Dimethyl sulfoxide (dmso)

Chitosan (Mw 320 kDa, degree of deacetylation 88.5%) was purchased from Bioprogress (Shchelkovo, Russia), hyaluronic acid sodium salt (Mw 5 and 30 kDa) was from Shiseido (Tokyo, Japan) and glutaraldehyde was from Merck (Darmstadt, Germany). Genipin, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and lysozyme from chicken egg white (lyophilized powder, 100,000 U/mg) were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), glutamine, sodium pyruvate, streptomycin, penicillin, phosphate buffered solution (PBS, pH 7.4) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were from PanEco (Moscow, Russia). Fetal bovine serum (FBS) was purchased from PAA Laboratories GmbH (Pasching, Austria), 2-mercaptoethanol was from Loba Feinchemie (Fischamend, Austria), dimethyl sulfoxide (DMSO) was from Helicon (Moscow, Russia) and Calcein AM was from eBioscience (San Diego, CA, USA).

All drugs were diluted to a final concentration in Liley solution. CGRP (rat peptide isoform which shares the same amino acid sequence with endogenous mouse peptide) and inhibitor of CGRP‐receptors—rat CGRP_8‐37—were obtained from Bachem (Bubendorf, Switzerland). Ryanodine (an activator of RyR) and an inhibitor of protein kinase A H‐89 were obtained from Enzo Life Sciences (Farmingdale, NY, USA). Inhibitor of vesicular ACh transporter—(±) vesamicol hydrochloride, inhibitors of CaMKII—KN‐62, KN‐93, and negative control for KN‐93—KN‐92—were all from Tocris Bioscience (Ellisville, MO, USA). Stock solutions of all drugs except H‐89 and inhibitors of CaMKII were prepared in deionized water, H‐89, KN‐62, KN‐93, and KN‐92 were dissolved in dimethylsulfoxide (DMSO) (Helicon, Moscow, Russia). The final DMSO concentration did not exceed 0.01% (v/v), and at this concentration, DMSO did not affect the parameters of spontaneous ACh release in mouse motor synapses. As KN‐62, KN‐93, and KN‐92 are reported to be light sensitive, efforts were made to minimize the exposure to light during the experiments involving these drugs.

The fast Ca²⁺ chelator (BAPTA-AM), the slow Ca²⁺ chelator (EGTA-AM) and P2X7 receptor antagonist A740003 were purchased from Tocris/Bio-Techne (Minneapolis, MN, USA) and Calbiochem Biochemicals (Nottingham, Great Britain), respectively. The P2X7 receptor agonist BzATP was purchased from Sigma-Aldrich (St. Louis, MO, USA). The L-type VDCC inhibitor nitrendipine was obtained from Biomol GmbH (Hamburg, Germany). Stock solutions of Ca²⁺ chelators, A740003 and nitrendipine were prepared using DMSO (Helicon, Moscow, Russia) as the solvent. BzATP was dissolved in deionized water. The final concentration of DMSO in the working solution did not exceed 0.01% (v/v) and did not affect spontaneous or evoked activity in mouse NMJs.