The study was approved by the Animal Care Committee of Rhineland-Palatinate, Germany (approval number: AZ 23 177-07/G 13-1-057), and all animals were treated in accordance with the EU Directive 2010/63/EU for animal experiments. The M3 receptor knockout mice (M3R−/−) and respective wild-type controls of the C57BL/6NTac background were used for the study. The generation of M3R−/− mice has been described previously [21]. The M3R−/− mice were backcrossed with C57BL/6NTac mice for 10 generations to obtain N10 congenic mice. Polymerase chain reaction (PCR) of DNA isolated from tail biopsies was used to identify the genotype of each animal. The M3R−/− mice are complete knockouts with no remaining M3 receptor activity. The mice were housed under pathogen-free conditions with a 12 h light/dark cycle, a temperature of 22 ± 2 °C, a humidity of 55 ± 10% and with free access to food and tap water. In all experiments, male mice at the ages of 3 and 15 months were used. The calculated number of animals per group was 8 assuming an effect size of d = 1.8 for tear secretion (power = 90%, α = 0.05).

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