Vglut2 ires cre mice

Adult male DAT-Cre (The Jackson Laboratory, stock number 006660) and vGlut2-ires-Cre mice (The Jackson Laboratory, stock number 016963), weighing 20–25 g, were used for all experiments. Mice were kept on a 12/12 h light/dark cycle (lights on at 7 A.M., lights off at 7 P.M.) with ad libitum access to food and water. Mice had a minimum of three weeks to recover after surgery, and at least 3 d of rest were provided after each experiment. All animal procedures were reviewed and approved by the authors Animal Care and Use Committee.

Animal care and procedures were approved by the University of Texas Health Science Center at Houston Institutional Animal Care and Use Committee. Mice were housed at 21–22°C on a 12/12 h light/dark cycle (7 A.M. to 7 P.M. light), with ad libitum access to standard pellet chow, unless otherwise stated during fasting experiments. Sim1-Cre mice (Balthasar et al., 2005 (link)) were bred to Ai9 reporter mice (Madisen et al., 2010 (link)) to generate Sim1-Cre::Ai9; some of the subjects used in behavioral experiments contained the reporter gene for post-hoc visualization purposes. Sim1-Cre::Vglut2^F/F mice were generated as previously described (Xu et al., 2013 (link)). Vglut2-ires-Cre mice (Vong et al., 2011 (link)) were purchased from The Jackson Laboratory (stock no. 016963) and bred to C57 mice to generate Vglut2-ires-Cre subjects used in the experiments. Mice were at least six weeks old before surgeries and testing, and were chosen from multiple litters. All experiments were done in males during the light cycle, between the early afternoon hours (12 P.M.) and early evening before the start of the dark cycle.

Ai9 tdTomato reporter mice (Allen Institute, The Jackson Laboratory; Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}) were crossed to Vglut2-ires-Cre/⁺ mice (The Jackson Laboratory; Slc17a6^tm2(cre)Lowl/J; Vong et al., 2011 (link)) to generate Vglut2-tdTomato compound mice for a developmental lineage trace of all neurons that have expressed the gene encoding the vesicular glutamate transporter Vglut2 at some point in their existence. Male (M) and female (F) mice were used in all experiments.
ArcPomc^+/- (ARC specific Cre-reversible Pomc KO or FneoΔ2) mice (Bumaschny et al., 2012 (link); Lam et al., 2015a (link); Chhabra et al., 2016a (link),b (link)) were crossed to Vglut2^Cre/+ mice to obtain compound heterozygous Vglut2^Cre/+; arcPomc^+/- mice. Those mice were back crossed to arcPomc^+/- mice to yield the control and experimental groups for POMC restoration and ISH studies. These three groups were: Vglut2^Cre/+; Pomc^+/+ (control), Vglut2^+/+; arcPomc^-/- (FNΔ2) and Vglut2^Cre/+; arcPomc^-/- (restored). FNΔ2 animals have a floxed-neomycin cassette inserted between neural Pomc enhancer 1 (nPE1) and the deleted neural Pomc enhancer 2 (ΔnPE2) locus, which prevents the transcription of Pomc in neurons, while leaving pituitary transcription intact. After Cre-mediated excision of the floxed-neomycin cassette, neuronal Pomc transcription is restored from the functional nPE1 enhancer.

Mice were group housed under a 12 h light-dark cycle with ad libitum access to food and water, 50–70% humidity, and 18–22 °C ambient temperature unless otherwise noted. Adult male and female (aged 2–3 months) C57BL/6J, Ai9 (Cre-dependent tdTomato reporter) mice, (Jackson Laboratories, Stock No. 007909), vGluT2-IRES-Cre mice (Jackson Laboratories, Stock No. 016963), and GAD2-IRES-cre mice (Jackson Laboratories, Stock No. 010802) were used in this study. All experimental procedures were approved by the Animal Care and Use Committee of Army Medical University.

Optogenetic manipulations, fiber photometry, in vivo multichannel recording, and RNA-inference experiments were performed in male GAD2-IRES-Cre mice (Jackson Laboratory stock 010802) or male Vglut2-IRES-Cre mice (Jackson Laboratory stock 016963). Descriptions in detail are in Supplementary methods.