Figure 1.

Breeding schematic and respiratory protocols. (A) The RC::P_hM4D allele is combined with 5 different rhombomere-specific-Cre lines to achieve cell specific expression of the hM4D receptor in embryonically-defined domains in the adult. Shown as an example is rhombomere 2 (HoxA2-Cre). (B) Hypercapnic respiratory protocol. The animal is placed into the respiratory chamber and allowed to acclimate under baseline room air conditions. The animal is then exposed to 20 minutes of 5% CO₂ followed by 20 minutes of room air. The animal is then injected intraperitoneally with 10 mg/kg of clozapine-N-oxide (CNO) followed by another 20 minutes of post-CNO room air, 20 minutes of 5% CO₂, and 20 minutes of room air. (C) Hypoxic respiratory protocol. This protocol is similar to the hypercapnic protocol except that no pre-CNO 10% O₂ measurement is taken to avoid potential confounds of hypoxic plasticity on the post-hypoxic response.