Journal: iScience

Article Title: Increased excitability of layer 5 neocortical pyramidal neurons and its contribution to seizure activity in Gabrg2 gene-deficient mice

doi: 10.1016/j.isci.2025.114421

Figure Lengend Snippet: The change of AP in L2/3 and L5 PNs (A) The SAP statistical pattern diagram. light gray line: WT L2/3 group, gray line: WT L2/3 -heat group, light green line: CKO L2/3 group, green line: CKO L2/3 -heat group, light blue line: WT L5 group, deep blue line: WT L5 -heat group, pink line: CKO L5 group, and red line: CKO L5 -heat group. Scale bars, 20 mV, 2s. (B) L2/3 and L5 PN morphology was observed by using a light microscope with Biocytin staining (white arrows). (C and D) Histograms and scatterplot of sAPs frequency of L2/3 (C, WT group: 0.029, 0.011–0.335, WT -heat group: 1.102, 0.626–1.840, CKO group: 0.217, 0.035–0.579, and CKO -heat group: 2.061, 1.485–3.447) and L5 (D, WT group: 0.030, 0.003–0.099, WT -heat group: 1.546, 0.524–1.810, CKO group: 0.290, 0.044–0.473, and CKO -heat group: 2.628, 1.790–3.731) PNs of 4 groups. n = 11 from 4 mice (male: female = 1:1), Mann Whitney test. (E) Histograms and scatterplot of sAPs frequency of L2/3 and L5 PNs of WT and CKO groups. (F) Histograms and scatterplot of sAPs frequency of L2/3 and L5 PNs of WT -heat and CKO -heat groups. n = 11 from 4 mice (male: female = 1:1), Mann Whitney test. (G and H) L5 PN with patch pipette, PN morphology was observed by using a light microscope with Biocytin staining. (I) AP potential statistical pattern diagram: action potential (AP) threshold (dV/dt > 10V/s), AP half-width (ms), AP amplitude (mV). Scale bars: 20 mV, 5 ms. (J) The phase plane plot (Vm versus dV/dt) for APs before and after heating. Voltage threshold was defined as dV/dt > 10V/s. The region around the threshold has been magnified for clearer image (Light-blue line: WT group, blue line: WT -heat group, pink line: CKO group, and red line: CKO -heat group). (K) The firing frequency of 4 groups for L5 PNs, current injections, from bottom to top, −60, 0, 100, 150, and 200 pA. Scale bars: see . (L–P) Histograms and scatterplot of the resting potential (L, WT group: −66.013 ± 1.188, WT -heat group: −65.954 ± 0.781, CKO group: −64.796 ± 1.052, and CKO -heat group: −65.394 ± 0.978), AP amplitude (M, WT group: 91.962 ± 2.328, WT -heat group: 89.707 ± 2.608, CKO group: 88.470 ± 1.862, and CKO -heat group: 88.539 ± 2.510), AP half-width (N, WT group: 0.810 ± 0.033, WT -heat group: 0.870 ± 0.061, CKO group: 0.785 ± 0.072, and CKO -heat group: 0.698 ± 0.027), threshold current (O, WT group: 133.889 ± 12.211, WT -heat group: 122.222 ± 10.180, CKO group: 113.889 ± 18.928, and CKO -heat group: 83.333 ± 11.771), and AP threshold (P, WT group: −35.153 ± 1.500, WT -heat group: −34.950 ± 1.289, CKO group: −34.011 ± 1.333, and CKO -heat group: −41.241 ± 0.643). n = 18 from 4 mice (male: female = 1:1), two-way ANOVA. (Q) The change of membrane voltage under step current (−60 to 20 pA) conditions of four groups n = 18, two-way ANOVA, ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. (Light-blue ∗: WT -heat group vs. WT group, black ∗: CKO -heat group vs. WT -heat group, and red ∗: CKO -heat group vs. CKO group). (R) The change of input resistance under step current (−60 to 200 pA) conditions of four groups. WT group (149.685 ± 15.732) vs. WT -heat group (227.759 ± 17.517), n = 18, two-way ANOVA. CKO group (180.546 ± 59.038) vs. CKO -heat group (241.583 ± 95.494), n = 18 from 4 mice (male: female = 1:1), Two-sided unpaired t test. (S) Heatmap image shows that the change in the frequency of AP under step current (−60 to 200 pA) conditions of four groups ( n = 15 from 4 mice, male: female = 1:1, see for detailed statistical information). (T) The summary plot of firing frequency over time for the four groups (WT group: 9, 0–15, WT -heat group: 17, 8–22, CKO group: 17, 2–24, and CKO -heat group: 31, 27–35). ∗∗∗∗ p < 0.0001 (firing frequency of 200 pA), n = 15 from 4 mice (male: female = 1:1), Friedman’s M test.

Article Snippet: For the miniature inhibitory postsynaptic currents (mIPSCs) recording, whole-cell currents were recorded from L5 PNs and an intracellular solution (in mM: 62.5 Potassiun D-gluconate, 8 NaCl, 62.5 KCl, 0.2 EGTA, 10 HEPES, 0.3 NaGTP and 2 MgATP, 0.3% biocytin, pH adjusted to 7.3 with NaOH). mIPSCs were recorded in the presence of TTX (1 μM), DL-2-amino-5-phosphonopentanoic acid (AP-V) (50 μM, MCE), and CNQX (20 μM, MCE), which were added to the ACSF.

Techniques: Light Microscopy, Staining, MANN-WHITNEY, Transferring, Membrane

Journal: iScience

Article Title: Increased excitability of layer 5 neocortical pyramidal neurons and its contribution to seizure activity in Gabrg2 gene-deficient mice

doi: 10.1016/j.isci.2025.114421

Figure Lengend Snippet: The changes of dendrites, dendritic spines, and synapses in L5 PNs (A) The changes of dendrites in L5 PNs from Golgi staining. Scale bars: 100μm. (B–D) Heatmap image shows the change of the mean intersections of dendrites 0 to 170 μm away from the soma of four groups (B). The mean intersections (C) and total intersections (D, WT group: 219.444 ± 14.527, WT -heat group: 194.056 ± 15.682, CKO group: 184.00 ± 9.316, and CKO -heat group: 128.389 ± 4.932) of L5 PNs from Golgi staining analysis in four groups ( n = 18 of each group, from 3 mice, male: female = 1:2. The gray ∗: WT group vs. CKO group, the black ∗: WT -heat group vs. CKO -heat group, the bluish ∗: WT group vs. WT -heat group, and the red ∗: CKO group vs. CKO -heat group. C, two-sided unpaired t test. D, two-way ANOVA. See for detailed statistical information. (E–G) Heatmap image shows the change of the mean length (μm) of dendrites 0 to 170 μm away from the soma of four groups (E). The mean length (μm) (F) and total length (μm) (G, WT group: 2801.693 ± 1 80.209, WT -heat group: 2521.529 ± 195.020, CKO group: 2387.626 ± 114.252, and CKO -heat group: 1687.768 ± 59.814) of L5 PNs from Golgi staining analysis in four groups ( n = 18 of each group, from 3 mice, male: female = 1:2. The gray ∗: WT group vs. CKO group, the black ∗: WT -heat group vs. CKO -heat group, the bluish ∗: WT group vs. WT -heat group, and the red ∗: CKO group vs. CKO -heat group. F, two-sided unpaired t test. G, two-way ANOVA. See for detailed statistical information. (H and I) The changes in dendritic spines of secondary branches of dendrites in L5 PNs from Golgi staining. White scale bars, 100 μm. Black scale bars, 10μm. (J–L) The mean spines (J, WT group: 1.135 ± 0.027, WT -heat group: 1.119 ± 0.038, CKO group: 1.145 ± 0.038, and CKO -heat group: 0.920 ± 0.029), mature spines (red arrows: mushroom, yellow arrows: stubby, green arrows: branched) (K, WT group: 0.588 ± 0.018, WT -heat group: 0.488 ± 0.021, CKO group: 0.525 ± 0.028, and CKO -heat group: 0.367 ± 0.017), and immature spines (L, WT group: 0.547 ± 0.029, WT -heat group: 0.631 ± 0.042, CKO group: 0.619 ± 0.036, and CKO -heat group: 0.553 ± 0.020) per 1 μm L5 PNs from Golgi staining analysis in 4 groups ( n = 32, from 4 mice, male: female = 1:2). two-way ANOVA. (M) The changes of synapses (red arrows) in L5 PNs from scanning electron microscopy (SEM). Scale bars: 1μm. (N–P) Average number of synapses per field (N, WT group: 9.667 ± 0.454, WT -heat group: 10.133 ± 0.350, CKO group: 8.600 ± 0.375, and CKO -heat group: 7.600 ± 0.190), total synaptic length per field (O, WT group: 7.728 ± 0.402, WT -heat group: 8.005 ± 0.286, CKO group: 6.839 ± 0.265, and CKO -heat group: 6.051 ± 0.229), and the average length of synapses per field (P, WT group: 0.839 ± 0.022, WT -heat group: 0.806 ± 0.026, CKO group: 0.801 ± 0.019, and CKO -heat group: 0.740 ± 0.027) of L5 PNs from SEM analysis in 4 groups ( n = 15, from 4 mice, male: female = 1:1). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, two-way ANOVA.

Article Snippet: For the miniature inhibitory postsynaptic currents (mIPSCs) recording, whole-cell currents were recorded from L5 PNs and an intracellular solution (in mM: 62.5 Potassiun D-gluconate, 8 NaCl, 62.5 KCl, 0.2 EGTA, 10 HEPES, 0.3 NaGTP and 2 MgATP, 0.3% biocytin, pH adjusted to 7.3 with NaOH). mIPSCs were recorded in the presence of TTX (1 μM), DL-2-amino-5-phosphonopentanoic acid (AP-V) (50 μM, MCE), and CNQX (20 μM, MCE), which were added to the ACSF.

Techniques: Staining, Electron Microscopy

Journal: iScience

Article Title: Increased excitability of layer 5 neocortical pyramidal neurons and its contribution to seizure activity in Gabrg2 gene-deficient mice

doi: 10.1016/j.isci.2025.114421

Figure Lengend Snippet: mEPSCs and mIPSCs of L5 PNs (A) Representative traces of mEPSCs and mIPSCs in the L5 PNs of WT, WT -heat , CKO, and CKO -heat groups (four groups). Scale bars, see . (B–E) The average amplitude (WT group: 9.925 ± 0.192, WT -heat group: 11.014 ± 0.216, CKO group: 10.937 ± 0.367, and CKO -heat group: 12.593 ± 0.464) and the frequency (WT group: 1.592 ± 0.131, WT -heat group: 2.321 ± 0.445, CKO group: 2.562 ± 0.429, and CKO -heat group: 7.045 ± 0.627) of mEPSCs in 4 groups. n = 26 from 4 mice, male: female = 1:1, two-way ANOVA. (F–I) The average amplitude (WT group: 13.385 ± 0.458, WT -heat group: 13.654 ± 0.506, CKO group: 13.997 ± 0.420, and CKO -heat group: 11.159 ± 0.575) and the frequency (WT group: 3.261 ± 0.365, WT -heat group: 4.972 ± 0.379, CKO group: 3.392 ± 0.347, and CKO -heat group: 0.570 ± 0.115) of mIPSCs in 4 groups. n = 23 from 4 mice, male: female = 1:1, two-way ANOVA. (J) Representative traces of mEPSCs and mIPSCs in the L5 PNs from the WT group, WT -heat group (Bar, 20 pA, 2 s) CKO group and CKO -heat group (Bar, 10 pA, 2 s) brain slices with bath solution containing GABA 0 μM, GABA 100 μM, GABA 100 μM + Zn 2+ 10 μM. CKO group and CKO-heat group, n = 8 from 3 mice, male: female = 2:1, WT group and WT-heat group, n = 9 from 3 mice, male: female = 2:1. (K and L) The average frequency of mIPSCs in four groups’ brain slices with bath solution contains GABA 100 μM (K, WT group: 2.208 ± 0.415, WT -heat group: 1.804 ± 1.226, CKO group: 1.747 ± 1.050, and CKO -heat group: 0.302 ± 0.362), GABA 100 μM + Zn 2+ 10 μM (L, WT group: 1.378 ± 0.701, WT -heat group: 1.160 ± 0.718, CKO group: 1.419 ± 0.967, and CKO -heat group: 0.287 ± 0.359). Two-sided unpaired t test. (M–P) The average frequency of mIPSCs in four groups’ brain slices with bath solution contains GABA 0 μM (WT group: 3.110 ± 1.897, WT -heat group: 2.012 ± 2.204, CKO group: 1.325 ± 0.776, and CKO -heat group: 0.300 ± 0.194), GABA 100 μM, GABA 100 μM + Zn 2+ 10 μM. Paired t test. (Q and R) The average amplitude of mIPSCs in four groups’ brain slices with bath solution contains GABA 100 μM (Q, WT group: 16.219 ± 4.382, WT -heat group: 17.814 ± 7.685, CKO group: 20.667 ± 12.024, and CKO -heat group: 8.535 ± 3.495), GABA 100 μM + Zn 2+ 10 μM (R, WT group: 15.332 ± 4.586, WT -heat group: 16.663 ± 5.576, CKO group: 18.52 ± 8.073, and CKO -heat group: 8.802 ± 3.711). Two-sided unpaired t test. (S–V) The average amplitude of mIPSCs in four groups’ brain slices with bath solution contains GABA 0 μM (WT group: 18.034 ± 3.997, WT -heat group: 18.316 ± 9.181, CKO group: 17.984 ± 7.263, and CKO -heat group: 9.832 ± 0.648), GABA 100 μM, GABA 100 μM + Zn 2+ 10 μM. Paired t test. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: For the miniature inhibitory postsynaptic currents (mIPSCs) recording, whole-cell currents were recorded from L5 PNs and an intracellular solution (in mM: 62.5 Potassiun D-gluconate, 8 NaCl, 62.5 KCl, 0.2 EGTA, 10 HEPES, 0.3 NaGTP and 2 MgATP, 0.3% biocytin, pH adjusted to 7.3 with NaOH). mIPSCs were recorded in the presence of TTX (1 μM), DL-2-amino-5-phosphonopentanoic acid (AP-V) (50 μM, MCE), and CNQX (20 μM, MCE), which were added to the ACSF.

Techniques:

Journal: iScience

Article Title: Increased excitability of layer 5 neocortical pyramidal neurons and its contribution to seizure activity in Gabrg2 gene-deficient mice

doi: 10.1016/j.isci.2025.114421

Figure Lengend Snippet: The protein expression of GABRG2 and other GABA A R major subunits (A) Representative Western blots show changes in GABRG2, GABRA1, GABRA2, GABRA5, GABRB1, and GABRB2 levels in WT, WT -heat , CKO, and CKO -heat mice. (B–G) The membrane protein expression of GABRG2 (B, WT group: 1.438 ± 0.026, WT -heat group: 0.964 ± 0.055, CKO group: 1.033 ± 0.078, and CKO -heat group: 0.565 ± 0.081, n = 4 from 4 mice, two-way ANOVA), GABRA1 (C, WT group: 1.325 ± 0.096, WT -heat group: 0.904 ± 0.061, CKO group: 1.017 ± 0.164, and CKO -heat group: 0.754 ± 0.081, n = 4 from 4 mice, CKO vs. CKO -heat , t test, two-tailed), GABRA2 (D, WT group: 2.267 ± 0.553, WT -heat group: 1.716 ± 0.443, CKO group: 2.764 ± 0.587, and CKO -heat group: 1.697 ± 0.472), GABRA5 (E, WT group: 1.176 ± 0.021, WT -heat group: 0.844 ± 0.076, CKO group: 1.298 ± 0.065, and CKO -heat group: 0.683 ± 0.073), GABRB1 (F, WT group: 1.341 ± 0.109, WT -heat group: 0.970 ± 0.013, CKO group: 0.899 ± 0.074, and CKO -heat group: 0.790 ± 0.089), and GABRB2 (G, WT group: 1.223 ± 0.118, WT -heat group: 0.874 ± 0.103, CKO group: 1.247 ± 0.036, and CKO -heat group: 0.655 ± 0.107), n = 4 from 4 mice, male: female = 1:1, two-way ANOVA. (H) The protein band of the cytosol protein of GABRG2. (I) The cytosol protein expression of GABRG2 (WT group: 0.977 ± 0.013, WT -heat group: 1.003 ± 0.066, CKO group: 0.900 ± 0.081, and CKO -heat group: 1.120 ± 0.023). n = 4 from 4 mice, male: female = 1:1, t test, two-tailed. (J) Schematic diagram of the distribution pattern of the GABRG2 subunit in the plasma membrane (light yellow), endoplasmic reticulum (light green), and nucleus (light blue) regions. (K) The schematic picture of immunofluorescence colocalization. Immunofluorescent triple-staining of GABRG2 (red), ATP1α1 (yellow), and DAPI (blue) in the L5, scale bars, 50 μm. The bottom row is the enlarged merge images, the membrane protein expression of GABRG2 in CKO -heat L5 PNs decreases significantly, scale bars, 10 μm. (L–O) Figures L, M, N, and O represent the immunofluorescence colocalization of WT, WT heat, CKO, and CKO heat groups L5 PNs, respectively (red line is GABRG2, yellow line is ATP1α1, and blue line is DAPI). The light-yellow box represents the expression of GABRG2 at the endoplasmic reticulum (ER) and nuclear peripheral GABRG2, respectively. (P) The differences in average optical density of GABRG2 (WT group: 67.23 ± 1.501, WT -heat group: 52.754 ± 5.342, CKO group: 45.793 ± 3.967, and CKO- heat group: 26.144 ± 1.513) in cerebral L5 among the four groups. n = 5 from 3 mice, male: female = 1:2, two-way ANOVA. (Q) The expression of GABRG2 (WT group: 244.471 ± 5.305, WT -heat group: 174.897 ± 14.855, CKO group: 218.372 ± 6.405, and CKO- heat group: 93.892 ± 5.010) at L5 PNs membrane in CKO -heat decreases significantly. n = 30 from 3 mice, male: female = 1:2, two-way ANOVA. (R) Immunofluorescent triple-staining of GABRG2 (red), HMOX1 (green), and DAPI (blue) in the L5, scale bars = 50 μm. The right rows are the enlarged merge images, scale bars = 20 μm. (S–V) Figures S, T, U, and V represent the immunofluorescence colocalization of WT, WT -heat , CKO, and CKO -heat groups L5 PNs, respectively (red line is GABRG2, green line is HMOX1, and blue line is DAPI). The light green box and light blue box represent the expression of GABRG2 at the cell membrane. (W–X) The expression of GABRG2 (WT group: 185.903 ± 14.108, WT -heat group: 123.359 ± 13.920, CKO group: 149.618 ± 13.163, and CKO -heat group: 9.638 ± 4.496) at endoplasmic reticulum (ER) and nuclear peripheral GABRG2 (WT group: 222.745 ± 13.929, WT -heat group: 192.793 ± 14.334, CKO group: 149.186 ± 18.028, and CKO- heat group: 105.868 ± 20.798) in CKO -heat L5 PNs decrease significantly. n = 20 from 3 mice, male: female = 1:2, two-way ANOVA. ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: For the miniature inhibitory postsynaptic currents (mIPSCs) recording, whole-cell currents were recorded from L5 PNs and an intracellular solution (in mM: 62.5 Potassiun D-gluconate, 8 NaCl, 62.5 KCl, 0.2 EGTA, 10 HEPES, 0.3 NaGTP and 2 MgATP, 0.3% biocytin, pH adjusted to 7.3 with NaOH). mIPSCs were recorded in the presence of TTX (1 μM), DL-2-amino-5-phosphonopentanoic acid (AP-V) (50 μM, MCE), and CNQX (20 μM, MCE), which were added to the ACSF.

Techniques: Expressing, Western Blot, Membrane, Two Tailed Test, Clinical Proteomics, Immunofluorescence, Staining