S 3 5 dihydroxyphenylglycine dhpg

Dulbecco’s modified Eagle’s medium (DMEM), Neurobasal medium, Fluo-4-AM and antibiotics were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was sourced from Thermo Electron Corporation (Melbourne, Australia). IP-ONE HTRF® assay kit was purchased from Cisbio, Genesearch (Arundel, Australia). Select mGlu₅ ligands: 2-(1,3-benzoxazol-2-ylamino)-4-(4-fluorophenyl)pyrimidine-5-carbonitrile (VU0366058), 2-[2-(3-methoxyphenyl)ethynyl]-5-methylpyridine (M-5MPEP), 3-Fluoro-N-(4-methyl-2-thiazolyl)-5-(5-pyrimidinyloxy)benzamide (VU0409106), and N-(3-chloro-2-fluorophenyl)-3-cyano-5-fluorobenzamide (VU0366248) were synthesized as previously described (Felts et al., 2013 (link); Mueller et al., 2012 (link); Rodriguez et al., 2005 (link); Sharma et al., 2008 (link)). (S)-3,5-dihydroxyphenylglycine (DHPG), 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP), and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) were purchased from Tocris Bioscience (Melbourne, Australia) and dipraglurant (ADX 48621) from ApexBio (Houston, TX). [³H]methoxy-PEPy was custom synthesized by Quotient Bioresearch (Rushden, Northamptonshire, UK) using the previously reported synthetic route (Cosford et al., 2003). Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and were of analytical grade.

The following drugs were used: cocaine (Sigma-Aldrich), (S)-3,5-Dihydroxyphenylglycine (DHPG, Tocris Bioscience), LY367385 (Tocris Bioscience), 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP, Tocris Bioscience), 1-Naphthyl acetyl spermine trihydrochloride (Naspm, Sigma-Aldrich), picrotoxin (Sigma-Aldrich), PKI6-22 (Sigma-Aldrich), QX-314 (Sigma-Aldrich), rapamycin (Sigma-Aldrich), Ro67-7476 (Tocris Bioscience), SCH23390 (Tocris Bioscience), SKF81297 (Sigma-Aldrich), tetrodotoxin (Sigma-Aldrich), and U73122 (Tocris Bioscience). picrotoxin was dissolved directly in the ACSF used for perfusion. LY367385 and MTEP were dissolved in DMSO with a 1:1000 ratio as stock solutions, which were added directly to ASCF to a desired final concentration during experiments. All other drugs were dissolved in distilled water with a 1:1000 ratio as stock solutions. Drugs were delivered to the recording chamber through a perfusion system (Harvard Apparatus).

(S)-3,5-Dihydroxyphenylglycine (DHPG) was obtained from Tocris Bioscience, and dissolved in water or ACSF for LTD experiments. 50 mM stock solutions were kept in aliquots at −80°C, except for LTD experiments (prepared freshly on the day of the experiment). Wortmannin (Tocris Bioscience) was dissolved in DMSO (10 µM) and kept in aliquots at −80°C. For metabolic labeling, EXPRE³⁵S³⁵S Protein Labeling Mix (Perkin Elmer) was used.