2.2. Preparing acute brain slices

All compounds were purchased from Sigma Aldrich, UK unless stated differently. For acute brain slices, rats were culled with isoflurane overdose and then decapitated. Horizontal brain slices comprising the hippocampus, dentate gyrus, and entorhinal cortex were prepared on the vibratome (Leica VT1200S, Leica Microsystems). All solutions were enriched with 95% O₂/5%CO₂ gas to maintain pH 7.4–7.35 and to provide sufficient tissue oxygenation. Slices were kept in artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 2.5 mM KCl, 0.9 mM NaH₂PO₄, 14 mM D‐glucose, 2.5 mM CaCl₂, 1 mM MgCl_2, and 2.6 mM NaHCO₃: osmolality 296 mOsm/kg. For recordings, slices were transferred to low Mg²⁺ ACSF solution, in which MgCl₂ was omitted and the KCl concentration was increased to 5 mM. Epileptiform activity in the pentylenetetrazole (PTZ) model of epilepsy was elicited by adding 2 mM of PTZ to high potassium (5 mM) ACSF solution. The submersion recording chamber was continuously perfused with solution enriched with 95%O₂/5%CO₂ at ~4 mL/min and temperature was controlled at ~33°C. Low Mg²⁺ or PTZ‐induced epileptiform discharges were recorded from CA1 region of the hippocampus. The recording electrode was filled with the perfusion solution. Recordings were obtained using a Multi‐Clamp 700B or Axopatch 200B amplifier (Molecular Devices), and were low‐pass filtered at 4 kHz. WinEDR (Strathclyde Electrophysiology Software) was used for data acquisition. Sampling rate was 10 kHz. Two TRPM7 channel blockers were used: carvacrol (Sigma Aldrich) and waixenicin A (kindly provided by DH from Hawaii Pacific University). ¹⁰

Carvacrol is an oil and so we initially compared carvacrol (1 mM) dissolved in DMSO (0.26%) prior to adding it to the perfusion solution in seven experiments and in the other seven experiments carvacrol was added without DMSO. In six control experiments DMSO was added as a vehicle to the perfusion solution (0.26%) after 20 min of the baseline recording to control for a potential effect of DMSO on epileptiform activity. There was no significant difference between these two sets of experiments (DMSO and carvacrol [1 mM] and carvacrol [1 mM] alone) and there was no effect of DMSO at this concentration on epileptiform activity in control conditions. Thus, experiments with and without DMSO were pooled for the final analysis. Based on these results lower concentrations of carvacrol (50–800 μM) were added without DMSO.

To block TRPM7 channel 10–20 μM of waixenicin A was used in the low Mg²⁺ experiments, and 10 μM (minimum effective dose) in the PTZ experiments. The specific protocol provided by DH from Hawaii Pacific University was used to dissolve waixenicin A. In brief, 50 μL of 99% methanol was added to the vial containing 50 μg waixenicin A, and then once half of methanol volume evaporated 1035 μL of a buffer (PTZ or low Mg²⁺ containing ACSF) was added to dissolve the compound completely. The final concentration of methanol in the perfusion solution was 0.23% in PTZ and 0.23%–0.46% in low Mg²⁺ experiments: these concentrations of methanol were added in control experiments with PTZ and low Mg²⁺.