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See Rogawski (doi:10.1093/awv369) for a scientific commentary on this article. The MCT ketogenic diet, an established treatment for drug-resistant epilepsy, leads to an elevation of plasma decanoic acid and ketones. Chang et al. show that decanoic acid, rather than ketones, provides anti-seizure activity in several ex vivo rat models of epilepsy, likely through the direct inhibition of AMPA receptors.
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Figure 1. Decanoic acid but not ketones acutely reduce epileptiform activity in two acute ex vivo models. Epileptiform activity was monitored using two ex vivo rat hippocampal slice models, following treatment with decanoic acid (DA, 1 mM) or ketones acetone and β-hydroxybutyrate (BHB) (both at 10 mM) or with solvent-only (Control). (A) Example trace recording of epileptiform activity (burst discharges) in hippocampal slices induced by application of pentylenetetrazol (PTZ, 2 mM, K+ 6 mM), a model for generalized seizures, and following treatment, where (B) the frequency of epileptiform activity is plotted against time, following treatment, and also shown as a comparison of the mean (±SD) frequency of burst discharges averaged from 20 to 40 min post-compound addition. (C) Example trace recording of epileptiform activity (burst discharges) in hippocampal slices induced by low-Mg2+ conditions, as a model for drug-resistant seizures, and following treatment, where (D) the frequency of epileptiform activity is plotted against time, and also shown as a comparison of the mean (±SD) frequency of burst discharges averaged from 20 to 40 min post-compound addition. Significance indicated by ***P < 0.001 compared to control (ANOVA with Tukey post hoc test). All data were normalized to baseline. Data are provided from between n = 4 and 7 repeats.
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Figure 2. The effect of decanoic acid on EPSCs and IPSCs. Currents were recorded from hippocampal CA1 pyramidal cells following exposure to decanoic acid (DA; 300 μM). (A) Representative electrophysiological recordings show reduced evoked EPSC amplitude following application of decanoic acid. The insert provides the average of 10 traces from the same cell indicating the shape of single EPSCs before (1), during (2) and after (3) application of decanoic acid. (B) Representative electrophysiological recordings show no effect of decanoic acid on IPSCs. (C) Summary data showing the effect of decanoic acid on mean normalized EPSCs ± SEM (n = 6). (D) In contrast, decanoic acid had no effect on mean normalized IPSCs ± SEM (n = 6). Decanoic acid did not change 1/CV2 (CV, coefficient of variation) of EPSC (E) or IPSC (F) amplitudes with means ± SEM pre- and post-decanoic acid treatment as indicated, consistent with a post-synaptic locus of action.
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Figure 3. The direct effect of decanoic acid on AMPA receptor-mediated currents. In these experiments, Xenopus oocytes were used to express AMPA (GluA2/A3 or GluA1) receptors, and currents were measured following application of L-glutamate (100 µM) with membrane potential clamped to −50 mV. (A) Representative trace recordings for GluA2/3, showing the effect of medium chain fatty acids decanoic acid (DA), octanoic acid (OA) and VPA (all at 1 mM) and the AMPA receptor inhibitor CNQX (30 μM) on inward currents; and (B) summary of mean normalized currents (±SEM). (C) Representative current traces of inhibitory dose-response curves for octanoic or decanoic acid. (D) Mean inhibitory dose-response curves for octanoic or decanoic acid, graphs show means ± SEM. (E) Representative trace recordings for GluA1, showing the effect of medium chain fatty acids and VPA on inward currents. (F) Summary of mean normalized currents (±SEM). Statistical analysis was performed using ANOVA with Dunnett’s post hoc test. *P < 0.05, ***P < 0.001, compared to control; +++P < 0.001, compared to solvent only (DMSO). Scale bars = 15 nA for octanoic acid and 5 nA for decanoic acid.
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Figure 4. The effect of AMPA receptor antagonism with GYKI 52466 on epileptiform activity. Epileptiform activity induced by application of pentylenetetrazol (2 mM, K+ 6 mM) was monitored following treatment with the selective AMPA receptor antagonist GYKI 52466 at a concentration (50 µM) that results in approximately the same (∼60–70%) decrease in AMPA receptor responses (± SD) as is observed with 1 mM decanoic acid (n = 4).
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Figure 5. The effect of 4-ethyloctanoic acid on AMPA (GluA2/A3) mediated current in Xenopus oocytes induced by L-glutamate. (A) Representative trace recording showing the effect of 4-ethyloctanoic acid at indicated concentrations on AMPA (GluA2/A3) current following application of glutamate (100 µM). (B) Inhibitory dose-response curves of 4-ethyloctanoic acid (n = 4) in the presence of glutamate (100 μM), with data present as means ± SEM. The responses are normalized to the maximal current response induced by application of L-glutamate for each recording.
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Figure 6. Characterization of decanoic acid-dependent AMPA receptor inhibition. In these experiments, Xenopus oocytes were used to express various AMPA receptor combinations, and glutamate-elicited currents were measured in the presence of varying decanoic acid concentrations. (A) Representative current trace showing different AMPA receptor subunit combinations (GluA1; GluA1/2; GluA2/3) following exposure to varying decanoic acid concentrations (0.001, 0.05, 0.3, 0.7, 1 and 3 mM) on current following application of glutamate (at 100 µM). Scale bars = 200 nA for GluA1 and GluA1/2; 20 nA for GluA2/3, and (B) mean inhibitory dose-response curves of decanoic acid from different AMPA receptor combinations (inset provides IC50 values) (n = 12). (C) Representative traces from electrophysiological recordings showing the effect of glutamate (0.001, 0.003, 0.01, 0.03, 0.1, 0.3 mM) on GluA2/3 currents in the presence of decanoic acid (at 0.3 mM or 1 mM). Scale bar = 5nA (no decanoic acid); 2 nA (0.3 mM decanoic acid); and 4 nA (1.0 mM decanoic acid). (D) Quantitative evaluation of decanoic acid potency against GluA2/3 currents showing mean glutamate concentration-response curves (n = 5 for each treatment) where responses are normalized to the maximal current for each recording in the absence of decanoic acid. (E) Representative current trace of the inhibitory dose–response curves for decanoic acid in the presence of glutamate (100 µM; n = 20 and 1 mM; n = 6) for GluA2/3 receptors and (F) mean inhibitory dose–response curves for decanoic acid against GluA2/3 in the presence of 100 µM or 1 mM glutamate show little change, suggesting a non-competitive inhibition of AMPA mediated current with respect to glutamate. (G) Representative current traces of the voltage dependence of decanoic acid inhibitory activity, where inhibition curves for decanoic acid in the present of glutamate (100 µM) for GluA2/3 receptors with voltage clamped to −80 and −40 mV. Scale bars = 200 nA and 20 nA, respectively. (H) Mean inhibitory dose-response curves at −80 and −40 mV (inset provides IC50 values) (n = 6).
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Figure 7. Decanoic acid binding of AMPA receptors. A molecular modelling approach was taken to investigate binding sites of decanoic acid on AMPA receptors using the active protein configuration (3KG2) of GluA2 and modelling residues within 6 Å of the ligand. (A) Decanoic acid is predicted to bind within the channel region of the receptor (magenta) on the M3 helices (overall structure shown in inset). Using this modelling approach, the known AMPA receptor agonist, perampanel, is predicted to bind to the linker region between the S1 glutamate binding domain and the channel pore (blue). (B) View from the intracellular side down the axis of the ion channel pore. Space-filled molecule is decanoic acid (magenta carbons and two red carboxylic acid oxygen molecules) binding at Pro584 (green) of one subunit with the equivalent binding of decanoic acid at the other three subunits omitted for clarity.
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See Rogawski (doi:10.1093/awv369) for a scientific commentary on this article. The MCT ketogenic diet, an established treatment for drug-resistant epilepsy, leads to an elevation of plasma decanoic acid and ketones. Chang et al. show that decanoic acid, rather than ketones, provides anti-seizure activity in several ex vivo rat models of epilepsy, likely through the direct inhibition of AMPA receptors.
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