Urb597

The antibodies were obtained from following sources: mouse anti-SMI-312 (Covance), rabbit anti-phospho-GAP43 (Ser-41; Sigma), rabbit anti-GAPDH, rabbit anti-sonic hedgehog, rabbit anti-phospho-PKAc (thr197), rabbit anti-PKAc (Cell Signaling), mouse anti-patched-1 (H-267, Santa-Cruz), rabbit anti-BMP4 (Abcam), rabbit anti-smoothened (Origene), goat anti-mouse Alexa Fluor 488 and 555 (Life Technologies). Empty construct pCDNA3.1 and EF1αLacZ for the expression of β-galactosidase have been described previously (Kharebava et al., 2008 (link)). Myc and FLAG-tagged human Shh expression construct pCMV6-Entry-myc-Flag-Shh from Origene. Vectors expressing mouse smoothened protein pEGFP-mSmo (Chen et al., 2002 (link)) is from Addgene (Plasmid 25395). For GLI1 transcriptional assays, the pGLI1-Luc Reporter plasmid was used (Signosis). Luciferase assay reagents and β-galactosidase quantification kits were from Promega. Cyclic AMP XP assay kit for the cAMP level quantification was from Cell Signaling Technology. Recombinant murine Shh was from Peprotech. Docosahexaenoic and other polyunsaturated fatty acids, essentially fatty acid-free BSA, SQ22536, and α-tocopherol were obtained from Sigma, and URB597 and cyclopamine were from Tocris; SAG was from Calbiochem.

The CB1 cannabinoid receptor antagonist SR141716A [5-(4-chloro-phenyl)−1-(2,4-dichlorophenyl)−4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] (National Institute of Mental Health, USA), the positive allosteric modulator of mGlu₅ receptors CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide) (National Institute of Mental Health, USA), the vanilloid TRPV1 antagonist capsazepine (N-[2-(4-Chlorophenyl)ethyl]−1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide) (Tocris) and the FAAH inhibitor URB597 (Cyclohexylcarbamic acid 3'-(Aminocarbonyl)-[1,1'-biphenyl]−3-yl ester) (Tocris) were dissolved in 5% Tween 80/5% polyethylene glycol/saline and given intraperitoneally (i.p.). CDPPB (0.75 mg/kg) or its vehicle were administered 30 min before testing; URB597 (0.1 mg/kg) or its vehicle were administered 2 hr before testing; SR141716A was administered 30 min before CDPPB or URB597 at a dose that did not induce effects by itself (1 mg/kg) in adult rats (Manduca et al., 2015 (link); Sciolino et al., 2011 (link)). capsazepine (5 mg/kg) was injected 30 min before CDPPB and URB597. Drug doses and pre-treatment intervals were based on the literature (Manduca et al., 2015 (link); Ratano et al., 2017 (link)) and on pilot experiments. Solutions were freshly prepared on the day of the experiment and were administered in a volume of 1 ml/kg.

The compounds which were tested:
URB 597 (0.1, 0.3, 1 mg/kg) (Tocris, USA)—FAAH inhibitor
JZL 184 (1, 4, 8, 20, 40 mg/kg) (Tocris, USA)—MAGL inhibitor
MK-801 (0.3, 0.6 mg/kg) (Tocris, USA)—NMDA receptor antagonist
All compounds were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in saline solution (0.9% NaCl) and administered intraperitoneally (ip) at a volume of 10 ml/kg. Fresh drug solutions were prepared on each day of experimentation. Control groups received injections of saline with Tween 80 (vehicle) at the same volume and by the same route of administration.
Experimental doses of drugs used and procedures were selected on the basis of literature data [23 (link)–28 (link)] and our previous experiments [16 (link), 17 (link), 29 (link)].

All drugs for performing electrophysiological studies were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and were added at the final concentration to the superfusion medium. CP 55,940, AMG9810, LY354740, AM251, URB597, JZL 184, tetrahydrolipstatin (THL), latrunculin A (LAT-A), and PTX were purchased from Tocris BioScience (Bristol, United Kingdom). S-Nitroso-N-acetylpenicillamine (SNAP) was purchased from Abcam (Cambridge, MA). All drugs were perfused at least 20 min before the LFS protocol apart from JZL 184. JZL 184 was preincubated at least 1 h before LFS protocol.