SYNGAP1 heterozygosity disrupts sensory processing by reducing touch-related activity within somatosensory cortex circuits

Abstract

In addition to cognitive impairments, neurodevelopmental disorders often result in sensory processing deficits. However, the biological mechanisms that underlie impaired sensory processing associated with neurodevelopmental disorders are generally understudied and poorly understood. We found that SYNGAP1 haploinsufficiency in humans, which causes a sporadic neurodevelopmental disorder defined by cognitive impairment, autistic features, and epilepsy, also leads to deficits in tactile-related sensory processing. In vivo neurophysiological analysis in Syngap1 mouse models revealed that upper-lamina neurons in somatosensory cortex weakly encode information related to touch. This was caused by reduced synaptic connectivity and impaired intrinsic excitability within upper-lamina somatosensory cortex neurons. These results were unexpected, given that Syngap1 heterozygosity is known to cause circuit hyperexcitability in brain areas more directly linked to cognitive functions. Thus, Syngap1 heterozygosity causes a range of circuit-specific pathologies, including reduced activity within cortical neurons required for touch processing, which may contribute to sensory phenotypes observed in patients.

Figure 1:. Reduced sensory-evoked brain activity in Syngap1 SSC.

(a-b) Stimulus paradigm used during IOS imaging. (c) Example IOS signals from one animal in each genotype obtained for β and C2 whiskers. (d-f) Scatter plots showing reduced IOS amplitudes in β (d) and C2 (e) whisker fields, but normal inter-barrel distance (f) in adult Syngap1 mutants. Unpaired two-sided t-Tests: t(20)=3.76 p=0.0014 for β amplitude (n=11 WT n=11 Het mice), t(16)=3.70 p=0.001 for C2 amplitude (n=9 WT, n=9 Het mice), t(19)=1.49 p=0.15 for inter-barrel distance(n=10 WT n=11 Het mice). (g, h) Quantification of the area responding to β or C2 whisker stimulation according to absolute (g) or relative thresholding methods (h). 2-way RM-ANOVA for absolute area F_(1,36)=11.18 p=0.002 for genotype, F_(3,108)=1.74 p=0.163 for genotype*threshold; 2-way RM-ANOVA for relative area F_(1,36)=2.8 p=0.1 for genotype, F_(3,108)=2.92 p=0.037 for genotype*threshold (n=20 WT n=20 Het IOS imaging sessions from different whiskers). For d-f, open circles represent animal means, black lines indicate population means and error bars indicate SEMs. For g-h, closed circles and squares represent population means and error bars indicate SEMs. Data in this figure were acquired from two independent cohorts of animals that were pooled together.

Figure 2:. Reduced ongoing and whisker-generated activity in SSC L2/3 neurons from awake Syngap1 mice.

(a) Experimental setup for awake, in vivo two-photon calcium imaging in Syngap1 crossed with Thy-1 Gcamp6s4.3 mice, with and without Botox injection. (b-c) Representative in vivo two-photon microscopy images (left) and ΔF/F traces (right) of spontaneous activity in 9 (1-9) neurons of L2/3 SSC of WT and Het Syngap1 X Thy-1 Gcamp6s4.3 mice, without (b) and with (c) Botox. ROI number 10 is the neuropil signal. Asterisks indicate detected calcium events (blue for WT, red for Het). (d-f) Cellular sensory properties from awake WT and Het mice without and with Botox injection. (d) Scatter plot showing fraction of spontaneously active cells in WT and Het mice (2-way RM-ANOVA, Genotype: F(1, 14) = 0.29, p = 0.60, Treatment: F(1,14) = 0.034, p = 0.86, Interaction: F(1, 14) = 0.012, p = 0.92; WT n=8 mice, Het n=8 mice). (e-f) Cumulative probability plots of ΔF/F amplitudes (e; KS Tests: WT/Botox- vs. Het/Botox-, p= 3.38E-7; WT/Botox+ vs. Het/Botox+, p=0.0002; WT/Botox- vs. WT/Botox+, p= 0.67; Het/Botox- vs. Het/Botox+, p= 0.078; WT: n_Botox-=1622 neurons, n_Botox+ = 1827 neurons; Het: n_Botox- = 1667 neurons, n_Botox+ = 1600 neurons) and spike counts (f, KS Tests, WT/Botox- vs. Het/Botox-, p= 1.82E-7; WT/Botox- vs. WT/Botox+, p= 7.99E-6; WT/Botox- vs. Het/Botox+, p= 2.87E-8; Het/Botox- vs. Het/Botox+, p= 0.99; WT/Botox+ vs. Het/Botox+, p=0.24). (g) Whisking behavior in head-fixed Syngap1 mice (Fraction of time whisking: Unpaired t-test t(12)=7.493, p= 7.0E-6; WT n=7 mice, Het n=7 mice. Bout frequency: Unpaired t-test t(12)=6.298, p= 4.0E-5; WT n=7 mice, Het n=7 mice. Mean bout duration: Unpaired t-test t(12)=0.8714, p=0.40; WT n=7 mice, Het n=7 mice). Data were pooled from two independent cohorts of animals and thus obtained from 2169 neurons in 56 imaging planes from 8 WT mice; 1971 neurons in 55 imaging planes from 8 Het mice. In scatter plots, open circles are animal means, black lines indicate population means, and error bars indicate SEMs. ***P<0.001, ****P<0.0001 for cumulative probability plots. All statistical tests were two-sided.

Figure 3:. Reduced whisker responsiveness of SSC neurons in behaving Syngap1 mice.

Representative in vivo two-photon microscopy images (a) and representative ΔF/F traces (b) of 9 (1-9) neurons in WT and Het (Botox-) mice in response to 5 passive whisker deflections at 5 Hz. Gray vertical lines indicate the timing of whisker stimuli. Green asterisks indicate calcium events within the response detection window. Red asterisks show spontaneous calcium events. ROI number 10 is the neuropil signal. (c-h) Cellular sensory properties from awake animals in response to 5 pulses at 5 Hz (c-e) and 60 pulses at 40 Hz whisker stimulation (f-h). (c) Scatter plot showing fraction of responding cells in WT and Het mice (Unpaired t-test t(14)=2.48, p=0.026; WT n=8 mice, Het n=8 mice). (d-e) Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (d, KS Test, p=0.13; WT: n=406 neurons, Het: n=330 neurons) and response probabilities (e, KS Test, p=0.23) in responding neurons. (f) Scatter plot depicting fraction of responding cells in WT and Het mice (Unpaired t-test t(14)=3.07, p=0.0084; WT n=8 mice, Het n=8 mice). (g, h) Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (g, KS Test, p=0.00050; WT: n=467 neurons, Het: n=368 neurons) and response probabilities (h, KS Test, p=0.0059) in responding neurons. (i-n) Cellular sensory properties from awake animals following Botox injection in response to 5 pulses at 5 Hz (i-k) and 60 pulses at 40 Hz whisker stimulation (l-n). (i) Scatter plot depicting fraction of responding cells in WT and Het mice (Unpaired t-test t(14)=0.35, p=0.73; WT n=8 mice, Het n=8 mice). (j, k) Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (j, KS test, p=0.32; WT: n=413 neurons, Het: n=416 neurons) and response probabilities (k, KS Test, p=0.0061) in responding neurons. (l) Scatter plot showing fraction of responding cells in WT and Het mice (Unpaired t-test t(14)=0.11, p=0.92; WT n=8 mice, Het n=8 mice). (m, n) Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (m, KS Test, p=0.39; WT: n=481 neurons, Het: n=445 neurons) and response probabilities (n, KS Test, p=0.010) in responding neurons. (c-h) Data obtained from 1921 neurons in 54 imaging planes from 8 WT mice; 2044 neurons in 54 imaging planes from 8 Het mice. (c-n) Data was pooled from two independent cohorts of animals and thus obtained from 2169 neurons in 56 imaging planes from 8 WT mice; 1971 neurons in 55 imaging planes from 8 Het mice. In scatter plots, open circles are animal means, closed circles are individual cells, black lines indicate population means, and error bars indicate SEMs. All statistical tests were two-sided.

Figure 4:. Reduced sensory responsiveness of L2/3 SSC neurons in Syngap1 mice is cortex-specific.

(a) Representative in vivo two-photon microscopy images of L2/3 SSC of WT and Het Emx1-Cre X Syngap1 cKO mice. (b) Representative ΔF/F traces of 9 (1-9) neurons in WT and Het mice in response to 5 passive whisker deflections at 5 Hz (from a). Gray vertical lines indicate the timing of whisker stimulus. Green asterisks indicate calcium events within the response detection window. Red asterisks show spontaneous calcium events. ROI number 10 is the neuropil signal. (c-e) Cellular sensory properties pooled from two independent cohorts of Emx1-Cre X Syngap1 cKO mice in response to 5 pulses at 5 Hz whisker stimulations under anesthesia. (c) Scatter plot showing fraction of responding cells in WT and Het mice (Unpaired t-test t(14)=0.85, p=0.41; WT n=8 mice, Het n=8 mice). (d, e) Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (d, KS Test, p=0.016; WT: n=327 neurons, Het: n=306 neurons) and response probabilities (e, KS Test, p= 8.2E-5) in responding neurons. Data obtained from 1671 neurons in 55 imaging planes from 8 WT mice; 1877 neurons in 58 imaging planes from 8 Het mice. (f-h) Cellular sensory properties pooled from two independent cohorts of Cux2-CreERT2 X Syngap1 cKO mice in response to 5 pulses at 5 Hz whisker stimulations under anesthesia. (f) Scatter plot showing fraction of responding cells in WT and Het mice (Unpaired t-test t(14)=0.56, p=0.58; WT n=8 mice, Het n=8 mice). (g, h) Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (g, KS Test, p=0.0002; WT: n=435 neurons, Het: n=445 neurons) and response probabilities (h, KS Test, p=0.67) in responding neurons. Data obtained from 2015 neurons in 57 imaging planes from 8 WT mice; 1901 neurons in 56 imaging planes from 8 Het mice. (i-k) Cellular sensory properties pooled from two independent cohorts of Rpb4-Cre X Syngap1 cKO mice in response to 5 pulses at 5 Hz whisker stimulations under anesthesia. (i) Scatter plot showing fraction of responding cells in WT and Het mice (Unpaired t-test t(13)=0.64, p=0.53; WT n=8 mice, Het n=7 mice). Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (j, KS Test, p=0.74; WT: n=340 neurons, Het: n=317 neurons) and response probabilities (k, KS Test, p=0.26) in responding neurons. Data obtained from 1684 neurons in 56 imaging planes from 8 WT mice; 1411 neurons in 46 imaging planes from 7 Het mice. In scatter plots, open circles are animal means, closed circles are individual cells, black lines indicate population means, and error bars indicate SEMs. All statistical tests were two-sided.

Figure 5:. Reduced sensory responsiveness in both excitatory and inhibitory neuronal populations in L2/3 SSC of Syngap1 mutants.

(a) Representative in vivo two-photon microscopy image of L2/3 SSC of a Gad2-T2A-NLS-MCherryXSyngap1 WT mouse expressing GCaMP6s (green) and Mcherry (red). White arrows indicate MCherry positive (inhibitory) neurons expressing GCaMP6s. (b) Representative ΔF/F traces of 9 (1-9) excitatory neurons in WT and Het mice in response to 5 passive whisker deflections at 5 Hz. Gray vertical lines indicate the timing of whisker stimuli. Green asterisks indicate calcium events within the response detection window. Red asterisks show spontaneous calcium events. ROI number 10 is the neuropil signal. (c-h) Cellular sensory properties of excitatory (c-e) and inhibitory (f-h) neurons. (c) Scatter plot showing fraction of excitatory neurons responsive to whisker stimulation (Unpaired t-test t(11)=1.891, p=0.0853; WT n=7 mice, Het n=6 mice). (d, e) Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (d, KS Test p=0.1543; WT n=200 neurons, Het n=104 neurons) and response probabilities (e, KS Test, p=0.0011) in responding excitatory neurons. (f) Scatter plot showing fraction of inhibitory neurons responsive to whisker stimulation (Unpaired t-test t(11)=1.164, p=0.2691; WT n=7 mice, Het n=6 mice). (g, h) Cumulative probability and scatter plots (inserts) of ΔF/F amplitudes (g, KS Test, p=0.9848; WT n=49 neurons, Het n=20 neurons) and response probabilities (h, KS Test, p=0.0176) in responding inhibitory neurons. Data in this figure was pooled from two independent cohorts of animals and thus obtained from 850 excitatory and 240 inhibitory cells in 48 imaging planes from 7 WT mice; 825 excitatory and 193 inhibitory cells in 45 imaging planes from 6 Het mice. Open circles are animal means, closed circles are individual cells, black lines indicate population means, and error bars indicate SEMs. All statistical tests were two-sided.

Figure 6:. In vivo patch clamp reveals that L2/3 SSC neurons in Syngap1 mutants have reduced sensory-evoked synaptic input.

(a) Representative in vivo traces for whole-cell patch clamp experiments in response to passive whisker stimulations. (b) Scatter plot showing the overall response peaks (t-test: t(15)=2.59, p=0.021; n=8 for WT n=9 for Het. (c) Individual response amplitudes (2-way RM-ANOVA, F(1,14)=4.82, p=0.045 for genotype effect, F(4,56)=0.72, p=0.58 for genotype and stimulus interaction) in response to whisker stimulation in L2/3 neurons from in vivo patch clamp recordings. For b, open circles represent animal means, black lines indicate population means and error bars indicate SEMs. For c closed circles represent population means and error bars indicate SEMs. Data obtained from two cohorts of Syngap1 animals. All statistical tests were two-sided.

Figure 7:. Syngap1 heterozygosity degrades synaptic connectivity and reduces intrinsic excitability of upper layer SSC neurons.

(a, c) Representative 3D reconstruction of L4 (a, left) and L2/3 (c, left) of SSC excitatory neurons depicting dendritic complexity and scatter plot (right) showing the total length and # of nodes by using Sholl analysis. (a) Total length: (WT = 5 mice, Het = 5 mice), unpaired t-Test, t(8)=4.002, p = 0.0030; # of Nodes: unpaired t-Test, t(8)=3.017, p = 0.0166. (c) Total length: (WT = 5 mice, Het = 6 mice), unpaired t-Test, t(9)=3.7713, p = 0.0044; # of Nodes: unpaired t-Test, t(9)=3.7090, p = 0.0048, from a single cohort of animals. (b, d) Examples of L4 (b, left) and L2/3 (d, left) apical dendrites and scatter plots (right) depicting the density of dendritic spines. (b) Layer 4 - spine density: (WT = 5 mice, Het = 5 mice), unpaired t-Test, t(8)=4.059, p = 0.0036. (d) Layer 2/3 - spine density: (WT = 5 mice, Het = 5 mice), unpaired t-Test, t(8)=7.80, p = 5.2E-5, from a single cohort of animals. (e) Representative traces depicting L2/3 excitatory neuron mEPSCs from acute WT and Het TC slices. (f, g) Cumulative probability and scatter plots (inserts) of mEPSC amplitudes (f, Kolmogorov-Smirnov test p= 2.2E-16) and mEPSC IEI (g, Kolmogorov-Smirnov test p= 2.1E-16). (f, g) Data was acquired from a single cohort of animals with n=7986 mEPSC events from16 neurons in 4 WT and n=6765 mEPSC events from 16 neurons in 4 Het mice. (h) Cartoon depicting experimental setup for investigating feed-forward excitation in L2/3 excitatory neurons from L4. (i) Representative traces depicting L2/3 excitatory neuron eEPSCs from acute WT and Het TC slices. (j) Scatter plot of eEPSC amplitudes in L2/3 following stimulation of L4 (Mann-Whitney test, U=14.00, p= 2.5E-5; Data obtained from a single cohort of animals; WT n=14 neurons from 4 mice, Het n=14 neurons from 4 mice). (k) Representative current-clamp traces from L2/3 excitatory neurons from acute WT and Het TC slices and (l) graph depicting a decrease in the number of spikes (2-way RM-ANOVA, F(1,46)=5.51, p=0.023 for genotype effect F(9,414)=5.46, 9.0E-9 for genotype and stimulus interaction, n=22 neurons from 5 WT mice, n=26 neurons from 6 Het mice) in response to current injections. (m) Scatter plot showing increased rheobase (Student’s t-test: t(44)=3.50, p = 0.0010) in the same set of neurons as in l. Data was obtained from a single cohort of animals. For morphology data, open circles are animal means, closed circles are individual cells, black lines indicate population means, error bars indicate SEMs, colored triangles represent spines (blue = WT, red = Het) and white triangles represent filopodia. For f, g, j, m, open circles are individual cells, black lines indicate population means, and error bars indicate SEMs. For l, circles represent population means and error bars indicate SEMs. All statistical tests were two-sided.

Figure 8:. Impaired texture discrimination and whisker-dependent Go/NoGo task performance in Syngap1 mice.

(a) Cartoons depicting different texture roughness of the objects used in Novel Texture Discrimination Task and relative protocol. (b) Scatter plot showing no preference in exploring T1 or T2. (T1 vs T2: n = 12 mice; unpaired t-Test, t(22) = 0.0016, p = 0.9986. (c-d) Box plots (solid line represents median, box represents interquartile range and whiskers represent maximum and minimum values) depicting time spent exploring identical textured objects during the learning phase and time spent exploring the novel (T2) and the old (T1) object for WT (c) and Het groups (d) [(WT mice Friedman test: χ2 (n = 18 mice, df = 3) = 28.87, exact sign. p = 1.4206 E-7; (HET mice, Friedman test: χ2 (n = 15 mice, df = 3) = 15.24, exact sign. p = 0.0016). Pairwise comparisons: (WT(learning) vs WT(testing), Sign test: n = 18 mice, Z = 4.007 p = 0.00006; HET(learning) vs HET(testing), Sign test: n = 15 mice, Z = 2.065, p = 0.04, ns). Statistical significance was accepted at the p < 0.03125. WT(learning) vs HET(learning), Mann-Whitney U test: Z = −2.567 p = 0.01; WT(testing) vs HET(testing), Mann-Whitney U test: Z = −2.821 p = 0.0048). (e) Scatter plot showing Exploration Index for animals in c and d: WT mice, paired t-Test: t(17) = 4.707, p = 0.0002; HET mice paired t-Test: t(14) = 1.641, p = 0.123; One sample test: WT(learning), t(17) = 1.555, p = 0.138; WT(testing), t(17) = 8.579, p = 1.39E-7; HET(learning), t(14) = −2.164, p = 0.048, ns; HET(testing), t(14) = −2.415, p = 0.03, ns. (f) Cartoon representation of Go/NoGo setup. Water-restricted, head-fixed mice were rewarded with water for licking a lick-port in response to a passive whisker (C2) deflection. (g) Detection task trial structure for Step 2 training. Go trials are identical to NoGo trials, except for the passive whisker deflection. Note, NoGo trials include activation of a “dummy piezo” not attached to any whisker to control for noise/vibration associated with piezo activation. (h) Step 2 training learning curve for WT mice showing the probability of licking (P(lick)) on Go (black, hit) or NoGo (blue, FA) trials (n=7 mice; 2-way RM-ANOVA with Bonferroni’s multiple comparison, Trial type: F_(1,6)=67.19 p= 0.0002; Session: F_(15,90)=0.4827 p=0.9437; Trial type*Session interaction F_(15,90)=5.86 p= 2.9E-8). (i, j) Reductions in angular velocity of whisker deflections impairs the ability of “Good Performing” WT mice to discriminate between trial types (i: n=6 mice; 2-way RM-ANOVA with Bonferroni’s multiple comparison, Trial type: F_(1,5)=471.1 p= 3.9E-6; Velocity: F_(3,15)=1.469 p=0.263; Trial type*Velocity interaction: F_(3,15)=30.12 p= 1.4E-6) and results in a reduced discrimination index (j: n=6 mice; RM-ANOVA with Bonferroni’s multiple comparison, F_(3,15)=24.52 p= 4.9E-6). (k) Proportion of mice to learn (performers, P vs. non-performers, Non-P) the task (WT n=7, Het n=7: Fisher’s Exact Test: p=0.0047). (l) Step 2 training learning curve for Het mice (n=7 mice; 2-way RM-ANOVA with Bonferroni’s multiple comparison, Trial type: F_(1,6)=8.44 p=0.027; Session: F_(15,90)=2.416 p=0.0054; Trial type*Session interaction F_(15,90)=0.8852 p=0.5825). (m) Learning curves depicting the fraction of total trials correct in Step 2 training (WT n=7, Het n=7; 2-way RM-ANOVA with Bonferroni’s multiple comparison, Genotype: F_(1,12)=10.13 p=0.0079; Session: F_(15,180)=3.665 p= 1.4E-5; Genotype*Session interaction: F_(15,180)=4.398 p= 5.2E-7). (n) Scatter plot showing the discrimination index at the completion of Step 2 training (WT n=7, Het n=7; Unpaired t-test, t(12) = 4.281, p=0.0011). Data for both Novel Texture Discrimination and Go/NoGo Tasks were obtained from two independent cohorts of animals. Open circles are individual animals, closed circles and solid black horizontal lines indicate population means and error bars or shaded area represent the SEMs, except for boxplots in c and d which are described above. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 for post-hoc multiple comparisons. (h, i, l) Solid black and blue dashed lines indicate performance criteria for hit and FAs, respectively. (m) Solid black line indicates performance criteria for total trials correct. All statistical tests were two-sided.