Loss of Tsc1 from striatal direct pathway neurons impairs endocannabinoid-LTD and enhances motor routine learning

Figures

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  Figure 1.. Developmental loss of Tsc1 from striatal projection neurons induces mTORC1 activation (A and B) Images of sagittal brain sections showing the expression patterns of a tdTomato Cre reporter (red) in a Drd1-Cre+;Ai9+/− mouse (EY217 GENSAT founder line, A) and an Adora2a-Cre+;Ai9+/− mouse (KG139 GENSAT founder line, B). Right panels show higher magnification images of the dorsal striatum (boxed regions). Neurons are labeled in blue using an anti-NeuN antibody. Str, striatum; SNr, substantia nigra pars reticulata; GP, globus pallidus. (C–E) Confocal images of dorsolateral striatum from a dSPN-Tsc1 WT (C), Tsc1 Het (D), and Tsc1 KO (E) mouse labeled with antibodies against phosphorylated S6 (p-S6, Ser240/244, green in the merged image) and NeuN (blue in the merged image). TdTomato (red in the merged imaged) is expressed in dSPNs. (F) Cumulative distributions of dSPN p-S6 fluorescence intensity per cell, expressed as a percentage of wild type (WT). 1,800 cells from 6 dSPN-Tsc1 WT mice, 1,200 cells from 4 dSPN-Tsc1 Het mice, and 1,500 cells from 5 dSPN-Tsc1 KO mice were analyzed. Inset boxplots display the 25%–75%-ile p-S6 per cell by genotype (line at the median, whiskers = min to…

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  Figure 2.. Mutations in Tsc1 or Tsc2 enhance motor routine learning (A) Accelerating rotarod learning curve across 12 trials (4 days) in dSPN-Tsc1 WT (n = 30), Het (n = 25), and KO (n = 29) mice. Circles represent mean ± SEM. Repeated-measures two-way ANOVA p values are shown; geno F (4, 110) = 7.034, trial F (5.092, 560.1) = 105.8, g × t F (44, 120) = 1.987. RPM, revolutions per minute. (B) Rotarod performance on trial 1 for dSPN-Tsc1 WT, Het, and KO mice quantified as terminal speed. Bars represent mean ± SEM. Dots represent values for individual mice (n is the same as in A). p = 0.7233, Kruskal-Wallis test. (C) Overall learning rate of dSPN-Tsc1 WT, Het, and KO mice calculated as the slope of the line of performance on the first trial (1) to the last trial (12) for each mouse (RPM/day). Bars represent mean ± SEM. Dots represent values for individual mice (n is the same as in A). p = 0.0120, Kruskal-Wallis test; dSPN WT versus KO, **p = 0.0071; dSPN WT versus Het, p = 0.1170, Dunn’s multiple comparisons test. (D) Percentage of dSPN-Tsc1 WT, Het, and KO mice with a positive learning curve (slope of performance) from trial 7 to trial 12 (10–80 RPM acceleration). (E) Accelerating rotarod…

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  Figure 3.. Tsc1 loss selectively increases cortico-dSPN synaptic drive (A) Confocal image of a sagittal brain section from a Tsc1wt/fl;Thy1-ChR2-EYFP+;Drd1-Cre+;Ai9+/− mouse. Right image shows cortical terminals expressing ChR2-EYFP (green) in the dorsal striatum. tdTomato (red) labels dSPNs. DAPI stained nuclei are in blue. (B) Schematic of the experiment. Cortical terminals expressing ChR2 were stimulated with 10 pulses of blue light at 20 Hz and responses were recorded from dSPNs in dorsolateral striatum. (C) Example traces of action potentials (AP) in dSPNs evoked by cortical terminal stimulation at 10% light intensity for the indicated genotypes. (D) Quantification (mean ± SEM) of the percentage of cortical terminal stimuli that evoked APs in dSPNs. dSPN-Tsc1 WT n = 5 neurons from 2 mice, dSPN Het n = 7 neurons from 2 mice, dSPN KO n = 5 neurons from 2 mice. Mixed-effects model (REML) p values are shown; geno F (2, 14) = 12.25, stim F (2.125, 26.35) = 45.79, g × s (10, 62) = 2.164. (E) Example traces of APs in iSPNs evoked by cortical terminal stimulation at 10% light intensity for the indicated genotypes. (F) Quantification (mean ± SEM) of the percentage of cortical terminal stimuli…

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  Figure 4.. Loss of Tsc1 increases excitatory synaptic transmission onto dSPNs (A) Example traces of miniature excitatory post-synaptic currents (mEPSCs) recorded from dSPN-Tsc1 WT, Het, and KO neurons. (B and C) Mean ± SEM mEPSC amplitude (B) and frequency (C) recorded from dSPN-Tsc1 WT, Het, and KO neurons. Dots represent individual neurons. For (B), p = 0.0337, F (2, 38) = 3.712, one-way ANOVA; p = 0.0766 (dSPN WT versus Het), *p = 0.0217 (dSPN WT versus KO), Holm-Sidak’s multiple comparisons tests. For (C), p = 0.0014, F (2, 38) = 7.822, one-way ANOVA; **p = 0.0049 (dSPN WT versus Het), **p = 0.0011 (dSPN WT versus KO), Holm-Sidak’s multiple comparisons tests. For both (B) and (C), dSPN WT n = 13 neurons from 3 mice, dSPN Het n = 15 neurons from 3 mice, dSPN KO n = 13 neurons from 3 mice. (D) Example traces of mEPSCs recorded from iSPN-Tsc1 WT, Het, and KO neurons. (E and F) Mean ± SEM mEPSC amplitude (E) and frequency (F) recorded from iSPN-Tsc1 WT, Het, and KO neurons. Dots represent individual neurons. For (E), p = 0.2942, F (2, 42) = 1.260, one-way ANOVA. For (F), p = 0.4083, F (2, 42) = 0.9151, one-way ANOVA. For both (E) and (F), iSPN WT n = 16 neurons from 3 mice, iSPN Het n = 15…

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  Figure 5.. dSPN-Tsc1 KO neurons have impaired eCB-LTD (A) Corticostriatal terminals in the dorsolateral striatum were stimulated with blue light to evoke EPSCs in dSPN-Tsc1 WT and KO neurons. DHPG (100 μM) was washed on for 10 min to induce eCB-LTD. Data are presented as mean ± SEM percentage of baseline (dashed line). Repeated-measures two-way ANOVA p values are shown; geno F (1, 10) = 15.93, time F (3.479, 34.79) = 7.993, g × t F (25, 250) = 2.563. dSPN WT n = 7 neurons from 3 mice, dSPN KO n = 5 neurons from 4 mice. Example traces on the right show the average EPSCs from the baseline period (“1”) and 35–40 min after DHPG application (“2”) for each genotype. (B) Corticostriatal terminals were stimulated with blue light to evoke EPSCs in dSPN-Tsc1 WT and KO neurons. WIN-2 (2 μM) was applied at time 0 to activate CB1 receptors. Data are presented as mean ± SEM percentage of baseline (dashed line). Mixed-effects model (REML) p values are shown; geno F (1, 4) = 0.2433, time F (2.410, 9.641) = 29.36, g × t F (1.551, 4.329) = 0.5214. dSPN WT n = 5 neurons from 4 mice, dSPN KO n = 4 neurons from 3 mice. Example traces on the right show the average EPSC from the last 5 min of the baseline period…

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  Figure 6.. dSPN Tsc1 KO cells exhibit reduced ribosome engagement of mRNAs involved in mGluR-eCB signaling (A) Schematic of the design of AAV-FLEX-TRAP, which allows Cre-dependent expression of a GFP-tagged ribosomal subunit (EGFP-L10a). (B) Schematic of the workflow for the Translational Ribosome Affinity Purification (TRAP) experiment. (C) Images of EGFP-L10a expression from an AAV5-hSyn-DIO-EGFP-L10a injection into the dorsolateral striatum of a Tsc1wt/wt;Drd1-Cre+;Ai9+/− mouse showing expression in tdTomato+ dSPNs. In the merged image, EGFP-L10 is in green, tdTomato is in red, and NeuN immunostaining is in blue to label neurons. (D and E) Quantification of relative Drd1 (D) and Drd2 (E) mRNA levels in TRAP samples (ribosome bound mRNA isolated from dSPNs) versus the unbound (“UN,” all striatal tissue) from the same mouse measured by qPCR. Bars represent mean ± SEM. Dots represent individual samples, taken from one mouse. For (D), ***p = 0.0009 (dSPN WT); **p = 0.0053 (dSPN KO), paired t tests. For (E), ***p = 0.0003 (dSPN WT); ***p = 0.0007 (dSPN KO), paired t tests. For (D) and (E), dSPN-Tsc1 WT n = 10 and dSPN-Tsc1 KO n = 7 mice. (F–J) Quantification of Grm1 (F), Grm5 (G), Homer1 (H),…