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Supplementary data
Table e-1. Sampling parameters used to estimate the hippocampus cell population.
Stereological Parameter / CA1 / CA2/3 / DENTATE GYRUSCounting frame (X x Y) (µm) / 30 x 30 / 30 x 30 / 30 x 30
Counting frame area (µm2) / 900 / 900 / 900
Sampling grid (X x Y) (µm) / 120 x 220 / 120 x 220 / 120 x 240
Sampling grid area (µm2) / 26400 / 26400 / 28800
Mean section thickness / 8 + 0.13 / 8 + 0.05 / 8 + 0.05
Section sampling frequency / 1:10 / 1:10 / 1:10
Average number of sections sampled / 10 + 1 / 10 + 1 / 10 + 1
Co-efficient of error / 0.04 / 0.04 / 0.04
Sampling parameters used for population estimates for pyramidal cells in the CA1, CA2/3 fields and granule cell layer in the dentate gyrus. Mean section thickness + SEM.
Figure e-1. Assessment of core body temperature and increased susceptibility to thermal-induced tonic-clonic seizures in R43Q mice.
The core temperature of R43Q (light grey line) and control animals was measured using a rectal probe. During 12 minutes in the heating chamber, animals in both groups showed a similar rate of core temperature increase to a final level of 41-42°C (A). A survival curve showed R43Q(black circles) had a significantly reduced time to thermal-induced tonic-clonic seizure compared to control mice (B); (n=12 R43Q; n=12control; Mantel-Cox test; p=0.0065).
Figure e-2. Comparison of electroencephalographic (ECoG) recordings from control and mutant mice in a C57Bl/6J genetic background.
No pathological spike-wave discharges were detected in ECoG recordings (example recording shown) from control and R43Q mutant animals. Control mice (n=4) were recorded for total of 2 hours. R43Q (n=5) were recorded for total of 6 hours (2 hour each x 3 days). All recordings exclude 30 minutes of habituation period.
Table e-2. Population estimates using NeuN versus Nissl labeling methods.
Animal ID / Marker / Estimated Neurons (103) / Marker / Estimated Neurons (103)BM57 / NeuN / 151 / Nissl / 151
BM58 / NeuN / 159 / Nissl / 152
BM59 / NeuN / 113 / Nissl / 113
Mean / 141+ 14 / Mean / 139+ 13
A comparison of the population estimates for pyramidal neurons in the CA1 region labeled using the neuronal marker NeuN and Nissl stained sections from the same animal. Estimates obtained for each cell marker and repeated for 3 mice were not significantly different (p=0.89; unpaired student t test). All final analysis included in the study were from Nissl stained sections. Mean + SEM).
Table e-3. Validation of the ratio of section sampling.
Sample Ratio / Genotype / Density (cells/mm3) / % Increase1:10 / control / 321389 / -
1:10 / R43Q / 446433 / 37
1:6 / control / 506294 / -
1:6 / R43Q / 693505 / 37
Population estimates for granule cells counted using Nissl stained sections were sampled using 1 in every 10th section then re-sampled at a higher ratio (1 in every 6th section) for comparison. An increase in granule cell density was found for the R43Q compared to the control mice using both sampling ratios.
Figure e-3. Comparison of granule cell density for mice with a pure C57Bl/6J background and control mice.
Granule cell estimates were obtained from an independent strain of C57Bl/6J mice and compared to control mice used in the study. Control mice are from crossing >15 generations to C57Bl/6J mice, the granule cell population estimates were similar for each strain (p= 0.69; n=10 C57Bl/6J and n=10 controlmice). Error bars SEM.