from four mice (Chat^Cre), 11 neurons from three mice (Mecp2^flox/y), 11 neurons from three mice (Chat-Mecp2^-/y). (E–H) Quantitative analysis of threshold, amplitude, half width, and after-hyperpolarization potential (ahp) for a single spike of cholinergic interneurons from NAc slices. P-values were calculated by one-way ANOVA with Bonferroni’s multiple comparisons test. F (2, 36)=1.222, p=0.3066 for threshold. F (2, 36)=0.7935, p=0.4600 for amplitude. F (2, 36)=0.1402, p=0.8697 for half width. F (2, 36)=0.1137, p=0.8929 for ahp. n = 15 neurons from four mice (Chat^Cre), 9 neurons from three mice (Mecp2^flox/y), 15 neurons from five mice (Chat-Mecp2^-/y). Data are means ± SEM. NS means no significance.

Figure 3—figure supplement 2. Depletion of MeCP2 Did Not Affect mIPSC Frequency of Cholinergic Interneurons, with Limited Expression of GABA_A Receptor α1 Subunits in Cholinergic Interneurons within NAc, Related to Figure 3.

(A) Cumulative distribution of mIPSC inter-event interval and average frequency of NAc cholinergic interneurons. P-values were calculated by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test. F (2, 39)=1.535, p=0.2282. n = 12 neurons from four mice (Chat^Cre), 14 neurons from four mice (Mecp2^flox/y), 16 neurons from four mice (Chat-Mecp2^-/y). (B) Fluorescence images showing NAc sections stained for the PV, ChAT, GABA_A receptor α1, and DAPI. (C) Immunoblotting of GABA_A receptor α1 in NAc extracts prepared from Chat^Cre and Chat-Mecp2^-/y mice. Each lane was loaded with 40 μg of protein, with GAPDH as loading control, and normalized to Chat^Cre levels. Data are means ± SEM. P-values were calculated by two-tailed unpaired t-test. t = 0.3411, df = 12, p=0.7390. n = 7 for Chat^Cre mice, seven for Chat-Mecp2^-/y mice. (D Example traces and statistical results of spontaneous spiking recorded from cholinergic interneurons in NAc of Chat-Mecp2^-/y mice. P-values were calculated by two-tailed unpaired t-test. t = 2.496, df = 19, p=0.0219. n = 11 neurons from three mice for ACSF, n = 10 neurons from three mice for L-838,417. (E) Example sIPSC traces and statistical results of amplitude and frequency recorded from cholinergic interneurons in NAc of Chat^Cre mice. P-values were calculated by two-tailed unpaired t-test. t = 2.688, df = 20, p=0.0141 for amplitude. t = 2.099, df = 20, p=0.0488 for frequency. n = 11 neurons from three mice for each group. (F) Example sIPSC traces and statistical results of amplitude and frequency recorded from cholinergic interneurons in NAc of Chat-Mecp2^-/y mice. P-values were calculated by two-tailed unpaired t-test. t = 2.108, df = 18, p=0.0493 for amplitude. t = 3.536, df = 18, p=0.0024 for frequency. n = 10 neurons from three mice for each group. Data are means ± SEM. NS means no significance.