Nifedipine

SLIGKV‐NH2 was purchased from Abcam. VKGILS‐NH2, U73122 and nifedipine were purchased from Tocris. BTP2 was obtained from EMD millipore and BAPTA‐AM from Caymen Chemicals. SLIGKV‐NH2 and VKGILS‐NH2 were dissolved in tissue culture grade water to make stock solutions. U73122, nifedipine, BTP2, and BAPTA‐AM were dissolved in DMSO to make stock solutions which were diluted in appropriate buffers before use in experiments.

JWH-018 was purchased from LGC Standards (LGC Standards S.r.L., Sesto San Giovanni, Milan, Italy) while amiodarone, atropine, nifedipine, and propranolol were from Tocris (Tocris, Bristol, UK). Drugs were initially dissolved in absolute ethanol (final concentration was 5%) and Tween 80 (2%) and brought to the final volume with saline (0.9% NaCl). The solution made with ethanol, Tween 80, and saline was also used as the vehicle. Amiodarone (5 mg/kg), atropine (5 mg/kg), nifedipine (1 mg/kg), and propranolol (2 mg/kg) were administered 60 min after JWH-018 injection. Drugs were administered by intraperitoneal injection (i.p.) in a volume of 4 µL/g.
The dose selection was evaluated based on a previous study by our group [21 (link)] that showed severe CV effects induced by 6 mg/kg JWH-018. In line with HED formula and with dosage scale reported by users [126 (link),127 ], this dose represents a toxic dose of JWH-018 in humans. The dosage of amiodarone [128 (link)], atropine [129 (link),130 (link)], nifedipine [131 (link)], and propranolol [132 (link)] were chosen from previous preclinical studies on rodents.

4-aminopyridine (4-AP) and pyruvate were purchased from Sigma (St. Louis, MO, USA). Hexahydro-1-[(5-iodo-1-naphthalenyl) sulfonyl]-1H-1,4-diazepine hydrochloride (ML-7) and (S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152) were purchased from Calbiochem (San Diego, CA, USA). Ethyleneglycol-bis (β-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) was purchased from Dojin (Kumamoto, Japan). Tetrodotoxin (TTX) was purchased from Wako (Osaka, Japan). D-(−)-2-amino-5-phosphonopentanoic acid (APV), bicuculline, (S)-(−)-blebbistatin, (R)-(+)-blebbistatin, thapsigargin and nifedipine were purchased from Tocris (Ellisville, MO, USA).

To increase neuronal activity, a high potassium solution (final concentration, 25 mM) was applied to the culture medium approximately 3 h after starting live imaging. To suppress neuronal firing, TTX (final concentration, 100 nM; Seikagaku-Kogyo) was applied to the medium. To block synaptic transmission, D-AP5 (100 μM) (Tocris) and DNQX (20 μM) (Tocris) were applied prior to imaging. To suppress spontaneous activity, the concentrations of Mg²⁺ and Ca²⁺ were raised to 4 mM (Gustafsson et al., 1987 (link)). Picrotoxin (final concentration, 100 μM; nacalai tesque) was also applied to suppress the effect of inhibitory neurons. To block the increase in intracellular Ca²⁺ concentration, D-AP5 and nifedipine (10 μM) (Tocris) were added to the culture medium prior to imaging.

Experiments were conducted with a Leica TCS SP8 confocal scanning microscope. Cultured neurons on coverslips were removed from the culture dish, placed in an imaging perfusion chamber with low calcium (1 mM), no magnesium ACSF and imaged using a ×63 oil immersion lens (Leica). Cells co-transfected with EGFP–GluN1 and RCaMP (pAAV.Syn.NES-jRCaMP1b.WPRE.SV40)⁴⁴ were imaged for 600 frames (2 min) at 5 frames per second. Low calcium, no magnesium ACSF containing 25 µM CNQX (Tocris, IL), 40 µM Nifedipine (Tocris, IL), and 1 µM Tetrodotoxin (Cayman, MI; ACSF*) was perfused onto the cells for 100 frames (20 s). After 100 frames, ASCF* was switched off and ASCF* plus 10 µM glutamate and 50 µM glycine was switched on for 50 frames (10 s). Glutamate was turned off and ACSF* was then switched back on for the remaining 450 frames (90 s). Each cell was imaged three times and response was averaged. ACSF* containing 100 µM APV (Tocris, IL) was then perfused on and the imaging process described above was repeated in the presence of APV to ensure cell response was NMDAR-specific.