Crf peptide

Alcohol for mouse drinking solution (20% v/v) was prepared using 95% ethyl alcohol (Pharmco Products Inc., Brookfield, CT) in tap water. DNQX, AP-5, CGP55845A, CRF peptide, and Astressin-2B were purchased from Tocris Bioscience (Ellisville, MO). SR-95531 (gabazine, GBZ; 100 μM) was purchased from Sigma-Aldrich. The CRF1 antagonist R121919 was supplied by Neurocrine Biosciences, Inc. (San Diego, CA). Stock solutions were prepared in ultra-pure water or DMSO (DNQX only), stored at 20°C, and diluted to final experimental concentration in aCSF on the day of testing. Doses for electrophysiology experiments were chosen based on their ex vivo effects in CeA neurons from prior reports (10 (link), 25 ). CP-154,526 was purchased from Tocris Bioscience and was selected for use in behavioral pharmacology experiments due to a robust literature describing its effects on alcohol drinking in mice (32 (link)–34 (link)).

Ethanol for acute application to slice was prepared using 95% ethyl alcohol (Pharmco Products Inc.) in aCSF. DNQX (20μM), AP-5 (50μM), CGP55845A (1μM) and CRF peptide (200nM) were purchased from Tocris Bioscience. The CRF1 antagonist R121919 (1μM) was supplied by Neurocrine Biosciences, Inc. (San Diego, CA.) All stock solutions were prepared in ultrapure water and diluted in aCSF on the day of recording, with the exception of AP-5, which was prepared in DMSO and diluted 1:1000 in aCSF. DNQX, AP-5, and CGP55845A were superfused in the bath solution continuously throughout all sIPSC recordings. Cell-attached firing was recorded in the presence of aCSF only, as previous work suggests that DNQX, AP-5 and CGP55845A application do not significantly alter the discharge rate of CeA CRF1+ neurons (Herman et al., 2013a (link)). Ethanol (5–7min total exposure time), CRF (10–12 min total exposure time) and R121919 (10–12 min total exposure time) were administered via a y-tube positioned for focal application to CeA neurons.