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21 protocols using ω-agatoxin IVA

All reagents were purchased from the Sigma Chemical Company (St. Louis, MO, USA) with the exceptions of ZD7288 from the Tocris company (UK), ω-Agatoxin IVA, ω-Conotoxin GVIA and pimozide from Alomone Labs (Isreal). All channel blockers were prepared as concentrated stock solutions in distilled water or DMSO and either added immediately to ACSF at working concentrations or stored at −20°C for subsequent utilization.
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The L-type HVA current blocker nifedipine (Sigma-Aldrich, St. Louis, MO) was diluted in DMSO as a 100 mM stock solution and stored at −20°C until use. ω-agatoxin IVA and ω-conotoxin GVIA (Alomone Labs, Jerusalem, Israel), which block N-type and P/Q-type HVA currents respectively, were diluted in diH2O as a 500 μM stock solution and stored at −20°C until use. CdCl2 (Thermo Fisher Scientific, Waltham, MA) was diluted in diH2O to a stock solution of 100 mM and stored at RT until use. DAMGO (Sigma-Aldrich, St. Louis MO) was diluted in diH2O to a stock solution of 1 mM and stored at −20°C until use. On the day of use, stock solutions were diluted in BaCl2 bath to a final concentration, indicated in parentheses: nifedipine (1 μm), ω-agatoxin IVA (200 nM), ω-conotoxin GVIA (200 nM), CdCl2 (100 μM), DAMGO (1 μM).
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For light response recordings, 50–100 nM LY341495 (Tocris) was added to Ames’ media after baseline experiments. At these concentrations, LY341495 is selective for Group II mGluRs (Kingston et al., 1998 (link)). Notably, mGluR3 has not been found in the mammalian retina (Brandstätter et al., 1998 (link)). For paired recordings and SAC pharmacology studies, the aCSF was perfused with 500nM-1μM LY354740 (Tocris), a highly selective Group II mGluR agonist (Schoepp et al., 1997 (link)) after whole-cell recording was established, and 3 μM LY341495 was subsequently used to competitively inhibit mGluR2 activity (Kingston et al., 1998 (link)). To block voltage-gated calcium channels, 300 μM CdCl2 (Sigma) or combinations of 1 μM ω-conotoxin GVIA (Abcam) and 250 nM ω-agatoxin IVA (Alomone) were added to the aCSF.
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To isolate presynaptic Ca2+ currents, aCSF was supplemented with 1 µM tetrodotoxin (TTX, Alomone labs, Jerusalem, Israel), 100 µM 4-aminopyridin (4-AP, Tocris, Bristol, UK) and 20 mM tetraethylammonium chloride (TEA, Sigma Aldrich, Darmstadt, Germany) to block Na+ and K+ conductance. Calyxes were whole-cell voltage-clamped at −80 mV. Current-voltage relationships were recorded in the presence of 1 mM CaCl2, pharmacological isolation of VGCC subtypes was performed in 2 mM CaCl2. We used 200 nM ω-agatoxin IVA (Alomone labs) to selectively block CaV2.1 and 2 µM ω-conotoxin GVIA (Alomone labs) for CaV2.2 VGCCs. Remaining current was blocked by 50 µM CdCl2. All experiments to isolate CaV2 subtypes were conducted in presence of cytochrome c (0.1 mg/ml). Presynaptic patch pipettes with open tip diameters 4–6 MΩ resistance were pulled from 2.0 mm thin-walled borosilicate glass (Hilgenberg, Malsfeld, Germany) and were filled with the following (in mM): 145 Cs-gluconate, 20 TEA-Cl, 10 HEPES, 2 Na2-phosphocreatine, 4 MgATP, 0.3 NaGTP, and 0.5 EGTA, pH 7.2, 325–340 mOsm). Pipettes were coated with Sylgard. Presynaptic series resistance was between 6 and 20 MΩ (usually between 10–15 MΩ) and was compensated online to 6 MΩ. Leak and capacitive currents were subtracted online with a P/5 routine. Cells with series resistance >20 MΩ and leak currents >100 pA were excluded from the analysis.
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All compounds were purchased from SigmaAldrich (Poole, U.K) unless otherwise stated. ω-Conotoxin MVIIC was purchased from Peptide Institute, Inc. (Osaka, Japan), ω-Agatoxin IVA was purchased from Alomone Labs. Calhex 231-HCl was purchased from Tocris Bioscience (Bristol, UK).
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Salbutamol (α-[(tert-butylamino)methyl]-4-hydroxy-m-xylene-α,α′-diol or albuterol; Sigma, cat. S8260) was used for in-vivo and ex-vivo experiments. For ex-vivo experiments, it was dissolved in Ringer solution to a final concentration of 0.06μM or 20μg/l. Clenbuterol (4-amino-α-(t-butylaminomethyl)-3,5-dichlorobenzyl alcohol hydrochloride; Sigma, cat. C5423) was diluted in Ringer solution and used exclusively in ex-vivo experiments at a final concentration of 1μM. Salbutamol and Clenbuterol concentrations used in this study are within those reported in the literature for the treatment of asthmatic patients.(Jacobson et al., 2003 (link); Yamamoto et al., 1985 (link)) ω-conotoxin GIIIB (a.k.a. myotoxin II, geographutoxin II, GTx-II; Alomone, cat. C270), a selective blocker of skeletal muscle Nav1.4 channels, was used at a concentration of 1μM. ω-conotoxin GVIA (a.k.a. SNX-124; Alomone, cat. C-300), which blocks N-type Ca2+ channels, was used at a final concentration of 3μM. ω-agatoxin IVA (a.k.a. ω-agatoxin-Aa4a, ω-AGTX-Aa4a, ω-aga-IVA, ω-agatoxin-4a; Alomone, cat. STA-500), which blocks P/Q-type Cav channels, was used at a concentration of 0.2 μM. Arvanil (N-arachidonoyl-vanillyl-amine; Sigma, cat. SML0520), which activates TRPV1, was used at a final concentration of 0.5 μM.
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Stocks of ω‐Conotoxin GVIA, ω‐agatoxin IVA, (R)‐baclofen, DNQX disodium salt, TTX, 4‐AP and DL‐AP5 sodium salt were dissolved in ddH2O. WIN55,212‐2 mesylate and nifedipine were dissolved in 100% DMSO. All drug stocks were stored at −20°C and diluted in aCSF just prior to superfusion. QX‐314 bromide was dissolved fresh in internal solution. ω‐Conotoxin GVIA and ω ‐agatoxin IVA were purchased form Alomone Labs (Jerusalem, Israel), QX‐314 bromide and 4‐AP from Millipore Sigma (St. Louis, MO, USA), and all other drugs from Tocris Biosciences (Minneapolis, MN, USA).
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For all experiments including the use of drug treatments, drug solvents (vehicle; EtOH or DMSO) were used as controls. Nifedipine (Sigma) was dissolved in EtOH and used at 50 μM for acute experiments, while 5 μM was used for chronic experiments. ω-agatoxin IVA (Alomone Labs, Jerusalem, Israel) was dissolved in distilled water and used at a final concentration of 100 nM. Tetrodotoxin (TTX; Alomone Labs) was dissolved in distilled water and used at final concentrations between 2.5 and 5 μM. D-tubocurarine chloride hydrate (Sigma) was dissolved in distilled water and used at 50 μM. Dynasore (Sigma) was dissolved in DMSO and used at a final concentration of 90 μM.
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Isosaponarin (purity > 98%) was purchased from ChemFace (Wuhan, China). The agents 4-AP, bafilomycin A1, GF109203X, Go6976, and rottlerin were purchased by Tocris Bioscience (Bristol, UK). ω-conotoxin GVIA and ω-agatoxin IVA were purchased from Alomone labs (Jerusalem, Israel). Isosaponarin dissolved in 0.1% dimethylsulfoxide (DMSO) was added 10 min before 4-AP addition, and other drugs (bafilomycin A1, ω-conotoxin GVIA, ω-agatoxin IVA, GF109203X, Go6976, and rottlerin) were added 10–20 min before this.
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BoNT/A1 (2.5 × 108 MLD50/mg), BoNT/D1 (2.8 × 107 MLD50/mg) and BoNT/E1 (3.0 × 107 MLD50/mg) were purchased from Metabiologics (Madison, WI, USA). For BoNT/E, toxin was activated by a 60 min incubation at 37 °C with 0.3 mg/mL TPCK-treated trypsin in 0.05 M sodium phosphate buffer (pH 6.5)19 (link). ω-agatoxin IVA, ω-conotoxin MVIIC and GV-58 were purchased from Alomone Labs (Jerusalem, Israel). FPL 64176 was purchased from Tocris Bioscience (Bristol, UK). All reagents for electrophysiology buffers were obtained from Sigma-Aldrich (St. Louis, MO, USA).
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