Acute naloxone-precipitated morphine withdrawal elicits nausea-like somatic behaviors in rats in a manner suppressed by N-oleoylglycine

Article abstract of DOI:10.1007/s00213-019-05373-2

Rationale: Acute naloxone-precipitated morphine withdrawal (MWD) produces a conditioned place aversion (CPA) in rats even after one or two exposures to high-dose (20 mg/kg, sc) morphine followed 24-h later by naloxone (1 mg/kg, sc). However, the somatic withdrawal reactions produced by acute naloxone-precipitated MWD in rats have not been investigated. A recently discovered fatty acid amide, N-oleoylglycine (OlGly), which has been suggested to act as a fatty acid amide hydrolase (FAAH) inhibitor and as a peroxisome proliferator-activated receptor alpha (PPARα) agonist, was previously shown to interfere with a naloxone-precipitated MWD-induced CPA in rats. Objectives: The aims of these studies were to examine the somatic withdrawal responses produced by acute naloxone-precipitated MWD and determine whether OlGly can also interfere with these responses. Results: Here, we report that following two exposures to morphine (20 mg/kg, sc) each followed by naloxone (1 mg/kg, sc) 24 h later, rats display nausea-like somatic reactions of lying flattened on belly, abdominal contractions and diarrhea, and display increased mouthing movements and loss of body weight. OlGly (5 mg/kg, ip) interfered with naloxone-precipitated MWD-induced abdominal contractions, lying on belly, diarrhea and mouthing movements in male Sprague–Dawley rats, by both a cannabinoid 1 (CB₁) and a PPARα mechanism of action. Since these withdrawal reactions are symptomatic of nausea, we evaluated the potential of OlGly to interfere with lithium chloride (LiCl)-induced and MWD-induced conditioned gaping in rats, a selective measure of nausea; the suppression of MWD-induced gaping reactions by OlGly was both CB₁ and PPARα mediated. Conclusion: These results suggest that the aversive effects of acute naloxone-precipitated MWD reflect nausea, which is suppressed by OlGly.

Full text of DOI:10.1007/s00213-019-05373-2

Psychopharmacology
https://doi.org/10.1007/s00213-019-05373-2
ORIGINAL INVESTIGATION
Acute naloxone-precipitated morphine withdrawal elicits nausea-like
somatic behaviors in rats in a manner suppressed by N-oleoylglycine
1
1
1
1
1
1
Erin M Rock & Samantha M Ayoub & Cheryl L Limebeer & Alexia Gene & Kiri L Wills & Marieka V DeVuono
&
2
3,4
5
2
1
Reem Smoum & Vincenzo Di Marzo & Aron H Lichtman & Raphael Mechoulam & Linda A Parker
Received: 17 June 2019 /Accepted: 30 September 2019
#
Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Rationale Acute naloxone-precipitated morphine withdrawal (MWD) produces a conditioned place aversion (CPA) in rats even
after one or two exposures to high-dose (20 mg/kg, sc) morphine followed 24-h later by naloxone (1 mg/kg, sc). However, the
somatic withdrawal reactions produced by acute naloxone-precipitated MWD in rats have not been investigated. A recently
discovered fatty acid amide, N-oleoylglycine (OlGly), which has been suggested to act as a fatty acid amide hydrolase (FAAH)
inhibitor and as a peroxisome proliferator-activated receptor alpha (PPARα) agonist, was previously shown to interfere with a
naloxone-precipitated MWD-induced CPA in rats.
Objectives The aims of these studies were to examine the somatic withdrawal responses produced by acute naloxone-precipitated
MWD and determine whether OlGly can also interfere with these responses.
Results Here, we report that following two exposures to morphine (20 mg/kg, sc) each followed by naloxone (1 mg/kg, sc) 24 h
later, rats display nausea-like somatic reactions of lying flattened on belly, abdominal contractions and diarrhea, and display
increased mouthing movements and loss of body weight. OlGly (5 mg/kg, ip) interfered with naloxone-precipitated MWD-
induced abdominal contractions, lying on belly, diarrhea and mouthing movements in male Sprague–Dawley rats, by both a
cannabinoid 1 (CB ) and a PPARα mechanism of action. Since these withdrawal reactions are symptomatic of nausea, we
1
evaluated the potential of OlGly to interfere with lithium chloride (LiCl)-induced and MWD-induced conditioned gaping in rats,
a selective measure of nausea; the suppression of MWD-induced gaping reactions by OlGly was both CB and PPARα mediated.
1
Conclusion These results suggest that the aversive effects of acute naloxone-precipitated MWD reflect nausea, which is sup-
pressed by OlGly.
.
.
.
.
.
.
Keywords N-oleoylglycine Acute naloxone-precipitated morphine withdrawal CB PPARα Nausea Somatic MWD
Gaping Rats
1
.
Abbreviations
AEA
AM
anandamide
AM251
*
Linda A Parker
parkerl@uoguelph.ca
ANOVA analysis of variance
1
2
3
4
Department of Psychology and Collaborative Neuroscience
Program, University of Guelph, Guelph, ON N1G 2W1, Canada
CB1
CB2
CPA
FAAH
ip
LiCl
LOB
M
cannabinoid 1
cannabinoid 2
Institute for Drug Research, Medical Faculty, Hebrew University,
Jerusalem, Israel
conditioned place aversion
fatty acid amide hydrolase
intraperitoneal
lithium chloride
lying on belly
Institute of Biomolecular Chemistry, Endocannabinoid Research
Group, Consiglio Nazionale delle Richerche, Pozzuli, Naples, Italy
Canada Excellence Research Chair on the Microbiome/
Endocannabinoid Axis in Metabolomic Health, University of Laval,
Quebec City, Canada
morphine
MK
MWD
N
MK886
morphine withdrawal
naloxone
5
Department of Pharmacology and Toxicology, Medical College of
Virginia Campus, Virginia Commonwealth University,
Richmond, VA, USA

Psychopharmacology
OEA
OlGly
PEA
PPARα
S
N-oleoylethanolamide
N-oleoylglycine
N-palmitoylethanolamide
peroxisome proliferator-activated receptor alpha
the potential of OlGly to interfere with naloxone-precipitated
MWD-induced CPA was instead prevented by pretreatment
with a CB receptor antagonist (AM251; Petrie et al. 2019),
1
suggesting that its action as a FAAH inhibitor may have been
sufficient to elevate AEA levels and reduce the aversive with-
saline
sc
TR
VEH
subcutaneous
taste reactivity
vehicle
drawal reactions via CB receptors (Ramesh et al. 2011).
1
Although Petrie et al. (2019) reported that OlGly interfered
with the aversive properties of acute naloxone-precipitated
MWD, it has not been determined if this lipid mediator would
also interfere with the somatic effects produced by acute
naloxone-precipitated MWD, which are mediated by action at
different neurobiological substrates (e.g., Koob et al. 1989;
Maldonado et al. 1996; Stinus et al. 1990). There has been little
investigation of the somatic behavioral effects produced by
acute naloxone-precipitated MWD. In a single study in mice,
Shoblock and Maidment (2006) reported that following a single
exposure to morphine, naloxone produced only the somatic
reaction of jumping. The present study first aimed to determine
the nature of somatic symptoms of acute naloxone-precipitated
MWD in rats. Following two trial cycles of morphine (20
mg/kg, sc) followed 24 h by naloxone (1 mg/kg), rats displayed
the nausea-like behaviors of abdominal contractions, diarrhea,
and the abnormal posture of lying on belly (a response seen in
rats experiencing LiCl malaise; Contrearas et al. 2007; Parker
et al. 1984; Meachum and Bernstein 1992; Tuerke et al. 2012),
as well as the behavior of mouthing movements (chewing) and
loss of body weight. Pretreatment with OlGly (5 mg/kg, intra-
peritoneal [ip]) interfered with these somatic withdrawal reac-
tions, without having any behavioral effect on its own. The
suppression of acute MWD by OlGly was reversed with either
Introduction
Acute naloxone-precipitated morphine withdrawal (MWD)
occurs even after one or two exposures to high-dose morphine
followed several hours later by naloxone in humans
(
(
Heishman et al. 1990; June et al. 1995) and other animals
Eisenberg 1982; Martin and Eades 1964). Using the acute
naloxone-precipitated MWD paradigm, it has been demon-
strated that the aversive properties of naloxone are dramatical-
ly enhanced when preceded 24–48 h by a high dose of mor-
phine (20 mg/kg, subcutaneous [sc]; Parker and Joshi 1998;
Parker et al. 2002; Shoblock and Maidment 2006; Wills et al.
2
016). Rats injected with naloxone 24 h after saline did not
display a conditioned place aversion (CPA). On the other
hand, rats injected with naloxone 24 h after morphine
displayed a dramatic naloxone-induced CPA, presumably be-
cause morphine treatment promotes constitutive activity of the
mu opioid receptors uncovering the aversive inverse agonist
property of naloxone (Shoblock and Maidment 2006). Indeed,
Wills et al. (2016) used this paradigm to investigate the role of
the endocannabinoid system in the regulation of the affective
properties of MWD.
We recently reported that N-oleoylglycine (OlGly), an en-
dogenous fatty acid amide mediator structurally similar to N-
acylethanolamines, which includes the endocannabinoid, anan-
damide (AEA), as well as the endogenous peroxisome
proliferator-activated receptor alpha (PPARα) agonists
N-oleoylethanolamide (OEA) and N-palmitoylethanolamide
a CB receptor antagonist or a PPARα antagonist. Given that
1
acute naloxone-precipitated MWD has been shown (McDonald
et al. 1997) to produce conditioned gaping reactions (Grill and
Norgren 1978) in rats, a selective measure of nausea-like
responding (Parker et al., 2014), and that a subset of the somatic
behaviors were indicative of nausea (abdominal contractions,
LOB, diarrhea), a reported withdrawal reaction in humans
(Jaffee and Martin 1990), we also determined that OlGly inter-
fered with conditioned gaping reactions produced by LiCl, as
well as with LiCl-induced vomiting in the Suncus murinus, an
animal model of emesis. Finally, we determined that OlGly
interfered with naloxone-precipitated MWD-induced condi-
(
(
PEA), interferes with nicotine reward and dependence in mice
Donvito et al. 2019) and with acute naloxone precipitated
MWD-induced conditioned place aversion (CPA) in rats
Petrie et al. 2019). OlGly does not bind cannabinoid 1 (CB1)
(
or cannabinoid 2 (CB ) receptors in vitro and does not produce
tioned gaping in rats, by both a CB and PPARα mechanism
2
1
the typical CB receptor tetrad (Martin et al. 1991) of behaviors
of action.
1
(
antinociception, hypothermia, catalepsy, and hypomobility)
characteristic of CB receptor agonists, but it weakly inhibits
1
fatty acid amide hydrolase (FAAH: IC50 8.65 μM; 89% inhibi-
tion of FAAH) and also binds PPARα (assessed by a modeling
experiment) and behaved as a PPARα agonist in a specific
luciferase assay for functional activity (Donvito et al. 2019). It
is of interest to note that, while the potential of OlGly to interfere
with nicotine reward in mice was prevented by pretreatment
with a PPARα antagonist (MK866; Donvito et al. 2019) and
Materials and methods
Subjects
The subjects were male Sprague–Dawley rats weighing be-
tween 257 and 297 g on the first treatment day. They were
obtained from Charles River QC and pair-housed (for CPA

Psychopharmacology
experiments) or individually housed (for conditioned gaping
response experiment) in a shoebox cage with ad lib access to
food and water. The colony room was on a 24-h light dark
cycle (7 AM; lights off; 7 PM lights on) such that behavioral
testing was conducted during the dark phase of the light cycle.
The Suncus murinus (house musk shrews) used in experiment
The product was extracted with ether (3 × 50 ml) and dried
(MgSO ), and solvent was evaporated under reduced pres-
4
sure. The crude material was chromatographed on silica
gel (eluting with chloroform/methanol) to yield a crystal-
line solid. Melting point 93–94 C (degradation); LC-MS:
+
(M-H) = 339 m/z; NMR (CD OH, ppm): 5.35–5.32
3
5
were bred and raised in the University of Guelph colony and
(m,2H), 4.45 (s,2H), 2.13–2.18 (m, 6H), 1.58 (m, 2H),
1.32–1.29 (m, 20H), 0.88 (t, 3H).
ranged from 123 to 376 days old at the time of testing. Naive
male (n = 7; 39.4–42.2 g) and female (n = 5; 25.9–26.5 g)
were individually housed in opaque white mouse cages in the
colony room at an ambient temperature of 21 °C on a 10/14-h
light-dark schedule (lights off at 7 PM). Shrews were tested in
their light cycle. They were provided with an open-ended
plastic cylinder (4 × 8 cm) containing enviro-paper and main-
tained on Medical/Royal Canin Feline Maintenance mixed
with Harlan Ferret dry chow and had ad libitum access to
water. All animal procedures were approved by the Animal
Care Committee of the University of Guelph and adhere to the
guidelines of the Canadian Council of Animal Care.
Apparatus
For investigation of the acute naloxone-precipitated MWD
somatic reactions in experiments 1 and 2, the observation
chamber consisted of four black Plexiglas boxes (22.5 × 26
× 20 cm) with an opaque lid, sitting on top of a clear glass-
topped table. There was a close circuit Panasonic WV-CP484
video camera placed below each of the chambers to video-
record somatic withdrawal behaviors that was fire-wired to a
computer for later scoring using “The Observer” Event re-
cording software (Noldus Information Technology Inc,
Leesburg, VA).
Drugs
For investigation of conditioned gaping reactions in exper-
iments 3 and 5, the rats were placed in taste reactivity (TR;
Grill and Norgren, 1978) chambers with their cannula attached
to an infusion pump (Model KDS100, KD Scientific,
Holliston, MA, USA) for fluid delivery. The TR chamber
was in a dark room next to a 25 W light source. The TR
chambers used were made of clear Plexiglas (22.5 × 26 × 20
cm) and sat on a table with a clear glass top. A mirror beneath
the chamber at a 45° angle facilitated viewing of the ventral
surface of the rat to observe orofacial responses. A Sony video
camera (Handycam, Henry’s Camera Waterloo ON) with a
fire-wire connection to computer was focused on the mirror
and used to record the rats from the mirror beneath the cham-
ber. The videos were later scored using “The Observer” Event
recording software (Noldus Information Technology Inc,
Leesburg, VA).
Morphine and naloxone were prepared in saline at a concen-
tration of 20 mg/ml and 1 mg/ml, respectively, and adminis-
tered subcutaneously (sc). Oleoyl glycine (OlGly) was dis-
solved in a vehicle (VEH) mixture of ethanol, Tween 80,
and physiological saline in a 1:1:18 ratio. OlGly was first
dissolved in ethanol, Tween 80 was then added to the solution,
and the ethanol was evaporated off with a nitrogen stream,
after which, the saline was added. The final VEH consisted
of 1:9 (Tween80/saline). OlGly was prepared at a concentra-
tion of 5 mg/ml and was administered intraperitoneally (ip).
MK886 (MK; Cayman Chemicals) and AM251 (Cayman
Chemicals) were prepared in the same VEH as the OlGly at
a concentration of 1 mg/ml and given ip at a dose of 1 mg/kg
(
Rock et al. 2017; Wills et al. 2014). LiCl (Sigma) was pre-
pared as a 0.15 M solution with sterile water and administered
ip at a volume of 20 ml/kg (for rats) and 60 ml/kg (for shrews;
the dose necessary to establish vomiting, Parker et al. 2004).
For investigation of shrew vomiting in experiment 4, the
clear Plexiglas observation chambers (22.5 × 26 × 20 cm)
were placed on a table with a clear glass top. A mirror at a
45° angle beneath the chamber facilitated viewing of the ven-
tral surface of each shrew.
Synthesis of Oleoyl glycine
A solution of oleic acid (1 g, 3.54 mmol) and N,N-
dimethylformamide (266 μl, 3.64 mmol) in dry methylene
chloride (10 ml) was added dropwise oxalyl chloride
Procedure
(
2.0 M solution in methylene chloride, 3.5 ml, 7 mmol)
Experiment 1: effect of OlGly on acute naloxone-precipitated
MWD somatic reactions. Upon arrival in the laboratory, the rats
were handled for 3 days prior to treatments. There were a total
of six groups (n = 8/group, S = saline, M = morphine, V =
VEH, OG = OlGly, N = naloxone): S-VN, S-VS, M-VN, M-
VS, S-OGS, M-OGN. The rats received two trial cycles 24 h
apart to evaluate the potential of naloxone to promote somatic
withdrawal 24 h following morphine. On the first day of each
under nitrogen atmosphere. The reaction mixture was
stirred for 1 h, and then, the solvent was evaporated under
a nitrogen flow. The crude material in methylene chloride
(
10 ml) was added to a solution of glycine (800 mg, 10.62
mmol) and 2 N potassium hydroxide in an ice bath. Then,
the reaction mixture was stirred for 1 h, water (10 ml) was
added, and the mixture was acidified to pH 3 with 1 N HCl.

Psychopharmacology
cycle, all rats were weighed and received either a 1-ml/kg sc
injection of saline or morphine (20 mg/kg, sc) and were placed
in an empty shoebox cage in the colony room. The rats were
monitored for signs of respiratory distress and returned to their
home cage once fully ambulatory. Two hours after morphine,
the rats were weighed again to determine potential weight loss.
On the second day of each cycle, 24 h after the morphine or
saline injections, all rats were weighed before they were
injected ip (1 ml/kg) with VEH or OlGly (5 mg/kg, sc) and
Experiment 3: effect of OlGly on LiCl-induced conditioned
gaping in rats All rats were surgically implanted with an
intraoral cannula under isofluorane anesthesia according to
the procedure described by Limebeer et al. (2010).
Following 3-day recovery from surgery, the rats received
an adaptation trial in which they were placed in the TR
chamber with their cannula attached to an infusion pump
(Model KDS100, KD Scientific, Hollliston, MA, USA) for
fluid delivery. Water was infused into their intraoral
cannuale for 2 min at the rate of 1 ml/min. The condition-
ing groups included V-LiCl (n = 7), OlGly-Saline (n = 8),
and OlGly-LiCl (n = 8). During conditioning, the rats were
injected with OlGly (5 mg/kg, ip) or VEH 20 min prior to
being individually placed in the TR chamber and
intraorally infused with 0.1% saccharin solution for 2 min
at the rate of 1 ml/min while the orofacial responses were
video recorded. Immediately after the saccharin infusion,
the rats were injected with 20 ml/kg of 0.15 M LiCl or
equivolume saline and returned to their home cage.
Seventy-two hours later, the rats were tested drug-free.
They were again intraorally infused with 0.1% saccharin
solution for 2 min at the rate of 1 ml/min while the
orofacial reactions were video recorded. The videotapes
were later scored by an observer blind to the experimental
conditions using The Observer for the behavior of gaping
(large openings of the mouth and jaw, with lower incisors
exposed).
10 min later were injected sc (1 ml/kg) with saline or naloxone
(1 mg/kg, sc). Ten minutes later, they were placed in the ob-
servation chamber for 30 min and monitored for presence of
diarrhea (yes/no). On the final trial, the rats’ somatic behaviors
were videotaped from below the chambers. Two hours after the
sc naloxone or saline injection, the rats were weighed again.
The video tapes were later scored using The Observer for the
behaviors listed in Table 1 which are typically produced by
withdrawal from chronic exposure to morphine (Gellert and
Sparber, 1977; Koob 2009; Maldonado et al. 1996).
Experiment 2: effect of a PPARα antagonist and a CB1 antag-
onist on OlGly inhibition of acute naloxone-precipitated MWD
somatic reactions We evaluated if the interference with somat-
ic withdrawal by OlGly was PPARα-mediated, using the
PPARα antagonist, MK886, or CB receptor mediated, using
1
the CB receptor antagonist, AM251 with rats assigned to one
1
of 6 groups: M-VN (n = 8), M-OGN (n = 8), M-MKVN (n =
8
(
), M-MKOGN (n = 7), M-AMVN (n = 7), and M-AMOGN
n = 8). Rats were treated as in experiment 1, but all rats were
injected with morphine (20 mg/kg, sc) 24 h prior to naloxone
1 mg/kg, sc). To determine the mechanism of the suppression
Experiment 4: effect of OlGly on LiCl-induced vomiting in
shrews Male (n = 7) and female (n = 5) shrews were trans-
ferred from the colony room to an empty cage in the ex-
perimental room that contained four meal worms. After 15
min, they were injected with VEH (3M/3F) or 5 mg/kg, ip,
OlGly (4M/2F). Twenty minutes later, they were injected
with LiCl and put into an observation chamber for 45 min.
An observer recorded the frequency of vomiting episodes
using The Observer.
(
of the somatic effects of MWD by OlGly, the rats were
pretreated with MK886 (1 mg/kg, sc) or AM251 (1 mg/kg
sc) 15 min prior to receiving an injection of OlGly (5 mg/kg,
sc) or VEH 10 min prior to naloxone. The videotapes of the
final trial were scored for the acute naloxone-precipitated
MWD responses found in experiment 1.
Table 1 Acute naloxone-precipitated MWD reactions
Behavior
Type
Definition
Weight loss
gm
Percentage of weight loss after 2 h
Body/genital grooming
Rearing
Duration
Duration
Duration
Frequency
Frequency
Frequency
Yes/no
Licking/grooming/sucking their fur/body/genitals
Lifting both paws off of the floor
Lying on belly (abnormal posture)
Abdominal contractions
Yawning
Lying flat on their belly with feet tucked under; belly flattened on floor
Both sides of the abdomen constricting inwards
A wide opening of the mouth, accompanied by a stretch upwards
Shaking out their fur from head to toe
Wet dog shakes
Diarrhea
Watery, non-solid feces
Mouthing movements (reflecting chewing)
Frequency
Opening their mouth or moving it around from side to side

Psychopharmacology
Experiment 5: effect of OlGly on naloxone-precipitated MWD-
induced conditioned gaping reactions in rats and mechanism
of action Experiment 5 was conducted in a similar manner as
was experiment 3, except that the rats received two naloxone-
precipitated MWD conditioning trials, to be consistent with the
procedures of Petrie et al. (2019) and those of experiments 1
and 2. Three days after intraoral cannulation surgery, the rats
were given an adaptation trial to the TR procedures (as in ex-
periment 3). Twenty-four hours later, conditioning began. They
were assigned to one of six groups: M-VN (n = 7), M-OGN (n
evidence of acute naloxone-precipitated withdrawal are re-
ported here. These behaviors included mouthing movements
(probably reflecting chewing), abdominal contractions, lying
on belly (LOB), instances of diarrhea (yes or no), and percent-
age body weight loss. OlGly interfered with naloxone-
precipitated MWD-induced mouthing movements, abdominal
contractions, lying on belly, and diarrhea, but not body weight
loss. Figure 1 presents each of these behaviors for the various
groups. The single factor ANOVA for each of these behaviors
revealed a significant main effect: mouthing movement, F(5,
42) = 15.7; p < 0.001; lying on belly, F(5, 42) = 13.6; p <
0.001; abdominal contractions, F(5, 42) = 47.5; p < 0.001;
percent body weight loss, F(5, 42) = 4.2; p = 0.004.
Subsequent Bonferroni post hoc tests revealed that group M-
VN displayed significantly more mouthing movements (p <
0.001), lying on belly (p < 0.001), abdominal contractions (p <
0.001) than all groups, including M-OGN. For the percentage
of body weight loss, group M-VN displayed more weight loss
than all groups (p < 0.025) except group M-OGN (which did
not significantly differ from any other groups).
=
8
8), M-MKVN (n = 8), M-MKOGN (n = 8), M-AMVN (n =
), and M-AMOGN (n = 8). We have previously demonstrated
that naloxone alone does not produce conditioned gaping in rats
McDonald et al. 1997; Parker and Rennie 1992). On the first
(
day of each cycle, all rats received morphine (20 mg/kg, sc) and
were placed in an empty shoebox cage in the colony room. The
rats were monitored for signs of respiratory distress and
returned to their home cage once fully ambulatory. On the sec-
ond day of each cycle, 24 h after the morphine injection, all rats
were injected ip (1 ml/kg) with VEH or OlGly (5 mg/kg, sc)
and 20 min later were individually placed in the TR chamber
and intraorally infused with 0.1% saccharin solution for 2 min
at the rate of 1 ml/min. Immediately after the saccharin infusion,
the rats were injected with naloxone (1 mg/kg) and returned to
their home cage. Additional rats were pretreated with MK886
Experiment 2: effect of a PPARα antagonist and a CB antag-
1
onist on OlGly inhibition of acute naloxone-precipitated MWD
somatic reactions The effect of OlGly on acute naloxone-
precipitated MWD somatic reactions was prevented by
(1 mg/kg) or AM251 (1 mg/kg) 15 min prior to administration
both the PPARα antagonist and by the CB antagonist,
1
of OlGly or VEH.
whereas neither antagonist per se produced any effect.
Figure 2 presents the mean number of each of the acute
naloxone-precipitated MWD somatic reactions in experi-
ment 2. The one-way ANOVAs revealed a significant ef-
fect among groups for mouthing movements, F(5, 40) =
9.4; p < 0.001, abdominal contractions, F(5, 50) = 17.4;
p < 0.001, and for lying on belly, F(5, 40) = 9.1; p < 0.001,
and for instances of diarrhea, F(5, 40) = 3.5, p = 0.01.
Bonferroni post hoc comparisons among groups revealed
that group MOGN displayed fewer mouthing movements,
abdominal contractions, and spent less time lying on belly
than all other groups (p < 0.001), which did not differ from
one another. For the measure of instances of diarrhea,
Bonferroni tests revealed that groups M-AMVN and M-
AMOGN differed significantly (p < 0.05) from group M-
OGN, and less conservative least significant difference
tests revealed that all groups differed significantly from
M-OGN.
Seventy-two hours after the second conditioning trial, the
rats were tested drug-free. They were again placed in the TR
chamber and intraorally infused with 0.1% saccharin solution
for 2 min at the rate of 1 ml/min while the orofacial reactions
were video recorded. The videotapes were later scored by an
observer blind to the experimental conditions using The
Observer for the behavior of gaping (large openings of the
mouth and jaw, with lower incisors exposed).
Analysis
In experiments 1 and 2, each measure described in Table 1 was
entered into a one-way analysis of variance (ANOVA), with
subsequent Bonferroni post hoc tests. In experiments 3 and 5,
the number of gaping reactions during the drug-free TR test
were entered into a one-way ANOVA with subsequent
Bonferroni post hoc tests. In experiment 4, the number of
LiCl-induced vomiting reactions displayed by male and female
shrews pre-treated with VEH or OlGly were entered into a 2 × 2
between factors ANOVA. Significance is defined as p < 0.05.
Experiment 3: effect of OlGly on LiCl-induced conditioned
gaping in rats OlGly interfered with LiCl-induced gaping
reactions. Figure 3 presents the mean number of gapes
displayed by LiCl-conditioned rats. The one-way
ANOVA revealed a significant effect of group, F(2, 20) =
Results
2
5.0; p < 0.001; subsequent Bonferroni tests revealed that
Experiment 1: effect of OlGly on acute naloxone-precipitated
group VEH-LiCl gaped significantly (p < 0.001) more than
MWD somatic reactions Only the behaviors that revealed
any other group.

Psychopharmacology
Fig. 1 Mean (±SEM) number of mouthing movements, number of
abdominal contractions, seconds of lying on belly, number of rats
displaying diarrhea, and percent body weight loss of rats in the various
groups in experiment 1. Rats in group M-VN displayed significantly
more of these withdrawal behaviors than any other group, except for body
weight loss in which rats in group M-VN did not differ from rats in group
M-OGN signified by . ***p < 0.001; *p < 0.05
1
Experiment 4: effect of OlGly on LiCl-induced vomiting in
shrews OlGly reduced LiCl-induced vomiting in the
S. murinus. Figure 4 presents the mean number of vomiting
episodes displayed by the male or female S. murinus follow-
ing VEH or OlGly (5 mg/kg) pretreatment injections. The
number of vomiting episodes was entered into a two-factor
between analysis of variance (ANOVA) with the factors of
pretreatment group and sex. The ANOVA revealed a
significant group effect, F(1, 8) = 29.7; p = 0.001 for
vomiting, and a non-significant sex effect F(1, 8) = 0.7; p >
0.05, and a non-significant group × sex interaction, F(1, 8) =
0.1; p > 0.05.
Experiment 5: effect of OlGly on acute naloxone-precipitated
MWD-induced conditioned gaping in rats and mechanism of
action OlGly interfered with MWD-induced conditioned

Psychopharmacology
Fig. 2 Mean (±SEM) number of
mouthing movements, number of
abdominal contractions, seconds
of lying on belly, and number of
rats displaying diarrhea in the
various groups in experiment 2.
MK = MK886, AM = AM251.
Rats in group M-OGN displayed
fewer of these withdrawal behav-
iors than any other group. ***p <
0
.001, *p < 0.05
gaping in rats in a manner mediated by both a CB receptor
Discussion
1
and a PPARα mechanism. Figure 5 presents the mean number
of gaping reactions displayed by the various groups during the
final TR test trial. A one-way ANOVA revealed a significant
effect of group, F(5, 41) = 3.5; p = 0.01. Subsequent
Bonferroni post hoc comparison tests revealed that group M-
OGN displayed significantly fewer conditioned gapes at test
than did all other groups (p’s < 0.05).
This was the first study to examine the somatic withdrawal
reactions displayed by male Sprague–Dawley rats in the acute
naloxone-precipitated withdrawal paradigm. Among the myriad
of withdrawal reactions reported in the literature following with-
drawal from chronic exposure to morphine, the only responses
evident from acute naloxone-precipitated MWD were the
Fig. 3 Mean (±SEM) number of gapes elicited by LiCl-paired saccharin
during the drug-free TR test of experiment 3. ***p < 0.001
Fig. 4 Mean (±SEM) number of LiCl elicited vomiting episodes by male
and female shrews over a 45-min test. ***p < 0.001

Psychopharmacology
appear to mediate the somatic MWD behavioral effects and
MWD-induced nausea, suggesting cross-talk between these
two systems in the regulation of nausea. Indeed, we have
previously reported that FAAH inhibition inhibits acute nau-
sea by its action on PPARα (Rock et al. 2015, 2017, 2019),
but anticipatory nausea by its action on CB receptors (Rock
1
et al. 2015). On the other hand, MAGL inhibition inhibits both
acute and anticipatory nausea by its action on CB receptors
1
(Parker et al. 2015; Sticht et al. 2016).
The somatic withdrawal responses elicited by acute
naloxone-precipitated MWD are reminiscent of those elicited
unconditionally by the nausea-inducing drug LiCl in rats
(Contreras et al. 2007; Parker et al. 1984; Tuerke et al. 2012).
That is, pretreatment with LiCl in rats can produce the abnor-
mal posture of lying on belly and abdominal contractions.
These reactions can be suppressed by the anti-emetic serotonin
(5-HT) 3 antagonist, ondansetron (Tuerke et al. 2012). Acute
naloxone-precipitated MWD has been shown to produce the
nausea-induced behavior of conditioned gaping in rats
(McDonald et al. 1997). Nausea is a primary symptom report-
ed by humans experiencing MWD (Jaffee and Martin 1990).
Conditioned gaping reactions (Grill and Norgren 1978) in
rats are only produced by emetic drugs and are selectively sup-
pressed by antiemetic drugs (see Parker 2014 for review). The
potential of OlGly to interfere with the somatic effects of acute
naloxone-precipitated MWD may therefore reflect its potential
to interfere with nausea. Accordingly, we found here that OlGly
interfered with LiCl-induced conditioned gaping reactions in
rats and with LiCl-induced vomiting in the Suncus murinus.
Additionally, OlGly interfered with acute naloxone-
precipitated MWD-induced conditioned gaping reactions in
rats. This latter effect of the mediator, like its effect on somatic
withdrawal elicited by acute naloxone-precipitated MWD, was
Fig. 5 Mean (±SEM) number of gapes elicited by acute naloxone-
precipitated MWD paired saccharin solution among the various groups
during the drug-free TR test of experiment 4. *p < 0.05
nausea-like behaviors of lying on belly, abdominal contractions,
and diarrhea, as well as mouthing movements (chewing) and
body weight loss. These reactions were not simply a response to
naloxone, because the S-VN group showed none of these be-
haviors. Shoblock and Maidment (2006) previously demon-
strated in mice that the predominant reaction to acute
naloxone-precipitated MWD was jumping; however, in rats,
we found no evidence of jumping behavior in any group. This
may have been due to the size of our chambers which may have
limited the display of this response, or due to species differences
in responding to acute naloxone-precipitated MWD.
Similar to the capability of OlGly to interfere with the af-
fective properties of acute naloxone-precipitated MWD-in-
duced CPA (Petrie et al. 2019), OlGly also interfered with
the nausea-like somatic withdrawal reactions (as well as
mouthing movements) of acute MWD. The suppression of
these MWD reactions by OlGly was reversed by both the
CB1 receptor antagonist, AM251, and the PPARα
antagonist, MK886. Petrie et al. (2019) previously reported
that OlGly interfered with acute naloxone-precipitated
again attenuated by both CB and PPARα receptor antagonists,
1
thus supporting our hypothesis that the inhibitory effect of
OlGly on the somatic responses might be a consequence of its
anti-nausea effects.
Our investigation of the potential of OlGly to interfere with
the aversive components of MWD has been limited to the
acute naloxone-precipitated MWD paradigm because of the
precision in timing of inducing the effect for the use in place
conditioning/nausea studies. A single exposure to high-dose
morphine (20 mg/kg, sc) is sufficient to demonstrate the aver-
sive effects of naloxone-precipitated MWD 24 h (and even 48
h; Parker et al. 2002) later, while multiple exposures produce
yet greater conditioned effects. However, it will also be im-
portant in future studies to investigate the potential of OlGly to
interfere with withdrawal from chronic exposure to morphine,
which has greater translational appeal. Understanding the neu-
ral substrates of both the positive and negative reinforcement
of motivational withdrawal from opiates is critical for
comprehending the chronic, relapsing compulsive use of opi-
ates which produce a significant dysregulation of brain
MWD-induced CPA in rats by a CB receptor mechanism of
1
action, whereas Donvito et al. (2019) previously reported that
OlGly interfered with nicotine-induced CPP in mice by a
PPARα mechanism of action. The present results suggest that
OlGly acts by both mechanisms to interfere with the somatic
properties of naloxone-precipitated MWD. OlGly both in-
hibits FAAH and activates PPARα (Donvito et al. 2019).
Therefore, it is likely that OlGly is acting as a FAAH inhibitor,
thereby elevating AEA, PEA, and OEA. The action of elevat-
ed AEA at CB receptors was therefore prevented with the
1
CB receptor antagonist (AM251), whereas the action of the
1
PPARα agonists, PEA and OEA, as well as the direct action of
OlGly as a PPARα agonist were prevented by the PPARα
antagonist (MK886). It is interesting that both mechanisms

Psychopharmacology
Koob GF (2009) Dynamics of neuronal circuits in addiction: reward,
antireward, and emotional memory. Pharmacopsychiatry 42:S32–
S41. https://doi.org/10.1055/s-0029-1216356
Koob GF, Wall TL, Bloom FE (1989) Nucleus accumbens as a substrate
for the aversive stimulus effects of opiate withdrawal.
Psychopharmacology 98:530–534
Limebeer CL, Vemuri VK, Bedard H, Lang ST, Ossenkopp KP,
Makriyannis A et al (2010) Inverse agonism of cannabinoid CB1
receptors potentiates LiCl-induced nausea in the conditioned gaping
model in rats. Br J Pharmacol 161:336–349. https://doi.org/10.1111/
j.1476-5381.2010.00885.x
Maldonado R, Stinus L, Koob GF (1996) Neurobiological mechanisms of
opiate withdrawal. R.G. Landes Company, Austin
Martin WR, Eades CG (1964) A comparison between acute and chronic
physical dependence in the chronic spinal dog. J Pharmacol Exp
Ther 146:385–394
Martin BR, Compton DR, Thomas BF, Prescott WR, Little PJ,
Razdan RK et al (1991) Behavioral, biochemical, and molecu-
lar modeling evaluations of cannabinoid analogs. Pharmacol
Biochem Behav 40:471–478. https://doi.org/10.1016/0091-
hedonic systems (Koob 2009). We have demonstrated that
endogenous OlGly is elevated in the nucleus accumbens in
rat brains undergoing acute naloxone precipitated MWD
(
Petrie et al. 2019). The nucleus accumbens is a region within
the extended amygdala that has been implicated in regulation
of the negative reinforcement of motivational withdrawal
from opiates, coined as the “dark side of addiction” (Koob
2
009; Koob et al. 1989) which drives drug seeking. OlGly
may be released to regulate the deviation from homeostasis
produced by acute naloxone-precipitated MWD. In future
studies we will extend this investigation to determine if
OlGly or other protective fatty acid amides are released in
the nucleus accumbens, amygdala, and bed nucleus of the stria
terminalis (extended amygdala) in rats experiencing with-
drawal from chronic exposure to morphine.
Funding information The research described here was funded by re-
search grants from the Natural Sciences and Engineering Council of
Canada (NSERC: 03629), Canadian Institutes of Health Research
3
057(91)90349-7
McDonald RV, Parker LA, Siegel S (1997) Conditioned sucrose aver-
sions produced by naloxone-precipitated withdrawal from acutely
administered morphine. Pharmacol Biochem Behav 58:1003–1008.
https://doi.org/10.1016/S0091-3057(97)00313-4
(CIHR:388239) and a research contract from PlantExt to Linda A Parker.
Compliance with ethical standards
Meachum C, Bernstein IL (1992) Behavioral conditioned responses to
contextual andodor stimuli paired with LiCl administration. Physiol
Behav 52:895–899. https://doi.org/10.1016/0031-9384(92)90368-C
Parker LA (2014) Conditioned flavor avoidance and conditioned gaping:
rat models of conditioned nausea. Eur J Pharmacol 722:122–133.
https://doi.org/10.1016/j.ejphar.2013.09.070
Conflict of interest The authors declare that they have no conflicts of
interest.
Parker LA, Cyr JA, Santi AN, Burton PD (2002) The aversive properties
of acute morphine dependence persist 48 h after a single exposure to
morphine: evaluation by taste and place conditioning. Pharmacol
Biochem Behav 72:87–92. https://doi.org/10.1016/S0091-
References
Contreras M, Ceric F, Torrealba F (2007) Inactivation of the interoceptive
insula disrupts drug craving and malaise induced by lithium. Science
3
057(01)00724-9
Parker LA, Hills K, Jensen K (1984) Behavioral CRs elicited by a
lithium- or an amphetamine-paired contextual test chamber. Anim
Learn Behav 12:307–315. https://doi.org/10.3758/BF03199972
Parker LA, Joshi A (1998) Naloxone-precipitated morphine withdrawal
induced place aversions: effect of naloxone at 24 hours
postmorphine. Pharmacol Biochem Behav 61:331–333. https://doi.
org/10.1016/S0091-3057(98)00104-X
Parker LA, Kwiatkowska M, Burton P, Mechoulam R (2004) Effect of
cannabinoids on lithium-induced vomiting in the Suncus murinus
(house musk shrew). Psychopharmacology 171:156–161. https://
doi.org/10.1007/s00213-003-1571-2
Parker LA, Niphakis MJ, Downey R, Limebeer CL, Rock EM,
Sticht MA, Morris H, Abdullah R, Lichtman AH, Cravatt BF
(2014) A new MAGL inhibitor, MJN110, reduces acute and
anticipatory nausea in rats and vomiting in Suncus murinus.
Psychopharmacology 232:583–593. https://doi.org/10.1007/
s00213-016-4277-y
2
6:655–658. https://doi.org/10.1126/science.1145590
Donvito G, Piscitelli F, Muldoon P, Jackson A, Vitale RM, D’Aniello E
et al (2019) N-Oleoyl-glycine reduces nicotine reward and with-
drawal in mice. Neuropharmacology 148:320–331. https://doi.org/
1
0.1016/j.neuropharm.2018.03.020
Eisenberg RM (1982) Further studies on the acute dependence produced
by morphine in opiate naive rats. Life Sci 31:1531–1540. https://doi.
org/10.1016/0024-3205(82)90043-1
Gellert VF, Sparber SB (1977) A comparison of the effects of naloxone
upon body weight loss and suppression of fixed-ratio operant be-
havior in morphine-dependent rats. J Pharmacol ExpTher 201:44–
5
4
Grill HJ, Norgren R (1978) The taste reactivity test: 1 Mimetic
responses to gustatory stimuli in neurologically normal rats.
Brain Res 143:263–279. https://doi.org/10.1016/0006-
8
993(78)90568-1
Heishman SJ, Stitzer ML, Bigelow GE, Liebson IA (1990) Acute opioid
physical dependence in humans: effect of naloxone at 6 and 24 hours
postmorphine. Pharmacol Biochem Behav 36:393–399. https://doi.
org/10.1016/0091-3057(90)90421-D
Parker LA, Rennie M (1992) Naltrexone-induced aversions: Assessment
by the placeConditioning, taste reactivity and taste avoidance para-
digms. Pharmacol Biochem Behav 41:559–565. https://doi.org/10.
1016/0091-3057(92)90373-n
Jaffee JH, Martin WR (1990) Opioid analgesics and antagonists. In:
Gilman AG, Rall TW, Nies AS, Taylor P (eds) The pharmaco-
logical basis of therapeutics (8th ed), Pergamon, New York pp
Petrie GN, Wills KL, Piscitelli F, Smoum R, Limebeer CL, Rock EM et al
(2019) Oleoyl glycine: interference with the aversive effects of acute
naloxone-precipitated MWD, but not morphine reward, in male
Sprague-Dawley rats. Psychopharmacology 236:2623–2633.
https://doi.org/10.1007/s00213-019-05237-9
Ramesh D, Ross GR, Schlosburg JE, Owens R, Abdullah R, Kinsey SG
et al (2011) Blockade of endocannabinoid hydrolytic enzymes at-
tenuates precipitated opioid withdrawal symptoms in mice. J
4
85-521
June HL, Stitzer ML, Cone E (1995) Acute physical dependence: time
course and relation to human plasma morphine concentrations. Clin
Pharmacol Ther 57:270–280. https://doi.org/10.1016/0009-
9
236(95)90152-3

Psychopharmacology
Pharmacol Exp Ther 339:173–185. https://doi.org/10.1124/jpet.111.
81370
Neuropharmacology 102:92–102. https://doi.org/10.1016/j.
neuropharm.2015.10.039
1
Rock EM, Limebeer CL, Ward JM, Cohen A, Grove K, Niphakis MJ,
Cravatt BF, Parker LA. (2015).Fatty acid amide hydrolase (FAAH)
inhibition interferes with acute nausea by a PPARα mechanism and
anticipatory nausea by a CB1 receptor mechanism in a double dis-
sociation. Psychopharmacology, 232: 3841-3848. https://doi.org/10.
Stinus L, Le Moal M, Koob GF (1990) Nucleus accumbens and amyg-
dala are possible substrates for the aversive stimulus effects of opiate
withdrawal. Neuroscience 37:767–773. https://doi.org/10.1016/
0306-4522(90)90106-E
Tuerke KJ, Winters BD, Parker LA (2012) Ondansetron interferes with
unconditioned lying-on belly and acquisition of conditioned gaping
induced by LiCl as models of nausea-induced behaviors in rats.
Physiol Behav 105:856–860. https://doi.org/10.1016/j.physbeh.
1
007/s00213-015-4050-7
Rock EM, Moreno-Sanz G, Limebeer CL, Petrie G, Angelini R, Piomelli
D et al (2017) Suppression of acute and anticipatory nausea by
peripherally restricted FAAH inhibitor in animal models: role of
PPARa and CB1 receptors. Br J Pharmacol 174:3837–3847.
https://doi.org/10.1111/bph.13980
Rock EM, Limebeer CL, Aliasi-Sanai L, Parker LA (2019) The ventral
pallidum as a critical region regulating fatty acid amide hydrolase
inhibition of nausea-induced conditioned gaping in male Sprague-
Dawley rats. Neuropharmacology 155:142–149. https://doi.org/10.
2
011.10.017
Wills KL, Petrie GN, Millett G, Limebeer CL, Rock EM, Niphakis MJ
et al (2016) Double dissociation of monoacylglycerol lipase inhibi-
1
tion and CB antagonism in the central amygdala, basolateral amyg-
dala, and the interoceptive insular cortex on the affective properties
of acute naloxone-precipitated morphine withdrawal in rats.
Neuropsychopharmacology 41:1865–1873. https://doi.org/10.
1
016/j.neuropharm.2019.05.031
1
038/npp.2015.356
Shoblock JR, Maidment NT (2006) Constitutively active mu opioid
receptors mediate the enhanced conditioned aversvie effect of
n a l o x o n e i n m o r p h i n e - d e p e n d e n t m i c e .
Neuropsychopharmacology 31:171–177. https://doi.org/10.
Wills KL, Vemuri K, Kalmar A, Lee A, Limebeer CL, Makriyannis A
et al (2014) CB1 antagonism: interference with affective properties
of acute naloxone-precipitated morphine withdrawal in rats.
Psychopharmacology 231:4291–4300. https://doi.org/10.1007/
s00213-014-3575-5
1
038/sj.npp.1300782
Sticht MA, Limebeer CL, Rafla BR, Abdullah RA, Poklis JL, Ho W,
Niphakis MJ, Cravatt BF, Sharkey KA, Lichtman AH, Parker LA
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
(
2016) Endocannabinoid regulation of nausea is mediated by 2-
arachidonolyglycerol (2-AG) in the rat visceral insular cortex.