Altered Fast Synaptic Transmission in a Mouse Model of DNM1-Associated Developmental Epileptic Encephalopathy

Synapse number is reduced by Dnm1^Ftfl/Ftfl, and accounts for reduced mPSC frequency. A, Images of biocytin-filled neurons (magenta) immunostained for vGlut1 (cyan), and vGAT (yellow). The top row shows representative excitatory neurons, and the bottom row shows representative inhibitory neurons. The left column shows representative WT neurons, and the right column shows representative Fitful neurons. Scale bars: 20 μm in length. B1–B4, Model effects tested using GEEs. C, Scatterplot showing excitatory synapse count (x-axis) and inhibitory synapse count (y-axis) onto excitatory neurons. Each dot represents an individual WT (black, N = 2, n = 11) or Fitful (chartreuse, N = 2, n = 15) neuron. Marginal boxplots define the median, IQR, and range along their respective axes. D, Plots showing synapse density (synapses per unit area) for the E-E (top) and I-E (bottom) motifs. WT swarm and box plots are in black and white, while Fitful swarm and box plots are colored for their respective motifs. Boxplots define the median, IQR, and range. E, Scatterplot showing excitatory synapse count (x-axis) and inhibitory synapse count (y-axis) onto inhibitory neurons. Each dot represents an individual WT (black, N = 2, n = 12) or Fitful (purple, N = 2, n = 13) neuron. Marginal boxplots define the median, IQR, and range along their respective axes. F, Plots showing synapse density (synapses per unit area) for the E-E (top) and I-E (bottom) motifs. WT swarm and box plots are in black and white, while Fitful swarm and box plots are colored for their respective motifs. Boxplots define the median, IQR, and range. G, Barplots showing the fraction of synapses located on the soma. Bars end at the mean, and error bars show the 95% confidence interval derived from bootstrap resampling. Dots represent individual neurons. H, Plots showing mPSC frequency normalized to synapse number for WT (white) and Fitful (colored) neurons; *pairwise comparison p < 0.05. For complete pairwise comparisons data, see Extended Data Figure 2-1.

Dnm1^Ftfl/Ftfl reduces ePSC amplitude at the I-E motif. A, Example ePSCs. B, Model effects assessed using GEEs. C, Effect sizes at each of the four motifs. Central marker and error bars represent mean effect size ± 95% CI calculated using hierarchical bootstrap resampling. D, Average evoked excitatory PSCs. Swarm plots show individual synapses, and central dots and error bars represent mean ± SD (E-E: N = 7, WT n = 22, Ftfl n = 15; E-I: N = 6, WT n = 26, Ftfl n = 23). E, Average evoked inhibitory PSCs. Swarm plots show individual synapses, and central dots and error bars represent mean ± SD (I-E: N = 7, WT n = 30, Ftfl n = 24; I-I: N = 5, WT n = 22, Ftfl n = 29); *pairwise comparison p < 0.05. For complete pairwise comparisons data, see Extended Data Figure 3-1.

Reduced eIPSC size at the I-E motif is because of reduced RRP size, and recovery from stimulation is impaired. A, Representative tetanic stimulation protocols from WT (black) and Fitful (orange) synapses. B, Example cumulative charge plot; x-axis shows stimulus number, and y-axis shows the cumulative charge evoked by that stimulus. Black line and shaded region represent mean ± SEM cumulative charge for WT synapses, and orange line and shaded region represents mean ± SEM cumulative charge for Ftfl synapses. Gray dashed lines are example fits of steady-state synaptic release during tetanus used to calculate RRP size. C, Scatterplot showing charge contained in the first stimulus (x-axis) and charge contained in the RRP (y-axis) of WT (black, N = 5, n = 14) and Fitful (orange, N = 5, n = 12) synapses. Marginal boxplots show median and IQR of their respective axes. Fliers are synapses that exceed 1.5× the IQR. D, Release probability at WT (black) and Fitful (orange) synapses. Dots represent individual synapses, and boxplots show range, median, and IQR. Fliers are synapses that exceed 1.5× the IQR. E, Example paired-pulse ratio IPSCs evoked with a 50-ms delay. Traces are normalized to the peak of the first IPSC. F, Box and swarm plots showing 50-ms paired-pulse ratios. Dots represent individual WT (black) and Fitful (orange) synapses, and boxplots show range, median, and IQR. G, Peak amplitudes of synaptic responses during 100-Hz stimulation for WT (black) and Fitful (orange) synapses, normalized to the peak of the first response. Lines and shaded regions represent mean ± SEM of responses. H, Timeline of synaptic recovery from 100-Hz stimulation for WT (black) and FItful (orange) synapses. Peak amplitudes of each response were normalized to the baseline response of the given neuron. Lines and shaded regions represent mean ± SEM of responses. I, Example IPSCs evoked by 10-Hz stimulation. Traces are normalized to the peak of the first IPSC. J, Peak amplitudes of synaptic responses during 10-Hz stimulation for WT (black) and Fitful (orange) synapses, normalized to the peak of the first response. Lines and shaded regions represent mean ± SEM of responses; *pairwise comparison p < 0.05. For complete pairwise comparisons data, see Extended Data Figures 4-1, 4-2.

Calcium Imaging shows little evidence of increased excitability. A, Example imaging field. Inset, Maximum fluorescence projection of the outlined region showing manually drawn ROIs. B, Example fluorescence traces from WT (black) and Fitful (red) neurons at DIV13. C, Average fluorescence events for Fitful (red, N = 3, n = 6788) and WT (black, N = 3, n = 8850) neurons across developmental epochs. Lines represent median fluorescence across all events, and the shaded region defines the IQR. D, Peak fluorescence boxplots across developmental epochs for WT (white) and Fitful (red) neurons. Boxplots define the range, median, and IQR for all observed neurons. Fliers represent neurons falling outside 3× of the IQR. E, Boxplots displaying the 80/20 fluorescence decay time for each cell in WT (white) and Fitful (red) cultures. Boxplots define the range, median, and IQR for all observed neurons. Fliers represent neurons falling outside 3× of the IQR. F, Boxplots displaying the number of events observed for each cell in WT (white) and Fitful (red) cultures. Boxplots define the range, median, and IQR for all observed neurons. Fliers represent neurons falling outside 3× of the IQR. G–I, Plots showing the effect size of the Fitful variant at each of the developmental epochs on peak change in flourescence (G), fluorescence decay (H), and events per cell (I). Central marker and error bars represent mean effect size ± 95% CI calculated using hierarchical bootstrap resampling; *pairwise comparison p < 0.05. For complete model effects and pairwise comparisons data, see Extended Data Figures 5-1, 5-2.

Spontaneous PSCs are larger and less frequent in Dnm1^Ftfl/Ftfl cultures. A, Representative voltage-clamp recordings of sEPSCs from WT (black) and Fitful (teal) neurons. B, Representative voltage-clamp recordings of sIPSCs from WT (black) and Fitful (orange) neurons. C, Box and swarm plots showing sEPSC frequency (top) and sEPSC charge (bottom) for WT (black, N = 3, n = 38) and Fitful (teal, N = 3, n = 38) neurons. Dots represent individual neurons. Boxplots show range, median, and IQR, with fliers representing neurons beyond 1.5× the IQR. D, Box and swarm plots showing sIPSC frequency (top) and sIPSC charge (bottom) for WT (black, N = 3, n = 53) and Fitful (orange, N = 3, n = 47) neurons. Dots represent individual neurons. Boxplots show range, median, and IQR, with fliers representing neurons beyond 1.5× the IQR. E, Model effects tested using GEEs; *pairwise comparison p < 0.05. For complete pairwise comparisons data, see Extended Data Figure 6-1.

Dnm1^Ftfl/Ftfl alters the frequency and timing of spontaneous APs. A, Example traces of the current response during cell-attached voltage clamp recordings of excitatory neurons from WT (black) and Dnm1^Ftfl (green) cultures. B1, Plot of the mean firing rate (MFR) of WT and Dnm1^Ftfl excitatory neurons. B2, Plot of the median ISI from each group. B3, Plot of the coefficient of variation (CV) of the ISI. C, A relative frequency histogram of all ISIs (log scale) collected from WT (left) and Dnm1^Ftfl (right) excitatory neurons. D, Example traces of the current response during cell-attached voltage clamp recordings of inhibitory neurons from WT (black) and Dnm1^Ftfl (orange) cultures. E1, Plot of the MFR of WT and Dnm1^Ftfl excitatory neurons. E2, Plot of the median ISI from each group. E3, Plot of the CV of the ISI. F, Relative frequency histogram of all ISIs (log scale) collected from WT (left) and Dnm1^Ftfl (right) inhibitory neurons. For all plots, each dot represents the mean or median value from one neuron; *pairwise comparison p < 0.05; **pairwise comparison p < 0.01, ***pairwise comparison p < 0.001. For complete pairwise comparisons data, see Extended Data Figure 7-1.

Bic-induced SLEs are shorter in Dnm1^Ftfl/Ftfl cultures. A, Representative Bic-induced SLEs from WT (black, N = 3, n = 48) and Fitful (red, N = 3, n = 50) neurons aligned to SLE onset. B, Plots showing latency to SLE onset. Boxplots define the range, median, and IQR of the distribution. Fliers are 1.5× the IQR. C, Plots showing duration of SLEs. Boxplots define the range, median, and IQR of the distribution. D, Plots showing charge evoked by SLEs. Boxplots define the range, median, and IQR of the distribution. Fliers are 1.5× the IQR; *pairwise comparison p < 0.05. For complete pairwise comparisons data, see Extended Data Figure 8-1.

Dnm1^Ftfl/Ftfl causes neuronal degeneration. A, Representative brightfield (left), PI (center), and merged (right) images for WT (top) and Fitful (bottom) cultures at DIV17. B, Representative brightfield (left), PI (center), and merged (right) images for WT (top) and Fitful (bottom) cultures at DIV21. C, Counts of PI-positive nuclei (average counts of two blind observers) for WT (black, DIV17: N = 2, n = 13; DIV21: N = 2, n = 15) and Fitful (red, DIV17: N = 2, n = 13; DIV21: N = 2, n = 15) images. Each dot represents the count for an individual field of view. Markers and error bars represent mean ± 95% CI calculated through bootstrap resampling; *pairwise comparison p < 0.05. For complete model effects and pairwise comparisons data, see Extended Data Figures 9-1, 9-2.