Cgp 52432

[2,3-³H]D-aspartate (specific activity 11.3 Ci/mmol) and [³H]GABA (specific activity 30.0 Ci/mmol) were from Perkin Elmer (Boston, MA, United States). (±)-baclofen, LY367385, (RS)-3,5 DHPG, and CGP 52432 were purchased from Tocris Bioscience (Bristol, United Kingdom). GF109203X, aminooxyacetic acid, naphtol blue black staining, horseradish peroxidase-coupled anti-mouse and anti-rabbit secondary antibodies were from Sigma (Milan, Italy). Donkey anti-mouse AlexaFluor-647, goat anti-guinea pig AlexaFluor-488, goat anti-rabbit AlexaFluor-555 were from Life Technologies Corporation (Carlsbad, CA, United States). Mouse anti-GABA_B1 and mouse anti-GABA_B2 antibodies were from Santa Cruz Biotechnology (Dallas, TX, United States). Rabbit anti-mGlu1 antibody, used in confocal analysis, was from Abcam (Cambridge, United Kingdom) and mouse anti-mGlu1 monoclonal antibody, used in Western blotting, was from BD Biosciences (San Jose, CA, United States). Guinea pig anti-vesicular glutamate transporters type 1 antibody was from Millipore (Temecula, CA, United States). Guinea pig anti-VGAT was from AlomoneLabs (Jerusalem, Israel). Bradford assay was from Bio-Rad (Segrate, Milan, Italy). KAPA Mouse Genotyping Kits were from Kapa Biosystems (Woburn, MA, United States). ECL Advance^TM was from Amersham Biosciences (Piscataway, NJ, United States).

Extracellular solution was prepared on the day of the experiment. Intracellular solution was prepared and frozen in aliquots and used within a week. Stock solutions of R-baclofen HCl (25 mM, Sigma-Aldrich, St. Louis, MO), GABA (50 mM, Sigma-Aldrich), CGP 52432 (10 mM, Tocris, UK) and R-isovaline HCl or S-isovaline HCl (100 mM, BioFine International, Vancouver, BC), were made using double distilled H₂0 and kept at 4°C. Final drug concentrations were prepared on the day of experiment.

CGP 52432 and 5′-Iodoresiniferatoxin were purchased from Tocris (Bristol) and AP5 was purchased from Ascent. Picrotoxin, BAPTA and L-NNA were purchased from Sigma-Aldrich.

For optogenetic experiments, acute brain slices were prepared from transgenic PV-IRES-Cre::Ai32 and SOM-IRES-Cre::Ai32 offspring as above in darkness. Whole-cell patch configuration was achieved and neuronal recordings were obtained at varying holding potentials as slices were illuminated with a blue LED pulse (a single pulse of 2 to 200 ms, or a train of 2 ms pulses at 10 to 100 Hz, power 11.5 mW) to elicit postsynaptic events. Where required, SR-95531 (2 µM; henceforth referred to as GABAzine), NBQX (10 µM) or CGP-52432 (10 µM) (all from Tocris, Bristol, UK) were washed into the perfusion bath via the perfusion pump.

D-AP5, CNQX and CGP52432 were purchased from Tocris Bioscience. Picrotoxin (PTX) was purchased from Sigma Aldrich. All drugs were prepared as stock solutions (CNQX was dissolved in DMSO and the other drugs in nanopure H₂O) and stored at − 20 °C; working aliquots were thawed and added to ACSF. Drugs were applied by bath perfusion at a flow rate of 1 ml/min.