Lidocaine hydrochloride

Lidocaine was dissolved in 0.9% saline and adjusted to a volume of 100 μl in each concentration group. It was intravenously administered via a tail vein catheter for 3 min during in vivo patch-clamp recordings. For spinal application, drugs were diluted in Krebs solution and superfused onto the spinal cord without altering the perfusion rate or temperature. The time necessary for the solution to flow from the stopcock to the spinal cord surface was approximately 10 s. Lidocaine hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA) and 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and tetrodotoxin (TTX) from Wako (Osaka, Japan).

2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ibuprofen, lidocaine hydrochloride, and caffeine were purchased from Sigma Aldrich (Poznań, Poland); disodium phosphate and potassium dihydrogen phosphate from Merck, Darmstadt (Germany); Biobase, beeswax were purchased from Mazidła.com (Poznań, Poland); grape seed oil was purchased from Monini (Spoleto, Italy); sodium acetate anhydrous, potassium persulfate, potassium acetate, xylene, 99.5% acetic acid, aluminium chloride, 36% hydrochloric acid, sodium chloride, potassium chloride, sodium sulphate anhydrous as well as propylene glycol, ethanol, and methanol were from Chempur (Piekary Śląskie, Poland), whereas acetonitrile for HPLC from J.T. Baker, (Landsmeer, The Netherlands). All reagents were of analytical grade.

Reference standards of lidocaine hydrochloride and monoethylglycinexylidide (≥95%) were purchased from Sigma Aldrich, Auckland, New Zealand. lidocaine hydrochloride for injection was purchased from Ethical agents Ltd., Auckland, New Zealand. Acetonitrile, methanol, water, and formic acid were mass spectrometry grade and were purchased from Fisher Scientific, Auckland, New Zealand. Heparin sodium and normal saline were purchased from Pfizer New Zealand Limited, Auckland, New Zealand, and Baxter Healthcare Pty Ltd., Old Toongabbie, NSW, Australia, respectively. Artificial colostrum and milk replacer were purchased from Farmlands Co-Operative Society Ltd., Palmerston North, New Zealand.

Reagents were ≥95% purity and were used as provided without further purification or characterisation. 2-chloro-N-phenylacetamide, 4-bromo-2-methoxy-N-phenylbenzamide, 3-fluoroacetanilide, N-methylacetanilide and 4-nitroacetanilide were purchased from Fluorochem (Hadfield, UK). 2-Acetamidophenol, 3-acetamidophenol, acetanilide, acetonitrile, ammonium acetate, ammonium formate, benzanilide, dimethyl sulphoxide (DMSO), formic acid, paracetamol (acetaminophen) and salicylanilide were purchased from Thermo Fisher Scientific (Hemel Hempstead, UK). Bicalutamide, flutamide, L-glutamine, 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid (HEPES), lidocaine hydrochloride, 3-methylacetanilide, 4-methylacetanilide, niclosamide, N-phenylurea, potassium dihydrogen phosphate, propanil, sodium phosphate dibasic, Trypan Blue and Williams’ Medium E were purchased from Sigma Aldrich (Gillingham, UK). 4-Fluoroacetanilide, 2-methylacetanilide, 3-nitroacetanilide and prilocaine hydrochloride were purchased from VWR International Ltd (Lutterworth, UK).

Adult mice (postnatal day 42–90) C57BL/6J wild-type (WT), both sex, n = 41; Kcng4-YFP (6–8 weeks, n = 11) mice [31 (link)], which labeled α-RGC (Supplementary Figure S1) were used in the study. The animals were maintained in a 12 h–12 h day–night cycle, and all experiments were performed during daylight hours. The mice were anaesthetized deeply with an intraperitoneal injection of ketamine (Vedno, St. Joseph, MO, USA) and xylazine (Akorn, Decatur, IL, USA) [80 and 10 mg (kg body weight)⁻¹, respectively], and lidocaine hydrochloride (20 mg mL⁻¹, Sigma-Aldrich, St. Louis, MO, USA) was applied locally to the eyelids and surrounding tissue. Eyes were removed under dim red illumination and hemisected anterior to the ora serrata. Anterior optics and the vitreous humor were removed, and the resultant retina–eyecup with sclera attached, either whole or in sections, was placed in a super-fusion chamber. For patch recordings, retinas were dissected into four equal quadrants and attached to a modified translucent Millicell filter ring (Millipore, Bedford, MA, USA). The flattened retina were superfused with oxygenated mammalian Ringer solution, pH 7.4, at 32 °C [32 (link)]. The anaesthetized animals were killed by cervical dislocation immediately after the enucleations.