Betaxolol

Cocaine HCl (National Institute on Drug Abuse) was dissolved in sterile 0.9% saline at a concentration of 10 mg/ml, and administered at a dose of 10 mg/kg (i.p.) for all experiments. The β1-AR antagonist betaxolol and the β2-AR antagonist ICI 118,551 were purchased from Tocris Bioscience (Minneapolis, MN), and were dissolved in sterile 0.9% saline. betaxolol and ICI 118,551 are commonly used to dissociate β1- and β2-ARs [14 (link)–16 (link)] and doses were chosen based on previous studies [10 (link),17 (link)].

Mixed glial cells were obtained from the cerebral cortex of Sprague Dawley rat pups at postnatal days 1–3 and cultured in DMEM supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. After 10 days in vitro, microglia were harvested by gentle shaking of the growth flask, plated in a 96 well plate at a density of 30,000 cells/well, and incubated at 37°C overnight. Microglia were pretreated with the ADRB1 antagonists CGP 20712A (0.1 μM; Tocris 1024) or betaxolol (10 μM; Tocris 0906), the ADRB2 antagonist ICI-118551 (0.1 μM; Tocris 0821), or vehicle for 15 min. Following the pretreatment, microglia were stimulated with LPS (10 ng/ml; Sigma Aldrich L4391) along with or without xamoterol (S-enantiomer; 1 μM) or isoproterenol (1 μM; Sigma Aldrich I5627) for 4 h at 37°C. Following the 4 h incubation, cell media was collected and concentration of TNF-α was measured by ELISA (Invitrogen, KRC3011) according to the manufacturer’s instruction.