Dantrolene

Normal aCSF was used in most cases for in vitro electrophysiological recordings. Ca²⁺-free solution was made by removing Ca²⁺ chloride from the recording solution and replacing it with an equimolar concentration of magnesium chloride. Low-Na⁺ solution was made by substituting equimolar concentrations of Na⁺ chloride by choline chloride. All solutions were oxygenated with 95% O₂/5% CO₂. All salt compounds, TEA (#T2265); TPPO (#T84603), L.A. (#L1376), BAPTA (#A9801); Caffeine (#C0750), NMA (#M2137), and 5-HT (#S2805) were obtained from Sigma-Aldrich. TTX (#1078), Chelerythrin (#1330), U73122 (#1268), Xestospongin-C (#1280), 9-Phenanthrol (#4999), Dantrolene (#0507), and Thapsigargin (#1138) were obtained from Tocris Bioscience. Dantrolene, Thapsigargin, Xestospongin-C, U73122, and 9-Phenantrol were dissolved in dimethylsulfoxide (DMSO) and added to the aCSF (final concentration of DMSO: 0.05–0.1%). L.A. was dissolved in ethanol and added to aCSF (final concentration of ethanol: 0.05–0.1%). Control experiments showed no effects of the vehicle. The other drugs were dissolved in water and added to the aCSF.

PD was purchased from ChemFaces (Wuhan, Hubei, China). Dantrolene, bafilomycin A1, dl-TBOA, CGP37157, PD98059, GF109203X, H89, ω-conotoxin GVIA and ω-agatoxin IVA were obtained from Tocris Cookson (Bristol, UK). 3′,3′,3′-Dipropylthiadicarbocyanine iodide (DiSC3(5)) and Fura-2-AM were purchased from Invitrogen (Carlsbad, CA, USA). EGTA). Nicotinamide adenine dinucleotide phosphate (NADP⁺), glutamate dehydrogenase (GDH), sodium dodecyl sulfate (SDS) and all other chemical reagents were supplied by Sigma-Aldrich Co. (St. Louis, MO, USA).
Sucrose buffer: 320 mM sucrose, 10 mM HEPES, pH 7.4; HEPES buffer medium (HBM): 10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM NaHCO₃, 1.2 mM NaH₂PO₄ and 10 mM glucose and, pH 7.4. Radioimmunoprecipitation assay buffer (RIPA): 50 mM NaCl, 1.0% IGEPAL^® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0 (Sigma-Aldrich Co., St. Louis, MO, USA). Tris-buffered saline (TBS-T): 20 mM Tris, 150 mM NaCl, 0.1% (w/v) Tween^® 20 detergent.

Worms were treated with various doses of dantrolene (Tocris Bioscience, Inc.) as described previously [35 (link)]. dantrolene was dissolved in DMSO and was both spread on unseeded NGM plates and added to the suspension of OP50 (the E. coli strain seeded onto the NGM plates). One day after seeding the NGM plates with the drugged OP50, L4 hermaphrodites were placed onto the dantrolene plates and raised at room temperature (20–22°C). Twenty-four to 36 hrs later, L1 larvae (F1 progeny) hatched within 1 hr were examined by both DIC and fluorescence imaging. dantrolene is likely affecting embryos through entry into the germline of adult hermaphrodites and subsequently being incorporated in the embryos. The 0 μM dantrolene control sample represent worms from NGM plates and seeded OP50 that were treated with DMSO, the solvant for dantrolene. The thapsigargin (Sigma-Aldrich, Inc.) treatment protocol is similar to that of dantrolene treatment, except that the worms were raised at 25°C instead of room temperature (20–22°C).

DL-TBOA, bafilomycin A1, dantrolene, CGP37157, KN62, H89, and GF109203X were purchased from Tocris (Bristol, UK). 3,3,3-Dipropylthiadicarbocyanine iodide [DiSC₃(5)] and fura-2-acetoxymethyl ester (Fura-2-AM) were purchased from Thermo (Waltham, MA, USA).ω-CgTX GVIA and ω-Aga IVA were purchased from Alomone lab (Jerusalem, Israel). 4-AP, dimethylsulfoxide (DMSO), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

KB-R7943 (Tocris Bioscience # 1244) was suspended in DMSO and applied to the axonal compartment at a final concentration of 10 μM for 1 h during axotomy (including 15 min pre-incubation before axotomy). Tetrodotoxin citrate (TTX; Tocris Bioscience #1078) was suspended in HBS and applied to the axonal compartment at a final concentration of 1 μM for 1 h during axotomy (beginning 15 min prior to axotomy). Veratridine (Tocris Bioscience #2918) was suspended in DMSO and applied to the axonal compartment at a final concentration of 10 μM for 10 min in the absence of axotomy/injury. Local ABS, which includes low-Ca²⁺, high-Mg²⁺, and TTX (0.5 mM CaCl₂, 10 mM MgCl₂, 1 μM TTX) was applied solely to the axonal compartment for 1 h during axotomy (15 min prior and 45 min after axotomy). Dantrolene (Tocris Bioscience #0507) and (-)-Xestospongin C (Tocris Bioscience #1280), stock concentrations prepared in DMSO, were diluted in HBS or ABS solution and added to axonal compartment at a final concentration of 20 and 1 μM respectively for 1 h during axotomy (with 15 min pre-treatment and 45 min treatment post-axotomy). DMSO or HBS was used as vehicles. Media stored from the axonal compartment prior to treatment was added back to the axonal compartment after treatment and washes with pre-warmed fresh neuron culture media.