Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KOleads to varying magnitudes of neuronal enlargement and mispositioning in the cortex.

(a) Diagram of the PI3K-mTORC1 pathway. Activation of mTORC1 signaling is controlled by positive (blue) and negative (purple) regulators within the pathway. (b) Diagram of overall experimental timeline and approach. IUE was performed at E15.5. A cohort of animals was used for patch clamp recording at P26-51 and another cohort was used for histology at P28-43. (c) Representative images of tdTomato+ cells (red) and p-S6 staining (green) in mouse mPFC at P28-43. (d) Quantification of p-S6 staining intensity (normalized to the mean control) in tdTomato+ neurons. (e) Quantification of tdTomato+ neuron soma size. (f) Representative images of tdTomato+ neuron (red) placement and distribution in coronal mPFC sections. Red square on the diagram denotes the imaged area for all groups. CC, corpus callosum. (g) Quantification of tdTomato+ neuron placement in layer 2/3. Left diagram depicts the approach for analysis: the total number of tdTomato+ neurons within layer 2/3 (white square) was counted and expressed as a % of total neurons in the imaged area. Right bar graphs show the quantification. (h) Quantification of tdTomato+ neuron distribution across cortical layers. Left diagram depicts the approach for analysis: the imaged area was divided into 10 equal-sized bins across the cortex, spanning the corpus callosum to the pial surface (white grids); the total number of tdTomato+ neurons within each bin was counted and expressed as a % of total neurons in the imaged area. Right bar graphs show the quantification. For graphs d, e: n = 4-8 mice per group, with 6-15 cells analyzed per animal. For graphs g, h: n = 3-7 mice per group, with 1 brain section analyzed per animal. Statistical comparisons were performed using (d, e) nested one-way ANOVA (fitted to a mixed-effects model) to account for correlated data within individual animals, (g) one-way ANOVA, or (h) two-way repeated measures ANOVA. Post-hoc analyses were performed using Holm-Šídák multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. All data are reported as the mean of all neurons or brain sections in each group ± SEM.

Statistical results (for main figures)

Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KOuniversally leads to decreased depolarization-induced excitability, but only RhebY35L, mTORS2215Y, and Tsc1KO expression leads to depolarized RMPs.

(a) Diagram of experimental approach for whole-cell patch clamp recording. Recordings were performed in layer 2/3 pyramidal neurons in acute coronal slices from P26-51 mice, expressing either control, RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, or Tsc1KO plasmids. (b-d) Graphs of membrane capacitance, resting membrane conductance, and RMP. (e) Representative traces of the AP firing response to a 300 pA depolarizing current injection. (f) Input-output curves showing the mean number of APs fired in response to 500 ms-long depolarizing current steps from 0 to 400 pA. Arrows point to initial spike doublets. (g-j) Graphs of rheobase, AP threshold, AP peak amplitude, and AP half-width. For all graphs: n = 5-10 mice per group, with 22-50 cells analyzed per animal. Statistical comparisons were performed using (b-d, g-j) nested one-way ANOVA (fitted to a mixed-effects model) to account for correlated data within individual animals or (f) mixed-effects ANOVA accounting for repeated measures. Post-hoc analyses were performed using Holm-Šídák multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, for the input-output curves in f: *p<0.05 (vs. control), #<0.05 (vs. Dedpdc5KO), Ɏp<0.05 (vs. Tsc1KO). All data are reported as the mean of all neurons in each group ± SEM.

Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KOleads to the abnormal presence of HCN4 channels with variations in functional expression.

(a) Representative images of tdTomato+ cells (red) and HCN4 staining (green) in mouse mPFC at P28-43. (b) Quantification of HCN4 intensity (normalized to the mean control) in tdTomato+ neurons. (c) Representative current traces in response to a series of 3 s-long hyperpolarizing voltage steps from -120 to -40 mV, with a holding potential of -70 mV. Current traces from the -40 and -50 mV steps were not included due to contamination from unclamped Na+ spikes. Orange lines denote the current traces at -90 mV. (d) IV curve obtained from Iss amplitudes. (e) ΔIV curve obtained from Ih amplitudes (i.e., ΔI, where ΔI=Iss – Iinst). (f) Graphs of Ih amplitudes at –90 mV. (g) Representative current traces in response to a series of 3-s long hyperpolarizing voltage steps from -110 mV to -50 mV in the mTORS2215Y condition pre- and post-zatebradine application. Orange lines denote the current traces at -90 mV. (h) IV curve obtained from Iss amplitudes in the mTORS2215Y condition pre- and post-zatebradine application. (i) ΔIV curve obtained from Ih amplitudes (i.e., ΔI) in the mTORS2215Y condition pre- and post-zatebradine application. Arrow points to the post-zatebradine Ih amplitude at -90 mV. (j) Representative traces of the zatebradine-sensitive current obtained after subtraction of the post-from the pre-zatebradine current traces in response to -110 mV to -50 mV voltage steps. Orange lines denote the current traces at -90 mV. (k) IV curve of the zatebradine-sensitive current obtained after subtraction of the post-from the pre-zatebradine current traces. (l) Graph of RMP in the control and mTORS2215Y conditions pre- and post-zatebradine application. Connecting lines denote paired values from the same neuron. For graph b: n = 4-8 mice per group, with 4-15 cells analyzed per animal. For graphs d, e, f: n = 5-10 mice per group, with 24-47 cells analyzed per animal. For graphs h, i, k, l: n = 4-6 neurons (paired). Statistical comparisons were performed using (b, f) nested ANOVA (fitted to a mixed-effects model) to account for correlated data within individual animals, (d, e) mixed-effects ANOVA accounting for repeated measures, or (h, i, l) two-way repeated measures ANOVA. Post-hoc analyses were performed using Holm-Šídák multiple comparison test. *p<0.05, **p<0.01, ****p<0.0001, for the IV curves in d, e, h, i: *p<0.05 (vs. control), #p<0.05 (vs. RhebY35L or Dedpdc5KO), Ɏp<0.05 (vs. PtenKO). All data are reported as the mean of all neurons in each group ± SEM.

Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KOleads to different impacts on sEPSC properties.

(a) Representative sEPSC traces recorded at a holding voltage of -70 mV. Top and bottom traces are from the same neuron. (b-d) Graphs of sEPSC frequency, amplitude, and total charge. For all graphs: n = 5-9 mice per group, with 17-34 cells analyzed per animal. Statistical comparisons were performed using a nested ANOVA (fitted to a mixed effects model) to account for correlated data within individual animals. Post-hoc analyses were performed using Holm-Šídák multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. All data are reported as the mean of all neurons in each group ± SEM.

Plasmid DNA information

Plasmid concentrations for control and experimental conditions

Distribution of p-S6 staining intensity and neuron soma size among individual animals P28-43.

(a) Distribution of p-S6 staining intensity among individual animals. (b) Distribution of neuron soma size among individual animals.

Neuron soma sizes at P7-9.

(a) Representative images of tdTomato+ cells (red) in mouse mPFC at P7-9. (b) Quantification of tdTomato+ neuron soma size. (c) Graph showing the distribution of neuron soma size among individual animals in each group. n = 3-8 mice per group, with 30 cells analyzed per animal. Statistical comparisons were performed using a nested t-test (control vs. mTORS2215Y) or nested one-way ANOVA (control vs. Dedpdc5KO vs. PtenKO, vs. Tsc1KO) (fitted to a mixed-effects model) to account for correlated data within individual animals. Post-hoc analyses were performed using Holm-Šídák multiple comparison test. *p<0.05, ***p<0.001, ****p<0.0001. Data are reported as the mean of all neurons in each group ± SEM.

Distribution of membrane and AP properties among individual animals P26-51. (a-c)

Distribution of membrane capacitance, conductance, and RMP among individual animals. (d-g) Distribution of rheobase, AP threshold, AP peak amplitude, and AP half-width among individual animals.

Distribution of HCN4 staining intensity and Ih amplitudes (at -90 mV) among individual animals at P28-43 and P26-51, respectively.

(a) Distribution of HCN4 staining intensity among individual animals. (d-g) Distribution of Ih amplitudes (at -90 mV) among individual animals.

Additional images of HCN4 staining in mPFC sections from P28-43 mice expressing mTORS2215Y.

(a) Representative images of tdTomato+ cells (red) and HCN4 staining (green). Top panels show overlay images. Bottom panels show HCN4 single-channel images. a1 and a2 show the ipsilateral and contralateral cortex, respectively. a3 and a4 are high-magnification images demonstrating HCN4 staining within the cell. (b) Representative images of immunostaining with (left) or without (right) HCN4 primary antibodies (control for secondary antibody specificity).

BaCl2 application decreases overall inward currents without affecting Ih in Tsc1KO neurons.

(a) Representative current traces in response to a series of 3-s long hyperpolarizing voltage steps from -130 mV to -40 mV in the Tsc1KO condition pre- and post-BaCl2 application. Orange lines denote the current traces at -90 mV. (b) IV curve obtained from Iss amplitudes in the Tsc1KO condition pre- and post-BaCl2 application. (c) IV curve obtained from Ih amplitudes (i.e., ΔI, where ΔI=Iss – Iinst) in the Tsc1KO condition pre- and post-BaCl2 application. (d) Representative traces of the BaCl2-sensitive current obtained after subtraction of the post-from the pre-BaCl2 current traces. Orange lines denote the current traces at -90 mV. (e) IV curve of the BaCl2-sensitive current obtained after subtraction of the post-from the pre-BaCl2 current traces. The isolated BaCl2-sensitive current reversed near -80 mV. (f) Graph of conductances (at - 500 pA) in the control and Tsc1KO conditions pre- and post-BaCl2 application. Connecting lines denote paired values from the same neuron. (g) Graph of % change in conductances pre- and post-BaCl2 application in the control and Tsc1KO conditions. (h) Graph of RMP in the control and Tsc1KO conditions pre- and post-BaCl2 application. Connecting lines denote paired values from the same neuron. (i) Graph of % change in RMP pre- and post-BaCl2 application in the control and Tsc1KO conditions. For all graphs: n = 10-13 neurons per group. Statistical comparisons were performed using (b, c, e) mixed-effects ANOVA, (f, h) two-way repeated measures ANOVA, or (g, i) unpaired t-test. Post-hoc analyses were performed using Holm-Šídák multiple comparison test. *p<0.05, **p<0.01, ****p<0.0001. All data are reported as the mean of all neurons or brain sections in each group ± SEM.

Distribution of sEPSC properties among individual animals at P26-51. (a-c)

Distribution of sEPSC properties among individual animals.

Statistical results (for supplemental figures)