9 phenanthrol

All chemicals were dissolved in extracellular solution at the final concentration. For drug application, the bath solution was exchanged twice with the drug-containing solution using a syringe, resulting in a final exchange of the bath solution by about 90–95%. For prolonged drug application (>15 min) the bath solution was exchanged with a drug-containing solution several times. The following agents were used: CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), D-APV (D-(2R)-amino-5-phosphonovaleric acid), riluzole (2-amino-6-(trifluoromethoxy)benzothiazole), flufenamic acid (FFA), 9-phenanthrol, gadolinium chloride, clemizole hydrochloride and ML204 (4-Methyl-2-(1piperidinyl)-quinoline; all Sigma); TTA-P2 (3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydropyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide): Alomone Labs; gabazine (2-(3-Carboxypropyl)-3-amino-6-(4methoxyphenyl)pyridazinium bromide: Abcam).

Patch pipettes were pulled from borosilicate glass capillaries with filament (1.5 mm outer diameter and 1.1 mm inner diameter; Sutter Instruments, BF150-110-7.5HP) with a resistance of 2–3 MΩ. The pipette recording solution contained (in mM) 10 KCl, 130 K-gluconate, 1.8 NaCl, 0.2 EGTA, 10 HEPES, 2 Na-ATP, 0.2% Biocytin, pH 7.3 adjusted with KOH; 290–300 mOsm. Whole-cell recordings were made with Axopatch 700B amplifier (with Clampex 10.7 and Axoclamp1.1, Molecular Devices) using an upright microscope (Nikon Eclipse FN1, with × 40, 0.8 NA water immersion objective lens) equipped with differential interference contrast (DIC) optics. DIC images were captured with an Andor Zyla 5.5 sCMOS camera. All recordings were performed at 32 °C in ACSF bubbled with 95% O₂ and 5% CO₂. For AP parameter determination experiments, 1 μM CNQX (Sigma-Aldrich, C-127) was applied in the bath solution to eliminate EPSPs. Cells with < 20 MΩ access resistance (continuously monitored) were accepted for analysis. Signals were low-pass filtered at 5 kHz and digitized at 20 kHz (Digidata 1550B, Molecular Devices). In vitro data analysis was performed with the help of Clampfit 10.7 (Molecular Devices) and Origin 8.6 (OriginLab Corporation). When it is indicated 30 μM 9-phenanthrol (Sigma-Aldrich, 211281) was applied into the bath solution.

For the activation of Akt, epidermal growth factor recombinant protein (R&D Systems, Minneapolis, MN, USA, 236‐EG) was added to the growth media (EGF, 100 ng·mL⁻¹/15 min). 9‐Phenanthrol (Sigma‐Aldrich, 211281) at 10 μm final concentration in the growth media for 2 h was used for the inhibition of TRPM4. DMSO was used as a vehicle. Before the experiments, tetracycline (Sigma‐Aldrich, T7660) was added to the growth media for TRPM4 induction at a final concentration of 1 μg·mL⁻¹ for 24 h. The calmodulin inhibitor W‐7 (Tocris Bioscience, Bristol, UK, 0369) was used at a final concentration of 100 μm for 1 h before the incubation with EGF.