Fryou, N. L.; Jiang, T.; Frick, N.; Kwasniewska, P.; Lipin, M. Y.; Kelz, M. B.; Thomas, S. A.; McKinstry-Wu, A. R.

Introduction. Here, we create a conditional Adra2a line and use it to show that sedative, hypnotic, and hypothermic effects of 2-agonists are neuronally mediated via the 2A adrenergic receptor. Methods. We generated mice with loxP sites flanking Adra2a using CRISPR/Cas9 gene targeting. This line was crossed with lines encoding Cre recombinase (Cre) under the control of the Vgat, Snap25, and Dbh promoters. Cell-specific knockout was confirmed using fluorescent in-situ hybridization demonstrating targeted reduction in Adra2a mRNA in the appropriate cell types. Mice were given intraperitoneal dexmedetomidine (0.3 or 1 mg/kg) or saline, and 20 minutes later righting reflex was assessed, followed by 3 rounds of rotarod testing, with fall time and end temperature recorded. Spontaneous activity was recorded using beam break for an hour after. Mice of each genotype were implanted with EEG leads and recorded while given 0.3 mg/kg IP dexmedetomidine. Results. We created a conditional knockout and demonstrated cell-type specific reduction of Adra2a mRNA in crossed lines with cell-specific Cre. The pan-neuronal Adra2a knockout showed resistance to all temperature, sedative, and hypnotic effect endpoints in response to the 2-agonist dexmedetomidine. Adrenergic knockout demonstrated resistance to 2-agonist hypnosis and moderate resistance to hypothermia and impaired coordination with forced movement. GABAergic knockout showed resistance only to impairment of spontaneous movement by 2-agonists. Spectral analysis of the EEG showed an increase in proportion of delta power with a sedative dose of dexmedetomidine in all lines except the pan-neuronal Adra2a knockout. Discussion. Future studies will pursue both the specific subtype(s) and location of neuronal populations responsible for sedative, hypnotic, and hypothermic effects of 2-agonists.