In vivo ketogenic diet treatment attenuates pathologic sharp waves and high frequency oscillations in in vitro hippocampal slices from epileptic Kv 1.1α knockout mice

Abstract

The ketogenic diet (KD) is an effective therapy for pediatric refractory epilepsies; however, whether the KD changes the pathologic network oscillations generated by an epileptic brain remains unknown. We have reported that hippocampal CA3 regions of epileptic Kv1.1α knockout (KO) mice generate pathologic sharp waves (SPWs) and high-frequency oscillations (HFOs) that have higher incidence, longer duration, and fast ripples compared to wild-type (WT). Synaptic activity of hyperexcitable KO mossy fibers significantly decreased CA3 principal cell spike-timing reliability, which contributed to this network pathology. In addition, we have demonstrated that the KD reduces seizures by 75% in KO mice. Here, we determined whether 10- to 14-day in vivo KD treatment exerts disease-modifying effects that alter the spontaneous SPW-HFO complexes generated by the hippocampal CA3 region of KO mice in vitro using extracellular multielectrode array recordings. We found that KD treatment significantly attenuated the pathologic features of KO SPWs and ripples and reduced the incidence of fast ripples. The KD also improved spike-timing reliability of KO CA3 principal cells, decreased mossy fiber excitability, increased mossy fiber-CA3 paired-pulse ratios, and reduced coupling of field excitatory postsynaptic potentials and population spikes in the CA3 region. Collectively, these data indicate that KD treatment modulates CA3-generated pathologic oscillations by dampening hyperactive mossy fiber synapses.

Keywords: Epilepsy; Fast ripple; High-frequency oscillations; Ketogenic diet; Kv1.1; Sharp wave.

Figure 1. The ketogenic diet diminishes pathologic features of SPW-HFO generated in KO hippocampal slices

A. Examples of SPW-HFOs recorded in the CA3 stratum radiatum region of hippocampal slices from WT, KO and KD treat KO (KO-KD) mice. Top trace: raw signal. Second trace: raw signal band pass filtered between 100-175 Hz. Third trace: raw signal band pass filtered between 200-600 Hz. Bottom trace: Time-frequency map of raw signal band pass filtered between 100-600 Hz. B. SPW intervals and durations are increased by KD treatment [n = 6 (WT), 11 (KO), 11 (KO-KD) slices]. Significance determined by an one-way ANOVA followed by Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001. C. KD treatment increases ripple interval and frequency, whereas duration is decreased [n = 584/6 (WT), 3971/11 (KO), 1311/11 (KO-KD) events/slices]. Significance determined by an one-way ANOVA followed by Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001. D. Fast ripple intervals are increased and durations are decreased by KD treatment, but internal fast ripple frequencies are unaffected [n = 6130/11 (KO), 1677/11 (KO-KD) events/slices]. Significance determined by an unpaired Student’s t-test, ***p<0.001.

Figure 2. Ketogenic diet treatment improves spike timing and neurotransmission

A. Example traces of doublet spikes and illustration of spike timing of ten subsequent doublets from the same neurons. B. Quantification of doublet CA3 principal cell inter-spike intervals (ISI) and spike jitter indicating KO cells have increased jitter when similar spikes are compared between groups. [n = 20 (WT), 19 KO, 18 (KO-KD) cells]. Significance determined by an one-way ANOVA followed by Tukey’s multiple comparison test, *p<0.05. C. ISIs and jitter of single units firing during SPWs are increased in KO and rescued with KD-treatment. [n = 20 (WT), 17 KO, 16 (KO-KD) cells]. Significance determined by an one-way ANOVA followed by Tukey’s multiple comparison test, **p<0.01, ***p<0.001.D. KD-treatment has no effect on KO field potential slopes of dendritic CA3 responses to mossy fiber stimulation. Fit with a Boltzmann equation. Significance determined with a two-way ANOVA for genotype/treatment and stimulation intensity (n = 6 slices). Genotype/treatment [F(2,270)=76.74, p<0.001) and stimulation intensity [F(17,270)=31.07, p<0.001] had significant effects and interaction [F(34,270)=1.904, p<0.01]. The two-way ANOVA was followed by Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001 (WT vs. KO); ⁺p<0.05, ⁺⁺p<0.01,⁺⁺⁺p<0.001 (WT vs. KO-KD). E. KD-treatment significantly increases the half maximal stimulation intensities (V₅₀) of the normalized I/O curves of field potential slopes. Fit with a Boltzmann equation. Significance for V₅₀’s determined with a one-way ANOVA followed by Tukey’s multiple comparison test, *p<0.05, ***p<0.001 (n = 6 slices). F. KD restores mossy fiber-CA3 paired pulse facilitation. Significance determined by a two-way ANOVA for genotype/treatment and stimulation interval (n = 6 slices). Genotype/treatment [F(2,54)=5.823, p<0.05) and stimulation interval [F(5,54)=38.83, p<0.001] had significant effects. The two-way ANOVA was followed by Tukey’s multiple comparison test, *p<0.05 (KO vs. KO-KD), ***p<0.001 (WT vs. KO). G. KD-treatment has no effect on KO fiber volley amplitudes of mossy fibers. Fit with a Boltzmann equation. Significance determined with a two-way ANOVA for genotype/treatment and stimulation intensity (n = 6 slices). Genotype/treatment [F(2,306)=110.6, p<0.001) and stimulation intensity [F(17,306)=27.84, p<0.001] had significant effects and interaction [F(34,306)=1.62, p<0.05]. The two-way ANOVA was followed by Tukey’s multiple comparison test: *p<0.05, **p<0.01, ***p<0.001 (WT vs. KO); ⁺⁺p<0.01,⁺⁺⁺p<0.001 (WT vs. KO-KD). H. KD-treatment significantly increases the half maximal stimulation intensities (V₅₀) of the normalized I/O curves of fiber volley amplitudes. Fit with a Boltzmann equation. Significance for V₅₀’s determined with a one-way ANOVA followed by Tukey’s multiple comparison test, *p<0.05, ***p<0.001 (n = 6 slices). I. KD-treatment has an intermediate effect on CA3 field EPSP slopes and population spike slopes compared to KO and WT. The net effect produces a shift in the extracellular field EPSP-population spike relationship to the right with a slope similar to WT indicating a decrease in the excitability of CA3 neurons (n = 6 slices).