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Cannabinoid CB 1 and CB 2 receptor mechanisms underlie cannabis reward and aversion in rats

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Background and purpose: Endocannabinoids are critically involved in brain reward functions, mediated by activation of CB1 receptors, reflecting their high density in the brain. However, the recent discovery of CB2 receptors in the brain, particularly in the midbrain dopamine neurons, has challenged this view and inspired us to re-examine the roles of both CB1 and CB2 receptors in the effects of cannabis. Experimental approach: In the present study, we used the electrical intracranial self-stimulation paradigm to evaluate the effects of various cannabinoid drugs on brain reward in laboratory rats and the roles of CB1 and CB2 receptors activation in brain reward function(s). Key results: Two mixed CB1 / CB2 receptor agonists, Δ9 -tetrahydrocannabinol (Δ9 -THC) and WIN55,212-2, produced biphasic effects-mild enhancement of brain-stimulation reward (BSR) at low doses but inhibition at higher doses. Pretreatment with a CB1 receptor antagonist (AM251) attenuated the low dose-enhanced BSR, while a CB2 receptor antagonist (AM630) attenuated high dose-inhibited BSR. To confirm these opposing effects, rats were treated with selective CB1 and CB2 receptor agonists. These compounds produced significant BSR enhancement and inhibition, respectively. Conclusions and implications: CB1 receptor activation produced reinforcing effects, whereas CB2 receptor activation was aversive. The subjective effects of cannabis depend on the balance of these opposing effects. These findings not only explain previous conflicting results in animal models of addiction but also explain why cannabis can be either rewarding or aversive in humans, as expression of CB1 and CB2 receptors may differ in the brains of different subjects.
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RESEARCH PAPER
Cannabinoid CB
1
and CB
2
receptor mechanisms underlie
cannabis reward and aversion in rats
Krista J. Spiller
1
|Guohua Bi
2
|Yi He
2
|Ewa Galaj
2
|Eliot L. Gardner
2
|ZhengXiong Xi
2
1
Department of Pathology and Laboratory
Medicine, Perelman School of Medicine,
University of Pennsylvania, Philadelphia,
Pennsylvania, USA
2
Molecular Targets and Medications
Discovery Branch, Intramural Research
Program, National Institute on Drug Abuse,
Baltimore, Maryland, USA
Correspondence
Krista Spiller, Department of Pathology and
Laboratory Medicine, Perelman School of
Medicine, University of Pennsylvania,
Philadelphia, PA 19104, USA.
Email: spillerk@upenn.edu
ZhengXiong Xi, Molecular Targets and
Medications Discovery Branch, NIDA IRP,
Baltimore, MD 21224, USA.
Email: zxi@intra.nida.nih.gov
Background and Purpose: Endocannabinoids are critically involved in brain reward
functions, mediated by activation of CB
1
receptors, reflecting their high density in the
brain. However, the recent discovery of CB
2
receptors in the brain, particularly in the
midbrain dopamine neurons, has challenged this view and inspired us to reexamine
the roles of both CB
1
and CB
2
receptors in the effects of cannabis.
Experimental Approach: In the present study, we used the electrical intracranial
selfstimulation paradigm to evaluate the effects of various cannabinoid drugs on
brain reward in laboratory rats and the roles of CB
1
and CB
2
receptors activation in
brain reward function(s).
Key Results: Two mixed CB
1
/CB
2
receptor agonists, Δ
9
tetrahydrocannabinol
(Δ
9
THC) and WIN55,2122, produced biphasic effectsmild enhancement of
brainstimulation reward (BSR) at low doses but inhibition at higher doses. Pretreat-
ment with a CB
1
receptor antagonist (AM251) attenuated the low doseenhanced
BSR, while a CB
2
receptor antagonist (AM630) attenuated high doseinhibited BSR.
To confirm these opposing effects, rats were treated with selective CB
1
and CB
2
receptor agonists. These compounds produced significant BSR enhancement and
inhibition, respectively.
Conclusions and Implications: CB
1
receptor activation produced reinforcing
effects, whereas CB
2
receptor activation was aversive. The subjective effects of can-
nabis depend on the balance of these opposing effects. These findings not only
explain previous conflicting results in animal models of addiction but also explain
why cannabis can be either rewarding or aversive in humans, as expression of CB
1
and CB
2
receptors may differ in the brains of different subjects.
1|INTRODUCTION
Marijuana or cannabis has now been legalized in many states of the
United States, although it is still unclear whether cannabis is entirely
safe (Schulden, Thomas, & Compton, 2009). In humans, cannabis
produces paradoxicaleffects that are often diametrically opposed.
For instance, cannabis is well known for its ability to produce eupho-
ria, pleasure, and relaxation (Fattore, Fadda, Spano, Pistis, & Fratta,
2008; Maldonado, Valverde, & Berrendero, 2006; Parsons & Hurd,
2015). However, not all users enjoy cannabis, and some experience
dysphoria, anxiety, and depression after its use (D'Souza et al.,
2004; Raft, Gregg, Ghia, & Harris, 1977). Even in the same person,
cannabis may produce positive effects at one time but negative
effects at another (Farris, Zvolensky, Boden, & BonnMiller, 2014;
Gregg, Small, Moore, Raft, & Toomey, 1976). Similar paradoxical
effects of Δ
9
tetrahydrocannabinol (Δ
9
THC, the major psychoactive
Abbreviations: ACEA, arachidonyl2′‐chloroethylamide; BSR, brainstimulation reward; ICSS,
intracranial selfstimulation; JWH 133, (6aR,10aR)3(1,1Dimethylbutyl)6a,7,10,10a
tetrahydro6,6,9trimethyl6Hdibenzo[b,d]pyran; NAc, nucleus accumbens; WIN55,2122,
(R)(+)[2,3Dihydro5methyl3(4morpholinylmethyl)pyrrolo[1,2,3de]1,4benzoxazin6yl]
1naphthalenylmethanone; Δ
9
THC, Δ
9
tetrahydrocannabinol
This article has been contributed to by US Government employees and their work is in the public
domain in the USA.
Received: 29 August 2018 Revised: 11 December 2018 Accepted: 30 January 2019
DOI: 10.1111/bph.14625
Br J Pharmacol. 2019;114. © 2019 The British Pharmacological Societywileyonlinelibrary.com/journal/bph 1
component of cannabis; Gaoni & Mechoulam, 1971) have been
found in nonhuman primates. Specifically, Δ
9
THC is self
administered by squirrel monkeys (Justinova, Tanda, Redhi, & Gold-
berg, 2003; Tanda, Munzar, & Goldberg, 2000), suggesting that it
has rewarding effects, but it is not selfadministered in rhesus mon-
keys (John, Martin, & Nader, 2017; Mansbach, Nicholson, Martin, &
Balster, 1994). In rodents (laboratory rats and mice), Δ
9
THC or
other cannabinoid compounds can be rewarding, ineffective or aver-
sive (Panagis, Vlachou, & Nomikos, 2008; Vlachou & Panagis, 2014).
For example, Δ
9
THC has been reported to facilitate electrical intra-
cranial brainstimulation reward (BSR; Gardner et al., 1988;
Katsidoni, Kastellakis, & Panagis, 2013; Lepore, Liu, Savage, Matalon,
& Gardner, 1996), while other groups and/or studies found depres-
sion of BSR (Kwilasz & Negus, 2012; Negus & Miller, 2014; Vlachou,
Nomikos, Stephens, & Panagis, 2007; Wiebelhaus et al., 2015). Con-
flicting findings have also been reported in studies using conditioned
place preference and intravenous selfadministration (Panagis,
Vlachou, & Nomikos, 2008; Vlachou & Panagis, 2014). The neurobi-
ological mechanisms underlying such paradoxical effects are poorly
understood.
With the identification of cannabinoid CB
1
and CB
2
receptors as
the major targets of cannabinoids (Matsuda, Lolait, Brownstein,
Young, & Bonner, 1990; Munro, Thomas, & AbuShaar, 1993) and
the finding that CB
1
receptors are highly expressed in the CNS and
CB
2
receptors are expressed predominantly in peripheral tissues, it
has generally been thought that the neurobehavioural and psychotro-
pic effects of cannabinoids are mediated by activation of CB
1
receptors
not CB
2
receptors (Mackie, 2005). This hypothesis is supported by
electrophysiological and neurochemical evidence demonstrating that
activation of CB
1
receptors on GABAergic neurons may increase mid-
brain dopaminergic neuron activity in the ventral tegmental area (VTA)
by dopamine neuron disinhibition (Lupica & Riegel, 2005; Szabo,
Siemes, & Wallmichrath, 2002) and that Δ
9
THC increases dopamine
release in the nucleus accumbens (NAc) as assessed by in vivo micro-
dialysis in rats (Chen, Paredes, Lowinson, & Gardner, 1991; Tanda,
Pontieri, & Di Chiara, 1997; although cf. Castaneda, Moss, Oddie, &
Whishaw, 1991). However, there is no direct behavioural evidence
in vivo demonstrating whether a CB
1
receptordependent mechanism
underlies cannabis reward. Moreover, we have recently reported that
activation of CB
1
receptors in glutamatergic neurons by Δ
9
THC pro-
duces aversive effects (Han et al., 2017).
In addition to CB
1
receptors, growing evidence indicates that CB
2
receptors are also expressed in the brain although the level is much
lower than CB
1
receptors in healthy subjects (Onaivi et al., 2006,
2008). Immunohistochemistry and in situ hybridization assays detect
CB
2
receptorimmunostaining or CB
2
receptor mRNA in various brain
regions (AracilFernandez et al., 2012; Ashton, Friberg, Darlington, &
Smith, 2006; Baek, Zheng, Darlington, & Smith, 2008; Brusco,
Tagliaferro, Saez, & Onaivi, 2008; Gong et al., 2006; Liu et al., 2009,
2017; Schmidt, Schafer, Striggow, Frohlich, & Striggow, 2012; Stempel
et al., 2016; Van Sickle et al., 2005; Zhang et al., 2019). Notably, CB
2
receptors were recently identified in VTA dopaminergic neurons
(Zhang et al., 2014, 2017, 2019) and dopaminergic terminals in the
NAc (Foster et al., 2016), two critical brain regions involved in drug
reward and addiction. Activation of CB
2
receptors in both brain
regions inhibits VTA dopaminergic neuron activity and NAc dopamine
release (Xi et al., 2011; Zhang et al., 2014, 2017). In addition, overex-
pression of brain CB
2
receptors inhibits cocaine selfadministration
and cocaineenhanced locomotion in mice (AracilFernandez et al.,
2012). These findings suggest that CB
2
receptors may also be involved
in cannabis reward or aversion.
In the present study, we used the electrical intracranial self
stimulation (ICSS) paradigm to evaluate the effects of various
cannabinoid ligands on ICSS and explored the roles of CB
1
and CB
2
receptors in these actions. Specifically, we used a wide range of doses
of Δ
9
THC, as well as the synthetic mixed CB
1
/CB
2
receptor agonist
WIN55,2122and then extended the findings using individual selec-
tive CB
1
and CB
2
receptor agonists, as well as pretreatment with
selective antagonists.
2|METHODS
Animals
All animal care and experimental procedures outlined in the animal
research protocol were approved by the Animal Care and Use Com-
mittee of the National Institute on Drug Abuse of the U.S. National
Institutes of Health under approved animal use protocol 07BNRB
47 and were carried out in compliance with applicable U.S. Federal
and Maryland state laws and regulations. Animal studies are reported
in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and
with the recommendations made by the British Journal of Pharmacol-
ogy. Adult male LongEvans rats (Charles River Laboratories, Raleigh,
NC; RGD Cat# 2308852, RRID:RGD_2308852), 300325 g at the
What is already known
Cannabis can produce both positive and negative effects
in different subjects or at different times.
The neurobehavioural effects of cannabinoids are
generally thought to be mediated by activation of CB
1
receptors.
What this study adds
In rats, CB
1
receptor activation produces reinforcing
effects, whereas CB
2
receptor activation is aversive.
These differential effects may explain the previous
conflicting results of Δ
9
THC treatment in animals.
What is the clinical significance
The subjective effects of cannabis may depend on the
balance of opposing CB
1
and CB
2
receptor effects.
2SPILLER ET AL.
BJP
time of surgery, were used. Animals were housed individually post
surgery in a climatecontrolled environment (70~74°F, humanity 40
50%, reverse 12 h light/dark cycle) with food (TestDiet, St. Louis,
MO, USA) and water freely available with the exception of the time
spent each day in the test chambers.
2.1 |Surgery
Under 60 mg·kg
1
sodium pentobarbital anaesthesia, rats were sur-
gically implanted with a unilateral monopolar stainless steel stimulat-
ing electrode (Plastics One, Roanoke, VA) targeted at the medial
forebrain bundle at the level of the lateral hypothalamus (stereotaxic
coordinates from bregma: AP + 2.5 mm, ML + 1.7 mm, and
DV 8.4 mm). A wire wrapped around a jeweller's screw implanted
in the skull and connected to a minipin in the electrical connector at
the top of the electrode was used to accommodate return electrical
current. The electrodes were cemented to the skull with acrylic resin
cement. Each animal was kept warm and under observation until all
effects of the anaesthetic had dissipated. Rats were monitored
closely and allowed a minimum of 7 days to recover, prior to the
start of experiments.
2.2 |ICSS apparatus
All training and testing occurred in standard operant chambers (MED
Associates, Georgia, VT), each of which contained a retractable wall
mounted lever and a cue light immediately above the lever. The oper-
ant chambers were enclosed in ventilated, soundattenuating cabinets.
Depression of the operant lever activated a brain stimulator.
2.3 |General ICSS procedure
The general BSR procedures were as reported previously (Pak et al.,
2006; Spiller et al., 2008; Xi et al., 2007, 2008). Briefly, rats were
allowed to selftrain to lever press for BSR. Each press on the operant
lever resulted in a 500ms train of 0.1ms rectangular cathodal pulses
through the electrode, followed by a 500ms timeoutin which fur-
ther presses did not produce brain stimulation. The initial stimulation
parameters were 72 Hz and 200 mA. If the animal did not learn to
lever press, the stimulation intensity was increased daily by 50 mA
until the animal learned to press (4560 responses per 30 s) or a max-
imum of 800 mA was reached. Animals (three of 50 rats) that did not
lever press at 800 mA or in which the stimulation produced unwanted
effects (e.g., head or body movements or vocalization) were removed
from the experiment.
2.4 |Ratefrequency ICSS procedure
Following establishment of lever pressing for BSR, animals were
presented with a series of 16 different pulse frequencies, ranging
from 141 to 25 Hz in descending order. At each pulse frequency,
animals responded for two 30s time periods, with the mean number
of lever responses recorded as the response rate. Between frequen-
cies, the lever retracted for 5 s. Animals were run for three sessions
per day; within each session, animals were run twice on the full
range of stimulation frequency over a 40min trial. The first session
was a warm up,the second session was the baseline session, and
the third session was the test session. The BSR threshold (θ
0
)
was defined as the minimum frequency at which the animal
responded for rewarding stimulation. Y
max
was defined as the maxi-
mal rate of lever responding. The BSR threshold (θ
0
) and Y
max
were
mathematically derived for each baseline run and each test session
run by analysing each ratefrequency BSR function generated by a
given animal over a given descending series of pulse
frequencies using bestfit mathematical algorithms as reported
previously (Spiller et al., 2009; Xi, Gilbert, et al., 2006; Xi, Newman,
et al., 2006).
2.5 |Testing the effects of Δ
9
THC, WIN55,2122,
AM251, AM630, ACEA, or JWH 133 on BSR
Once a baseline value was achieved (<10% variation over five contin-
uous days), the rats were randomly divided into five experimental
groups and treated with different test compounds (Table 1) to assess
the effects of Δ
9
THC, cocaine, WIN55,2122, AM251,AM630,
ACEA,orJWH 133 on BSR. All animals were injected, between the
baseline and test BSR sessions with an i.p. injection of sterile water,
0.5% Tween80 or tocrisolve vehicle (i.e., the 0 mg·kg
1
dose in each
group) or one of various doses of test compounds. Thirty minutes
after test compound injection, the test sessions began. After each test,
animals received an additional 57 days of BSR restabilization until a
new baseline θ
0
was established. The order of testing of various drug
TABLE 1 Experimental groups and the drug treatments in each
group of rats
Group # Test drugs Treatment (mg·kg
1
)
a
1Δ
9
THC (n= 14) Δ
9
THC (0, 0.3, 1, 3, 5),
(AM251 + THC),
(AM630 + THC)
2 WIN55,2122(n= 11) WIN (0, 0.3, 1, 3),
(AM251 + WIN),
(AM630 + WIN)
3 ACEA (n= 8) ACEA (0, 0.1, 0.3, 1),
(AM251 + ACEA),
(AM630 + ACEA)
4 JWH 133 (n= 7) JWH (0, 3, 10, 20),
(AM251 + JWH),
(AM630 + JWH),
Δ
9
THC (1)
5 Cocaine (n= 7) Cocaine (0, 3), Δ
9
THC (1),
AM251 (0, 1, 3),
AM630 (0, 1, 3, 10)
a
The order of testing for the various drug doses in each group was
counterbalanced according to a Latin square design.
SPILLER ET AL.3
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doses was counterbalanced according to a Latin square design. To
monitor potential drug effects on motor behaviour, the maximum rate
of lever pressing (Y
max
) was measured, and any treatment that altered
this significantly in either direction was eliminated from the study (see
also Section 2.8).
2.6 |Testing the effects of AM251 or AM630
pretreatment on drugenhanced or druginhibited BSR
For pretreatment studies, rats were injected, between the baseline
and test BSR sessions, with 0.5% Tween80 vehicle, AM251
(3 mg·kg
1
), or AM630 (3 mg·kg
1
) 10 min prior to the second drug
injection. Then, a dose of the second drug (Δ
9
THC, WIN55,2122,
ACEA, or JWH 133) was administered 30 min before the test session
began. After each test, animals received an additional 37 days of BSR
restabilization until a new baseline θ
0
was established.
2.7 |Locomotor activity
Four additional groups of rats (n= 8 each) were used to evaluate the
locomotor effects of cannabinoid compounds. These additional drug
naive rats were placed in locomotor detection chambers (Accuscan,
Columbus, OH) and habituated for 1 hr. Each group then randomly
received one dose of Δ
9
THC (0, 1, 3, or 5 mg·kg
1
i.p.),
WIN55,2122 (0, 0.3, 1, or 3 mg·kg
1
i.p.), JWH 133 (0, 10,
20 mg·kg
1
), or ACEA (0, 0.3, 1, 3 mg·kg
1
). The Δ
9
THC and
WIN55,2122 groups of rats were also used to observe the effects
of AM251 (3 mg·kg
1
i.p.) or AM630 (3 mg·kg
1
i.p.) on openfield
locomotion. Following injection, locomotor activity was recorded
for 2 hr in 10min intervals. Each animal was tested three to five
times with different drug doses in a counterbalanced manner. The
time interval was 13 days between each test. Distance counts per
10 min bin (cm) were used to evaluate the effects of each cannabi-
noid compound on locomotion.
2.8 |Data and statistical analysis
The data and statistical analysis comply with the recommendations
of the British Journal of Pharmacology on experimental design and
analysis in pharmacology. Experiments showing biphasic effects on
BSR by Δ
9
THC and WIN55,2122 were performed in two indepen-
dent groups of rats, with seven to 14 animals per group as shown in
figure legends. All other experiments were performed once, with
seven to 14 rats per treatment. Though the experimenter was not
blinded to the animals' identity and treatment condition during data
collection, the data were blinded during analyses. No data points
were excluded from the analysis in any experiment. Data were
checked for normality using the ShapiroWilk method and for equal
variance by the BrownForsythe method. Statistical significance was
determined using paired twotailed ttests when comparing two
groups, and oneway ANOVAs for repeated measures when compar-
ing multiple groups, using SigmaPlot. For significant results by one
way ANOVA, all pairwise multiple comparisons were made using
the HolmSidak method. A Pvalue of less than 0.05 was considered
significant.
2.9 |Materials
Δ
9
THC and cocaine (provided by the National Institute on Drug
Abuse, Intramural Research Program, Baltimore, MD) were dissolved
in sterile 0.5% Tween80 (SigmaAldrich) and saline, respectively.
WIN55,2122, AM251, AM630, and ACEA (Tocris) were dissolved
in sterile 0.5% Tween80. JWH 133 (Tocris) was dissolved in
Tocrisolve(Tocris Bioscience brand of BioTechne Corporation,
Minneapolis, MN).
2.10 |Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corre-
sponding entries in http://www.guidetopharmacology.org, the com-
mon portal for data from the IUPHAR/BPS Guide to
PHARMACOLOGY (Harding et al., 2018), and are permanently
archived in the Concise Guide to PHARMACOLOGY 2017/18
(Alexander, Christopoulos et al., 2017).
3|RESULTS
3.1 |Mixed CB
1
receptor/ CB
2
receptor agonists
have biphasic effects on BSR
Systemic administration of a wide range of doses of Δ
9
THC pro-
duced biphasic effects (Figure 1a,b). A low dose of Δ
9
THC
(1.0 mg·kg
1
) significantly enhanced BSR (i.e., reduced the minimum
frequency at which the animal responded for rewarding stimulation)
by 79%, while the highest dose tested (5.0 mg·kg
1
) significantly
inhibited BSR by about 9%. No dose of Δ
9
THC affected the maxi-
mal operant response (Y
max
; Figure 1a), suggesting no significant
sedation or locomotor impairment by Δ
9
THC administration (see
also Section 2.8).
Because there has been controversy over the effect of Δ
9
THC
on reward, we treated a separate group of rats with the enhancing
dose of Δ
9
THC as well as a low dose of the appetitive drug,
cocaine, for comparison (Figure 1c). The facilitating effect of cocaine
was roughly twice as much as that of Δ
9
THC, even at the dose of
2 mg·kg
1
, which is a moderately low dose compared to the doses
of cocaine used to produce reward ICSS in other recent studies
(Bauer, Banks, & Negus, 2014; Yang et al., 2017). The 1 mg·kg
1
dose of Δ
9
THC significantly facilitated ICSS in this second indepen-
dent cohort of rats, relative to vehicle treatment (difference of
means = 8.8%).
Similar to Δ
9
THC, systemic administration of the synthetic
high affinity CB
1
/CB
2
receptor agonist WIN55,2122 (Compton,
Gold, Ward, Balster, & Martin, 1992) produced biphasic effects
(Figure 1d), with similar percent shifts. We also found that doses of
4SPILLER ET AL.
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5 mg·kg
1
i.p. and above of WIN55,2122 produced a sedative effect
in the rats as assessed by a decrease in the maximum frequency of
lever pressing (Y
max
). Those animals were excluded from the study in
order to differentiate treatment effects on reward from potentially
confounding effects on motor function.
3.2 |Effects of Δ
9
THC or WIN55,2122on
openfield locomotion
To further determine the potential involvement of locomotor effects,
we observed the effects of the same doses of Δ
9
THC or
WIN55,2122 on openfield locomotion in rats. Δ
9
THC produced a
trend towards reduction (not significant) in openfield locomotion
(Figure 2a,b). However, systemic administration of WIN55,2122 pro-
duced a significant, dosedependent reduction in basal levels of loco-
motor activity (Figure 2c,d), suggesting locomotor depression or
sedation. Posthoc individual group comparisons revealed a significant
reduction in locomotion only after 3 mg·kg
1
WIN55,2122 adminis-
tration (Figure 2d).
3.3 |Effects of selective CB
1
or CB
2
receptor
antagonists on Δ
9
THCor WINaltered BSR
In order to understand the nature of the biphasic effects produced by
these mixed CB
1
/CB
2
receptor agonists, we next pretreated Δ
9
THC
or WIN55,2122 with the selective CB
1
or CB
2
receptor antagonists
AM251 and AM630, respectively. We first confirmed that neither
AM251 nor AM630 itself altered BSR or Y
max
levels at the doses
tested (AM251, 1 and 3 mg·kg
1
i.p. or AM630, 1, 3, and 10 mg·kg
1
i.p.; Figure 3).
We then selected doses in the middle of the ranges that have been
previously shown to be effective in antagonizing CB
1
receptors
FIGURE 1 Biphasic effects of Δ
9
THC or WIN55,2122 on electrical BSR low doses enhance, whereas high doses inhibit BSR. (a)
Representative stimulationresponse curves, indicating that a low dose of Δ
9
THC (1 mg·kg
1
i.p.) shifted the stimulationresponse curve to the
left and decreased the stimulation threshold (θ
0
) value, while a higher dose of Δ
9
THC (5 mg·kg
1
i.p.) significantly shifted the curve to the right
and increased the stimulation threshold (θ
0
). Δ
9
THC did not affect maximal operant responses (Y
max
level) at any dose tested. (b) Summary of all
Δ
9
THC doses tested, with both rewarding and aversive effects apparent as percentage enhancement or inhibition of θ
0
(n= 14, oneway ANOVA
for repeated measures, F
4, 52
= 11.9). (c) The rewarding effects of low dose Δ
9
THC are only about half of those produced by 2 mg·kg
1
cocaine
(n= 7, oneway ANOVA for repeated measures, F
2, 12
= 23.6 ). (d) The synthetic full CB
1
/CB
2
receptor agonist WIN55,2122 had similar effects to
Δ
9
THC, wherein a low dose of WIN55,2122 (0.3 mg·kg
1
) enhanced brain reward, and the highest dose tested (3 mg·kg
1
) inhibited brain reward
(n= 11, F
3, 30
= 9.3). For all panels, individual data points are shown as black circles, with bars indicating group means ± SD shown to the right.
*P< 0.05, significantly different from vehicle treatment groups
SPILLER ET AL.5
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(3 mg·kg
1
of AM251; Xi, Gilbert, et al., 2006) and CB
2
receptors
(3 mg·kg
1
of AM630; Rahn et al., 2014). Pretreatment with AM251
10 min prior to a 1.0 mg·kg
1
Δ
9
THC injection moderately attenuated
the Δ
9
THCenhanced BSR, with a change in BSR facilitation from
about 7.4% after the Vehicle + Δ
9
THC treatment to 2.1% after the
AM251 + Δ
9
THC treatment (Figure 4a, left panel), although
this change was not statistically significant. Given the low level of
Δ
9
THCenhancement in this group of rats, this result may have been
due to a floor effect. However, compared to the vehicle control group,
1 mg·kg
1
Δ
9
THCenhanced BSR was clearly blocked by AM251
(Figure 1b vs. Figure 4a). In contrast to Δ
9
THC, AM251 significantly
blocked the 0.3 mg·kg
1
WINenhanced BSR (Figure 4b, left panel),
from mean value of 10.5% to 1.2%. AM251 pretreatment had no
effect on the inhibition of BSR by the 5.0 mg·kg
1
dose of Δ
9
THC
(Figure 4a, right panel) or the 3 mg·kg
1
dose of WIN55,2122
(Figure 4b, right panel). These data suggest that the enhancement of
BSR by low doses of CB
1
/CB
2
receptor agonists is driven by action
on the CB
1
receptor.
To assess the inhibitory effects on ICSS of high dose Δ
9
THC or
WIN55,2122, we pretreated the animals with the selective CB
2
antagonist AM630 (3 mg·kg
1
i.p., 10 min prior). The inhibition pro-
duced by both 5.0 mg·kg
1
Δ
9
THC (Figure 4c) and 3 mg·kg
1
WIN55,2122 (Figure 4d) was attenuated to baseline levels by this pre-
treatment, with a 13% and 10% shift, respectively, compared to when
the rats were pretreated with vehicle. AM630 did not alter the low dose
facilitation of ICSS by either compound. Taken together, these results
suggest that the biphasic effects of Δ
9
THC or WIN55,2122 result
from differential CB
1
or CB
2
receptormediated effects.
3.4 |CB
1
receptor activation is rewarding, whereas
CB
2
receptor activation is dysphorogenic
To further investigate our hypothesis that it is the actions on different
cannabinoid receptor subtypes that drive Δ
9
THC's and WIN55,212
2's biphasic effects on BSR in rats, we used selective agonists in differ-
ent groups of rats. First, we treated rats with the highly selective CB
1
receptor agonist ACEA (Figure 5a, 0.1, 0.3, 1.0 mg·kg
1
) and found
that this treatment produced only a monophasic enhancement of
BSR, which was significantly attenuated by pretreatment with
FIGURE 2 High doses of WIN55,2122 decrease spontaneous locomotion. (a, b) The time course and AUC measurements after systemic
administration of different doses of Δ
9
THC or vehicle in the openfield test revealed a significant time main effect (twoway ANOVA for
repeated measures, F
11, 77
= 5.93), but no significant Δ
9
THC treatment main effect (F
3, 21
= 2.30) or Treatment × Time interaction (F
33, 231
= 0.39).
(c, d) In contrast, WIN55,2122 administration produced a more pronounced impairment of spontaneous movement in the openfield test (twoway
ANOVA for repeated measures revealed a significant Time main effect (F
11,77
= 6.35), treatment main effect (F
3,21
= 7.08), and Treatment X Time
interaction (F
33,231
= 1.97)), shown here by the time course for individual doses (c) and the AUC summary data (d)
6SPILLER ET AL.
BJP
AM251 (3 mg·kg
1
) but not by the CB
2
receptor antagonist AM630
(Figure 5b). Consistent with these data, the selective CB
2
receptor
agonist, JWH 133, increased BSR threshold at 20 mg·kg
1
(Figure 5c)
by about 14% from baseline. This inhibition was attenuated by AM630
(Figure 5d) but not AM251, confirming the CB
2
receptor specificity of
this effect.
3.5 |Effects of ACEA and JWH 133 on openfield
locomotion
Finally, we observed the effects of ACEA and JWH 133 on openfield
locomotion. We found that systemic administration of the same doses
of ACEA that enhanced BSR had no effect (Figure 6a,b), while JWH
133 produced a dosedependent reduction, on openfield locomotion
(Figure 6c,d). Post hoc individual group comparisons revealed a signif-
icant reduction in locomotion after 20 mg·kg
1
JWH 133 administra-
tion (Figure 6d).
4|DISCUSSION
The major findings in the present study are that systemic administra-
tion of Δ
9
THC or the synthetic cannabinoid agonist WIN55,2122
produced dosedependent biphasic effects lower doses enhanced,
while high doses inhibited BSR, as assessed by electrical ICSS. The
selective CB
1
receptor agonist, ACEA, produced a BSRenhancing
effect, while the selective CB
2
receptor agonist, JWH 133, produced
a dosedependent inhibition of BSR. The BSRenhancing effect
produced by low doses of Δ
9
THC, WIN55,2122, or ACEA was
blocked selectively by the CB
1
receptor selective antagonist,
AM251, while the inhibition of BSR produced by high doses of Δ
9
THC or WIN55,2122 or by JWH 133 was blocked by the selective
CB
2
receptor antagonist AM630. Together, these data suggest that
brain cannabinoid CB
1
and CB
2
receptors modulate brain reward
function in opposite directions, that is, CB
1
receptor activation
producing enhancement and CB
2
receptor activationproducing inhibi-
tion of BSR.
It is well known that cannabis can be rewarding or aversive in
both humans and experimental animals (Panagis, Vlachou, &
Nomikos, 2008; Vlachou & Panagis, 2014). ICSS is a commonly used
behavioural paradigm to study brain reward functions (Bauco &
Wise, 1997; Peng et al., 2010; Wise, 1996). In this model, animals
press a lever to deliver brief electrical pulses to a discrete brain
region such as the VTA of the midbrain or the middle forebrain bun-
dle via an implanted electrode. Most drugs of abuse such as cocaine,
heroin, or nicotine lower the stimulation threshold for electrical BSR,
indicating enhanced BSR and implying a summation between the
BSR and the drug reward (Bauco & Wise, 1997; Peng et al., 2010).
However, the effects of cannabinoids on BSR have been controver-
sial. In some studies, Δ
9
THC produced a significant reduction in the
electrical stimulation threshold in rats (Gardner et al., 1988; Lepore,
Liu, Savage, Matalon, & Gardner, 1996), suggesting enhanced BSR.
However, in other studies, Δ
9
THC or other cannabinoid agonists
either had no effect on electrical BSR (Vlachou et al., 2007) or pro-
duced a reduction in electrical BSR (i.e., aversion) in rats (Katsidoni,
Kastellakis, & Panagis, 2013; Vlachou, Nomikos, & Panagis, 2005,
FIGURE 3 Neither AM251 nor AM630 produced a significant alteration in BSR (a, b) or openfield locomotion as assessed by the time course of
locomotion (c) or the AUC after each drug administration (d) at any dose tested
SPILLER ET AL.7
BJP
2006). An important finding in the present study is that the hedonic
effects of cannabis or cannabinoids depend on a drug doselower
doses are rewarding, while higher doses are aversive. This may in
part explain the previous conflicting findings regarding cannabis
actions in humans and experimental animals.
We note that our results on the enhancing effects of low dose
Δ
9
THC differ from some previous studies showing Δ
9
THCinduced
inhibition (Kwilasz & Negus, 2012; Negus & Miller, 2014; Vlachou
et al, 2007; Wiebelhaus et al., 2015). This could be related to smaller
sample sizes used in these studies. For example, Vlachou and col-
leagues tested all Δ
9
THC doses (0, 0.5, 1 or 2 mg·kg
1
i.p.) using only
five animals (Vlachou et al., 2007). The power analysis performed for
the present study suggests at least n= 7 are needed to detect an
8% change in BSR with a power of 0.79 (α= 0.05). Assuming that
the Vlachou et al. study had similar low levels of variability between
rats as in the present study, a sample size of five would be underpow-
ered to detect an 8% change.
Nevertheless, because of the negative findings in those previous
studies, we repeated the low dose Δ
9
THC treatment in three inde-
pendent groups of rats (see Table 1, n=714). We found the same
moderate but significant enhancement in all groups with low dose
Δ
9
THC treatment. This is further supported by a similar level of
enhancement after treatment with the CB
1
receptor agonist, ACEA.
Our results also fit well with observations in other animal models of
drug reward, in which CB
1
receptor agonists increase the motivational
and reinforcing effects of alcohol, nicotine, and opiates, whereas
diminished CB
1
receptor signalling diminishes the rewarding effects
of these drugs (Parsons & Hurd, 2015). In all of the figures presented
in the present study, we show individual data points for each compar-
ison to allow for greater future reproducibility.
Another important finding in the present study is that different
receptor mechanisms may underlie cannabis reward versus aversion.
This is supported by several lines of evidence. First, the selective
CB
1
receptor agonist, ACEA, enhanced electrical BSR, an effect that
FIGURE 4 Pretreatment with AM251, a selective CB
1
receptor antagonist, blocks low dose WIN55,2122induced enhancement of BSR but
not high doseinduced BSR inhibition. (a) Pretreatment with AM251 (3 mg·kg
1
i.p., 10 min prior to Δ
9
THC) appeared to attenuate Δ
9
THC
mediated enhancement and inhibition of BSR although paired ttests did not reveal significant differences. (b) Pretreatment with the same dose
of AM251 significantly blocked WINenhanced BSR (n= 12) but not high dose WINinduced inhibition of BSR (n= 10, paired ttest). (c, d)
Pretreatment with AM630, a selective CB
2
receptor antagonist, blocks high dose Δ
9
THCor WIN55,2122mediated BSR inhibition. When
AM630 (3 mg·kg
1
i.p.) was injected 10 min prior to Δ
9
THC (c) or WIN55,2122 treatment (d), the high dose BSR inhibition was significantly
attenuated (5 mg·kg
1
Δ
9
THC, n= 12, paired ttest 3 mg·kg
1
WIN55,2122, n= 10). For all panels, individual data points are shown as black
circles, with bars indicating group means ± SD shown to the right. *P< 0.05, significantly different from vehicle + WIN treatment; #P< 0.05,
significantly different from vehicle + Δ
9
THC group
8SPILLER ET AL.
BJP
was blocked by the selective CB
1
receptor antagonist AM251 but
not by the selective CB
2
receptor antagonist, AM630. Second, the
selective CB
2
receptor agonist, JWH 133, dosedependently
inhibited electrical BSR, an effect that was selectively blocked by
AM630 not by AM251. Third, the BSRenhancing effect produced
by low doses of WIN55,2122 was also blocked by AM251 not
AM630, while the BSRsuppressing effect produced by higher doses
of Δ
9
THC or WIN55,2122 was blocked by AM630 not AM251.
We note that the BSRenhancing effect produced by low dose Δ
9
THC appeared to be reduced by both AM251 and AM630
(Figure 4a,c). This may be related to the fact that (a) Δ
9
THC
enhanced BSR is moderate (~7%) and marginally significant and (b)
brain levels of CB
2
receptors are much lower than those of CB
1
receptors. Thus, AM630 may also bind to brain CB
2
receptors to
affect Δ
9
THCenhanced BSR to a certain extent. Compared to Δ
9
THC, WIN55,2122 produced more potent biphasic effects on BSR,
which were blocked by AM251 and AM630, respectively.
WIN55,2122 also produced more potent locomotor reduction than
Δ
9
THC. The mechanisms underlying the different pharmacological
efficacies or potencies of Δ
9
THC and WIN55,2122 on BSR and
locomotion are unclear. They may be related to different receptor
binding profilesΔ
9
THC may act as a CB
1
/CB
2
receptor partial ago-
nist, while WIN55,2122 may act as a CB
1
/CB
2
receptor full agonist
(Paronis, Nikas, Shukla, & Makriyannis, 2012; Pertwee, 2010; Tai &
Fantegrossi, 2017). Whatever the mechanisms, the present findings
with both Δ
9
THC and WIN55,2122 suggest that activation of
CB
1
receptors is rewarding, while activation of CB
2
receptors is
aversive. This means that the final subjective effect of cannabis
depends on the balance of two opposite actions on brain reward
function. Individual differences in brain CB
1
receptor and CB
2
recep-
tor expression may in part explain why cannabis is rewarding in
some subjects but aversive in others. These findings may also relate
to our previous reports that both CB
1
receptor antagonists and CB
2
receptor agonists produce inhibitory effects on cocaine self
administration and reinstatement of drugseeking behaviour (Xi, Gil-
bert, et al., 2006; Xi et al., 2008, 2011; Zhang et al., 2014, 2015).
Similarly, overexpression of CB
2
receptors in the brain inhibits
cocaine selfadministration and attenuates cocaineinduced locomo-
tor sensitization (AracilFernandez et al., 2012). Consistent with
these findings, several recent reports indicate that CB
1
receptors
FIGURE 5 Effects of selective CB
1
or CB
2
receptor agonists on electrical brainstimulation reward. (a) ACEA, a selective CB
1
receptor agonist,
produced a significant enhancement in BSR (oneway ANOVA for repeated measures, F
3, 21
= 4.6, n= 8). (b) Pretreatment with AM251 (3 mg·kg
1
)
but not AM630 (3 mg·kg
1
) blocked ACEAenhanced BSR (n= 8, oneway ANOVA for repeated measures, F
2, 14
= 11.8). (c) JWH 133, a selective
CB
2
receptor agonist, dosedependently inhibited BSR (n=7,F
3, 27
= 12.5). (d) Pretreatment with AM630 (3 mg·kg
1
) but not AM251 (3 mg·kg
1
)
blocked 20 mg·kg
1
JWH 133induced BSR inhibition (n=7,F
2, 12
= 9.5). For all panels, individual data points are shown as black circles, with bars
indicating group means ± SD shown to the right. *P< 0.05, significantly different from vehicle; #P< 0.05, significantly different from
vehicle + ACEA treatment; P< 0.05, significantly different from VEH + JWH 133 treatment
SPILLER ET AL.9
BJP
and CB
2
receptors may play opposing roles in modulating cocaine's
action, e.g., CB
2
receptor agonism exerting behavioural effects simi-
lar to those of CB
1
receptor antagonism on acquisition and expres-
sion of cocaineinduced conditioned place preference, cocaine
induced locomotion, cocaineinduced cFos expression and MAPK
expression (Delis et al., 2017; GarciaCabrerizo & GarciaFuster,
2016). Such differential CB
1
versus CB
2
receptor effects may also
partially explain some of the difficulty in parsing the neurological
effects of cannabis use (Filbey et al., 2014).
We note that, while WIN55,2122 or JWH 133 did produce a sig-
nificant reduction in openfield locomotion in a dosedependent man-
ner, neither Δ
9
THC nor ACEA produced such effects, suggesting
possible involvement of locomotor suppression in high dose
WIN55,2122or high dose JWH 133inhibited BSR. Although we
cannot completely exclude it, such a possibility could be low since
WIN55,2122 or JWH 133, at the same high doses, did not alter max-
imal operant lever responses (Y
max
). In addition, we have previously
reported that JWH 133, at the same high doses (10 and 20 mg·kg
1
)
did not alter cocaine selfadministration under fixed ratio 1 schedule
of reinforcement but produced an increase in break point (maximal
lever response to receive a drug infusion) for cocaine self
administration under progressive ratio schedule of reinforcement in
rats (Zhang et al., 2015). These findings suggest that in the presence
of drug or BSR, animals still worked very hard to get the reward and
displayed high motivation to overcome druginduced locomotor inhi-
bition or sedation to get reward.
The cellular mechanisms underlying CB
1
receptormediated
reward are not fully understood. We have recently reported that
CB
1
receptor mRNA is expressed in VTA GABAergic and glutamater-
gic neurons in mice (Han et al., 2017), which may project to VTA
dopaminergic neurons and modulate dopaminergic neuron activity
(Lupica, Riegel, & Hoffman, 2004). CB
1
receptormediated inhibition
of VTA GABAergic neurons may disinhibit dopaminergic neuron
activity, producing rewardenhancing effects, while CB
1
receptor
inhibition of VTA glutamatergic neurons may be aversive by decreas-
ing VTA glutamate release and thereby decreasing dopaminergic
neuronal activity (Han et al., 2017). We have hypothesized that the
hedonic effect of CB
1
receptor activation may depend on the net
FIGURE 6 Effects of ACEA and JWH 133 on openfield locomotion. (a) Time courses of basal levels of locomotor activity after systemic
administration of different doses of ACEA or vehicle, illustrating that ACEA did not significantly alter openfield locomotor activity. A twoway
ANOVA for repeated measures over time and drug dose revealed a significant time main effect (F
11, 77
= 5.93, P< 0.001) but no significant ACEA
treatment main effect (F
3, 21
= 2.230) or Treatment × Time interaction (F
33, 231
= 0.38). (b) The AUC data from (a) after ACEA administration. A
oneway ANOVA for repeated measures over Δ
9
THC dose failed to show a significant treatment main effect (F
3, 21
= 2.30). (c) Time course of the
effects of JWH 133 on openfield locomotion. A twoway ANOVA for repeated measures over time and drug dose revealed a significant treatment
main effect (F
2,14
= 6.74), time main effect (F
11, 77
= 1.68), and Treatment × Time interaction (F
22,154
= 2.86). (d) AUC data from (c) after JWH 133
administration. A oneway ANOVA for repeated measures over dose revealed a significant JWH 133 treatment main effect (F
2,14
= 6.74). *P< 0.05,
significantly different from vehicle group
10 SPILLER ET AL.
BJP
effect of these two opposing actions (Han et al., 2017). In the pres-
ent study, we found that activation of CB
1
receptors was rewarding
in rats. This would be congruent with a supposition that more CB
1
receptors are expressed in VTA GABAergic neurons or GABAergic
afferents or that CB
1
receptormediated GABAergic disinhibition of
VTA dopaminergic neurons is dominant when animals are exposed
to low loses of cannabis or Δ
9
THC. Conversely, at high doses, can-
nabis or Δ
9
THC may activate CB
1
receptors on VTA glutamatergic
neurons or glutamatergic afferents, producing aversive or reward
depressing effects.
In addition to CB
1
receptors, CB
2
receptors are found in VTA
dopaminergic neurons in both rats and mice (Zhang et al., 2014,
2017). Given that activation of CB
2
receptors inhibits dopaminergic
neuronal activity in the VTA, dopamine release in the NAc (Foster
et al., 2016; Xi et al., 2011; Zhang et al., 2014, 2017), and
dopaminerelated behaviours such as intravenous cocaine self
administration, cocaineinduced conditioned place preference, and
cocaineinduced hyperactivity (Delis et al., 2017; Liu et al., 2017; Xi
et al., 2011), we believe that the aversive or rewarddepressing effects
produced by the selective CB
2
receptor agonist, JWH 133, or high
doses of Δ
9
THC or WIN55,2122 are mediated at least in part by
direct activation of CB
2
receptors on VTA dopaminergic neurons.
In conclusion, CB
1
receptor activation produces reinforcing effects,
whereas CB
2
receptor activation is aversive. These opposing effects
may not only explain the conflicting findings in previous ICSS studies
but also explain why cannabis is rewarding or aversive in different
subjects under different circumstances.
ACKNOWLEDGEMENT
This work was supported by the Intramural Research Program (IRP) at
the National Institute on Drug Abuse (NIDA) National Institutes of
Health (NIH), U.S. Public Health Service.
AUTHOR CONTRIBUTIONS
K.S., E.L.G., and Z.X.X. designed the experiments. K.S., G.H.B., E.G.,
and H.Y. conducted the experiments. K.S., G.H.B., E.G., and Z.X.X.
analysed the data and made the figures. K.S. and Z.X.X. wrote the
manuscript. E.L.G. revised the manuscript.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
DECLARATION OF TRANSPARENCY AND SCIENTIFIC
RIGOUR
This Declaration acknowledges that this paper adheres to the princi-
ples for transparent reporting and scientific rigour of preclinical
research as stated in the BJP guidelines for Design & Analysis, and
Animal Experimentation, and as recommended by funding agencies,
publishers and other organisations engaged with supporting research.
ORCID
Krista J. Spiller https://orcid.org/0000-0002-1980-694X
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... THC activates CB1 which produces psychoactivity, including the "drug liking" effects sought after with street and recreational cannabis [269]. This "drug liking" effect is a result of inhibition of GABAergic neurotransmission within the nucleus accumbens which facilitates dopamine release [96]. ...
... The topic of cannabinoid/opioid pharmacological interactions is complex and has been covered only in broad strokes here. These interactions are important to pursue in research since this will have important clinical and policy outcomes [34,105,225,269,324,365]. ...
... THC activates CB1 which produces psychoactivity, including the "drug liking" effects sought after with street and recreational cannabis [269]. This "drug liking" effect is a result of inhibition of GABAergic neurotransmission within the nucleus accumbens which facilitates dopamine release [96]. ...
... The topic of cannabinoid/opioid pharmacological interactions is complex and has been covered only in broad strokes here. These interactions are important to pursue in research since this will have important clinical and policy outcomes [34,105,225,269,324,365]. ...
... There is significant euphoria/positive reinforcement during the intake of psychoactive cannabinoids combined with an anxiolytic effect [3,119,153,273,274]. The mechanisms involved with the activation of CB1 receptors by ligands (most commonly 9-THC) result in the reward's reinforcement [119,275]. Activation of CB2 receptors seems to have the opposite effect [275]. Consequently, the effect of the cannabinoid compound will depend on the composition of its preparation (potency, enantiomer composition, and pharmacokinetics). ...
... The mechanisms involved with the activation of CB1 receptors by ligands (most commonly 9-THC) result in the reward's reinforcement [119,275]. Activation of CB2 receptors seems to have the opposite effect [275]. Consequently, the effect of the cannabinoid compound will depend on the composition of its preparation (potency, enantiomer composition, and pharmacokinetics). ...
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... Within the brain, CB2 receptors are expressed predominantly on microglia (Cabral et al., 2008;Maresz et al., 2005;Núñez et al., 2004) and are involved in the regulation of chemotaxis and cytokine release (Cabral et al., 2008;Klegeris et al., 2003). Neuronal CB2 receptor expression has been reported Onaivi et al., 2006;Zhang et al., 2021a;Zhang et al., 2021b;Zhang et al., 2017), and while the contribution of CB2 receptors to neuronal signalling and behavioural regulation is largely unknown (Malfitano et al., 2014), recent evidence has demonstrated their contributions to reward processing (Galaj et al., 2020;Spiller et al., 2019) and motor behaviour (Zhang et al., 2021b) in rodents. CB1 receptors, on the other hand, show greatest expression in the central nervous system, with the densest neuronal expression in the cerebellum and in corticolimbic regions involved in emotion and cognition, such as the medial prefrontal cortex (mPFC), the hippocampus, and the amygdala (Egertová and Elphick, 2000;Herkenham et al., 1991;Hohmann and Herkenham, 1999;Tsou et al., 1998;Tsou et al., 1999). ...
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