Tubocurarine

During this study the following drugs were used: diethylenetriamine/nitric oxide adduct (DETA-NO, 250–500 µM, Sigma), Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 500 µM-1 mM, Sigma), 8-(4-Chlorophenylthio)-guanosine 3′, 5′-cyclic monophosphate sodium salt (8-pCPT-cGMP, 500–750 µM, Sigma), 1 H-[1] (link), [2] (link), [4] (link)Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 500 µM, Ascent Scientific), tetrodotoxin (TTX, 1 µM, Ascent Scientific), (+)-tubocurarine hydrochloride (tubocurarine, 3 µM, Sigma), formamide (2 M, Sigma) and 18-β-glycyrrhetinic acid (18βGA, 100 µM, Sigma).

In gramicidin perforated experiments and in experiments performed for assessing the KCC2 efficacy, GABA_AR responses were isolated by using a cocktail of drugs containing 0.2 μM tetrodotoxin (TTX, Latoxan Laboratory, France), 4 mM kynurenic acid (Millipore Sigma), 10 μM (+)-tubocurarine (Millipore Sigma), 5 μM Dihydro-β-erythroidine hydrobromide (DHβE, Bio-techne, France), and 3 μm strychnine (Millipore Sigma) that respectively blocked voltage-dependent Na⁺ action potentials, and glutamate, cholinergic, and glycinergic input to MNs. In CsCl experiments, IPSCs were isolated pharmacologically using DL-AP5 40 μM ((2R)-amino-5-phosphonovaleric acid, Bio-techne, France) and CNQX 20 μM (6-cyano-7-nitroquinoxaline-2,3-dione, Bio-techne, France). mIPSCs were isolated in the presence of 0.2 μM TTX (Latoxan, France). GABA and glycine mIPSCs were isolated by adding 3 μM strychnine or 3 μM GABAzine (SR 95531 hydrobromide, Bio-techne, France), respectively. These blockers were added to the cocktail containing 0.2 µM TTX, 4 mM kynurenic acid, 10 μM (+)-tubocurarine and 5 μM DHβE. In experiments assessing the KCC2 efficacy, 10 µM bumetanide (Millipore Sigma) was applied to block NKCC1 and 10 µM VU0240551 (Bio-techne, France) (10 µM) to block KCC2. Serotonin (5-HT, 10 µM), dopamine (DA, 100 µM) and N-Methyl-D-aspartic acid (NMDA, 10 µM) were from Millipore Sigma.

Matrigel basement membrane matrix was purchased at Corning Life Sciences (354230). Matrigel protein concentration as obtained by the Lowry method ranged between 9.2 and 10.4 mg/ml and endotoxin as measured by Limulus amoebocyte lysate assay was <1.5 EU/ml. Collagenase, recombinant BDNF and insulin were purchased from Sigma (C9263, B3795 and I1882, respectively). Dispase II was purchased from Roche (Neutral protease, grade II, 04942078001). Chicken embryo extract was purchased at MP Biochemicals (MP Biochemicals, 2850145). Recombinant rat CNTF and recombinant rat GDNF were purchased at R&D Systems (557-NT-010 and 512-GF-010, respectively). Tetrodotoxin (TTX) was purchased at Alomone Labs (T-550). Tubocurarine was purchased at Sigma-Aldrich (T2379).

2,4,6-dinitrobenzenesulfonic acid (DNBS), croton oil, fluorescein isothiocyanate (FITC)-conjugated dextran (molecular mass 3–5 kDa), betamethasone, atropine, tubocurarine and neutral red (NR) solution were purchased from Sigma (Milan, Italy). TNF-α was obtained from R&D Systems, Space Import-Export SRL (Milano, Italy). All reagents for cell cultures were obtained from Sigma, Bio-Rad Laboratories (Milan, Italy) and Microtech Srl (Naples, Italy). All chemicals and reagents employed in this study were of analytical grade.

Human myotubes and Motor Neurons were cultured in 96-well plates for 15 days. For recording of contractions, cells were placed in a Spinning Disk microscope system (Zeiss) under physiological conditions (37°C and 5% CO₂). Recording of contractions was performed in basal condition (before drugs addition) during 1min30 in phase contrast timelapse imaging (10X magnification) with time interval of 600ms, then after addition of 150 µM Tubocurarine (Sigma-Aldrich), 2 µM Tetrodotoxin (TTX, Tocris), BoNT/A and BoNT/A (0) during 1 min 30 in phase contrast timelapse imaging (10X magnification) with time interval of 600 ms at different timepoints. Analysis was performed with the open-source software tool MUSCLEMOTION (Sala et al., 2018 (link)) on ImageJ software, to quantify contractions according to instructions.

Fish were immobilized by an initial intramuscular injection of Tubocurarine (Sigma-Aldrich, Steinheim, Germany; 25–50 μL of 5 mg/mL solution). For the recordings fish were fixated on a stage in a tank, with a major part of the body immersed in water. Analgesia was refreshed in intervals of two hours by cutaneous Lidocaine application (2 %; bela-pharm, Vechta, Germany) around the operation wound and the head mounting rod. Electrodes (borosilicate; 1.5 mm outer diameter; GB150F-8P; Science Products, Hofheim, Germany) were pulled to a resistance of 50–100  $\mathrm{M}\mathrm{\Omega }$ (model P-97; Sutter Instrument, Novato, CA) and filled with 1 M KCl solution. Electrodes were fixed in a microdrive (Luigs-Neumann, Ratingen, Germany) and lowered into the nerve. Recordings of electroreceptor afferents were amplified (SEC-05, npi-electronics, Tamm, Germany, operated in bridge mode). All signals, P-unit recording, local and global EOD (see below) and the generated stimulus, were digitized with a sampling rate of 40 kHz (PCI-6229, National Instruments, Austin, TX). RELACS (www.relacs.net) running on a Linux computer was used for online spike and EOD detection, stimulus generation, and calibration.