sEPSCs and mEPSCs in PV+ cells from control Vs PV+ cells from cHet mice. Related to

Fig. 1

Syngap1 haploinsufficiency in Nkx2.1+ interneurons is associated with reduced sEPSC amplitude and mEPSC frequency in LIV PV+ cells.

(a) Representative traces of sEPSCs recorded in PV+ cells from control Tg(Nkx2.1-Cre):RCEf/f:Syngap1+/+(blue, n=28 cells, 11 mice) and cHet Tg(Nkx2.1-Cre):RCEf/f:Syngapf/+ (red, n=28 cells, 11 mice) mice. (b) , Cumulative probability plots show a significant decrease in the amplitude (p=0.048, LMM) and no change in the inter-sEPSC interval (LMM, p=0.186). Insets illustrate significant differences in the sEPSC amplitude for inter-cell mean comparison (*p=0.044, LMM) and no difference for inter-sEPSC interval (p=0.676, LMM). (c) Representative examples of individual sEPSC events (100 pale sweeps) and average traces (bold trace) detected in PV cells of control and cHet mice. (d) Superimposed scaled traces (left top) and summary bar graphs for a group of cells (bottom) show no significant differences in the sEPSCs kinetics (p=0.350, LMM for rise time, and p=0.429, LMM for decay time). (e) Summary bar graphs showing no differences for inter-cell mean charge transfer (p=0.064, LMM, left) and for the charge transfer when frequency of events is considered (p=0.220, LMM). (f) Representative traces of mEPSCs recorded in PV+ cells from control (blue, n=18 cells, 7 mice) and cHet (red, n=8 cells, 4 mice) mice. (g) Cumulative probability plots show no change in the amplitude of mEPSC (p=0.135, LMM) and an increase in the inter-mEPSC interval (*p=0.027, LMM). Insets illustrate summary data showing no significant differences in the amplitude (p=0.185, LMM) and the inter-mEPSCs interval for inter-cell mean comparison (p=0.354, LMM). (h) Representative examples of individual mEPSC events (100 pale sweeps) and average traces (bold trace) detected in PV+ cells of control and cHet mice. (i) Summary bar graphs for a group of cells show no significant differences in the quantal content (p=0.222, LMM) and mEPSCs kinetics (p=0.556, LMM for rise time, and p=0.591, LMM for decay time). (j) Summary bar graphs showing no differences for inter-cell mean charge transfer (p=0.069, LMM, left) and for the charge transfer when frequency of events is considered (p=0.137, LMM).

The amplitude of sIPSCs, but not mIPSCs, in LIV PV+ cells is reduced in Tg(Nkx2.1-Cre):RCEf/f:Syngap1f/+mice

(a) Representative traces of sIPSCs recorded in PV+ cells from control (blue, n=27 cells, 8 mice) and cHet (red, n=24 cells, 7 mice) mice. (b) , Cumulative probability plots show a significant decrease in the amplitude (**p=0.003, LMM) and no change in the inter-sIPSC interval (LMM, p=0.069). Insets illustrate significant differences in the sIPSC amplitude for inter-cell mean comparison (**p=0.002, LMM) and no difference for inter-sIPSC interval (p=0.102, LMM). (c) Representative examples of individual sIPSC events (100 pale sweeps) and average traces (bold trace) detected in PV cells of control and cHet mice. (d) Summary bar graphs for a group of cells show no significant differences in the sIPSCs kinetics (p=0.113, LMM for rise time, and p=0.602, LMM for decay time). (e) Summary bar graphs showing no differences for inter-cell mean charge transfer (p=0.234, LMM, left) and for the charge transfer when frequency of events is considered (p=0.273, LMM). (f) Representative traces of mIPSCs recorded in PV+ cells from control (blue, n=25 cells, 8 mice) and cHet (red, n=24 cells, 7 mice) mice. (g) Cumulative probability plots show no change in the amplitude of mIPSC (p=0.118, LMM) and in the inter-mIPSC interval (p=411, LMM). Insets illustrate summary data showing no significant differences in the amplitude (p=0.195, LMM) and the inter-mIPSCs interval for inter-cell mean comparison (p=0.243, LMM). (h) Representative examples of individual mIPSC events (100 pale sweeps) and average traces (bold trace) detected in PV+ cells of control and cHet mice. (i) Summary bar graphs for a group of cells show no significant differences in the mIPSCs kinetics (p=0.103, LMM for rise time, and p=0.597, LMM for decay time). (j) Summary bar graphs showing no differences for inter-cell mean charge transfer (p=0.374, LMM, left) and for the charge transfer when frequency of events is considered (p=0.100, LMM).

sIPSCs and mIPSCs in PV+ cells from control Vs PV+ cells from cHet mice. Related to

Fig. 2

eAMPA and eNMDA currents in LIV PV+ cells from control Vs cHet mice. Related to

Fig. 3

Thalamocortical eAMPA transmission is decreased in LIV PV+ cells from Tg(Nkx2.1-Cre):RCEf/f:Syngap1f/+mice.

(a) Representative examples of individual eAMPA (negative deflections) and eNMDA (positive deflections) (5-10 pale sweep) and average traces (bold trace) recorded in PV cells from control (blue, n=16 cells, 7 mice) and cHet mice (red, n=14 cells, 7 mice). (b) Summary plots showing no change in the failure rate of eAMPA (left, p=0.550, LMM) and a significant increase in the minimal (including failures and successes) eAMPA amplitude (*p=0.031, LMM) and (c, left) charge transfer (*p=0.033, LMM) in cHet mice. (c, right) Summary bar graph illustrating the percentage of PV+ cells containing eNMDA in the thalamocortical evoked EPSC. (d) Synaptic latency histograms (bottom) of thalamocortical eEPSC from control and cHet mice, and summary bar graph (top) illustrating an increase in the onset latencies of eEPSC in cHet mice (*p=0.023, LMM). For both histograms, bins are 0.1 msec wide. (e) Summary plots showing a significant increase in the potency of eAMPA (successes only, **p=0.003, LMM, left) in cHet mice with no change in eNMDA (p=0.969, LMM), A significant increase is present in the NMDA/AMPA ratio (i.e. ratio of the peak for eNMDA and eAMPA, **p=0.001, LMM, right) in cHet mice. (f) Summary plots showing no significant differences in the eAMPA (p=0.177, LMM for rise time, and p=0.608, LMM for decay time) and eNMDA kinetics (p=0.228, LMM for rise time, and p=0.221, LMM for decay time).

Morphological properties of total PV+ cells population in control Vs cHet mice. Related to

Fig. 4.

Syngap1 haploinsufficiency in Nkx2.1+ interneurons does not appear to alter the dendritic arbour of PV+ cells.

(a) Anatomical reconstructions of PV+ cells filled with biocytin in control (left, n=10 cells, 6 mice) and cHet (right, n=12 cells, 7 mice) during whole-cell patch-clamp recordings and post hoc immunohistochemical validation of BC interneurons confirming the positivity for PV. (b) Summary data showing no differences in somatic (p=0.636, LMM) and dendritic parameters between the two genotypes (p=0.456 for terminals #, p=0.887 for total dendritic length, p=0.100 for branching points # and p=0.460 for dendritic surface area, LMM). (c, left) Representative reconstruction of PV+ cells and superimposed concentric circles used for Sholl analysis, with a radius interval of 10 µm from the soma. (c, center) Sholl analysis of PV+ cells dendritic branch patterns revealed no differences in the number of dendritic intersections (Two-way Repeated Measure ANOVA with Sidak’s multiple comparison post hoc test, p=0.937). (c, right) Summary bar graph showing no differences in the total number of dendritic intersections obtained from Sholl analysis in control and cHet mice (p=0.502, LMM).

Membrane properties of total PV+ cells population in control Vs cHet mice. Related to

Fig. 5

PV+ cells intrinsic excitability is decreased in Tg(Nkx2.1-Cre):RCEf/f:Syngap1f/+ mice.

(a) Summary data showing no changes in the passive membrane properties between control (blue, n=33 cells, 15 mice) and cHet mice (red, n=40 cells, 17 mice) (p=0.081 for Vm, p=0.188 for Rin, p=0.188 for Cm, p=0.199 for Tm, LMM) (b) Summary data showing no differences in AP half-width (p=0.111, LMM) but a significant decrease in AP amplitude (*p=0.032, LMM) and a significant increase in AP latency (*p=0.009, LMM) from PV+ cells recorded in cHet mice. (c, left) Summary bar graph shows a significant decrease in AP threshold from cHet mice (***p<0.001, LMM) and phase plane (Vm versus dV/dt, c, right bottom) for the first AP generated confirmed this result with a more hyperpolarized value (the area squared) of AP threshold for generation of AP. (c, right top) Representative single APs evoked by rheobase currents from control and cHet mice. Action potentials are aligned at 50% of the rising phase on X axis and peak on Y axis. Note the more hyperpolarized AP with consequent reduction in AP amplitude in PV+ cells from cHet mice. (d) Summary bar graph shows a significant increase in the rheobase current (**p=0.004, LMM). (e, left) Summary plot showing a reduction of averaging number of APs per current step (40 pA) amplitude recorded from LIV PV+ cHet (red circles, n=38 cells; 18 mice) compared to control (blue circles, n=30 cells, 15 mice) neurons, including group averages (± S.E.M., Two-way Repeated Measure ANOVA with Sidak’s multiple comparison post hoc test, ****p<0.0001). (e, right) Representative voltage responses indicating the typical FS firing pattern of PV+ cells in control and cHet mice in response to depolarizing (+120 pA and +240 pA) current injections corresponding to rheobase and 2x rheobase current.

Syngap1 haploinsufficiency in Nkx2.1+ interneurons affects the denritic arbour of a specific subpopulation of LIV PV+ cells.

(a, left) Strong negative correlation of Fmax with AP half-width in PV+ cells from control (blue, n=33 cells, 15 mice) and cHet mice (red, n=40 cells, 17 mice). (a, right) Hierarchical clustering based on Euclidean distance of PV+ cells from control mice. Clustering is based on AP-Half width and Max frequency. Asterisks indicate cells with longer AP-half width felling into the cluster including PV+ cells with higher of values of Fmax. (b, left) Correlation of parameters describing membrane properties of PV+ interneurons. The 13 passive and active membrane properties used for PCA analysis (derived from 27 PV+ cells from control mice; see materials and methods) are arrayed against each other in a correlation matrix with the degree of correlation indicated by the shading: white is uncorrelated (correlation index of 0) and black is perfectly correlated (correlation index of 1, diagonal squares). PCA on the 13 parameters to reduce the dimensionality. (b, right) The first (PC1) and second (PC2) PC values derived for each interneuron are plotted against each other. No clear separation of subgroups in scatterplot of first 2 PCs is present when genotype is taken into consideration. (c) Cumulative histograms of AP half widths in control (n=33 cells, 15 mice) and cHet mice (n=40 cells, 17 mice) fitted with two Gaussian curves. Vertical line indicates the cutoff value at intersection between the two curves. For both histograms, bins are 0.05 msec wide. (d) PCA analysis using the cutoff value of 0.78 ms and the 13 passive and active membrane properties distinguish two subgroups of PV+ cells with short (black circles) and broad (turquoise circles) AP-half width duration in both genotypes. Insets illustrate pie charts describing the % of two subgroups of PV+ cells in the control and cHet mice. (e) Anatomical reconstructions of a BC-short and (f) a BC-broad filled with biocytin in control mice during whole-cell patch-clamp recordings and post hoc immunohistochemical validation for PV. (g) Summary data in control mice (gray, BC-short n=5 cells, 4 mice; turquoise, BC-broad n=5 cells, 4 mice) showing no significant difference in terms of distance from pia (p=0.856, LMM) for both subtypes of PV+ cell analyzed indicating LIV location and significant differences in dendritic parameters between the two subpopulations of PV+ cells (*p= 0.016 for dendr. surface area, *p=0.043 for # branching points, LMM) and no change in total dendritic length (p=0.057, LMM). (h) Summary data in cHet mice (gray, BC-short n=6 cells, 4 mice; turquoise, BC-broad n=6 cells, 3 mice) showing no significant difference in terms of distance from pia (p=0.594, LMM) for both subtypes of PV+ cell and all dendritic parameters (p= 0.062 for total dendritic length, p=0.731 for dendr. surface area, p=0.081 for # branching points, LMM). (i) Summary data showing a significant increase in dendritic complexity between control (gray, n=5 cells, 4 mice) and cHet (white, n=6 cells, 4 mice) for the subpopulation of BC-short (*p=0.009 for dendr. surface area, *p=0.048 for # branching points, LMM) and no difference for the total dendritic length (p=0.070, LMM). (j) Summary data showing preserved dendritic parameters in cHet (turquoise filled with pattern, n=6 cells, 3 mice) Vs control (turquoise, n=5 cells, 4 mice) (p= 0.967 for total dendritic length, p=0.784 for dendr. surface area, p=0.290 for # branching points, LMM). (k) The strong positive correlation of dendritic surface area with AP half-width is present only in PV+ cells from control mice (blue, n=10 cells, 8 mice) and disappears in cHet mice (red, 12 cells, 7 mice).

Morphological properties of BC-short Vs BC-broad in control mice. Related to Fig. 6.

Morphological properties of BC-short Vs BC-broad in cHet mice. Related to Fig. 6.

Morphological properties of BC-short in control Vs cHet mice. Related to Fig. 6.

Morphological properties of BC-broad in control Vs cHet mice. Related to Fig. 6.

Membrane properties of BC-short PV+ cells in control Vs cHet mice . Related to Fig. 7.

Membrane properties of BC-broad PV+ cells in control Vs cHet mice. Related to Fig. 7.

Intrinsic excitability is decreased in both subpopulations of PV+ cells in cHet mice

(a) Summary data showing no changes in the passive membrane properties of BC-short between control (blue, n=12 cells, 9 mice) and cHet mice (red, n=24 cells, 13 mice) (p=0.189 for Vm, p=0.856 for Rin, p=0.188 for Cm, p=0.077 for Tm, LMM) (b) Summary data showing no differences in AP half-width (p=0.386, LMM) and AP latency (p=0.210, LMM) but a significant decrease in AP amplitude (*p=0.024, LMM) of BC-short recorded in cHet mice. (c, left) Summary bar graph shows a significant decrease in AP threshold from cHet mice (**p=0.002, LMM) and phase plane (c, center) for the first AP generated confirmed this result with a more hyperpolarized value (the area squared) of AP threshold for generation of AP. (c, right) Representative single APs evoked by rheobase currents from control and cHet mice. (d) Summary bar graph shows a significant increase in the rheobase current (*p=0.015, LMM). (e, left) Summary plot showing a reduction of averaging number of APs per current step (40 pA) amplitude recorded from LIV BC-short in cHet (red circles, n = 22 cells; 13 mice) compared to control (blue circles, n = 11 cells, 9 mice) neurons, including group averages (± S.E.M., Two-way Repeated Measure ANOVA with Sidak’s multiple comparison post hoc test, **p=0.005). (e, right) Representative voltage responses indicating the typical FS firing pattern of BC-short in control and cHet mice in response to depolarizing (+120 pA and +240 pA) current injections corresponding to rheobase and 2x rheobase current. (f) Summary data showing a significant decrease in RMP (*p=0.023, LMM) of BC-broad from cHet mice (red, n=16 cells, 11 mice) but no changes in the other passive membrane properties compared to control mice (blue, n=21 cells, 12 mice) (p=0.244 for Rin, p=0.170 for Cm, p=0.639 for Tm, LMM). (g) Summary data showing no differences in AP half-width (p=0.593, LMM) and AP amplitude (p=0.713) and a significant increase in AP latency (*p=0.035, LMM) from BC-broad cells recorded in cHet mice. (h, left) Summary bar graph shows a significant decrease in AP threshold from cHet mice (*p=0.010, LMM) and phase plane (h, center) for the first AP generated confirmed this result with a more hyperpolarized value (the area squared) of AP threshold for generation of AP. (h, right). Representative single APs evoked by rheobase currents from control and cHet mice. (i) Summary bar graph shows no difference in the rheobase current (p=0.402, LMM). (j, left) Summary plot showing no difference in the averaging number of APs per current step (40 pA) amplitude recorded from LIV BC-broad in cHet (red circles, n=16 cells, 11 mice) compared to control (blue circles, n=18 cells; 11 mice) neurons, including group averages (± S.E.M., Two-way Repeated Measure ANOVA with Sidak’s multiple comparison post hoc test, p= 0.333). (j, right) Representative voltage responses indicating the typical FS firing pattern of BC broad in control and cHet mice in response to depolarizing (+120 pA and +240 pA) current injections corresponding to rheobase and 2x rheobase current.

Membrane properties of total SST+ cells population in control Vs cHet mice. Related to

Fig. 8

Intrinsic excitability of SST+ cells is less affected by embryonic-onset Syngap1 haploinsufficiency in Nkx2.1 interneurons.

(a) Representative voltage responses indicating the typical regular adapting firing pattern of SST+ in control mice in response to hyperpolarizing (-40 pA) and depolarizing (+80 pA and +160 pA) current injections corresponding to Ih associated voltage rectification, rheobase and 2x rheobase current respectively. (b) Post hoc immunohistochemical validation of these interneurons confirming their positivity for SST+ and negativity for PV-. (c) PCA using the 13 parameters previously described clearly separate the cluster of SST+ cells (pink circles) from BC-short (black circles) having however some overlaps with BC-broad (turquoise circles) in control mice. (d) Summary data showing no changes in the passive (p=0.469 for Vm, p=0.681 for Rin, p=0.922 for Cm, p=0.922 for Tm, LMM) and (e) active membrane properties (p=0.675 for AP half width, p=0.342 for AP amplitude, p=0.081 for AP latency, p=0.119 for AP threshold, LMM) between SST+ cells from control (blue, n=10 cells, 7 mice) and cHet mice (red, n=12 cells, 7 mice) (f) Summary plot showing a reduction of averaging number of APs per current step (40 pA) amplitude recorded from LIV SST+ in cHet (red circles, n= 12 cells, 8 mice) compared to control (blue circles, n=8 cells, 7 mice) neurons, including group averages (± S.E.M., Two-way Repeated Measure ANOVA with Sidak’s multiple comparison post hoc test, ****p<0.0001).

Membrane properties of PV+ in not-treated and α-DTX-treated PV+ cells in control and cHet. Related to

Fig. 9

Syngap1 haploinsufficiency alters the intrinsic excitability of LIV PV+ cells through Kv1 channels.

(a, left) Summary bar graph shows a significant decrease in AP half-width from cHet mice in absence (*p=0.034, LMM) or presence of α-DTX (*p=0.039, LMM). (a, right) Summary bar graph shows a significant decrease in AP threshold of PV+ cells from cHet mice (*p=0.049, LMM) and the rescue of this deficit in presence of α-DTX (p=0.940, LMM). (b) delta (Δ) value was calculated for AP threshold by subtracting individual values of α-DTX-treated cells from the average of their respective control group. A significant increase in AP threshold Δ number was found for cHet treated PV+ cells (*p=0.015, LMM). (c. left) Phase plane for the first AP generated confirmed this result with more depolarized value (the area squared) of AP threshold in cHet treated cells (red dotted line). (c, center and right) Representative single APs evoked by rheobase currents from control and cHet mice (center), and control and cHet treated PV+ cells (blue and red dotted lines) . Of note the more hyperpolarized AP in cHet PV+ cells and the rescue of this deficit in presence of α-DTX. (d) Summary plot showing no difference in the averaging number of APs per current step (40 pA) amplitude recorded from LIV PV+ in cHet treated and not treated PV+ cell compared to control neurons in presence or absence of α-DTX , including group averages (± S.E.M., Two-way Repeated Measure ANOVA with Sidak’s multiple comparison post hoc test, p>0.05). (e, left) Summary bar graph shows a significant difference in AP latency Δ number (*p=0.006, LMM); (e, right) representative voltage traces clearly show a reduction in the AP onset for PV+ cells treated with α-DTX (pink trace). (Control, n=9 cells, 4 mice; control α-DTX-treated, n=11 cells, 6 mice; cHet, n=23 cells, 10 mice; cHet α-DTX-treated, n=18 cells, 8 mice).

sEPSC amplitude is reduced in LIV SST+ cells in Tg(Nkx2.1-Cre):RCEf/f:Syngap1f/+mice

(a) Representative traces of sEPSCs recorded in SST+ cells from control (blue, n=8 cells, 5 mice) and cHet mice (red, n=7 cells, 5 mice). (b) Cumulative probability plots show a significant decrease in the amplitude (***p<0.001, LMM) and no change in the inter-sEPSC interval (LMM, p=0.124). Insets illustrate significant differences in the sEPSC amplitude for inter-cell mean comparison (*p=0.010, LMM) and no difference for inter-sEPSC interval (p=0.236, LMM). (c) Representative examples of individual sEPSC events (100 pale sweeps) and average traces (bold trace) detected in SST+ cells of control and cHet mice. (d) Superimposed scaled traces (left top) and summary bar graphs for a group of cells (bottom) show no significant differences in the sEPSCs kinetics (p=0.562, LMM for rise time, and p=0.281, LMM for decay time). (e) Summary bar graphs showing a significant decrease in cHet mice for inter-cell mean charge transfer (*p=0.015, LMM, left) and for the charge transfer when frequency of events is considered (*p>0.030, LMM).

Syngap1 haploinsufficiency reduces the density of local vGlut1 excitatory inputs without affecting VGlut2 thalamocortical inputs to PV+ cell somata.

(a) Representative images of auditory cortex immunolabelled for PV (grey), VGlut1 (cyan), PSD95 (magenta) in control (Nkx2.1 Cre; Syngap1+/+) and cHet (Nkx2.1 Cre; Syngap1flox/+) adult mice. Scale bar: 10 µm (b) Quantification of the density of perisomatic puncta colocalizing both VGlut1 and PSD95 normalised to controls (Unpaired t-test *p= 0.0447). Number of mice: n=5 mice for control and n=7 for cHet. (c) Representative images of auditory cortex immunolabelled for PV (grey), VGlut2 (cyan), PSD95 (magenta). Scale bar: 10 µm (d) Quantification of of the density of perisomatic puncta colocalizing both VGlut2 and PSD95 normalised to controls (Unpaired t-test p= 0.3345). Number of mice: n=5 mice for control and n=8 for cHet. Yellow arrows indicate PV cell somata. Bar graphs represent mean ± SEM. ns p > 0.05 ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

sEPSCs in SST+ cells from control Vs SST+ cells from cHet mice. Related to Fig. S1

Membrane properties of BC-broad PV+ Vs BC-short PV+ in control mice. Related to Fig. 6