S 3 5 dhpg

Stock solutions were prepared as follows: thapsigargin (Sigma, 1 mM, DMSO), (S)-3,5-DHPG (Tocris, 20 mM, H₂O), ionomycin (Sigma, 5 mM, DMSO), A23187 (Sigma, 30 mM, DMSO), 4-chloro-m-cresol (Sigma, 1 M, DMSO), cyclopiazonic acid (Sigma, 25 mM, DMSO), caffeine (Sigma, 5 mM, H₂O). Antibodies used were chicken polyclonal anti-GFP (Aves # GFP-1020), rabbit monoclonal anti-RCAS1 (Cell signaling # 6960), and mouse monoclonal anti-PDI (Abcam # ab2797). Secondary antibodies were Alexa Fluor 488 and Alexa Fluor 568 (Life Technologies).

Drugs used for the electrophysiological experiments were applied by dissolving them to the desired final concentration in the bathing ACSF. The concentrations of the various drugs were as follows: AM281 (2 μM), 5′-Iodoresiniferatoxin (I-RTX, 1 μM), CNQX (10 μM), MK-801 (25 μM), orlistat (5 μM), N-palmitoylethanolamine (PEA, 10 μM), O-1602 (10 μM), CID-16020046 (CID-1602, 20 μM), (S)-3,5-DHPG (50 μM) (from Tocris, Bristol, UK). Bicuculline (10 μM) (from Sigma-RBI, St. Louis, USA). Bicuculline, CNQX, (S)-3,5-DHPG and MK-801 were dissolved in water. AM281, I-RTX, orlistat, O-1602 and CID-16020046 were dissolved in DMSO. PEA was dissolved in ethanol. The intraneuronal delivery of orlistat was performed dissolving the drug (orlistat, 5 µM) in the internal solution.