All animal care and experimental procedures were approved in advance by the University of Virginia Institutional Animal Care and Use Committee. The sNuc-Seq experiments used adult male Chat-Cre mice (Jackson Laboratories, JAX, stock # 28861; 28 weeks old). The fluorescent in situ hybridization experiments used C57BL/6J mice from the Jackson Laboratory (JAX, 000664). The following mouse lines were used for optogenetic studies: Chat-Cre (Rossi et al., 2011 (link); JAX, 028861); Crfr2α-eGFPCre bacterial artificial chromosome (BAC) transgenic mice (“Crhr2-Cre mice”; Anthony et al., 2014 (link)); Vipr2-Cre (Daigle et al., 2018 (link); JAX, 31332); Phox2b-Flp (Hirsch et al., 2013 (link); JAX, 22407); CaTCh (Daigle et al., 2018 (link); JAX, 025109); Ai32 (RCL-ChR2(H134R)/EYFP) mice (Madisen et al., 2012 (link); JAX, 024109); and a Chat-p2a-Flp mouse line (see Method Details below). The anterograde tracing experiments used Chat-Cre, Crhr2-Cre, Vipr2-Cre, and C57BL/6J mice. Unless otherwise specified, all experiments used adult mice with approximately equal numbers of male and female mice. Mice were housed at 22-24°C with a 12-h light:12-h dark cycle and ad libitum access to standard mouse chow and water.
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