Psychopharmacology
in counterbalanced order) 20 min (Petrie et al. 2019) prior to
placement in the conditioning box lined with the grid or hole
floor (counterbalanced) for 20 min, while their locomotion
was tracked by EthoVision. Three days after the final condi-
tioning day, the rats received a 10-min drug-free as described
previously.
final saline or naloxone injection where all rats received two
bottles (saccharin (0.1%) and reverse osmosis water) in their
home cage and consumption of both liquids was measured at
the same intervals used during pretest. As in the pretest, the
amounts consumed were converted to saccharin PRs.
Experiment 7: Effect of oleoyl alanine (HU595) and HU596
on the rat serine hydrolase proteome for FAAH activity
and on luciferase assay for PPARα activity
Experiment 4: Potential of dimethyloleoyl glycine (HU596)
to interfere with MWD CPA
The rats (n = 32) were treated as in experiment 2, except that
they were pretreated with VEH (n = 12), 1 mg/kg HU596 (n =
Competitive activity-based protein profiling (ABPP) assay of
activity at FAAH For preparation of proteomes, rat brains were
dounced homogenized in an isotonic buffer consisting of
8
), or 5 mg/kg HU596 (n = 12) 10 min prior to naloxone.
20 mM HEPES, 2 mM DTT, 0.25 M sucrose, and 1 mM
Experiment 5: Mechanism of action of oleoyl alanine (HU595)
interference with acute naloxone-precipitated MWD-induced
CPA
MgCl , pH 7.2. Lysed proteomes were then subjected to a
2
low-speed spin (1400×g, 5 min) to remove debris, and ultra-
centrifugation (100,000×g, 45 min) to separate membrane and
cytosolic fractions. The supernatant was removed and saved
as the soluble proteome, while the pellet was washed and
resuspended in isotonic resuspension solution (20 mM
HEPES, 2 mM DTT) by pipetting and saved as the membrane
proteome. Rat brain membranes were diluted to 1 mg/ml prior
to use (Chang, et al. 2012). Proteomes (50 μl) were preincu-
bated with either DMSO or 10–30 μM concentrations of
oleoyl glycine (OG), HU595, and HU596 at 37 °C. After
20 min, FP-Rh (1.0 μl, 50 μM in DMSO, a kind gift from
Ben Cravatt) was added and the mixture was incubated for
another 30 min at 37 °C. Reactions were quenched with SDS
loading buffer (12.5 μl, 5×) and run on SDS-PAGE (sodium
dodecyl sulfate polyacrylamide gel electrophoresis; Chang
et al., 2013). Following gel imaging, serine hydrolase activity
was determined by measuring fluorescent intensity of gel
band corresponding to FAAH using ImageJ 1.43u software.
The 33 rats were treated as in experiment 2 (with only the 10 min
pretreatment time) except that, on the naloxone trials, they also
received an injection of VEH, MK886 (1 mg/kg, i.p.; PPARα
antagonist), or AM251 (1 mg/kg, i.p.; CB receptor antagonist)
1
20 min prior to an injection of HU595 (5 mg/kg, i.p.). Ten
minutes following pretreatment with HU595, the rats were
injected with naloxone (1 mg/kg, s.c.) and 10 min later were
placed in the MWD chamber. The groups were VEH-HU595
(n = 10), MK886-HU595 (n = 11), and AM251-HU595 (n = 12).
Petrie et al. (2019) previously demonstrated that at the doses
employed here neither AM251 nor MK886 modified the strength
of CPA produced by acute naloxone-precipitated MWD.
Experiment 6: Effect of oleoyl alanine (HU595) on saccharin
preference following acute naloxone-precipitated MWD
In order to familiarize the rats with saccharin, the rats (n = 80)
were pre-exposed to two bottles (saccharin (0.1%) and reverse
osmosis water) in their home cage and consumption of both
liquids was measured at 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h. The
cumulative amount of saccharin and water consumed at each
of the six intervals was converted into a cumulative saccharin
preference ratio (PR) (cumulative saccharin consumed/(cumu-
lative saccharin + cumulative water consumed)). Using pretest
PR scores, rats were assigned to one of 4 groups (N = 20/
group): saline–saline, saline–naloxone, morphine–saline, or
morphine–naloxone. Seventy-two hours following the pretest,
the rats received two cycles of acute naloxone-precipitated
MWD. On day 1, rats were injected s.c. with saline or
Luciferase assay of PPARα activity Cell culture, reagents, and
transfections
Fibroblast-like (COS-7) cells were propagated in
Dulbecco’s modified Eagle medium (DMEM; Cat. no.
11965092, Life Technology, Milan, Italy) supplemented
with 10% FBS, and 1% penicillin and streptomycin (Cat.
no. 15070063, Life Technology, Milan, Italy) in a humidi-
fied atmosphere of 95% air/5% CO at 37 °C. For cell
2
transfection, cells were seeded onto 24-well plastic plates
2
at 2 × 103 cells/cm density. After plating, the cells were
transfected on the next day with (a) pM1-hPPARα-Gal4,
(b) TK-MH100x4-Luc containing the UAS enhancer ele-
ments, and (c) Renilla luciferase (pRL, cat. E2231;
Promega, Milan, Italy). The combination of plasmids was
transfected into the cells by use of Lipofectamine LTX (Life
Technology, Milan, Italy) following the manufacturer’s in-
struction. After 24 h, the cells were treated with vehicle
(DMSO 0.003%) and HU595 and HU596 (up to 50 μM).
2
0 mg/kg morphine and were monitored in empty cages for
signs of respiratory distress. Twenty four hours later, the rats
in each group were pretreated with an i.p. injection of VEH
(n = 10) or 5 mg/kg HU595 (n = 10) 10 min prior to receiving
a s.c. injection of saline or naloxone. Over the next 48 h, this
cycle was repeated once more, except 10 min following the