Fig. 6
From: Neuregulin 1/ErbB4 signaling contributes to the anti-epileptic effects of the ketogenic diet
Knockdown of ErbB4 from hippocampus diminishes the anti-seizure effects of KD. a Schematic of virus injection. hSyn promoter-driven Cre virus was injected bilaterally into hippocampal region. b Representative images of mcherry expression in virus-injected mice. Hippocampal sections were collected 3 weeks after stereotaxic microinjection of AAV-hSyn-cre-mcherry virus. Enlarged images of the dotted area were shown on the bottom. Scale bar: 500 µm (top) and 100 µm (bottom). c Reduced ErbB4 expression in hippocampus of ErbB4f/f mice injected with AAV-hSyn-Cre-mcherry virus. Shown were representative blots. ErbB4 band density was normalized with the loading control GAPDH. d Quantitative analysis of the Western blot data in (c). n = 6 mice per group. Student’s t test, t(10) = 8.679, P < 0.0001. e Diagram of hippocampal slice recording. Pyramidal neurons in the CA1 region were clamped in whole-cell configuration. Evoked postsynaptic currents were recorded under stimulation by using a concentric bipolar electrode. f Representative eIPSC traces. Scale bars, 100 ms and 1000 pA. g Knockdown of ErbB4 in the hippocampus attenuates the effect of KD on eIPSC amplitudes. n = 13 neurons from 3 mice of “Control + CD” group; n = 15 neurons from 3 mice of “Control + KD” group; n = 13 neurons from 3 mice of “Cre + CD” group; n = 13 neurons from 3 mice of “Cre + KD” group. Two-way ANOVA, “Control + CD” vs “Control + KD”: F(1,234) = 51.25, P < 0.0001; “Cre + CD” vs “Cre + KD”: F(1,216) = 0.1648, P = 0.6852. h Representative sIPSC traces. Scale bars, 2 s and 50 pA. i Knockdown of ErbB4 in the hippocampus reduces the effect of KD on sIPSC frequency. n = 13 neurons from 3 mice of “Control + CD” group; n = 15 neurons from 3 mice of “Control + KD” group; n = 13 neurons from 3 mice of “Cre + CD” group; n = 13 neurons from 3 mice of “Cre + KD” group. Student’s t test, “Control + CD” vs “Control + KD”: t(26) = 4.979, P < 0.0001; “Cre + CD vs “Cre + KD”: F(24) = 0.2191, P = 0.8284. j Diagram of experimental design. AAV virus was injected into hippocampus 2 weeks prior to KD feeding. Three weeks later, mice were subjected to electrophysiological recordings or KA infusion to induce seizures. k Knockdown of ErbB4 in hippocampus diminishes the effect of KD on the development of seizure. Mice were subject to KA infusion and scored for seizure stage every 5 min. n = 8 mice of “Control + CD” group; n = 9 mice of “Control + KD” group; n = 8 mice of “Cre + CD” group; n = 9 mice of “Cre + KD” group. Repeated two-way ANOVA, “Control + CD vs “Control + KD”: F(1,105) = 6.351, P = 0.0235; “Cre + CD vs “Cre + KD”: F(1,105) = 2.382, P = 0.1435. l The averaged seizure score is decreased by Knockdown of ErbB4 in the hippocampus. n = 8 mice of “Control + CD” group; n = 9 mice of “Control + KD” group; n = 8 mice of “Cre + CD” group; n = 9 mice of “Cre + KD” group. Student’s t test, “Control + CD” vs “Control + KD”: t(15) = 3.873, P = 0.0015; “Control + CD” vs “Cre + CD”: t(14) = 2.522, P = 0.0244; “Cre + CD” vs “Cre + KD”: t(15) = 1.733, P = 0.1035; “Control + KD” vs “Cre + KD”: t(16) = 4.771, P = 0.0002