PMC

(A,B) pS6 (red) immunolabeling in E14.5 Tsc1^ΔE12/ΔE12 embryos. (C,D) E17.5 Tsc1^ΔE12/ΔE12 embryos had a robust increase in pS6 (red) compared to controls. (E–G) Tsc1^ΔE12/ΔE12 mutants had high pS6 levels (red). R26^LacZ (β-gal, green) independently showed similar recombination efficiency across genotypes. Control and mutant sections were imaged with identical exposure settings. n≥3 animals per genotype per stage. Scale bars: (A,B) 30µm, (C,D) 15µm, (E–G) 30µm.

(A) Control and Tsc1^ΔE18/ΔE18 sections were immunostained for MAP2 (green) and pS6 (red). Soma size was graphed by genotype and pS6 expression and showed no significant difference. Note: pS6+ neurons were rarely observed in Tsc1^+/+ (2 cells) and Tsc1^ΔE18/ΔE18 brains (8 cells) and were not graphed for clarity. (B,C) Tsc1^+/+ and Tsc1^ΔE18/ΔE18 sections were immunolabeled for RFP (red) and pS6 (green). Tsc1^ΔE18/ΔE18 TCAs were superfluous and disorganized in deep cortical layers (region 1, arrow) and internal capsule (region 2, arrow). PV (region 3, green) was absent from Tsc1^ΔE18/ΔE18 and Tsc1^+/+ TCAs (red). Scale bars: (A) 8 µm (B,C) 240µm; (B1,B2,C1,C2) 61µm; (B3,C3) 57µm. See also .

(A) Thalamocortical regions of interest. (B) Sections were immunolabeled for MAP2 (green), pS6 (red), and counterstained with Hoechst (blue). Thalamic neurons of Tsc1^+/+ and Tsc1^ΔE12/+ mice were pS6-. Recombination produced a mosaic thalamus of unaffected (pS6-, open arrowhead) and affected (pS6+, filled arrowhead) neurons. Soma area is plotted by genotype and pS6 status. Numbers of neurons are listed and geometric means indicated by horizontal lines. (C–F) Analysis of PV (green) and RFP (red) revealed PV+ fibers in the internal capsule of Tsc1^ΔE12/ΔE12 mice (E, arrowheads), but not in controls (C). Soma of Tsc1^ΔE12/ΔE12 RFP+ neurons were also PV+ (F, arrowheads). n=3 animals per genotype. Scale bars: (B) 32µm (C–F) 48µm. **p<0.005. See also .

(A–F) Expression of β-gal (green) in sagittal sections of thalamus. Co-localization with parvalbumin (PV, red, A–C) or calbindin (Calb, red, D–F) is indicated by arrowheads. (G–L) Recombination in PV+ (G–I) or Calb+ (J–L) neurons. Scale bar in A (270µm) applies to low-magnification panels; scale bar in A3 (30µm) applies to high magnification panels. Thalamic nuclei: anteromedial (AM), laterodorsal (LD), lateral geniculate (LG), mediodorsal (MD), medial geniculate (MG), posterior (Po), peripeduncular (PP), subgeniculate (SubG), thalamic reticular (TRN), ventrobasal (VB), ventrolateral (VL), ventromedial (VM). Additional abbreviations: anterior commissure (a.c.), fornix (f), fasciculus retroflexus (fr), hippocampus (hipp), internal capsule (i.c.), intergeniculate leaflet (IGL); See also .

(A–C) LFPs in primary somatosensory neocortex were altered in Tsc1^ΔE12/ΔE12 and Tsc1^ΔE18/ΔE18 mutants. (D) Average power spectra of mutants and controls (E12.5 and E18.5 experiments were pooled together) shows increased power between 2–4Hz. Lines and shading represent mean ± SEM (E) 3Hz power was normalized to 1Hz power for controls (black, E12.5; gray, E18.5) and mutants (red, Tsc1^ΔE12/ΔE12; pink, Tsc1^ΔE18/ΔE18). Data points with black outlines represent recordings performed on aged animals (>8 months). (F) ≥20-second epochs of high-power 3Hz activity are plotted. (G) The percentage of time spent grooming is plotted by genotype. E12.5 and E18.5 graphs share a y-axis. Inset: A Tsc1^ΔE12/ΔE12 mouse that developed a wound from over-grooming (H) Number of seizures per hour of observation time is plotted by genotype. E12.5 and E18.5 graphs share a y-axis. Inset: Contorted posture typically observed during seizures. *p<0.05, **p<0.005. See also .

(A,B) RFP+ TCAs (red) delineated individual vibrissa barrels in Tsc1^+/+ neocortex, but were diffuse in Tsc1^ΔE12/ΔE12 mutants (region 1). Mutants had excess axonal processes in deep cortical layers (arrow) and RFP+ TCA fascicles that were less defined in the internal capsule (region 2). (C–J) Cortical vibrissa barrels stained with cytochrome oxidase (CO). (C,D) Controls had well-defined CO+ barrels (brown) separated by CO-septa. (G,H) Tsc1^ΔE12/ΔE12 cortex had misshapen barrels (brown) and small vibrissa barrels were nearly indistinguishable (gray). (E,I) Tsc1^+/+ RFP+ TCAs (red) targeted the CO+ barrel hollows (black, asterisks) but were less restricted in Tsc1^ΔE12/ΔE12 mice. (F,J) Barrel neurons (NeuN+, green) clustered around the perimeter of CO+ barrel hollows (black). Dashed line: extent of CO+ barrel hollow. Solid line: 15mm outer perimeter (“wall”) used for quantification in M. (K) Average CO+ barrel size was larger in Tsc1^ΔE12/ΔE12 mutants. (L) The septa proportion showed no difference. (M) Tsc1^ΔE12/ΔE12 mice had lower neuron density in the barrel wall and hollow versus Tsc1^+/+ animals. Scale bars: (A,B) 240µm; (A1,A2,B1,B2) 61µm; (F,J) 130mm. thal, thalamus; str, striatum; ctx, neocortex. *p<0.05, **p<0.005. Data are represented as mean ± s.d. See also .

(A) DIC/fluorescence shows electrode (yellow dashed lines) targeted to a RFP+ (red) VB neuron. Neurons were filled with biocytin (green) and immunostained for pS6 (white, insets). Morphology was reconstructed as shown below each filled neuron. (B) Tsc1^ΔE12/ΔE12 neurons (red) had lower membrane input resistance and higher input capacitance, but unchanged time constants compared to littermate controls (black), and to E18.5 mutants (pink) and controls (gray). (C) Representative traces from E12.5 control and mutant (left) shows that Tsc1^ΔE12/ΔE12 action potentials were faster and larger. Tsc1^ΔE12/ΔE12 action potential dynamics (right) were significantly different with respect to depolarization rate, maximum amplitude, and repolarization rate. Tsc1^ΔE18/ΔE18 neurons were similar to controls (). (D) Tsc1^ΔE12/ΔE12 spike afterpotentials (red) were more negative during the fast (fAHP) and during the slow phase (sAHP) compared to controls (black). Total post-spike membrane potential was integrated over time and quantified by integrating the voltage signal over 280 ms (right). (E) Representative tonic voltage response of a Tsc1^+/+ and Tsc1^ΔE12/ΔE12 neuron to current injections (400 pA, top and 200 pA, bottom). (F) Peak firing frequency per current step (F/I) is plotted for Tsc1^+/+ (black, n=12) and Tsc1^ΔE12/ΔE12 neurons (red, n=17) (G) Linear slopes of the F/I curves are quantified. (H) Representative voltage response of a Tsc1^+/+ and a Tsc1^ΔE12/ΔE12 thalamic neuron to hyperpolarizing current step. Insets show rebound bursts. (I) Intraburst firing frequency as a function of spike number within each burst is plotted for Tsc1^+/+ (black, n=11) and Tsc1^ΔE12/ΔE12 neurons (red, n=18). (J) Mean intraburst firing frequencies are quantified. Box plots represent minimum, first quartile (Q1), median, Q3, and maximum. Outliers (open circles) were >Q3+1.5*IQR or <Q1−1.5*IQR. Scale bars: (A) 20 µm (DIC), 30 µm (biocytin/morphology). *p<0.05, **p<0.005. See also .