Article Figures & Data
Figures
- Figure 1.
Verification of reported Scn1a+/− phenotypes. A, Survival plot of Scn1a+/− (blue; n = 25) and Scn1a+/+ WT litter mates (WT; gray; n = 30) from seven litters, plotted as probability of survival per day. ***p < 0.001, χ2 test. All electrophysiology experiments were done during the period shaded in orange (P21–P38). B, Example whole-cell recordings of neuron membrane potential to depolarizing current steps (100-, 200-, and 300-pA steps). C, Spike number plotted as a function of current step size (main effect of current step: F(12,180) = 62.73; p < 0.0001; genotype: F(1,15) = 0.03; p = 0.87; interaction: F(12,180) = 0.16; p = 0.99). Error bars for this and all subsequent figures indicate standard error of the mean, n = (cells/mice).
- Figure 2.
Similar PSPs in CA1 pyramidal neurons from WT and Scn1a+/− mice. A, Example PSPs recorded in CA1 pyramidal cells from Scn1a+/+ (WT; gray) and Scn1a+/− (Het; blue) mice in response to single stimulations of SC axons at 1×, 3×, and 5× the minimal stim intensity in ACSF (top) and 3× the minimal stim in 10 μm gabazine (gbz; bottom). B, The peak amplitude across a range of stim intensities is similar between genotypes [main effect of genotype: F(1,19) = 0.62; p = 0.44, main effect of stim intensity: F(1.57,29.83) = 51.18; p < 0.0001, interaction: F(8,152) = 1.11; p = 0.36; two-way RM ANOVA; n = (cells/mice)]. C, Firing probability across stim intensities is similar between genotypes (main effect of genotype: F(1,38) = 0.09; p = 0.77, main effect of stim intensity: F(2.68,101.7) = 7.09; p < 0.001; three-way RM ANOVA) and is similarly increased by gbz application in both genotypes (main effect of drug: F(1,38) = 21.98; p < 0.0001, genotype × drug: F(1,38) = 0.76; p = 0.39). D, Area under the curve, with spikes truncated, across stim intensities is similar between genotypes (main effect of genotype: F(1,38) = 0.96; p = 0.33, main effect of stim intensity: F(2.68,101.7) = 19.23; p < 0.0001, main effect of drug: F(1,38) = 24.21; p < 0.0001; genotype × drug: F(1,38) = 1.11; p = 0.30; three-way RM ANOVA). E, The number of spikes fired in gbz was also similar between genotypes (main effect of genotype: F(1,19) = 0.01; p = 0.90, main effect of stim intensity: F(1.57,29.83) = 6.96; p < 0.01, interaction: F(8,152) = 0.69; p = 0.70; two-way RM ANOVA).
- Figure 3.
Normal temporal synaptic integration in Scn1a+/− neurons. A, Example PSPs recorded in CA1 pyramidal cells from Scn1a+/+ (top, gray) and Scn1a+/− (bottom, blue) mice in response to theta burst stimulation (hash marks below) of SC axons at 1×, 3×, and 5× the minimal stim intensity with no synaptic blockers. B, No change in amplitude of PSPs, measured as area under the curve, between genotypes [main effect of genotype: F(1,19) < 0.41; p > 0.53; n = (cells/mice)]. C, No difference in number of action potentials fired in response to theta stimulation between genotypes (main effect of genotype: F(1,19) = 0.06; p = 0.81, main effect of stim intensity: F(1.04,19.82) = 4.40; p = 0.048, interaction: F(2,38) = 0.19; p = 0.83; two-way RM ANOVA).
- Figure 4.
Reduced feedforward inhibitory input to Scn1a+/− CA1 pyramidal neurons. A, Experimental design. We performed whole-cell voltage-clamp recordings of CA1 pyramidal cells. SC axons were stimulated upstream from the CA1 pyramidal neuron, which directly release glutamate onto the CA1 pyramidal neuron to generate an EPSC, and onto inhibitory interneurons that then provide GABAergic IPSCs to the pyramidal cells. B, Example traces of EPSCs (recorded at Egaba = −70 mV) and IPSCs (recorded at Eglutamate = 0 mV) in WT (left, gray) and Scn1a+/− (right, blue) neurons evoked at the minimal stimulation intensity required to evoke an EPSC (1×), 3×, and 5× the minimal intensity. C, Example traces of IPSCs recorded in ACSF (black), in the presence of glutamate blockers (10 μm NBQX, 50 μm D-AP5, red) and in the presence of glutamate blockers + gabazine (10 μm, gray), demonstrating that the majority of the GABAergic input using this paradigm was feedforward inhibition. D, 79.4% in WTs; 86.7% in Scn1a+/−; p = 0.38, t test. E1, Peak amplitude of ESPCs by stimulation intensity is similar between genotypes [main effect of genotype: F(1,16) = 0.03; p = 0.87, main effect of stimulation intensity: F(1.158,18.53) = 17.45; p < 0.001, interaction: F(8,128) = 0.13; p = 0.99; two-way RM ANOVA n = (cells/mice)]. E2, The CV measured using 50 stimulations at 2.5× the minimal stim intensity (p = 0.33; n = 11 WT and n = 11 Scn1a+/−) and (E3) and PPR of EPSCs (p = 0.11; n = 24 WT and n = 31 Scn1a+/; t test) are not different between WT and Scn1a+/− neurons. F1, The IPSC growth curve is reduced in Scn1a+/− neurons compared with WT (main effect of genotype: F(1,16) = 3.03; p = 0.10, main effect of stimulation intensity: F(1.158,18.53) = 39.11; p < 0.001, interaction: F(8,128) = 4.10; p < 0.001). F2, The CV of IPSCs is increased in Scn1a+/− neurons (*p = 0.02; n = 11 WT and n = 11 Scn1a+/−), and (F3) PPR is increased (*p = 0.02; n = 26 WT and n = 31 Scn1a+/−). G, Excitatory to inhibitory ratio, measured as the [area of the EPSC/(area of EPSC + area of IPSC)] was similar in WT and Scn1a+/− neurons across stim intensities (main effect of genotype: F(1,16) = 1.29; p = 0.22, stimulation intensity: F(1.158,18.53) = 0.83; p = 0.44, interaction: F(8,128) = 0.11; p = 0.99; two-way RM ANOVA).
- Figure 5.
Reduced miniature IPSC frequency to Scn1a+/− CA1 pyramidal neurons. A, Example raw traces of mEPSCs, recorded in gabazine (10 μm) and 1 μm TTX (left) and averages (right). B, Example mIPSCs recorded in glutamate blockers (10 μm NBQX, 50 μm D-AP5). C, The frequency of mEPSCs in unchanged, but mIPSCs are reduced in Scn1a+/− neurons (***p < 0.001; t test; N = 10 cells from 4 mice WT; n = 13 cells from 6 mice Scn1a+/−). D, The lack of change in mEPSC frequency between WT and Scn1a+/− neurons is demonstrated in the similar cumulative frequency of interevent intervals plot. E, The cumulative frequency plot of mIPSCs is shifted to the right for Scn1a+/− neurons (K-S test). The amplitude (F) and kinetics (G, H) of mEPSCs and mIPSCs are similar between genotypes (p > 0.05; t test).
- Figure 6.
Direct activation of inhibitory interneurons reveals normal amplitude but more facilitating GABAergic responses in CA1 pyramidal neurons. A, Experimental design. We stimulated near the recorded CA1 pyramidal cell (PC) in the presence of glutamatergic blockers (10 μm NBQX, 50 μm D-AP5) to directly activate local GABAergic interneurons (IN) and axons. B, Example traces of IPSCs from WT (left, gray) and Scn1a+/− (right, blue) neurons evoked at the minimal stimulation intensity required to evoke an EPSC (1×), 3×, and 5× the minimal intensity (top). Bottom, Overlaid paired-pulse stimulation (50-ms isi) traces recorded at 2.5× the minimal stim intensity, normalized to the peak of the first stimulation. C, IPSC growth functions showed no deficits in Scn1a+/− neurons (main effect of genotype: F(1,20) = 0.03; p = 0.86, main effect of stim intensity: F(1.63,32.60) = 15.09; p < 0.0001, interaction: F(8,160) = 1.05; p = 0.40; two-way RM ANOVA). D, No difference in CV of IPSCs measured using 50 stimulations at 2.5× the minimal stim intensity (p = 0.25, t test). E, IPSC PPR is increased in Scn1a+/− neurons (*p = 0.03; t test). F, No difference in IPSC kinetics between genotypes (rise: p = 0.15; decay: p = 0.12; Mann–Whitney tests).
- Figure 7.
IPSPs are less depressing in Scn1a+/− animals and provide increased inhibition at higher frequency stimulation. A, Example traces of IPSPs in response to theta-burst stimulation from WT (top, gray) and Het (bottom, blue) neurons. B, Overlaid traces of the first burst from the theta burst paradigm demonstrating that responses in WT neurons were much more depressing than those from Scn1a+/− neurons. Stim intensity was normalized to generate the same amplitude (∼2 mV) for the first IPSP across cells. C, IPSP maximum hyperpolarization for each stimulation in the theta burst paradigm in increased in Scn1a+/− neurons [main effect of genotype: F(1,14) = 6.00; *p = 0.03, main effect stim number: F(1.60,22.38) = 18.51; p < 0.0001, interaction: F(24,336) = 3.00; p < 0.0001; two-way RM ANOVA; n = (cells/mice)]. D, PPR of IPSPs (amplitude of second IPSP/first IPSP) across a range of stim intensities showing that IPSPs are less depressing in Scn1a+/− neurons at higher frequencies (main effect of genotype: F(1,9) = 1.18; p = 0.31; main effect frequency: F(1.69,15.17) = 2.03; p = 0.17, interaction: F(4,36) = 2.29; p < 0.08; two-way RM ANOVA; **p < 0.01 Sidak’s multiple comparisons test). E, IPSP peak hyperpolarization in response to 20 stimulations at 10 Hz (main effect of genotype: F(1,280) = 8.86; **p = 0.003, stim number: F(19,280) = 0.02; p > 0.99, interaction: F(19,280) = 0.01; p > 0.99; two-way RM ANOVA), (F) 50 Hz (main effect of genotype: F(1,280) = 0.41; p = 0.52, stim number: F(19,280) = 0.18; p > 0.99, interaction: F(19,280) = 0.02; p > 0.99), and (G) 100 Hz (main effect of genotype: F(1,280) = 7.49; **p = 0.006, stim number: F(19,280) = 0.58; p = 0.92, interaction: F(19,280) = 0.03; p > 0.99).
Tables
WT (n = 7 cells from 5 mice) Het (n = 10 cells from 7 mice) p value (unpaired t test) Resting membrane potential (mV) −60.59 ± 1.83 −58.40 ± 1.61 0.385 Input resistance (MΩ) 136.60 ± 16.90 118.00 ± 10.34 0.335 Membrane time constant (ms) 17.19 ± 2.12 12.64 ± 6.06 0.138 Capacitance (pF) 132.00 ± 13.6 112.10 ± 18.00 0.428 Voltage sag (mV) 5.81 ± 0.52 6.72 ± 0.44 0.190 Rheobase (pA) 153.60 ± 24.7 140.00 ± 13.50 0.611 Voltage threshold (mV) −39.22 ± 1.70 −38.25 ± 1.05 0.617 1st spike rise time (μs) 269.40 ± 21.21 255.50 ± 22.68 0.674 1st spike half width (ms) 1.49 ± 0.12 1.36 ± 0.09 0.375
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