Figures and data
Cntnap2-/- SPNs exhibit increased cortical drive.
(A) Left: schematic of the corticostriatal connectivity experiments. For corticostriatal excitability, cortical terminals expressing ChR2 were stimulated with 10 pulses of blue light at 10 Hz and responses were recorded from dSPNs (red) and iSPNs (grey) in dorsolateral striatum. For corticostriatal synaptic strength, cortical terminals expressing ChR2 were stimulated with blue light at increasing intensity and synaptic currents were recorded from dSPNs (red) and iSPNs (grey) in dorsolateral striatum. Center: 10x confocal image of the striatum from a Cntnap2+/+;D1- tdTomato;Thy1-ChR2 mouse. Right: 20x confocal image of dorsolateral striatum from a Cntnap2+/+;D1-tdTomato;Thy1-ChR2 mouse. YFP (green) labels cell bodies and axons of a subset of layer V pyramidal neurons, tdTomato (red) labels dSPNs, and DAPI stained nuclei are in blue.
(B) Example single traces of action potentials (APs) in dSPNs evoked by cortical terminal stimulation at 20% light intensity for the indicated genotypes.
(C) Quantification (mean ± SEM) of the number of APs evoked in dSPNs at different light intensities. Cntnap2+/+ n=9 mice, 24 cells, Cntnap2-/-n=10 mice, 22 cells. Two-way ANOVA p values are shown; geno F (1, 221) = 16.75, stim F (12, 221) = 34.19, g x s F (12, 221) = 0.7682.
(D) Example single traces of action potentials (APs) in iSPNs evoked by cortical terminal stimulation at 20% light intensity for the indicated genotypes.
(E) Quantification (mean ± SEM) of the number of APs evoked in iSPNs at different light intensities. Cntnap2+/+ n=9 mice, 23 cells, Cntnap2-/-n=10 mice, 21 cells. Two-way ANOVA p values are shown; geno F (1, 234) = 8.067, stim F (12, 234) = 24.88, g x s F (12, 234) = 0.2057.
(F) Average EPSC traces from example dSPNs of each genotype induced by optogenetic cortical terminal stimulation at 14% light intensity.
(G) Quantification of EPSC amplitude evoked in dSPNs at different light intensities (line represents the mean, dots/squares represent average EPSC amplitude for each mouse). Cntnap2+/+ n=8 mice, 17 cells, Cntnap2-/- n=5 mice, 13 cells. Two-way ANOVA p values are shown; geno F (1, 33) = 0.6527, stim F (2, 33) = 11.80, g x s F (2, 33) = 0.05117.
(H) Average EPSC traces from example iSPNs of each genotype induced by optogenetic cortical terminal stimulation at 14% light intensity.
(I) Quantification of EPSC amplitude evoked in iSPNs at different light intensities (line represents mean, dots/squares represent average for mouse). Cntnap2+/+ n=6 mice, 13 cells, Cntnap2-/-n=4 mice, 11 cells. Two-way ANOVA p values are shown; geno F (1, 24) = 2.770, stim F (2, 24) = 3.466, g x s F (2, 24) = 0.1408.
Inhibition is not reduced in Cntnap2-/- SPNs.
(A) Top: schematic of the experiment. A bipolar stimulating electrode was placed roughly 200 μm from the recording site. A range of electrical stimulation intensities was applied to the tissue while IPSCs were recorded from dSPNs (red) and iSPNs (grey) in dorsolateral striatum. Bottom: 20x confocal image of dorsolateral striatum from a Cntnap2+/+;D1-tdTomato mouse. tdTomato (red) labels dSPNs, DAPI stained nuclei are in blue.
(B) Average IPSC traces from example dSPNs of each genotype evoked by electrical stimulation at 1.5 (x0.01 mA) intensity for the indicated genotypes.
(C) Quantification (mean ± SEM) of IPSC amplitude in dSPNs at different stimulation intensities. Cntnap2+/+ n=17 cells from 9 mice, Cntnap2-/- n=16 cells from 9 mice. Two-way ANOVA p values are shown; geno F (1, 248) = 0.004612, stim F (7, 248) = 16.09, g x s F (7, 249) = 0.3164.
(D) Average IPSC traces from example iSPNs of each genotype evoked by electrical stimulation at 1.5 (x0.01 mA) intensity for the indicated genotypes.
(E) Quantification (mean ± SEM) of IPSC amplitude in iSPNs at different stimulation intensities. Cntnap2+/+ n=16 cells from 9 mice, Cntnap2-/- n=16 cells from 10 mice. Two-way ANOVA p values are shown; geno F (1, 240) = 11.25, stim F (7, 240) = 10.17, g x s F (7, 240) = 0.1947.
(F) Top: schematic of the experiment. PV interneuron terminals expressing ChR2 were stimulated with blue light at a range of intensities and optically-evoked IPSCs were recorded from dSPNs (red) and iSPNs (grey) in dorsolateral striatum. Bottom: 20x confocal image of dorsolateral striatum from a Cntnap2+/+;D1-tdTomato;PV-Cre;Ai32 mouse. YFP (green) labels PV interneurons, tdTomato (red) labels dSPNs, DAPI stained nuclei are in blue.
(G) Average IPSC traces from example dSPNs of each genotype evoked by optogenetic PV interneuron stimulation at 30% light intensity.
(H) Quantification (mean ± SEM) of IPSC amplitude in dSPNs at different light intensities. Cntnap2+/+ n=29 cells from 15 mice, Cntnap2-/- n=23 cells from 11 mice. Two-way ANOVA p values are shown; geno F (1, 504) = 0.006524, stim F (7, 504) = 14.26, g x s F (7, 504) = 0.1124.
(I) Average IPSC traces from example iSPNs of each genotype evoked by optogenetic PV interneuron stimulation at 30% light intensity.
(J) Quantification (mean ± SEM) of IPSC amplitude in iSPNs at different light intensities. Cntnap2+/+n=24 cells from 14 mice, Cntnap2-/- n=27 cells from 13 mice. Two-way ANOVA p values are shown; geno F (1, 392) = 2.638, stim F (7, 392) = 13.59, g x s F (7, 392) = 0.5088.
Intrinsic excitability is increased in Cntnap2-/- SPNs.
(A) Example AP traces in dSPNs evoked by a 200 pA current step for the indicated genotypes.
(B) Quantification (mean ± SEM) of the number of APs evoked in dSPNs at different current step amplitudes. Cntnap2+/+ n=22 cells from 8 mice, Cntnap2-/- n=23 cells from 8 mice. Two-way ANOVA p values are shown; geno F (1, 688) = 48.80, stim F (15, 688) = 107.5, g x s F (15, 688) = 1.737.
(C) Quantification (mean ± SEM) of the rheobase current in dSPNs. Dots/squares represent the rheobase current for each neuron. n is the same as in panel B. **p=0.0016, two-tailed unpaired t test.
(D-F) Quantification (mean ± SEM) of dSPN membrane resistance (D), *p=0.0328, Mann- Whitney test; membrane capacitance, p=0.2182, Mann-Whitney test; and resting membrane potential (F), p=0.9914, two-tailed unpaired t test. Dots/squares represent the average value for each neuron. n is the same as in panel B.
(G) Example AP traces in iSPNs evoked by a 200 pA current step for the indicated genotypes.
(H) Quantification (mean ± SEM) of the number of APs evoked in iSPNs at different current step amplitudes. Cntnap2+/+ n=22 cells from 8 mice, Cntnap2-/-n=21 cells from 8 mice. Two-way ANOVA p values are shown; geno F (1, 656) = 4.186, stim F (15, 656) = 118.7, g x s F (15, 656) = 0.7780.
(I) Quantification (mean ± SEM) of the rheobase current in iSPNs. Dots/squares represent the rheobase current for each neuron. n is the same as in panel H. p=0.0923, two-tailed unpaired t test.
(J-K) Quantification (mean ± SEM) of iSPN membrane resistance (J), p=0.8193, Mann-Whitney test; membrane capacitance (K), p=0.6886, two-tailed unpaired t test; and resting membrane potential (L), p=0.4859, two-tailed unpaired t test. Dots/squares represent the average value for each neuron. n is the same as in panel H.
Cntnap2-/- mice exhibit increased repetitive behaviors.
(A-D) Schematics of the behavioral assays used to measure repetitive behaviors in Cntnap2+/+ and Cntnap2-/- mice, created with BioRender.com. Male and female mice were used for all tests. (E-H) Quantification (mean ± SEM) of open field activity over 60 minutes. (E) total distance traveled, p= 0.3538, Mann-Whitney test; (F) average speed, p=0.3832, Mann-Whitney test; (G) number of rears, p=0.1892, two-tailed unpaired t test; (H) number of center entries, *p=0.0101, two-tailed unpaired t test. Cntnap2+/+ n=41 mice, Cntnap2-/-n=34 mice.
(I) Quantification (mean ± SEM) of the number of manually scored grooming bouts in the first 20 minutes of the open field test, **p=0.0034, Mann-Whitney test, Cntnap2+/+ n=41 mice, Cntnap2-/-n=34 mice.
(J) Quantification (mean ± SEM) of total marbles buried in the marble burying assay. Cntnap2+/+ n=33 mice and Cntnap2-/- n=33 mice, *p=0.0396, two-tailed unpaired t test.
(K-M) Quantification (mean ± SEM) of performance in the holeboard assay. (K) Total number of nose pokes in 10 minutes, *p=0.0212, Mann-Whitney test; (L) nose pokes in the first five minutes, p=0.4811, two-tailed unpaired t test; and (M) nose pokes in the last five minutes, *p=0.0116, two-tailed unpaired t test. Cntnap2+/+ n=25 mice, Cntnap2-/- n=22 mice.
(N) Quantification (mean ± SEM) of the frequency of movement syllables (top 25 most frequent syllables) in the open field assay defined by Keypoint-MoSeq. Cntnap2+/+n=13 mice and Cntnap2-/- n=11 mice, **p=0.0013 for grooming 6, **p=0.0013 for grooming 7, Kruskal-Wallis test with Dunn’s correction for multiple comparisons.
(O) Quantification (mean ± SEM) of the entropy of syllable transitions in the open field assay. Cntnap2+/+ n=13 mice and Cntnap2-/- n=11 mice, *p=0.0236, two-tailed unpaired t test.
(P) Accuracy of a Random Forest decoder trained on DeepLabCut basic locomotor data (Movement), Keypoint-MoSeq syllable usage data (Syllable usage), or Keypoint-MoSeq syllable transition data (Syllable transition) in distinguishing between Cntnap2+/+ and Cntnap2-/-mice. Dotted line represents chance performance.
For panels E-M and O, dots/squares represent the value for each mouse.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
Cntnap2-/- mice exhibit enhanced motor learning.
(A) Schematic of the rotarod apparatus (top), and design of the task (bottom), created with BioRender.com. Mice walk to stay on the rotating rod for three 5-minute trials a day for two days at 5-40 RPM acceleration over 5 minutes, followed by three trials a day for two days at 10-80 RPM.
(B) Quantification (mean ± SEM) of accelerating rotarod performance across 12 trials for the indicated genotypes. Cntnap2+/+ n=30 mice, Cntnap2-/- n=29 mice. Two-way ANOVA p values are shown; geno F (1, 683) = 92.17, trial F (11, 683) = 58.45, g x t F (11, 683) = 2.820.
(C) Quantification (mean ± SEM) of rotarod performance on trial 1 quantified as terminal speed. Dots/squares represent the performance of individual mice. n is same as in panel B, p=0.1518, Mann-Whitney test.
(D) Quantification (mean ± SEM) of learning rate (RPM/day) calculated as the slope of the line of performance from the first trial (1) to the last trial (12) for each mouse. Dots/squares represent the learning rate for individual mice. n is same as in panel B, ***p=0.0002, two-tailed unpaired t test.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
Cntnap2-/- mice exhibit cognitive inflexibility.
(A) Schematic of the four-choice odor-based reversal learning task, created with BioRender.com. Colored circles represent pots with different scented wood shavings. Yellow ring represents the food reward. Red circle in the Day 2 Reversal panel indicates a novel odor. (B-D) Quantification of parameters during acquisition learning. Mean ± SEM number of trials to reach criterion (B, at least 8 out of last 10 trials correct), p=0.5397, Mann-Whitney test; number of quadrant entries before making a choice (C), p=0.9118, Mann-Whitney test; and latency to make a choice (D), p=0.7224, two-tailed unpaired t test. Dots/squares represent the value for each mouse.
(E) Quantification (mean ± SEM) of the number of trials to reach criterion (at least 8 out of last 10 trials correct) during the recall test on day 2, p=0.3737, Mann-Whitney test.
(F-I) Quantification of parameters during reversal learning. Mean ± SEM number of trials to reach criterion (F, at least 8 out of last 10 trials correct), **p=0.0048, two-tailed unpaired t test; number of quadrant entries before making a choice (G), *p=0.0158, two-tailed unpaired t test; latency to make a choice (H), **p=0.0013, two-tailed unpaired t test; and number of trials to reach criterion after the first correct choice (I), *p=0.0183, two-tailed unpaired t test.
(J) Quantification (mean ± SEM) of the total number of errors made during reversal learning, **p=0.0034, Mann-Whitney test.
(K) Quantification (mean ± SEM) of the different error types made during reversal learning. Perseverative errors, ***p=0.0005, regressive errors **p=0.0068, novel errors, p=0.9955, irrelevant errors p=0.9988, omissions, p=>0.9999, repeated measures two-way ANOVA with Šídák’s multiple comparisons test; geno F (1, 18) = 10.91, error F (4, 72) = 53.49, g x e F (4, 72) = 4.292, animal F (18, 72) = 0.9638.
(L) Quantification (mean ± SEM) of the percent of choices made for each odor, binned across three trials, during reversal learning. Odor 1 was rewarded during acquisition learning and odor 2 was rewarded during reversal learning.
For panels B-L, n=10 Cntnap2+/+ mice and 10 Cntnap2-/- mice.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
Summary of mouse lines used.
Cntnap2-/- SPNs do not have altered spine density.
(A) Representative deconvoluted images of dendritic spines from dSPNs for the indicated genotypes.
(B) Quantification (mean ± SEM) of dendritic spine density per 10 μm of dendrite in Cntnap2+/+and Cntnap2-/- dSPNs. Dots/squares represent the average spine density per neuron. Cntnap2+/+ n=9 neurons (15 dendrites) from 6 mice, Cntnap2-/- n=8 neurons (15 dendrites) from 6 mice. p=0.9964, two-tailed unpaired t test.
(C) Representative deconvoluted images of dendritic spines from iSPNs for the indicated genotypes.
(D) Quantification (mean ± SEM) of dendritic spine density per 10 μm of dendrite in Cntnap2+/+and Cntnap2-/- iSPNs. Dots/squares represent the average spine density per neuron. Cntnap2+/+ n=7 neurons (15 dendrites) from 4 mice, Cntnap2-/-n=10 neurons (15 dendrites) from 7 mice. p=0.5362, Mann-Whitney test.
PV interneuron intrinsic excitability is unchanged in Cntnap2-/-mice.
(A) Top: schematic of the experiment. tdTomato-expressing PV interneurons were recorded in the dorsolateral striatum and injected with current steps of increasing magnitude to evoke firing. Bottom: 20x confocal image of dorsolateral striatum from a Cntnap2+/+;PV-Cre;Ai9 mouse. tdTomato (red) labels PV interneurons, DAPI stained nuclei are in blue.
(B) Example traces of APs in PV interneurons evoked by a 200 pA current step for the indicated genotypes.
(C) Quantification (mean ± SEM) of the number of APs evoked in PV interneurons at different current step amplitudes. Cntnap2+/+ n=28 cells from 6 mice, Cntnap2-/-n=23 cells from 4 mice. Two-way ANOVA p values are shown; geno F (1, 784) = 2.728e-006, stim F (15, 784) = 67.25, g x s F (15, 784) = 0.1039.
(D) Quantification (mean ± SEM) of the rheobase current. Dots/squares represent the rheobase current for each neuron. n is the same as in panel C. p=0.8852, two-tailed unpaired t test.
(E-G) Quantification (mean ± SEM) of the membrane resistance (E), p=0.3422, Mann-Whitney test; membrane capacitance (F), p=0.9055, two-tailed unpaired t test; and resting membrane potential (G), p=0.3141, two-tailed unpaired t test. Dots/squares represent the average value for each neuron. n is the same as in panel C.
(H) Phase plane plot of a single AP evoked in PV interneurons from Cntnap2+/+ (black) and Cntnap2-/- (red) mice.
(I-K) Quantification (mean ± SEM) of the AP threshold (I), p=0.6025, two-tailed unpaired t test; AP height (J), p=0.2850, two-tailed unpaired t test; and AP width at half max (K), p=0.2561, two- tailed unpaired t test. Dots/squares represent the value for the first spike evoked at rheobase for each neuron. n is the same as in panel C.
Cntnap2-/- mice do not exhibit changes in Parvalbumin positive cell number or expression.
(A) Representative confocal images of dorsolateral striatum labeled with an antibody against Parvalbumin (PV) protein for the indicated genotypes, mice aged P21.
(B) Quantification (mean ± SEM) of the number of PV positive cells per ROI at P21, p=0.2554, two-tailed unpaired t test. Cntnap2+/+ n=8 sections from 4 mice (2 sections imaged per mouse), Cntnap2-/-n=8 sections from 4 mice (2 sections imaged per mouse).
(C) Representative confocal images of dorsolateral striatum labeled with an antibody against PV for the indicated genotypes, mice aged postnatal day (P) 40-50.
(D) Quantification (mean ± SEM) of the number of PV positive cells per ROI, p=0.5144, two- tailed unpaired t test. Cntnap2+/+ n=24 sections from 12 mice (2 sections imaged per mouse), Cntnap2-/-n=24 sections from 12 mice (2 sections imaged per mouse).
(E) Representative confocal images of dorsal striatum labeled with an antibody against PV for the indicated genotypes, mice aged P40-50.
(F) Quantification (mean ± SEM) of the number of PV positive cells in the whole dorsal striatum at P40-50, p=0.4201, two-tailed unpaired t test. Cntnap2+/+ n=8 sections from 4 mice (2 sections imaged per mouse), Cntnap2-/- n=8 sections from 4 mice (2 sections imaged per mouse).
(G) Quantification (mean ± SEM) of the average single cell fluorescence of PV antibody staining in dorsal striatum, p=0.9996, two-tailed unpaired t test. Cntnap2+/+n=8 sections from 4 mice (2 sections imaged per mouse), Cntnap2-/-n=8 sections from 4 mice (2 sections imaged per mouse).
(H) Quantification (mean ± SEM) of the average bulk fluorescence of PV antibody staining in dorsal striatum per section, p=0.4878, two-tailed unpaired t test. Cntnap2+/+ n=8 sections from 4 mice (2 sections imaged per mouse), Cntnap2-/-n=8 sections from 4 mice (2 sections imaged per mouse).
(I) Representative western blots for CASPR2, PV, and GAPDH protein in dorsal striatal tissue punches. 10 independent samples per genotype are shown.
(J) Quantification (mean ± SEM) of PV protein expression normalized to GAPDH. Data are presented as a percentage of Cntnap2+/+ levels, p=0.1485, two-tailed unpaired t test. Cntnap2+/+ n=10 mice, Cntnap2-/-n=10 mice.
(K) Representative confocal images of in situ hybridization for Pvalb in dorsolateral striatum for the indicated genotypes, mice aged P40-50.
(L-N) Quantification (mean ± SEM) of the number of Pvalb positive cells in the dorsolateral striatum per section (J), p>0.9999, Mann-Whitney test; dorsomedial striatum (K), p=0.7429, Mann-Whitney test; and central striatum (L), p=0.6286, Mann-Whitney test. Cntnap2+/+ n=4 sections from 2 mice (2 sections imaged per mouse), Cntnap2-/- n=4 sections from 2 mice (2 sections imaged per mouse).
Action potential properties are unchanged in Cntnap2-/- SPNs.
(A) Phase plane plot of a single AP evoked in dSPNs from Cntnap2+/+ (black) and Cntnap2-/- (orange) mice.
(B-D) Quantification (mean ± SEM) of dSPN AP threshold (B), p=0.0516, two-tailed unpaired t test; AP height (C), p=0.0833, two-tailed unpaired t test; and AP width at half max (D), p=0.3792, two-tailed unpaired t test. Dots/squares represent the value for the first spike evoked at rheobase for each neuron. Cntnap2+/+ n=22 cells from 8 mice, Cntnap2-/-n=23 cells from 8 mice.
(E) Phase plane plot of a single AP evoked in iSPNs in Cntnap2+/+ (green) and Cntnap2-/- (blue) mice.
(F-H) Quantification (mean ± SEM) of iSPN AP threshold (F), p=0.1495, two-tailed unpaired t test; AP height (G), p=0.1794, Mann-Whitney test; and AP width at half max (H), p=0.2409, two- tailed unpaired t test. Dots/squares represent the value for the first spike evoked at rheobase for each neuron. Cntnap2+/+n=22 cells from 8 mice, Cntnap2-/- n=21 cells from 8 mice.
DeepLabCut and Keypoint-MoSeq analysis of Cntnap2-/- mice.
(A) Quantification (mean ± SEM) of the total distance traveled in the open field measured by DeepLabCut, p=0.8201, Mann-Whitney test; Cntnap2+/+ n=13 mice and Cntnap2-/- n=11 mice.
(B) Quantification (mean ± SEM) of the number of grooming bouts in the open field assessed by Keypoint-MoSeq, calculated as the number of instances of all grooming syllables. Cntnap2+/+ n=13 mice and Cntnap2-/- n =11 mice, *p = 0.0352, Mann-Whitney test.
(C) Map depicting the difference in transitions between syllables (top 25 most frequent syllables) for Cntnap2+/+ and Cntnap2-/-mice (Cntnap2-/- minus Cntnap2+/+). A red circle around a syllable number indicates increased usage of that syllable in Cntnap2-/- mice, a blue circle indicates decreased usage of that syllable in Cntnap2-/- mice. A red line between syllable numbers indicates increased usage of that syllable transition in Cntnap2-/-mice, a blue line indicates decreased usage of that syllable transition in Cntnap2-/-mice. The thickness of the line scales with the size of the difference and the size of the circle surrounding the syllable number scales with usage of the syllable.
