Cgp 55845a

We purchased CGP55845A, DNQX, and AP-5 from Tocris Bioscience (Ellisville, MI), recombinant mouse IL-1β from Biolegend (San Diego, CA), recombinant human IL-1ra from Peprotech (Rocky Hill, NJ), and ethanol from Remet (La Mirada, CA). Drugs were dissolved in ACSF by adding a known concentration of the stock solutions. Drug concentrations for IL-1β, IL-1ra, and ethanol were selected based on our previous reports^{33 (link),53 (link)}.

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)).

Rats were anesthetized and brain slices prepared (25 (link),27 (link)). Neuronal intrinsic membrane properties and excitability, spontaneous/miniature inhibitory postsynaptic currents (s/mIPSCs) and spontaneous cell firing were recorded in 236 cells in the medial central amygdala (CeA) (25 (link)–28 (link)). Each experiment includes data from a minimum of 4 rats, with 1–2 cells per animal. Recordings were analyzed with pClamp (Molecular Devices, Sunnyvale, CA), MiniAnalysis (Synaptosoft Inc., Fort Lee, NJ) or NeuroExpress software by Dr. A. Szucs (26 (link),29 (link)). Cells were classified as increased, decreased, or no change based on ±20% change in the maximum drug effect from baseline.
DL-AP5, CGP 55845A, DNQX, noradrenaline bitartrate, prazosin hydrochloride and propranolol hydrochloride (Tocris, Bristol, UK); bicuculline and tetrodotoxin (TTX; Sigma. St. Louis, MO); and alcohol (Remet, La Mirada, CA) were dissolved in ACSF.

We purchased ω-Agatoxin TK, AP-5, CGP 55845A and DNQX from Tocris (Bristol, UK); 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), Nifedipine and TTX from Sigma (St. Louis, MO); ω-Conotoxin GVIA from AnaSpec (Fremont, CA); and ethanol from Remet (La Mirada, CA). CRF was synthesized by Dr. Jean Rivier at the Salk Institute for Biological Studies, and R121919 was synthesized by Dr. Kenner Rice at the Drug Design and Synthesis Section of the National Institute on Drug Abuse. Drugs were dissolved in aCSF and applied locally by Y-tubing (Murase et al., 1989 (link)) or bath perfusion.

Taurine (Tau) and strychnine (Strych) were purchased from Sigma (St. Louis, MO, USA). CGP 55845A, DL-2-amino-5-phosphonovalerate (DL-AP5), and 6,7-dinitroquinoxaline-2,3-dione (DNQX) were obtained from Tocris (Ellisville, MO). Ethanol (EtOH) was purchased from Remet (La Mirada, CA, USA). All drugs were added to aCSF from stock solutions to obtain known concentrations in the superfusate, and only applied once per CeA slice.

We purchased BAPTA (1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) from Sigma (St. Louis, MO), CGP 55845A, DL-AP5 and DNQX from Tocris (Ellisville, MO), ethanol from Remet (La Mirada, CA) and TTX from Biotium (Hayward, CA). WIN (WIN55,212-2; [(3R)-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone, monomethanesulfonate) and AM251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-2,4-dichloro phenyl)-4-methyl-1H-pyrazole-3-carboxa-mide) were purchased from Cayman Chemical (Ann Arbor, MI), and dissolved in dimethylsulfoxide (final concentrations of 0.05–0.1%), which had no effect on synaptic responses in control experiments.