CNO-hM4D mediated perturbation of noradrenergic DBH-Cre neurons whole animal plethysmography recordings

Plethysmography on conscious, unrestrained mice was carried out as described on 6-12 week old adult animals with at least 12 animals in both the experimental and control group for a given experiment. Mice were subjected to a five-day habituation protocol with each day consisting of several minutes of handling, temperature taken by rectal probe, intraperitoneal saline injection, and 30 minutes in the plethysmography chamber. Mice were then tested within one week of the last day of conditioning.

On the day of testing, mice were taken from their home cage, weighed, and rectal temperature was taken. Animals were then placed into an airtight, temperature controlled (~32°C) plethysmography chamber and allowed to acclimate for at least 20 minutes in room air (21% O₂/79% N₂) conditions. After acclimation and measurement under room air, the chamber gas was switched to a hypercapnic mixture of 5% CO₂/21% O₂/74% N₂ for 20 minutes. Chamber gas was then switched back to room air for 20 minutes. The mice were briefly removed for rectal temperature measurement and intra-peritoneal injection of clozapine-N-oxide (CNO, National Institute of Mental Health Chemical Synthesis and Drug Supply Program) dissolved in saline (0.1 mg/mL) for an effective concentration 1 mg/kg. The animal was returned to the chamber for another 20 minutes of room air, 20 minutes of hypercapnia, and 20 minutes of room air. The animal was then removed from the chamber and rectal temperature was taken immediately afterwards and again 30 minutes after the termination of the experiment. The animal was placed in its own cage during these 30 minutes at the ambient room temperature (~23°C).

Individual recordings for all animals expressing hM4D receptors in NA neurons are in this repository.