Kw6002

KW6002 (Istradefyline, Sigma-Aldrich) is a selective adenosine A2a receptor antagonist with a molecular weight of 384 and a Ki of 29.6 nM in rats. It’s half-life of 110 min, 97% bio-availability after intraperitoneal injection, and ability to cross the blood brain barrier (Yang et al., 2007 ), make it a suitable drug for our in vivo experiments. The drug was dissolved in DMSO at 9.3 mg/ml, sonicated and stored at 4°C in a dark vial protected from light. The day of the experiment, the drug was administrated via intraperitoneal injection at a dose of 0.5 mg/kg.

CtB-Saporin (50 μg, Catalog No: IT-14, Advanced Targeting Systems, San Diego, CA) was dissolved in 80 μl phosphate buffered saline (PBS) and administered intrapleurally to target phrenic motor neurons (25 μg per side) using 50 μl Hamilton syringes having semi-blunt needles of 23-gauge and 6 mm length through the fifth intercostal space anterior axillary line (Mantilla et al., 2009 (link)). Additionally, 50 μg CtB (Catalog No: 227039, EMD Millipore, Billerica, MA, USA) was dissolved in 25 μl sterile water and injected intrapleurally (12.5 μl on each side) to label the spared phrenic motor neurons.
Rats were administered with A2a receptor antagonist starting 36 hours after CtB-Saporin injections, until 6 hours before perfusion at day 5 or 24 hours before EMG recordings (to minimize acute effects of the drug on recordings) and subsequent perfusion. KW6002 (Istradefylline, Sigma-Aldrich) is a selective, blood-brain-barrier-penetrable A2a receptor antagonist (Yang et al., 2007 (link)). KW6002 was dissolved in DMSO (37°C) at 9.3 mg/ml concentration, sonicated, aliquoted and stored at 4°C in dark. Prior to use, KW6002 was re-warmed and administered intraperitoneally twice daily at a dose of 0.5 mg/kg (1.0 mg/kg per day total). The volume of DMSO administered to the vehicle rats was also normalized to body weight (~17–20 μl DMSO per injection, ~50 μl/kg).