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Endocannabinoid signaling in reward and addiction

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Abstract

The brain reward system is critical for survival. The hedonic effects produced by eating, exercise and sexual activity provide important motivational effects that increase the likelihood of future engagement in these criti­ cal activities (that is, positive reinforcement). The reward system is also essential for important negative hedonic responses, in which aversive or unpleasant events (for example, sickness or bodily harm) increase the likeli­ hood of behaviours that will avoid or relieve these negative states (that is, negative reinforcement). Seminal discoveries demonstrating that the effects of marijuana (cannabis sativa) are mediated by canna­ binoid receptors in the brain propelled significant research initiatives that expanded our knowledge about the body's endogenous cannabinoid system (termed the endocannabinoid (eCB) system (ECS)), which is now acknowledged to have a prominent role in modulat­ ing brain reward function and maintaining emotional homeostasis. This Review examines the evidence for an eCB influence in the positive­reinforcing effects of natural rewards and drugs of abuse. In contrast to the initial pleasurable experience of rewarding stimuli, pro­ longed drug exposure contributes to aberrant synaptic plasticity, negative emotional states and impaired learn­ ing and memory processes that sustain compulsive drug consumption, which is characteristic of the addicted state. We explore the ECS signalling underlying these maladaptive processes and provide an overview of the existing literature regarding the genetic factors that are associated with the ECS to gain insight about the potential contribution of ECS signalling dysregulation to addiction disorders.
The brain reward system is critical for survival. The
hedonic effects produced by eating, exercise and sexual
activity provide important motivational effects that
increase the likelihood of future engagement in these criti-
cal activities (that is, positive reinforcement). The reward
system is also essential for important negative hedonic
responses, in which aversive or unpleasant events (for
example, sickness or bodily harm) increase the likeli-
hood of behaviours that will avoid or relieve these negative
states (that is, negative reinforcement).
Seminal discoveries demonstrating that the effects
of marijuana (cannabis sativa) are mediated by canna-
binoid receptors in the brain propelled significant
research initiatives that expanded our knowledge about
the body’s endogenous cannabinoid system (termed the
endocannabinoid (eCB) system (ECS)), which is now
acknowledged to have a prominent role in modulat-
ing brain reward function and maintaining emotional
homeostasis. This Review examines the evidence for
an eCB influence in the positive-reinforcing effects of
natural rewards and drugs of abuse. In contrast to the
initial pleasurable experience of rewarding stimuli, pro-
longed drug exposure contributes to aberrant synaptic
plasticity, negative emotional states and impaired learn-
ing and memory processes that sustain compulsive drug
consumption, which is characteristic of the addicted
state. We explore the ECS signalling underlying these
maladaptive processes and provide an overview of the
existing literature regarding the genetic factors that
are associated with the ECS to gain insight about the
potential contribution of ECS signalling dysregulation
to addiction disorders.
The ECS and reward circuits
The ECS comprises G protein-coupled receptors and
small neuromodulatory lipid ligands, as well as bio-
synthetic and metabolic enzymes for the synthesis and
degradation of the ligands, respectively. Two major
types of cannabinoid receptor have been characterized
and cloned: cannabinoid1 receptors (CB1Rs; encoded
by CNR1) and CB2Rs (encoded by CNR2). CB1Rs are
the most-abundant G protein-coupled receptors that are
expressed in the adult brain, and they show particularly
dense expression in regions that have a known involve-
ment in reward, addiction and cognitive function,
including the amygdala, cingulate cortex, pre frontal cor-
tex (PFC), ventral pallidum, caudate putamen, nucleus
accumbens (NAc), ventral tegmental area (VTA) and
lateral hypothalamus1,2. CB2Rs are expressed mainly
by immune cells, although recent evidence suggests
that such receptors are also expressed in neurons, glia
and endothelial cells in the brain3. CB1Rs and CB2Rs
are coupled to similar transduction systems, primarily
through Gi or Go proteins. CB1Rs directly inhibit the
release of GABA, glutamate and acetylcholine, which
produce widespread effects on neural signalling across
many neurotransmitter systems.
To date, the best-characterized eCB ligands are
N-arachidonylethanolamide (anandamide (AEA)) and
2-arachidonoylglycerol (2-AG). Owing to their lipid
nature, AEA and 2-AG are not stored in vesicles but are
synthesized ‘on demand’ by cleavage of membrane pre-
cursors and immediate release through Ca2+-dependent
mechanisms. AEA is derived from the phospholipid
precursor N-arachidonoyl-phosphatidylethanolamine
Synaptic plasticity
The process by which synaptic
communication strengthens or
weakens as a result of changes
in morphology, composition or
signal-transduction efficiency
in response to intrinsic or
extrinsic signals.
Endocannabinoid signalling
in reward and addiction
Loren H.Parsons1 and Yasmin L.Hurd2
Abstract | Brain endocannabinoid (eCB) signalling influences the motivation for natural
rewards (such as palatable food, sexual activity and social interaction) and modulates the
rewarding effects of addictive drugs. Pathological forms of natural and drug-induced
reward are associated with dysregulated eCB signalling that may derive from pre-existing
genetic factors or from prolonged drug exposure. Impaired eCB signalling contributes to
dysregulated synaptic plasticity, increased stress responsivity, negative emotional states
and cravings that propel addiction. Understanding the contributions of eCB disruptions
to behavioural and physiological traits provides insight into the eCB influence on
addiction vulnerability.
1Committee on the
Neurobiology of Addictive
Disorders, The Scripps
Research Institute, 10550
North Torrey Pines Road, La
Jolla, California 92037, USA.
2Friedman BrainInstitute,
Departmentsof Psychiatry
and Neuroscience, Icahn
School of Medicine at Mount
Sinai, 1470 Madison Avenue,
New York City, New York
10029, USA.
e‑mails: lparsons@scripps.
edu; yasmin.hurd@mssm.edu
doi:10.1038/nrn4004
Published online
16 September 2015
THE ENDOCANNABINOID SYSTEM
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CB1R ABHD6
AA
glycerol
Glutamate
GABA
AA
ethanolamine
VTA
Dopaminergic VTA neuron
DAGLα or
DAGLβNAPE-PLD
2-AG DAG NAPE AEA
2-AG
2-AG
2-AG AEA
AEA AEA
AEA
2-AG
2-AG
MAGL
FAAH
MAGL
2-AG
GABAergic
terminal
Limbic system
A collection of brain structures
that includes the amygdala,
hippocampus, limbic cortex,
limbic midbrain areas and
anterior thalamic nuclei,
regulates autonomic and
endocrine function and
participates in the control of
emotion, motivation, long-term
memory and olfaction.
(NAPE) and, although the precise mecha-
nisms for AEA formation are not known, N-acyl-
phosphatidylethanolamine-specific phospholipase D
(NAPE-PLD) is likely to have a role in this process.
2-AG derives primarily from the hydrolytic metabolism
of 1,2-diacylglycerol (DAG) by the sn-1-selective DAG
lipases (DAGLs) DAGLα and DAGLβ. AEA is primar-
ily catabolized by fatty acid amide hydrolase 1 (FAAH1),
and 2-AG is catabolized by monoacylglycerol lipase
(MAGL) and, to a lesser extent, by α,β-hydrolase 6
(ABHD6), cyclooxygenase 2 (COX2) and FAAH1. The
eCB catabolic enzymes have distinct cellular anatomical
locations, with MAGL being localized predominantly
in presynaptic terminals and FAAH1 being localized to
the postsynaptic domain of neurons (FIG.1). AEA and
2-AG both exert agonist activity at CB1R and CB2R.
AEA binds with slightly higher affinity to CB1R than to
CB2R and, like Δ9-tetrahydrocannabinol (Δ9-THC; the
main psychoactive component of the cannabis plant),
AEA exhibits low efficacy as an agonist at both recep-
tors, producing sub-maximal signalling on binding.
2-AG binds with essentially equal affinity at CB1R and
CB2R and exhibits greater agonist efficacy than AEA.
AEA and 2-AG also exhibit agonist properties at several
secondary receptors, including peroxisome proliferator-
activated receptors (PPARs), G protein-coupled receptor
55 (GPR55) and GPR119, and AEA exerts potent agonist
effects at transient receptor potential (TRP) ion channels,
includingTRPV1.
Neurobiology of reward
Mesocorticolimbic dopamine (DA) pathways, which
arise from the midbrain VTA, have a critical role in the
mediation of reward. In particular, the VTA DA projec-
tion to the NAc (part of the ventral striatum) has a prom-
inent role in positive reinforcement (FIG.2), that is, the
recognition of rewards in the environment and promo-
tion of goal-directed behaviour (approach behaviour),
resulting in reward acquisition. Natural rewards, such as
food, sex and exercise, and drugs of abuse — including
psychostimulants (such as cocaine and amphetamine),
nicotine, alcohol, opiates and cannabinoids — increase
NAc DA levels, and this neurochemical response con-
tributes to subjective reward and positive reinforcement4.
Components of the limbic system are also innervated
by VTA DA neurons, including the amygdala, hippo-
campus, orbitofrontal cortex and parts of the PFC.
These regions are interconnected in complex circuits
that involve excitatory (primarily glutamatergic) and
inhibitory (primarily GABAegic) projections5. In broad,
simplistic terms, amygdala circuits contribute to the for-
mation of associative reward- and fear-related memories,
hippocampal circuits are critical for declarative memory
functions and frontal cortical circuits mediate control
of executive functions. In turn, innervation of the NAc
by each of these circuits allows sensory and emotional
information to be converted into motivational actions
through the output to extrapyramidal motor systems. DA
signalling in the dorsal striatum does not have a major
influence in processing acute reward but has a key role in
the development of compulsive forms of reward seeking
and consumption6.
These same circuits participate in negative-reinforce-
ment mechanisms that promote behaviours for avoiding
or relieving aversive states. In general, NAc DA levels
are decreased by aversive conditions, such as unavoid-
able shock, chronic pain, certain patterns of over- or
under-eating and withdrawal from addictive drugs, and
the resultant increased activity of medium spiny out-
put neurons contributes to aversive states5,7. Negative-
reinforcement mechanisms associated with abstinence
from long-term access to palatable food or abused drugs
are mediated in part by excessive influence of pro-stress
signalling systems (such as corticotropin-releasing fac-
tor and dynorphin) and impaired function of anti-stress
signalling systems (such as neuropeptide Y and nocicep-
tin) in stress circuits that involve the central nucleus of
the amygdala (CeA), bed nucleus of the stria terminalis
(BNST), frontal cortex and medial shell of the NAc5,8.
Thus, reward processing is mediated in large part
through an interconnected network of structures,
including the VTA, NAc, ventral pallidum, CeA, BNST
and PFC. In addition to the well-known involvement of
DA described above, reward processing is heavily influ-
enced by many other systems, including the cholinergic,
opioid peptide, glutamatergic and GABAergic systems.
CB1Rs are present in each of the interconnected struc-
tures involved in reward1,2,9, where they exert widespread
modulatory influences on excitatory and inhibitory
signalling in a manner that influences reward process-
ing10,11. In particular, eCBs have a prominent role in
Figure 1 | Endocannabinoid biosynthesis, signalling and clearance. The
most-commonly accepted route for N-arachidonylethanolamide (anandamide (AEA))
synthesis is from catalysis of N-arachidonoyl-phosphatidylethanolamine (NAPE) via a
specific phospholipase D, N‑acyl‑phosphatidylethanolamine phospholipaseD
(NAPE-PLD). 2-arachidonoylglycerol (2-AG) derives from the hydrolysis of
1,2-diacylglycerol (DAG) via the sn-1-selective DAG lipases (DAGLs) DAGLα and DAGLβ.
DAGLα is found on the plasma membranes of both dopaminergic and non-dopaminergic
neurons in the ventral tegmental area (VTA), opposite cannabinoid1 receptor
(CB1R)-expressing glutamate and GABA axon terminals200. Termination of
endocannabinoid (eCB) signalling is initiated by cellular reuptake followed by
enzyme-mediated hydrolytic cleavage. 2-AG hydrolysis is primarily mediated by
presynaptic monoacylglycerol lipase (MAGL), although postsynaptic enzymes, including
α,βhydrolase 6 (ABHD6), also contribute to 2-AG clearance. AEA hydrolysis occurs in
postsynaptic cells through fatty acid amide hydrolase (FAAH). Although these
mechanisms are depicted here in the VTA, the pre- and postsynaptic organization of eCB
biosynthetic and hydrolytic enzymes is generally conserved throughout the brain. AA,
arachidonic acid.
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BLA
HIPP
DLStr GP
BNST
CeA
Glutamate
GABA
Dopamine
CRF
VP
PFC
VTA
Nature Reviews | Neuroscience
NAc
Level of CB1R
Low High
Intracranial self-stimulation
An operant behavioural
paradigm in which subjects
produce a behavioural
response (such as a lever press
or wheel turn) to receive brief
electrical pulses into specific
regions in the brain reward
pathways.
Conditioned place
preference
(CPP).A behavioural paradigm
used to study the rewarding
and aversive effects of drugs
through Pavlovian conditioning.
Self-administered
In a medical sense, when a
pharmacological substance is
purposefully delivered by test
subjects to themselves.
Operant self-administration is
a behavioural procedure in
which experimental subjects
learn to produce an operant
response (for example, a lever
press or nose poke) to receive
a drug reward (such as an
intravenous infusion, a small
bolus for oral consumption or
delivery of a discrete bolus of
vapour that is inhaled).
Discriminative stimulus
A stimulus in a drug-discrimina-
tion paradigm that the animal
has learned to associate with a
predictable consequence
(whether rewarding or
unrewarding) and that increases
the elicitation of a specific
behaviour by the animal.
fine-tuning the activity of the VTA–NAc DA projection
and its influence on approach and avoidance behaviours
that govern reward acquisition (FIG.3).
eCB signalling and reward
Exogenous AEA and 2-AG both increase extracellular
DA levels in the NAc in a CB1R-dependent manner12,
and the ECS exerts a strong influence on the fine-tuning
of midbrain DA-cell activity13. Through these and other
interactions, the ECS has a prominent influence on the
hedonic effects of natural rewards, such as food14, sexual
activity15 and social interaction16. This is mediated in
part through a direct CB1R modulation of the mesolim-
bic DA response to natural reward16 and through the
interactions between the ECS and other signalling sys-
tems (such as those involving endogenous opioids and
hypothalamic signalling molecules, among others)17,18.
The rewarding effects of cannabinoid receptor
activation are underscored by the fact that cannabis is
one of the most-widely used illicit substances world-
wide. Δ9-THC is the primary psychoactive constituent
in cannabis and exhibits low efficacy as an agonist at
CB1R and CB2R19. In animal models, both Δ9-THC and
synthetic CB1R agonists enhance brain reward func-
tion (as indexed by intracranial self-stimulation), produce
rewarding effects in the paradigm of conditioned place
preference (CPP) and are voluntarily self-administered
(intravenously and also directly into the NAc shell
and posterior VTA)20. These effects are critically reli-
ant on CB1R signalling and are highly dose-sensitive,
with a rapid shift to negative-reinforcing effects with
increasingdose.
In contrast to exogenous cannabinoid receptor ago-
nists, pharmacological enhancement of eCB levels gen-
erally does not produce rewarding effects perse. For
example, in most animal studies, selective eCB-clearance
inhibitors do not support operant self-administration,
do not produce CPP and do not alter brain stimula-
tion reward thresholds in rats and mice21. Similarly,
exogenously administered AEA or 2-AG, or selective
FAAH or MAGL inhibitors, do not produce Δ9-THC-
like discriminative stimulus effects. However, exogenous
AEA and 2-AG both support operant self-administra-
tion in squirrel monkeys21 and produce rewarding and
Δ9-THC-like effects in rats when they are administered
after eCB-clearance inhibition9,22. Concurrent FAAH and
MAGL inhibition in mice produces Δ9-THC-like dis-
criminative stimulus and behavioural effects22,23. These
findings suggest that robust engagement of eCB signal-
ling is needed to evoke rewarding effects. However, recent
evidence indicates that squirrel monkeys with a history
of AEA, nicotine or cocaine self-administration will self-
administer the FAAH inhibitor URB694, although this
compound does not produce Δ9-THC- or nicotine-like
discriminative stimulus effects and does not increase
mesolimbic DA release24. Although it remains to be
determined whether URB694 will be self-administered
by drug-naive monkeys or other species, this observation
indicates that FAAH inhibition is not aversive and may
produce mildly rewarding effects.
Cannabinoid receptor involvement in non-cannabinoid
drug reward. The presence of CB1Rs throughout brain
reward circuits and the rewarding effects produced
by CB1R activation allow for the possible influence
of eCB signalling on the acute rewarding effects pro-
duced by non-cannabinoid substances (the effects of
CB1R and FAAH manipulations on non-cannabinoid
drug reward are summarized in TABLE1). In general,
drugs that activate CB1Rs do indeed seem to facilitate
the rewarding effects of non-cannabinoid drugs. CB1R
agonists increase the motivational and reinforcing effects
of alcohol, nicotine and opiates, as indexed by animal
models of drug reward (including the CPP and oper-
ant self-administration assays), whereas diminished
Figure 2 | Distribution of endocannabinoid signalling
mechanisms within the brain reward circuits.
Cannabinoid1 receptors (CB1Rs) are expressed throughout
the regions implicated in reward and addiction, including
the basolateral amygdala (BLA), prefrontal cortex (PFC),
hippocampus (HIPP), ventral pallidum (VP), globus pallidus
(GP), dorsolateral striatum (DLSTr), nucleus accumbens
(NAc), ventral tegmental area (VTA), bed nucleus of the stria
terminalis (BNST) and central nucleus of the amygdala
(CeA)1,2,9. In general, the expression patterns of
endocannabinoid (eCB)-biosynthetic enzymes (for
example, N-acyl-phosphatidylethanolamine
phospholipaseD (NAPE‑PLD) and 1,2‑diacylglycerol
lipase-α (DAGLα)) and hydrolytic eCB-clearance enzymes
(for example, fatty acid amide hydrolase (FAAH) and
monoacylglycerol lipase (MAGL)) are similar to those for
CB1Rs across the regions depicted here201,202. Within the
amygdala, CB1R, DAGLα, MAGL and FAAH expression is
highest in the lateral and basolateral nuclei, with
substantially lesser expression in the CeA56,201. In the dorsal
striatum, there is a comparable medial-lateral gradient of
CB1R and DAGLα expression, with greater levels of
expression evident in lateral aspects201,203. Comparatively
weaker CB1R, DAGLα and FAAH expression is observed in
the NAc201. Although little to no CB1R expression is found in
dopamine cells in the NAc, DAGLα has been found in both
dopaminergic and non-dopaminergic cells in this region204.
CRF, corticotropin-releasing factor.
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a VTA
GABA
2-AG 2-AG
2-AG AEA
AEA AEA
AEA
2-AG
2-AG
MAGL
2-AG
iMSN
dMSN
Dopamine
2-AG
2-AG
2-AG Gi/o
D2R
D1R
b NAc
Activation leads
to stimulus avoidance
To VP
Indirect
pathway
Activation leads to approach
or appetitive behaviour
To VTA
eCB-LTD
Glutamate
CB1R
Glutamatergic
cortical neuron
AEA and 2-AG
synthesis
Dopaminergic
VTA neuron
Suppression of
glutamatergic signalling
Arachidonic acid
Glycerol
MAGL
GABAergic input
from the pallidus,
the RMTg or a
local interneuron
MAGL
Suppression of GABAergic signalling
GABAergic terminal of the
dMSN projection from the NAc
FSI
Direct
pathway
Glutamatergic
neuron from the
PFC, BLA or vHIPP
From VTA
CB1R signalling (through either genetic deletion or
pharmacological antagonism) attenuates the motiva-
tional and rewarding effects of these drugs11,25,26. The
effects of CB1R antagonism on alcohol and nicotine
reward result in part from a diminished ability of these
drugs to increase NAc DA release27. Blockade of CB1Rs
specifically in the VTA decreases alcohol and nicotine
self-administration28,29, and blockade of CB1Rs spe-
cifically in the NAc reduces alcohol consumption28,30.
However, whereas nicotine reward is critically depend-
ent on the mesolimbic DA system31, the motivational
and rewarding effects of alcohol and opiates are less
Figure 3 | Endocannabinoid influences in the VTA and
NAc contributing to approach and avoidance
behaviours. a | Endocannabinoids (eCBs) influence
ventral tegmental area (VTA) synaptic signalling.
2-Arachidonoylglycerol (2-AG) produced by dopaminergic
VTA neurons acts on cannabinoid1 receptors (CB1Rs) on
nearby glutamatergic and GABAergic terminals before
being degraded by α,βhydrolase 6 (ABHD6) or
monoacylglycerol lipase (MAGL). CB1Rs mediate robust
inhibition of GABA inputs arising from the pallidus,
rostromedial tegmental (RMTg) nucleus and local
interneurons onto VTA dopamine (DA) cells205, and most
evidence points to a role for 2-AG but not
N-arachidonylethanolamide (anandamide (AEA)) in these
processes109,206. CB1Rs are also localized on glutamatergic
terminals synapsing on VTA DA neurons, with relatively
greater expression on vesicular glutamate transporter1
(VGLUT1)-positive terminals of cortical origin compared
with VGLUT2-expressing terminals of subcortical origin207.
Extensive evidence demonstrates eCB-mediated
suppression of glutamate signalling in the VTA208. Thus,
eCBs have a prominent role in fine-tuning the activity of the
mesolimbic DA projection through modulation of both
excitatory and inhibitory signalling in the VTA. b | The eCBs
also influence nucleus accumbens (NAc) synaptic signalling.
The majority of NAc neurons (>90%) are GABAergic
medium spiny neurons (MSNs) that comprise the direct-
and indirect-projection pathways. Direct-pathway MSNs
(dMSNs) project to midbrain regions, including the VTA,
and activation of this pathway increases behaviour towards
a stimulus (approach or appetitive behaviour).
Indirect-pathway MSNs (iMSNs) project to the ventral
pallidum (VP), and activation of this pathway increases
stimulus avoidance209. dMSNs express excitatory D1 DA
receptors (D1Rs), whereas iMSNs express inhibitory D2Rs;
thus, reward-related phasic DA release activates the direct
pathway and inhibits the indirect pathway, thereby
increasing approach behaviour and reducing avoidance
behaviour210. NAc MSN activity is also heavily modulated by
glutamatergic inputs from the prefrontal cortex (PFC),
basolateral amygdala (BLA) and ventral hippocampus
(vHIPP), which express CB1Rs211. CB1R-mediated
suppression of excitatory signalling (eCB-mediated
long-term depression (eCB-LTD)) is preferentially active at
iMSN synapses212, possibly resulting from D2R-mediated
eCB production from iMSN cell bodies213. Thus, increased
NAc eCB formation preferentially reduces excitatory input
to iMSNs versus dMSNs, resulting in decreased avoidance
behaviour. Through these mechanisms, increased eCB
signalling in the NAc increases approach behaviour while
reducing avoidance-related processing, thereby enhancing
appetitive responses towards a stimulus. CB1Rs are also
expressed on terminals of fast-spiking interneurons (FSIs) in
the NAc, the majority of which are electrically and
chemically coupled and provide direct innervation to
adjacent MSNs214. FSIs exert important influence on the
synchronization of neural ensemble activity and thus eCB
signalling may also exert critical influence on NAc output
through feedforward modulation of MSN network activity214.
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Non-contingent
Drug delivery that is
involuntary (experimenter-
administered) or is not
dependent on a behavioural
response by an experimental
subject; sometimes referred to
as forced administration.
DA-dependent32,33, and the CB1R modulation of the
rewarding effects of these drugs probably involves non-
dopaminergic mechanisms. Indeed, CB1R antagonism
does not alter opiate-induced increases in NAc DA
levels but reduces opiate reward through the preven-
tion of opiate-induced reductions in ventral pallidal
GABA release34. In comparison to these drugs, CB1R
manipulations on psychostimulant reward have mod-
est and less-consistent effects. CB1R agonists reduce
the facilitation of brain stimulation reward produced
by cocaine and reduce cocaine self-administration35,36.
Most reports indicate that CB1R antagonism does not
affect psychostimulant reward (as assessed by cocaine-
induced enhancement of brain stimulation reward, CPP
and self-administration) or cocaine-induced increases in
NAc DA levels37 (but see REFS27,37).
Recent evidence in mice also implicates CB2Rs in
the modulation of drug reward, including an inhibi-
tory influence on cocaine and alcohol reward38,39 but a
facilitatory influence on nicotine reward40,41. However,
disparate observations have been made in rats39,42,43, and
it is possible that these findings are influenced by species
differences in CNR2 splicing that confer distinct CB2R
structure, function or pharmacology39.
Alterations in brain eCB levels elicited by drugs of abuse.
Given the on-demand nature of eCB production and the
associated modest eCB signalling tone under baseline
conditions, the robust influence of cannabinoid receptor
signalling on non-cannabinoid drug reward has led to
the hypothesis that drug exposure increases brain eCB
formation. Substantial evidence demonstrates that there
are alcohol-induced alterations in post-mortem eCB con-
tent in the rodent brain, although inconsistencies among
studies cloud definitive conclusions regarding the direc-
tion of change and the regional nature of the effects44.
For example, alcohol exposure increases extracellular
2-AG levels in rat NAc (measured by invivo microdi-
alysis), and this is more pronounced following voluntary
self-administration than after non-contingent alcohol
exposure28,30. By contrast, extracellular AEA levels in the
NAc are unaltered by alcohol self-administration and are
decreased by non-contingent alcohol administration.
Alcohol also seems to induce region-specific changes
in brain-tissue eCB levels, with alcohol-induced
disruptions being consistently observed in striatal
regions30,45,46 but not in frontal cortical areas28. This is
consistent with evidence that alcohol consumption is
reduced by CB1R antagonism in the VTA and NAc but
not in the PFC28,30,47.
Similarly to alcohol, nicotine alters eCB levels in the
rodent brain, with factors such as the brain region evalu-
ated and the voluntary nature of drug exposure having
important relevance to the effects observed. Repeated
non-contingent nicotine injections increase AEA lev-
els in rat limbic forebrain and dorsal striatal tissue but
decrease both AEA and 2-AG levels in cortical tissue48.
Intravenous nicotine self-administration increases extra-
cellular levels of both AEA and 2-AG in the rat VTA,
and the effect on 2-AG is sensitized by chronic nicotine
exposure49. Interestingly, although VTA 2-AG levels are
elevated by both voluntary and non-contingent nicotine
exposure, VTA AEA levels are increased only by vol-
untary nicotine self-administration49. Together with the
evidence of distinct patterns of brain eCB levels induced
by volitional versus non-contingent alcohol exposure28,30,
these data suggest that brain eCB production is influ-
enced not only by drug-related pharmacological effects
but also by neural activity engaged by active drug self-
administration (possibly related to the motivation for
drug consumption).
Relatively less is known regarding the effects of other
rewarding drugs on brain eCB levels. Available evi-
dence consistently indicates that opiates increase AEA
but decrease 2-AG tissue concentrations in the striatum,
limbic forebrain and hippocampus50,51. Similarly, heroin
self-administration increases extracellular AEA with a
concomitant decrease of extracellular 2-AG levels in the
Table 1 | Summary of CB1R and FAAH influences on non-cannabinoid drug reward
Genetic or
pharmacological
manipulation
Drug
Ethanol Nicotine Opiates Stimulants
CB1R knockout CPP
Operant self-administration
Ethanol-induced NAc DA
CPP
Operant self-administration
CPP
Operant self-administration
No change in CPP
No change in operant
self-administration
CB1R antagonist Operant self-administration
Preference
Ethanol-induced NAc DA
CPP
Operant self-administration
Nicotine-induced NAc DA
CPP
Operant self-administration
No change in
morphine-induced NAc DA
Operant self-administration
(AM251)
Cocaine effects on ICSS
No change in
cocaine-induced NAc DA
CB1R agonist Operant self-administration
Motivation for ethanol
CPP CPP
Motivation for heroin
Operant self-administration
Cocaine effects on ICSS
FAAH inhibition Operant self-administration
Preference for ethanol
versus water
CPP (mouse, CB1R)
CPP (rat, non-CB1R)
Operant self-administration
(rat, non-CB1R)
Nicotine-induced NAc DA
(rat, non-CB1R)
No change in operant
self-administration
No change in
morphine-induced VTA DA
excitation
No change in
cocaine-induced VTA DA
excitation
No change in operant
self-administration
CB1R, cannabinoid 1 receptor; CPP, conditioned place preference; DA, dopamine; FAAH, fatty acid amide hydrolase; ICSS, intracranial self-stimulation (an index of
brain reward function); NAc, nucleus accumbens; VTA, ventral tegmental area.
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Epigenetic mechanisms
Methods by which functionally
relevant changes to the
genome occur that do not
involve disruptions in the
nucleotide sequence of DNA;
these include DNA
methylation, histone
modification and non-coding
RNA-associated gene silencing
Extended amygdala
A grouping of brain regions
that orchestrate emotional
behavioural responses and
includes the central nucleus of
the amygdala, sublenticular
substantia innominate, nucleus
accumbens shell and the bed
nucleus of the stria terminalis.
rat NAc30. Psychostimulants generally produce modest
disruptions in brain eCB content, with subtle increases
and decreases in 2-AG concentration in forebrain fol-
lowing non-contingent acute and chronic cocaine
exposure, respectively (no other alterations are evident
regardless of region analysed)48,52. Moreover, voluntary
cocaine self-administration does not alter rat extracel-
lular NAc eCB levels30 but decreases 2-AG content in
frontal cortex and hippocampal tissue53–55.
Collectively, these findings indicate that alcohol,
nicotine and opiates alter brain eCB content, consonant
with the CB1R influence on the behavioural effects pro-
duced by these drugs. The generally modest effects of
psychostimulants on brain eCB levels are in-line with
the subtle CB1R influence on psychostimulant-induced
behaviours. Similarly to that seen with multiple biologi-
cal conditions, drug exposure often produces distinct
and sometimes opposite effects on brain AEA and 2-AG
levels. This suggests differential regulation of the synthe-
sis and/or degradation of these eCB moieties at specific
synapses that may arise from the segregation of MAGL
and FAAH in the pre- and postsynaptic compartments56,
or the hypothesized role of AEA and 2-AG in regulating
‘tonic’ and ‘phasic’ signalling in the ECS, respectively57.
Although a general picture of drug-induced alterations
in brain eCB levels is emerging, experimental differ-
ences between studies — including the drug dose used,
the method of drug exposure and the duration of treat-
ment — make it difficult to draw strong conclusions, and
additional studies are warranted.
Influence of eCB tone on drug reward. eCBs are rap-
idly degraded, so strategies that reduce eCB clear-
ance have been used as a means to further investigate
the eCB influence on drug reward. Most investiga-
tions have focused on the effects of FAAH inhibition,
because selective tools for inhibiting MAGL and
other eCB-clearance enzymes were not available until
recently. Such studies have shed light on important spe-
cies differences that confound the overall conclusions
that can be made from existing data. For example,
FAAH inhibition in mice increases nicotine reward in
the CPP paradigm58,59, but FAAH inhibition in rats pre-
vents nicotine-induced CPP, diminishes nicotine self-
administration and blunts nicotine-induced increases in
NAc DA release60. The potentiation of nicotine reward
in mice by FAAH inhibition is CB1R-mediated, whereas
the reduction in nicotine reward in rats results from
activation of PPARα by non-cannabinoid lipids, such
as oleoylethanolamide and palmitoylethanolamide, that
are hydrolytically cleared by FAAH61. FAAH inhibition
also produces distinct species-related alterations in alco-
hol consumption, with increased intake being observed
in mice but not in rats62–65. The mechanisms underlying
these differences are not understood. Brain region-spe-
cific disruptions in FAAH activity may be an important
factor. In regard to alcohol reward, inhibition of FAAH
activity specifically in the PFC results in increased alco-
hol consumption, and rats selectively bred for high alco-
hol intake and preference are characterized by reduced
FAAH activity specifically in the PFC64,65. The effects of
FAAH inhibition on opiate and psychostimulant reward
have primarily been studied in rats. FAAH inhibition
does not alter morphine- or cocaine-induced disrup-
tions in VTA DA-cell firing or the self-administration
of either drug66,67. However, FAAH inhibition diminishes
cocaine-induced alterations in NAc medium-spiny neu-
ron activity68, and this may contribute to enhanced sen-
sitization of both cocaine-induced motor activity and
mesolimbic DA responses following repeated cocaine
exposure69. Other studies have investigated the effects of
putative eCB transport inhibitors, such as AM404 and
VDM11, and the findings thus far suggest that these
compounds produce subtle and inconsistent effects on
nicotine and cocaine reward70–73.
Although growing evidence implicates ECS influences
in the modulation of acute drug reward, additional efforts
are needed to further clarify the nature of eCB disrup-
tions caused by different classes of abused drug and the
neural mechanisms through which these eCB influences
are mediated. Selective inhibitors of 2-AG clearance have
recently been developed, but studies using them are still in
their infancy and there are presently no published reports
on the effects of enhanced 2-AG tone on drug reward
and related physiological events. As such, there remains a
substantial gap of knowledge, given the prominent 2-AG
influence on neural signalling and plasticity related to
both drug and natural rewards. Nevertheless, the role of
the CB1R in drug reward is unequivocal and, although
there is evident complexity related to the effects produced
by eCB-clearance inhibition (producing discrete modula-
tion of eCB tone in specific synapses and circuits when
compared with broad CB1R activation by exogenous
CB1R agonists), the extant evidence strongly supports an
eCB influence on the sensitivity to, and motivation for,
several drugs ofabuse.
eCB signalling in addiction
Numerous factors influence the transition from inter-
mittent, controlled drug use to the compulsive forms of
drug-seeking and drug-taking behaviour that charac-
terize addiction. Substantial evidence implicates genetic
influences in the development of substance-use disor-
ders (SUDs) and pathological forms of eating, sexual
behaviour and gambling74, and it is increasingly recog-
nized that epigenetic mechanisms drive lasting changes
in addiction-related gene expression75. Long-term drug
exposure induces lasting neuroadaptations in motiva-
tional mechanisms that propel drug-seeking behaviour
and drug use. Although initial drug use is motivated by
hedonic processes, prolonged drug exposure progres-
sively blunts reward system function, thereby leading to
escalated frequency and amount of drug consumption,
resulting in a dependent state wherein negative affective
symptoms (for example, dysphoria, anxiety and irritabil-
ity) emerge during abstinence. These negative emotional
states arise from the recruitment of stress signalling sys-
tems (such as corticotropin releasing factor and dynor-
phin) and dysregulation of mechanisms that constrain
these responses (such as neuropeptide Y and nociceptin)
within the extended amygdala5. Renewed drug consump-
tion alleviates these negative affective states, and this is
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Conditioned reinforcement
The process through which
neutral stimuli acquire
motivational properties
through association with a
primary reinforcer.
Stochastic optical
reconstruction microscopy
A super-resolution imaging
technique that uses sequential
activation and time-resolved
localization of photoswitchable
fluorophores to create
high-resolution images
enabling precise fluorophore
localization with nanometre
resolution.
conceptualized to motivate compulsive drug use through
negative reinforcement5. Superimposed on these pro-
cesses is a dysregulation of corticostriatal mechanisms
mediating stimulus–response learning, constraint of
impulsivity, conditioned reinforcement and incentive moti-
vation, resulting in a narrowed focus on drug-seeking at
the expense of natural rewards6.
eCBs exert prominent modulatory influence on the
extended amygdala and corticostriatal circuits5,32, and
increasing evidence suggests that pre-existing genetic
influences on the ECS and/or drug-induced dysregula-
tion of eCB function participate in the development and
maintenance of addictions, including pathological forms
of eating (BOX1). The following sections consider the
consequences of chronic drug exposure on eCB signal-
ling within the reward circuitry and related disruptions
in synaptic plasticity, affective state and learning and
memory mechanisms related to extinction and relapse.
Finally, evidence is discussed for an influence of innate
disruptions in ECS function (eCB gene polymorphisms)
as vulnerability factors for substance abuse and addictive
disorders inhumans.
Chronic drug exposure and eCB function. It is unsur-
prising that chronic cannabis use disrupts brain can-
nabinoid receptor availability and function. Using the
inv ivo technique of positron emission tomography (PET)
imaging, one study76 reported that there was downregu-
lation of brain CB1Rs in daily cannabis users, that the
level of downregulation correlated with the number of
years of cannabis use and that this downregulation was
reversed after 1month of monitored abstinence. Another
PET study reported a global reduction in CB1R avail-
ability77 driven by differences in the temporal lobe, ante-
rior and posterior cingulate cortex and NAc. Similarly,
animals given non-contingent chronic cannabinoid
exposure exhibit decreased CB1R function throughout
the brain78,79. Recent experiments using stochastic optical
reconstruction microscopy demonstrate that chronic expo-
sure to clinically relevant doses of Δ9-THC results in a
startling loss of CB1Rs on terminals of perisomatically
projecting GABAergic interneurons in the mouse hippo-
campus and internalization of the remaining CB1Rs80.
The resulting deficits in inhibitory CB1R control over
hippocampal GABA release persisted during several
weeks of Δ9-THC abstinence, and this may underlie the
enduring loss of hippocampal long-term potentiation
(LTP) in rodents and memory deficits in humans evident
following chronic cannabinoid exposure81.
Surprisingly little is known of the effect of chronic
cannabinoid exposure on other facets of ECS function.
Chronic cannabinoid exposure increases enzymatic
clearance of AEA and reduces brain tissue AEA content
in rodents82,83, and frequent cannabis smokers present
decreased AEA and increased 2-AG levels in blood84,85,
although increased serum AEA levels are evident follow-
ing at least 6months of cannabis abstinence86. The con-
tribution of these disruptions to cannabis-use disorder
and related physiological and behavioural disruptions
is presently unexplored. However, as discussed below,
eCBs provide important homeostatic constraint over
emotional state87 and sleep function88, and it is conceiv-
able that Δ9-THC-induced impairment of eCB signal-
ling contributes to the negative emotional states and
sleep disturbances present during protracted cannabis
abstinence89,90.
Several findings support the hypothesis that chronic
exposure to non-cannabinoid drugs disrupts eCB signal-
ling and processing. Chronic alcohol exposure in rodents
alters eCB-related gene expression in a manner sensitive
to the intermittent nature of alcohol exposure and post-
alcohol abstinence period91 and downregulates CB1R
expression and function45,92. Post-mortem studies of
alcohol-dependent humans also demonstrate disrupted
CB1R expression in the ventral striatum and corti-
cal regions93, and invivo imaging studies demonstrate
decreased CB1R availability in heavy-drinking alco-
holics that persists for at least 1month of abstinence94,95
(but see REF.96). Although a potential contribution of
variants of CNR1 (which encodes CB1R) to these obser-
vations cannot be excluded, a common interpretation
based on animal studies is that these CB1R adaptations
in humans with alcoholism are a consequence of pro-
longed alcohol-induced increases in brain eCB levels.
This is supported by evidence of transient recovery (and
perhaps eventual upregulation) of CB1R function in
humans during protracted alcohol abstinence92,97.
In rodents, chronic nicotine exposure induces distinct
age-related disruptions in CB1R binding, with increased
levels being evident in the PFC, VTA and hippocampus
Box 1 | Endocannabinoid signalling and eating disorders
Food and drug addiction derive in part from aberrant brain reward function and share
overlapping neuroadaptations in the mesolimbic system176. Similarly to drug addiction,
food addiction is defined by compulsive over‑ or under‑eating, aberrant feeding despite
negative consequences and unsuccessful attempts to ‘normalize’ dysfunctional eating.
The rewarding effects of self‑starvation or binge eating may involve disrupted
endocannabinoid (eCB) function177. Anorexia nervosa and binge‑eating disorder are
associated with increased blood N‑arachidonylethanolamide (anandamide (AEA)) levels
and diminished levels of leptin178 (an anorectic hormone that inhibits AEA synthesis).
Anorexia is associated with a CNR1 (the gene encoding cannabinoid1 receptor (CB1R))
AAT‑triplet repeat ((AAT)n) polymorphism179 (but see REF.180) and a synergistic
association between the C385A fatty acid amide hydrolase (FAAH) and CNR1 rs1049353
polymorphisms181. The C385A FAAH polymorphism is also associated with obesity182,
although it is unknown whether this may influence hedonic mechanisms, metabolism or
both. Anorexia and bulimia nervosa are also associated with elevated plasma CNR1
mRNA levels183, increased CB1R availability in frontal cortical areas184 and a
nonsynonymous CNR2 (the gene encoding CB2R) polymorphism185. Preclinical models
of anorexia suggest therapeutic effects for Δ9‑tetrahydrocannabinol (Δ9‑THC)186 (but
see REF.187). Although two small clinical trials did not show Δ9‑THC efficacy176, a larger
trial demonstrated small but significant weight gain in women with severe anorexia
nervosa following 4weeks of treatment with dronabinol188. Conversely, in binge‑eating
disorder, the CB1R antagonist rimonabant significantly reduces binge eating and
promotes weight loss, with only modest presentation of psychiatric side effects189,190.
A withdrawal state similar to that associated with drugs of abuse is evident in rats
during forced abstinence from highly palatable food191,192, with symptoms including
increased anxiety and excessive consumption on renewed access to palatable food.
These symptoms result from increased corticotropin-releasing factor1 (CRF1; also
known as CRHR1) signalling in the central nucleus of the amygdala (CeA)8 and are
reversed by increased CeA 2-arachidonoylglycerol (2-AG)–CB1R signalling193. This
suggests that dysregulated CeA function is a common factor contributing to
pathological motivation for drugs and food, and that CeA eCB signalling may counter
withdrawal‑related stress signalling.
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of adolescent, but not adult, rats98 and increased hip-
pocampal and decreased striatal CB1R binding being
seen in adult rats during protracted nicotine absti-
nence99. Few studies have investigated altered CB1R
binding following chronic opiate or psychostimulant
exposure, but findings in rodents implicate impaired
CB1R function in the development and expression of
opiate dependence100,101 and demonstrate that chronic
cocaine use increases CB1R binding in the dorsal stria-
tum, NAc and cortical areas102. Interestingly, detoxified
cocaine addicts present significant increases in plasma
AEA and decreases in plasma 2-AG content103, but the
functional consequence of these disturbances is not
known. Overall, accruing data suggest that long-term
exposure to various drug classes compromises eCB
processing and CB1R expression and function. As dis-
cussed below, these perturbations may contribute to
aberrant neural signalling during acute and protracted
drug abstinence.
Addiction-related synaptic plasticity. The development
and persistence of addiction is attributed to maladaptive
synaptic plasticity evident in the neuronal reorganization
(molecular, cellular and functional activity) of meso-
corticolimbic and striatal pathways. eCB signalling at
CB1Rs is implicated in several forms of synaptic plasticity,
most commonly in depolarization-induced suppression
of excitatory transmission (DSE) or inhibitory transmis-
sion (DSI), short-term depression (STD) and long-term
depression (LTD; a prolonged form of weakened synaptic
strength)68,104. STD, DSE and DSI are mediated primarily
by 2-AG signalling, typically persist for a minute or less,
and have been observed in brain areas relevant to reward
and addiction, including the VTA, basolateral amyg-
dala, hippocampus, neocortex and substantia nigra10. By
comparison, eCB-mediated LTD can persist for hours or
weeks, is particularly important in learning and memory,
and has also been observed in addiction-related regions,
including the NAc, VTA, amygdala, PFC, hippocampus
and dorsal striatum10.
Acute and chronic alcohol exposure reduces
CB1R-dependent plasticity, resulting in long-lasting
disinhibition of striatal output neurons and diminished
eCB-mediated LTD (eCB-LTD) at inhibitory striatal
synapses105,106. Because the dorsal striatum mediates
reward-guided learning and habitual behaviour, these
eCB disruptions may contribute to maladaptive habitual
behaviour that perpetuates addiction107. Cocaine dimin-
ishes eCB-LTD of excitatory transmission in the NAc108
and facilitates eCB-LTD of inhibitory signalling at
VTA DA synapses109,110, resulting in diminished inhibi-
tory control over VTA DA-cell activity and heightened
excitatory signalling in the NAc (FIG.4). Cocaine also
disrupts eCB-LTD of excitatory transmission in the
BNST111, a component of the extended amygdala, and
this may contribute to aberrant stress–reward inter-
actions (via projections to the VTA)112 and excessive
anxiety-like behaviour. Similarly, chronic Δ9-THC or
synthetic CB1R agonist exposure abolishes eCB-LTD
of excitatory and inhibitory signalling in the NAc
and hippocampus113,114, which may significantly affect
reward processing mediated by these regions. Little is
known regarding opiate- or nicotine-induced disrup-
tions in eCB-mediated synaptic plasticity, although
cue-induced reinstatement of nicotine-seeking behav-
iour (an animal model of relapse) relies in part on the
induction of CB1R-mediated LTP of cortical synapses in
the BNST115. Thus, chronic drug exposure disrupts eCB-
mediated forms of synaptic plasticity in several regions
involved in reward processing. As discussed below,
impaired eCB-mediated plasticity may also contribute
to dependence-related affective disruptions that serve
to sustain drug dependence.
Withdrawal-related affective disruption. Stress has a
prominent role in the development of addiction116, and
stress exposure disrupts eCB-mediated plasticity in
regions that participate in emotional control, includ-
ing the NAc, amygdala and BNST117. Withdrawal from
most drugs of abuse is associated with increased stress
responsivity and persistent negative affective symp-
toms, such as anxiety and depression, the severity of
which are closely associated with relapse susceptibility.
Comorbidity of affective disorders and SUDs is preva-
lent, and pre-existing negative affective traits may be
an antecedent to addiction. The ECS participates in a
negative-feedback system that constrains emotional
distress under stressful circumstances and contrib-
utes to the suppression of aversive memories117,118. This
function is reliant on eCB-mediated forms of synaptic
plasticity, and deficient eCB signalling is associated with
increased anxiety and depression. As such, impaired
eCB function may contribute to the negative affective
states and increased stress responsivity that underlie
negative-reinforcement mechanisms driving drug use
by dependent individuals and that contribute to drug
relapse following periods of abstinence.
Mice lacking CB1Rs exhibit greater anxiety-like
behaviour than normal animals during nicotine with-
drawal119, although innate anxiety-like behaviour in the
knockout mice clouds interpretations. Studies evaluating
eCB-clearance inhibition provide more-direct insight
into withdrawal-related eCB disruption and negative
affect. Acute FAAH inhibition reverses enhanced anx-
iety-like behaviour that is normally present during both
nicotine and alcohol withdrawal65,120, and the eCB-trans-
port inhibitor AM404 attenuates depression-like behav-
iour during nicotine withdrawal121. Post-traumatic stress
disorder is particularly prevalent among individuals
with alcohol-use disorders, and this is often modelled
in rodents using the fear-potentiated startle paradigm
to study reflexive physiological reaction to a stimulus.
Rodents selectively bred for high alcohol consumption
exhibit greater fear-potentiated startle than corollary
lines bred for low alcohol consumption122,123. In addi-
tion, acute FAAH inhibition by LY2183240 reduces
fear-potentiated startle in high alcohol-preferring, but
not low alcohol-preferring, mice124, consistent with the
efficacy of FAAH inhibition for accelerating the extinc-
tion of aversive memory125 . LY2183240 also enhances the
conditioned rewarding effects of alcohol without alter-
ing alcohol consumption itself, suggesting that FAAH
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Nature Reviews | Neuroscience
MAGL
2-AG
MAGL
2-AG
GABA
Glutamate
eCB
GABA
synapse
Glutamate
synapse
GABAAR
mGluR
AMPAR
b Drug-induced enhancement of eCB-LTDi in VTA
Enhanced excitability of DA VTA neurons
c Drug-induced loss of of eCB-LTDe in NAc
NAc
MSN
Enhanced excitation of NAc cells
MAGL
2-AG
MAGL
2-AG
eCB
2-AG
AEA
NAPE DAG
NAPE-PLD DAGα or
DAGβ
FAAH
eCB-LTDe eCB-LTDi
VTA DA cell
MAGL
2-AG
MAGL
2-AG
eCB
2-AG
AEA
NAPE DAG
NAPE-PLD DAGα or
DAGβ
FAAH
eCB-LTDe eCB-LTDi
2-AG
AEA
NAPE DAG
NAPE-PLD DAGα or
DAGβ
FAAH
a
eCB-LTDe eCB-LTDi
eCB-mediated plasticity of excitatory and inhibitory signalling
inhibition influences memory-related processes (condi-
tioned fear and conditioned alcohol reward) in animals
predisposed towards high alcohol consumption.
Addiction-related learning and memory
Both positive and negative memories and conditioned
cues associated with drug use perpetuate drug-seeking
behaviour and the continued cycle of abuse. The ECS has
a prominent role in learning and memory processes126,
and CB1R signalling is strongly linked to the conditioned
rewarding effects of alcohol, nicotine and opiates11,25.
Although drug-induced conditioning effects are gener-
ally interpreted in the context of drug reward, a CB1R
influence on the associative learning aspects of drug
exposure is also possible, which as discussed below may
have relevance to the persistent reactivity to drug-related
memories that characterizes addiction.
Drug-seeking (relapse). Drug exposure produces pow-
erful interoceptive effects that become associated with
environmental cues, such that these cues alone can
induce craving and promote relapse following periods of
abstinence127. In addition to conditioned drug memories,
acute exposure to a preferred drug or pharmacologically
related agent (that is, drug priming) and stressful events
can precipitate relapse116.
Animal models of relapse demonstrate an impor-
tant cannabinoid influence on the reinstatement of
extinguished drug-seeking and drug-taking behav-
iours. Δ9-THC and synthetic CB1R agonists reinstate
drug-seeking for cannabinoids, alcohol, nicotine, opi-
ates and cocaine, whereas CB1R antagonists attenuate
drug-seeking behaviour associated with each of these
drugs25,128,129. CB1Rs in the PFC and NAc shell influence
cue-induced reinstatement of both heroin- and nicotine-
seeking behaviour, whereas CB1Rs in the basolateral
amygdala contribute to cue-induced nicotine- but not
heroin-seeking behaviour130,131. Despite the subtle effects
of CB1R inactivation on psychostimulant self-adminis-
tration, CB1R antagonism attenuates drug-primed, cue-
induced and some forms of stress-induced reinstatement
of cocaine- and methamphetamine-seeking behaviour in
Figure 4 | Drug-induced alterations in endo-
cannabinoid-mediated synaptic plasticity.
Simplified summary of the effects of cocaine,
Δ9-tetrahydrocannabinol (Δ9-THC) and possibly other
drugs of abuse on endocannabinoid-mediated
long-term depression (eCB-LTD). a | Under normal
circumstances, eCB-LTD is induced by afferent
stimulation with or without postsynaptic depolarization,
resulting in 2-arachidonoylglycerol (2-AG) formation
from postsynaptic cells. 2‑AG activates cannabinoid1
receptors (CB1Rs) on stimulated or neighbouring,
non-stimulated neurons, which together with other
events (for example, increased [Ca+2], NMDA receptor
(NMDAR) stimulation and dopamine (DA) D2 receptor
stimulation) results in persistently decreased
neurotransmitter release. The presynaptic signalling
mechanisms contributing to eCB-LTD are not fully
understood. Depending on brain region, eCB-LTD of
both excitatory (glutamatergic; eCB-LTDe) and
inhibitory (GABAergic; eCB-LTDi) afferents has been
described. b | Repeated cocaine exposure facilitates
eCB-LTDi in the ventral tegmental area (VTA)109,110,
resulting in diminished inhibitory constraint of VTA
DA-cell activity and increased excitability. c | By
contrast, eCB-LTDe is lost in the nucleus accumbens
(NAc) medium spiny neurons (MSN) following exposure
to either cocaine or Δ9-THC108,113,114, resulting in
diminished constraint of glutamatergic release and
increased excitation of NAc cells. Thus, drug exposure
results in concurrent loss of eCB-mediated plasticity
that normally provides inhibitory control over VTA
DA-cell excitation and that normally constrains
excitatory signalling in the NAc terminal region,
conferring an overall enhancement of mesolimbic
signalling. AEA, N-arachidonylethanolamide
(anandamide); AMPAR, AMPA receptor; DAGL,
1,2-diacylglycerol lipase; FAAH, fatty acid amide
hydrolase; GABAAR, GABAA receptor; mGluR,
metabotropic glutamate receptor; NAPE,
N-arachidonoyl-phosphatidylethanolamine; NAPE-PLD,
N-acyl-phosphatidylethanolamine-specific
phospholipase D.
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rats25. Thus, CB1R signalling modulates drug-seeking for
various pharmacologically distinct drugs. There is also
evidence that CB1R antagonism blocks both cue- and
priming-induced reinstatement of seeking behaviour
for non-drug rewards, such as sucrose and corn oil132,133
(but see REF.134). Accordingly, CB1R signalling seems
to participate in the modulation of conditioned reward
in general.
Drug-primed and cue-induced nicotine- and cocaine-
seeking behaviour are reduced following acute FAAH
inhibition that leads to elevated AEA levels25,60, which may
be surprising considering that CB1R agonists enhance
both nicotine- and cocaine-seeking behaviour25. However,
inhibition of eCB clearance probably amplifies eCB sig-
nalling preferentially in circuits or synapses activated by
a given stimulus (in this case, drug-seeking behaviour),
rather than inducing more-widespread indiscriminate
CB1R activation, as produced by exogenous CB1R ago-
nists. Moreover, FAAH hydrolyses a large range of fatty
acid moieties, and the effects of FAAH inhibition on
drug-seeking behaviour may involve non-cannabinoid
lipid signalling. In this regard, it is notable that the eCB
transport inhibitor VDM11 attenuates both nicotine-
and cue-induced nicotine-seeking behaviour71, and this
compound may preferentially block AEA clearance with
weaker effects on non-cannabinoid lipids135,136. Similarly,
the eCB transport inhibitor AM404 dose-dependently
attenuates nicotine- and cue-induced nicotine-seeking
behaviour without altering nicotine self-administration72.
In contrast to nicotine- and cocaine-seeking behav-
iours, neither FAAH inhibition nor eCB transport
inhibition alter cue- or stress-induced reinstatement
of alcohol-seeking behaviour65,137. However, studies in
humans with alcoholism suggest a relationship between
eCB tone and craving that may relate to the degree of
dependence and possibly inherent factors contributing
to alcoholism vulnerability. In social drinkers, alcohol-
related cues increase both craving and plasma AEA levels,
and the relative magnitude of cue-induced increases in
AEA is significantly correlated with the degree of crav-
ing138. However, recently detoxified individuals with alco-
holism present significantly lower baseline plasma AEA
levels than non-dependent social drinkers and, although
alcohol-related cues elicit more-intense cravings in alco-
holics, these individuals do not present significant cue-
induced increases in plasma AEA. This blunted AEA
response may reflect aberrant eCB processing in people
with alcoholism, but further investigations are needed to
confirm a direct link between this potential peripheral
biomarker and brain eCB signalling, as well as possible
causal relationships between dysregulated eCB processing
and behaviour.
Extinction learning. The potent motivational effects of
drug-related cues create substantial difficulties during
periods of attempted drug abstinence and are causal in the
reinstatement of drug intake (for example, relapse)127. One
approach for reducing the motivational impact of drug-
associated cues is through extinction training, in which
a subject learns that these cues no longer have predictive
value. However, extinction therapy is generally ineffective
for reducing relapse in both humans139 and rodents140,
and it is conceivable this is a consequence of diminished
learning mechanisms required to override the original
cue-association memory. The ECS has a prominent role in
memory extinction, and deficient CB1R signalling results
in impaired extinction of cued fear memory, contextual
fear memory, fear-potentiated startle and spatial memory
under mildly aversive conditions141,142. Moreover, as previ-
ously noted, FAAH inhibition facilitates the extinction of
fearful memory in mice selectively bred for high levels of
alcohol preference and consumption124. Because aversive
memory may be involved in relapse to drug taking143,
deficient eCB signalling following long-term drug expo-
sure may contribute to the limited efficacy of extinction
therapy for addiction.
eCB gene polymorphisms and addiction
Approaches to explore the contribution of the ECS to
addiction disorders in humans often involve heritability
considerations, as it is now acknowledged that genet-
ics plays an important part in drug addiction vulner-
ability, accounting ~30–80% for risk depending on the
drug class144,145. Based on the growing evidence of a role
for the ECS in regulating reward, mood and cognition
and owing to its prominent expression within neuronal
systems related to these functions, the ECS has been
viewed as a central target for candidate-gene studies
of addictive disorders. Similarly to the preclinical ani-
mal studies described above, most investigations have
focused on CNR1 and FAAH146,147. Consistent with most
genetic investigations, important confounding factors
include race, ethnicity, type of drug, polysubstance use
and population sample size. Nevertheless, although
they are not all equivocal, what can be garnered from
existing genetic studies (although limited) suggests
that genomic heterogeneity of the eCB-related genes
may influence in part substance abuse vulnerability
and relate to behavioural and pathophysiological traits
that are highly associated with addictive disorders in
humans (FIG.5).
CNR1. Human CNR1 is located on chromosome 6
(6q14-q15), with the coding region situated at the
5ʹ-end of exon4. Several different CNR1 isoforms vary
in expression across brain regions, although each of the
main mRNA variants expresses the same exon4 that
encodes the CB1R protein148. Indeed, CNR1 exhibits
substantial functional conservation, with few common
missense variants in the CB1R protein being expressed148.
One of the first CNR1 variants explored in relation
to drug abuse was the AAT-triplet repeat ((AAT)n)
microsatellite polymorphism in the 3ʹ-untranslated
region, located close to the exon4 translational start
site148. Unfortunately, direct functional evidence is lack-
ing to understand its relevance to eCB processing, but
the increased number of repeats is speculated to result
in reduced CB1R expression149. Increased frequency of
long (AAT)n was initially observed in an intravenous
drug-dependent non-Hispanic US white population150,
and this was partially supported in subsequent evalua-
tions of Afro-Caribbean individuals151. Some reports
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Nature Reviews | Neuroscience
6q15
Chromosome 6
a CNR1
b
FAAH
Chromosome 1
1p35-p34
Cytogenetic band
A distinct region on the
chromosome (visible
microscopically after special
staining).
Endophenotype
A term used to separate
behavioural symptoms into
stable phenotypes with a clear
genetic basis, typically
applicable to heritable
disorders.
Haplotype blocks
Sets of DNA variations (or
polymorphisms) that tend to
be inherited together.
failed to replicate the original finding148,152, but a meta-
analysis of multiple variants of CNR1 in white popula-
tions specifically identified the (AAT)n polymorphism
(n > 16 repeats) as the only significant association with
illicit SUDs153. Interestingly, the (AAT)n polymorphism
has been linked with reduced amplitude of the frontal
lobe P300 event-related brain potential, a disruption that
has been suggested as a neurobiological endophenotype of
impaired cortical processing in drug abusers146.
Additional single nucleotide polymorphisms (SNPs) of
CNR1 have been investigated, the most frequent of which
is a silent intragenic biallelic polymorphism (G1359A;
rs1049353). This exon 4 synonymous mutation does not
change the amino acid sequence of the mature protein,
but the SNP is speculated to affect mRNA stability or pro-
tein translation in a way that could alter CB1R function.
Several investigations, although not all congruent, sug-
gest an association of the CNR1 G1359A polymorphism
with substance abuse. For example, the A-allele is asso-
ciated with severe alcoholism, specifically in relation to
enhanced withdrawal delirium in white patients154 and
enhanced impulsivity in Native Americans with a high
lifetime prevalence of substance dependence155. The
G1359A variant has also been associated with heroin
abuse in a white population, but with the A-allele con-
ferring protection and the G/G genotype conferring
addiction risk156. Additional studies are clearly needed
to determine whether the risk-versus-protection profile
might depend on the drugclass.
Aside from the G1359A SNP, most of the other
CNR1 variants reported to be associated with addiction
are not within the coding region; this is not surprising,
considering that it is now evident that most variation in
the genome falls outside protein-coding regions157. The
rs2023239 variant, representing a T to C polymorphism
in the intronic region upstream of exon3, has been shown
to relate to CB1R levels measured in post-mortem brain
tissue158 and inviv o using PET imaging94, with the C-allele
being associated with enhanced CB1R levels in the normal
human brain. As discussed above, increased CB1R in ani-
mal models is predictive of addiction vulnerability and,
indeed, the rs2023239 SNP has been linked to a gen-
eral liability for substance abuse148. Individuals with the
C-allele use greater amounts of cannabis, exhibit higher
cannabis dependency and experience greater negative
affect and craving for cannabis following withdrawal159.
The rs2023239 minor allele also associates with increased
activation in reward-associated brain areas (as measured
by blood oxygenation level-dependent (BOLD) imaging)
to cannabis-related cues160. rs2023239 C-allele carriers
also have enhanced alcohol cue-elicited brain activa-
tion in the PFC, NAc and midbrain (consistent with the
VTA and surrounding regions), greater subjective reward
when consuming alcohol, a strong correlation between
cue-elicited brain activation and alcohol consumption
measures, and a strong association with alcohol-use
disorder and craving measures161.
Several CNR1 haplotype blocks have also been linked
with addiction. When analysed as a haplotype (TAG),
three SNPs (rs806379, rs1535255 and rs2023239) in
the distal region of intron 2 of the CNR1 gene were sig-
nificantly associated with polysubstance abuse in adults
from different ethnicities148. Moreover, Agrawaletal.162
demonstrated an association between a CNR1 haplo-
type (rs806380, rs806368 and rs754387) and cannabis
dependence (the majority of these individuals also met
criteria for alcohol dependence). The rs806380 SNP
proximal to the TAG haplotype has also been linked with
the development of cannabis-dependence symptoms
(protective effect of G-allele). Other haplotypes have
been reported to associate with either low (rs6454674,
rs806380, rs806377 and rs1049353: GGCC) or increased
(TACC and GACC) risk for cannabis dependence163.
However, inconsistent and nominal significance has been
reported in replication studies of cannabis dependence in
adolescent and young adult populations164 and for other
haplotypes in substance abuse populations165. Overall,
although the majority of genetic investigations suggest
Figure 5 | CNR1 and FAAH genes and genetic variants associated with addiction. The endocannabinoid (eCB)
genes primarily studied to date in relation to genetic associations with addiction are CNR1 (part a) and fatty acid amide
hydrolase (FAAH) (part b). Human CNR1, which encodes cannabinoid1 receptor (CB1R), maps to chromosome 6,
specifically in the cytogenetic band 6q14-q15, and is transcribed from the minus strand (3ʹto-5ʹ orientation) of the DNA.
The gene contains four exons, with the protein-coding region being located at the 5ʹend of exon 4 (REFS146–148). There
are multiple mRNA variants of CNR1, with the prominent form encoding the canonical 472-amino-acid-long protein. The
FAAH gene is located on human chromosome 1, 1p35-p34, and is transcribed from the plus strand (5ʹto-3ʹ orientation).
The gene contains 15 exons, with functional protein domains being encoded across multiple exons. Recently, another
FAAH gene, FAAH2, was identified on chromosomeX in cytogenetic band Xp11.21; the encoded protein is composed of
532 amino acids and shares about 20% sequence identity with the canonical protein FAAH1 (REF.147). There is evidence,
although not all consistent, that genetic variants (arrows) associated with addiction and related phenotypes, such as
reward sensitivity, impulsivity and negative affect, are located within exon 4 (rs1049353), introns (rs2023239, rs1535255
and rs806380) and the 3ʹuntranslated region (AAT-triplet repeat; rs806368) of CNR1 (REFS159,146–148). For the FAAH
gene, the polymorphic variant most associated with substance-use disorders is rs324420 (exon 3)166,146,147.
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Post-translational histone
modification
A covalent modification of
histones that package and
order DNA into nucleosomes.
These modifications occur
during or after histone
biosynthesis.
an association between CNR1 variants and aspects of
SUDs, the data are not definitive and no causative loci
have been described to date. What seems most consist-
ent in the human genetic studies is a relevance to drug
cue sensitivity and craving, which would complement the
preclinical animal studies that demonstrate their direct
link to theECS.
FAAH. Few genetic investigations have focused on other
components of the ECS, with FAAH being the second
gene most-often studied in relation to addiction, based
on AEA’s important functional role. Human FAAH is
located on chromosome 1p35-p34 and has 15 exons,
with functional protein domains being encoded across
multiple exons. A SNP that has been highly investigated
is rs324420, which is located in exon 3 and results in
a missense mutation of a C–A replacement at position
385, leading to a proline to threonine change at protein
position 129 (REF.166). This C385A SNP is functional
and is thought to result in reduced FAAH expression
and enzyme activity, such that individuals with the A/A
genotype have enhanced plasma concentrations of AEA
and other N-acylethanolamine FAAH substrates167.
Although some studies have not observed associa-
tions between the C385A polymorphism and SUDs,
existing evidence implicates this genetic disruption
in addiction-related behaviours in different races and
ethnicities147. Specifically, the A/A genotype associates
with reduced vulnerability for cannabis dependence in
white adults, whereas the C/C genotype associates with
increased craving and negative affect during cannabis
withdrawal. Initial studies failed to detect a link between
the A/A genotype and alcohol or nicotine dependence168,
although recently an over-representation of C/C carri-
ers was observed among individuals consuming levels of
alcohol that put them at increased risk of alcohol-related
problems169. Carriers of the FAAH C385A SNP display
increased ventral striatal reactivity associated with delay
discounting, a behavioural index of impulsivity and
reward sensitivity170 and a markedly decreased relation-
ship between threat-related amygdala reactivity and trait
anxiety, similar to patterns observed in individuals with
high familial risk for alcoholism171. These findings sug-
gest that dysregulation of FAAH function through the
C385A polymorphism confers increased impulsivity and
increased anxiety sensitivity.
Collectively, recent studies of CNR1 and FAAH
genetic variants generally suggest an association with
endophenotypes implicated in addiction susceptibility,
including reward sensitivity, impulsivity and negative
affect, consistent with preclinical evidence linking the
ECS to such behaviours. Gene–gene interactions within
the ECS may also be relevant to vulnerability, as there
seem to be additive interactions between variants of the
FAAH (C385A; rs324420) and CNR1 (rs2023239) genes,
resulting in heightened neural responses in reward-
related brain areas to cannabis cues and more-severe
negative affect during cannabis abstinence159,160. Growing
evidence of an eCB influence on epigenetic mechanisms
suggests an additional but understudied way in which EC
signalling may contribute to addiction (BOX2). Clearly, a
major confounding factor of most investigations to date
is the small population size used, emphasizing the need
for replication studies and studies using larger popula-
tions. The few existing agnostic genome-wide approaches
have not identified eCB-related genes in relation to SUDs
interrogated thus far. However, the contribution of endo-
phenotypes along the continuum between genotype and
drug-abuse phenotype has not been interrogated, despite
the complex nature of non-Mendelian addictive disor-
ders. The lack of systematic consideration of behavioural
traits limits the possibility to understand the full reper-
toire of the ECS to individual vulnerability to addiction.
Moreover, the functional consequences of the variants
(causal or correlated) are unknown, which makes coming
to definitive conclusions challenging.
Summary and future directions
Although enhancement of eCB levels does not produce
rewarding effects perse, eCB signalling at cannabinoid
receptors participates in the mediation and modulation of
both natural and drug-induced reward. Brain eCB content
is modulated by most drugs of abuse and natural rewards,
and a robust CB1R influence on the motivation to con-
sume distinct classes of abused drugs and the association
of CNR1 polymorphisms with aberrant reward process-
ing and addictive behaviours strongly implicate CB1Rs in
the aetiology of addiction. Long-term drug use leads to
neuroadaptive downregulation of eCB signalling resulting
from diminished CB1R and/or CB2R function as well as
possible disruptions in eCB biosynthesis and/or clearance.
This blunting of eCB function may contribute to known
susceptibility factors for relapse, namely, increased stress
Box 2 | Endocannabinoid influence on epigenetic mechanisms
Epigenetic influences are functionally relevant changes to the genome that do not
involve disruptions in the nucleotide sequence of DNA. Examples of epigenetic
mechanisms include DNA methylation, post-translational histone modification,
nucleosome positioning and silencing associated with small non-coding RNAs (such as
microRNAs and small interfering RNAs). Recent evidence demonstrates that epigenetic
factors can regulate the expression of endocannabinoid (eCB)‑related genes and that
eCBs may themselves induce epigenetic alterations194. For example, DNA
hypermethylation of CNR1 (the gene encoding cannabinoid1 receptor (CB1R)) results
in downregulation of transcription in the CNS and immune system, whereas decreased
DNA methylation can result in increased fatty acid amide hydrolase (FAAH)
transcription; these processes have been implicated in several pathologies, including
colon cancer and late‑onset Alzheimer disease. Conversely, eCB‑induced alterations in
enzymes influencing histone modification may disrupt the transcription of several
genes, including those encoding various neurotransmitter systems. In rodents, early life
stress (maternal separation) is associated with elevated DNA methylation of the CNR1
promoter195, which, through a resultant decrease in CB1R expression, could contribute
to affective dysregulation and addiction susceptibility later in life. Several studies
implicate increased epigenetic influences following chronic Δ9‑tetrahydrocannabinol
(Δ9‑THC) exposure. Cannabis‑dependent patients present robust methylation of the
CNR1 promoter in association with diminished CNR1 mRNA in peripheral blood cells196.
Furthermore, offspring of maternal cannabis users exhibit histone methylation and
dysregulated mesolimbic dopamine D2 receptor expression197, and adolescent Δ9‑THC
exposure is associated with nucleus accumbens (NAc) chromatin modifications and
concurrent upregulation of the opioid neuropeptide proencephalin gene198. Prenatal
alcohol exposure increases expression of the regulatory microRNA miR-26b (which
targets the 3ʹ‑untranslated region of the CNR1 transcript) and decreased CNR1
transcription in the adult mouse brain199. Thus, growing evidence suggests that there
are eCB‑related epigenetic influences following drug exposure.
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responsivity, increased negative affect, inefficient extinc-
tion of drug-related memories and increased drug-seeking
behaviour and drug craving. Recent preclinical evidence
demonstrates the efficacy of eCB-clearance inhibitors
for ameliorating these behavioural abnormalities, which
might offer future therapeutic interventions for addic-
tion disorders. Importantly, because eCBs are generally
produced in a synapse-specific manner, eCB-clearance
inhibitors may preferentially facilitate eCB signalling in
specific circuits engaged by distinct stimuli (for example,
stress- or drug-associated cues) and therefore could pre-
sent fewer unwanted behavioural effects than are pro-
duced by exogenous agonists that produce widespread
cannabinoid receptor activation.
Despite growing attention being given to the cannab-
inoids, there are still notable gaps in our understand-
ing of the eCB influence on reward and addiction. The
ECS plays a prominent part in neuronal guidance and
brain development172 and, as such, disruptions in eCB
function at an early age probably have substantial con-
sequences for adult brain function. This is underscored
by increasing evidence of the long-term consequences
of prenatal or adolescent cannabinoid exposure173,174.
Although the effects of early life exposure to non-
cannabinoid drugs are well studied, the specific con-
tributions of persistent drug-induced disruptions in
eCB signalling on adult neural function and behaviour
are not understood. Robust bidirectional interactions
between the ECS and sex hormones are now recog-
nized175, but few studies have characterized possible
sex differences in the eCB influence on reward func-
tion, addiction and cognitive processing. There are also
substantial limitations in the interpretation and replica-
tion of genetic analyses of the eCB influence in addic-
tion, owing to heterogeneity of the populations, drug
classes, polysubstance use and even drug-use pheno-
types examined. Large-scale future studies across dif-
ferent populations and drug classes will be critical to
understanding the relative effect and causal nature of
ECS-related genetic mutations in the vulnerability to
addictive disorders. Filling these gaps of knowledge is
critical, given the important need for scientific data to
help guide current discussions and changes being made
in marijuana-legalization policies.
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Acknowledgements
This work was supported by grants from the US National
Institutes of Health to L.H.P. (AA020404, AA006420,
AA022249 and AA017447) and Y.L.H. (DA023214,
DA030359 and DA033660). This is manuscript number
29049 from The Scripps Research Institute. The authors thank
D. Lewis for his help during the preparation of this manuscript.
Competing interests statement
The authors declare no competing interests.
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... The endocannabinoid system, including arachidonoyl ethanol amide (anandamide/AEA), 2-arachidonoylglycerol (2-AG), and cannabinoid receptor 1 (CB1R), achieves Giulia Margiani and Maria Paola Castelli equal contribution. maximum expression during adolescence with a subsequent decline at adulthood (Glass et al. 1997;Meyer et al. 2018); this system plays an important role in cognitive and rewarding functions (Koob and Volkow 2010;Parsons and Hurd 2015). Indeed, the EC system modulates dopamine (DA) response to natural reward, as it finely controls the activity of DAergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), modulating rewarding and reinforcing behaviors (De Luca et al. 2014) and, ultimately, affecting drug use and abuse (Parsons and Hurd 2015). ...
... maximum expression during adolescence with a subsequent decline at adulthood (Glass et al. 1997;Meyer et al. 2018); this system plays an important role in cognitive and rewarding functions (Koob and Volkow 2010;Parsons and Hurd 2015). Indeed, the EC system modulates dopamine (DA) response to natural reward, as it finely controls the activity of DAergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), modulating rewarding and reinforcing behaviors (De Luca et al. 2014) and, ultimately, affecting drug use and abuse (Parsons and Hurd 2015). ...
... As shown in Fig. 7(a) and (b), no significant differences in EEAT2 and Cox-2 levels between groups were observed in either the cortex or striatum. Moreover, considering the crucial role of CB1Rs in brain areas involved in addiction and motivated behaviors (López-Gallardo et al. 2012;Parsons and Hurd 2015;Zamberletti et al. 2015) and that IVSA of JWH-018 in adolescent mice is mediated by the activation of CB1Rs (Fig. 1(f)), we examined whether changes in CB1Rs expression were present in the adult cortex and striatum following adolescent JWH-018 IVSA. As shown in Fig. 7(c), no significant differences were observed in the cortex and striatum, although there is an upward trend of CB1R levels in the striatum of JWH-018 mice. ...
Article
Full-text available
Rationale The use of synthetic cannabinoid receptor agonists (SCRAs) is growing among adolescents, posing major medical and psychiatric risks. JWH-018 represents the reference compound of SCRA-containing products. Objectives This study was performed to evaluate the enduring consequences of adolescent voluntary consumption of JWH-018. Methods The reinforcing properties of JWH-018 were characterized in male CD1 adolescent mice by intravenous self-administration (IVSA). Afterwards, behavioral, neurochemical, and molecular evaluations were performed at adulthood. Results Adolescent mice acquired operant behavior (lever pressing, Fixed Ratio 1–3; 7.5 µg/kg/inf); this behavior was specifically directed at obtaining JWH-018 since it increased under Progressive Ratio schedule of reinforcement, and was absent in vehicle mice. JWH-018 IVSA was reduced by pretreatment of the CB1-antagonist/inverse agonist AM251. Adolescent exposure to JWH-018 by IVSA increased, at adulthood, both nestlet shredding and marble burying phenotypes, suggesting long-lasting repetitive/compulsive-like behavioral effects. JWH-018 did not affect risk proclivity in the wire-beam bridge task. In adult brains, there was an increase of ionized calcium binding adaptor molecule 1 (IBA-1) positive cells in the caudate-putamen (CPu) and nucleus accumbens (NAc), along with a decrease of glial fibrillary acidic protein (GFAP) immunoreactivity in the CPu. These glial alterations in adult brains were coupled with an increase of the chemokine RANTES and a decrease of the cytokines IL2 and IL13 in the cortex, and an increase of the chemokine MPC1 in the striatum. Conclusions This study suggests for the first time that male mice self-administer the prototypical SCRA JWH-018 during adolescence. The adolescent voluntary consumption of JWH-018 leads to long-lasting behavioral and neurochemical aberrations along with glia-mediated inflammatory responses in adult brains.
... CB1 receptors are coupled to G i/o G-proteins and are expressed widely within the cortex, hippocampus, amygdala, cerebellum and basal ganglia, as well as more modestly in the NAc [68], to mediate a variety of physiological and behavioural effects of eCBs. In the context of reward-related neural plasticity, the role of eCBs have been investigated for food [69,70], sport [71], sex [72] and drug rewards [73][74][75]. In many cases, eCBs are released in the NAc and act on CB1 receptors to modulate the local release of dopamine, presumably regulating the rewarding properties of associated stimuli [76,77]. ...
... How eCBs regulate the rewarding nature of social behaviours has only been sparsely studied, but what we know so far reflects the typical sources of variation discussed earlier. Social reward is an important aspect of fostering prosocial behaviour, and because eCBs are important for the regulation of reward in general [74], it is likely they play a part in the emergence of rewarding social behaviours as well [78]. For example, the distribution of the CB1 receptor across brain areas correlates with species-level differences in social organization. ...
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Oxytocin modulates social behaviour across diverse vertebrate taxa, but the precise nature of its effects varies across species, individuals and lifetimes. Contributing to this variation is the fact that oxytocin's physiological effects are mediated through interaction with diverse neuromodulatory systems and can depend on the specifics of the local circuits it acts on. Furthermore, those effects can be influenced by both genetics and experience. Here we discuss this complexity through the lens of a specific neuromodulatory system, endocannabinoids, interacting with oxytocin in the nucleus accumbens to modulate prosocial behaviours in prairie voles. We provide a survey of current knowledge of oxytocin–endocannabinoid interactions in relation to social behaviour. We review in detail recent research in monogamous female prairie voles demonstrating that social experience, such as mating and pair bonding, can change how oxytocin modulates nucleus accumbens glutamatergic signalling through the recruitment of endocannabinoids to modulate prosocial behaviour toward the partner. We then discuss potential sex differences in experience-dependent modulation of the nucleus accumbens by oxytocin in voles based on new data in males. Finally, we propose that future oxytocin-based precision medicine therapies should consider how prior social experience interacts with sex and genetics to influence oxytocin actions. This article is part of the theme issue ‘Interplays between oxytocin and other neuromodulators in shaping complex social behaviours’.
... The antineoplastic role of cannabinoids in malignancy of the immune system, [13,14] as well as in many other tumors, i.e., osteosarcoma, is well documented [15,16]. Cannabinoids derive from the Cannabis plant, and interact with the cannabinoid receptors CB1 and CB2, principally expressed in the central nervous system and in peripheral and immune cells, respectively [17]. These receptors, together with their specific ligands (endocannabinoids) and the enzymes involved in their own synthesis and degradation, constitute the endocannabinoid system (ECS). ...
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Acute lymphoblastic leukemia type B (B-ALL) is the most common kind of pediatric leukemia, characterized by the clonal proliferation of type B lymphoid stem cells. Important progress in ALL treatments led to improvements in long-term survival; nevertheless, many adverse long-term consequences still concern the medical community. Molecular and cellular target therapies, together with immunotherapy, are promising strategies to overcome these concerns. Cannabinoids, enzymes involved in their metabolism, and cannabinoid receptors type 1 (CB1) and type 2 (CB2) constitute the endocannabinoid system, involved in inflammation, immune response, and cancer. CB2 receptor stimulation exerts anti-proliferative and anti-invasive effects in many tumors. In this study, we evaluated the effects of CB2 stimulation on B-ALL cell lines, SUP-B15, by RNA sequencing, Western blotting, and ELISA. We observe a lower expression of CB2 in SUP-B15 cells compared to lymphocytes from healthy subjects, hypothesizing its involvement in B-ALL pathogenesis. CB2 stimulation reduces the expression of CD9, SEC61G, TBX21, and TMSB4X genes involved in tumor growth and progression, and also negatively affects downstream intracellular pathways. Our findings suggest an antitumor role of CB2 stimulation in B-ALL, and highlight a functional correlation between CB2 receptors and specific anti-tumoral pathways, even though further investigations are needed.
... Reward is mediated by the mesocorticolimbic dopamine system, in which dopaminergic cell bodies in the ventral tegmental area (VTA) project reward-related information to the nucleus accumbens, amygdala, hippocampus, orbitofrontal cortex, and prefrontal cortex [102]. CB1R is highly expressed throughout these regions [103][104][105], and various drugs of misuse rely on CB1R signalling to exert their rewarding effects. ...
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Suicide is a devastating complication of psychiatric disorders characterized by despair, hopelessness, and overwhelming mental distress or pain. Non-suicidal self-injury (NSSI), suicide ideation (SI) and suicide attempts (SA) all fall under the umbrella of suicide behaviour (SB). Several biopsychological theories describe SB as an attempt to relieve mental pain. They comment on the role of tolerance-habituation to the rewarding effects of SB-induced emotional regulation, as well as increasing physical or somatic pain tolerance, both of which contribute to the escalating patterns of repetitive SB. The endocannabinoid system (ECS) is involved in a wide range of homeostatic and neuromodulatory functions including appetite/feeding, sleep, motor control, pain perception, cognition, mood/affect, and reward processing. The downregulation of endocannabinoid signalling has major implications for affective disorders, pain disorders, and substance use disorders. SB can be seen as a manifestation of these disorders and has also been linked to ECS dysfunction. Drawing from both animal and human studies, we aim to understand repetitive SB as an endocannabinoid-mediated pain and reward disorder. We hypothesize that mental distress triggers the first incidence of NSSI or SB, from which patients derive stress-induced endocannabinoid-mediated analgesia. As patients become increasingly tolerant to this mechanism of analgesia, SB escalates to override increasing mental distress. This hypothesis calls for more research on endocannabinoid-based therapies to prevent the progression from NSSI or SI to fatal SA.
... Of all the several roles that the endocannabinoid system portrays, signaling on the brain reward circuitry might explain its conception as a promising target for the treatment of SUD. CB 1 receptors are extensively located in the brain and their modulation over striatal regions such as the VTA and NAc pathways regulate reward tone [263] . Cannabinoids, especially Δ 9 -THC, but also CBD and synthetic analogues, are being extensively studied as a potential cannabinoid-receptor agonist therapy for SUD, perhaps following the same trend of nicotine [ 264 , 265 ] and opioid [266] agonist and antagonist pharmacotherapies. ...
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Substance use disorder (SUD) is a global public health concern that affects millions of people worldwide. Considering current research, addiction has been noted as the last stage of a chronic disease that may impair brain reward circuit responses and affects personal and social life. Treatments for SUD face challenges including availability and limited pharmacological response, often resulting in low retention of patients. A growing number of studies from the 'psychedelic renaissance' have highlighted the therapeutic potential of psychedelics for several psychiatric disorders, including SUD. In this non-systematic review we discuss past and current clinical and observational studies with classic (LSD, DMT, psilocybin and mescaline) and non-classic (ibogaine, ketamine, MDMA, salvinorin A and THC) psychedelics for the treatment of SUD published until December 2021. Although results are still inconclusive for LSD, DMT, mescaline, MDMA and Salvinorin A, in general, the literature presents moderate evidence on the controlled use of psilocybin and ketamine for Alcohol Use Disorder, ketamine for management of opiate and alcohol withdrawal, and THC preparations for reducing withdrawal symptoms in Cannabis and possibly in Opioid Use Disorder. Importantly, studies suggest that psychedelics should be more effective when employed as an adjunct therapy. Extensive research is warranted to further elucidate the role of psychedelics in the treatment of SUD.
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Background: Circular RNAs (circRNAs) have been crucially implicated in various diseases, however, their involvement in chronic intermittent ethanol (CIE) exposure remains unclear.Objective: The present study was conducted to evaluate the circular RNA expression alteration in brain samples and to identify the molecular mechanisms underlying chronic intermittent ethanol exposure.Methods: Male C57BL/6J mice (10 for each group) were given 4 weeks of chronic intermittent ethanol exposure. Whole brain samples were collected for high-throughput sequencing and circRNA bioinformatic analysis. Real-time quantitative PCR (RI-qPCR) and agarose electrophoresis were used to validate the differentially expressed circRNAs. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis were performed. A p level < 0.05 was considered statistically significant.Results: Compared with the control group and baseline values, the CIE group showed a significant increase in ethanol intake. High-throughput sequencing revealed 399 significantly different circRNAs in CIE mice, including 150 up-regulated circRNAs and 249 down-regulated circRNAs. GO analysis showed that the most significantly enriched term for biological process, cellular component, and molecular function were GO:0050885, GO:0016020 and GO:0005515, respectively. The most enriched pathways in KEGG analysis were GABAergic synapse (mmu04727), followed by retrograde endocannabinoid (eCB) signaling (mmu04723) and morphine addiction (mmu05032). Among the circRNAs, RT-qPCR confirmed 14 upregulated and 13 downregulated circRNAs in the brain tissues with 9 upregulated and 10 downregulated circRNAs being observed in blood samples.Conclusions: Our study suggests that chronic ethanol exposure upregulates or downregulates circRNAs in the brain, which, in turn, could alter neurotransmitter release and signal transduction.
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Social behaviour is an essential component of human life and deficits in social function are seen across multiple psychiatric conditions with high morbidity. However, there are currently no FDA-approved treatments for social dysfunction. Since social cognition and behaviour rely on multiple signalling processes acting in concert across various neural networks, treatments aimed at social function may inherently require a combinatorial approach. Here, we describe the social neurobiology of the oxytocin and endocannabinoid signalling systems as well as translational evidence for their use in treating symptoms in the social domain. We leverage this systems neurobiology to propose a network-based framework that involves pharmacology, psychotherapy, non-invasive brain stimulation and social skills training to combinatorially target trans-diagnostic social impairment. Lastly, we discuss the combined use of oxytocin and endocannabinoids within our proposed framework as an illustrative strategy to treat specific aspects of social function. Using this framework provides a roadmap for actionable treatment strategies for neuropsychiatric social impairment. This article is part of the theme issue ‘Interplays between oxytocin and other neuromodulators in shaping complex social behaviours’.
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Objective The purpose of this study was to describe the management of 2 long-term users of cannabis with nutrition and psychotherapy. Clinical Features A 28-year-old man presented with a medical history of asthma, depression, anxiety, and smoking, and was a long-term user of cannabis for 9 years (usually 3 times a week). A 39-year-old man presented with a medical history of anxiety and fatigue, and was a long-term user of cannabis for 14 years (usually twice a week). Laboratory tests showed altered blood levels of homocysteine, vitamins, and cortisol. Intervention and Outcome Both patients were given supplements of vitamins (folic acid, methylcobalamin, and pyridoxine), vitamin D, Rhodiola rosea, and L-tyrosine. Psychotherapy also was provided to both patients. After 2 months of treatment, both patients improved and reduced their cannabis consumption. Conclusion This study describes vitamin deficiencies, low cortisol levels, and hyperhomocysteinemia in 2 cannabis users who were managed with a combination of nutritional supplements and psychotherapy.
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In psychostimulant drug addiction, relapse is the most concerning outcome to be managed, considering there is no approved treatment for this neuropsychiatric condition. Here, we investigated the effects of the CBD treatment on the relapse behavior triggered by stress, after being submitted to the amphetamine (AMPH)-induced conditioned place preference (CPP) in rats. To elucidate the mechanisms of action underlying the CBD treatment, we evaluated the neuroadaptations on dopaminergic and endocannabinoid targets in the ventral striatum (VS) and ventral tegmental area (VTA) of the brain. Animals received d,l-AMPH (4 mg/kg, i.p.) or vehicle in the CPP paradigm for 8 days. Following the first CPP test, animals were treated with CBD (10 mg/kg, i.p.) or its vehicle for 5 days and subsequently submitted to forced swim stress protocol to induce AMPH-CPP relapse. Behavioral findings showed that CBD treatment prevented AMPH-reinstatement, also exerting anxiolytic activity. At the molecular level, in the VTA, CBD restored the CB1R levels decreased by AMPH-exposure, increased NAPE-PLD, and decreased FAAH levels. In the VS, the increase of D1R and D2R, as well as the decrease of DAT levels induced by AMPH were restored by CBD treatment. The current outcomes evidence a substantial preventive action of the CBD on the AMPH-reinstatement evoked by stress, also involving neuroadaptations in both dopaminergic and endocannabinoid systems in brain areas closely involved in the addiction. Although further studies are needed, these findings support the therapeutic potential of CBD in AMPH-relapse prevention.
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A potent, synthetic cannabinoid was radiolabeled and used to characterize and precisely localize cannabinoid receptors in slide-mounted sections of rat brain and pituitary. Assay conditions for 3H-CP55,940 binding in Tris-HCl buffer with 5% BSA were optimized, association and dissociation rate constants determined, and the equilibrium dissociation constant (Kd) calculated (21 nM by liquid scintillation counting, 5.2 nM by quantitative autoradiography). The results of competition studies, using several synthetic cannabinoids, add to prior data showing enantioselectivity of binding and correlation of in vitro potencies with potencies in biological assays of cannabinoid actions. Inhibition of binding by guanine nucleotides was selective and profound: Nonhydrolyzable analogs of GTP and GDP inhibited binding by greater than 90%, and GMP and the nonhydrolyzable ATP analog showed no inhibition. Autoradiography showed great heterogeneity of binding in patterns of labeling that closely conform to cytoarchitectural and functional domains. Very dense 3H-CP55,940 binding is localized to the basal ganglia (lateral caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), cerebellar molecular layer, innermost layers of the olfactory bulb, and portions of the hippocampal formation (CA3 and dentate gyrus molecular layer). Moderately dense binding is found throughout the remaining forebrain. Sparse binding characterizes the brain stem and spinal cord. Densitometry confirmed the quantitative heterogeneity of cannabinoid receptors (10 nM 3H-CP55,940 binding ranged in density from 6.3 pmol/mg protein in the substantia nigra pars reticulata to 0.15 pmol/mg protein in the anterior lobe of the pituitary). The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.
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Sleep problems during withdrawal from cannabis use are a common experience. The details regarding how abstinence from cannabis impacts sleep are not well described. This paper reviews the literature including a measure of cannabis withdrawal and sleep in humans. A literature search using a set of cannabinoid and sleep-related terms was conducted across eight electronic databases. Human studies that involved the administration of cannabinoids and at least one quantitative sleep-related measure were included. Review papers, opinion pieces, letters or editorials, case studies (final N < 8), published abstracts, posters, and non-English papers were excluded. Thirty six publications were included in the review. Sleep was frequently interrupted during cannabis withdrawal, although the specific mechanisms of disruption remain unclear. Methodological issues in the majority of studies to date preclude any definitive conclusion on the specific aspects of sleep which are affected.
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Inhibition of the enzyme fatty acid amide hydrolase (FAAH) counteracts reward-related effects of nicotine in rats, but has not been tested for this purpose in non-human primates. Therefore, we studied the effects of the first- and second-generation O-arylcarbamate-based FAAH inhibitors, URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) and URB694 (6-hydroxy-[1,1'-biphenyl]-3-yl-cyclohexylcarbamate), in squirrel monkeys. Both FAAH inhibitors: 1) blocked FAAH activity in brain and liver, increasing levels of endogenous ligands for cannabinoid and alpha-type peroxisome proliferator-activated (PPAR-α) receptors; 2) shifted nicotine self-administration dose-response functions in a manner consistent with reduced nicotine reward; 3) blocked reinstatement of nicotine seeking induced by re-exposure to either nicotine priming or nicotine-associated cues; and 4) had no effect on cocaine or food self-administration. The effects of FAAH inhibition on nicotine self-administration and nicotine priming-induced reinstatement were reversed by the PPAR-α antagonist, MK886. Unlike URB597, which was not self-administered by monkeys in an earlier study, URB694 was self-administered at a moderate rate. URB694 self-administration was blocked by pretreatment with an antagonist for either PPAR-α (MK886) or cannabinoid CB1 receptors (rimonabant). In additional experiments in rats, URB694 was devoid of THC-like or nicotine-like interoceptive effects under drug-discrimination procedures, and neither FAAH inhibitor induced dopamine release in the nucleus accumbens shell-consistent with their lack of robust reinforcing effects in monkeys. Overall, both URB597 and URB694 show promise for the initialization and maintenance of smoking cessation, due to their ability to block the rewarding effects of nicotine and prevent nicotine priming-induced and cue-induced reinstatement.Neuropsychopharmacology accepted article preview online, 10 March 2015. doi:10.1038/npp.2015.62.
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A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell type- and subcellular compartment-specific manner. We developed a new approach to this problem by combining cell-specific physiological and anatomical characterization with super-resolution imaging and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically projecting GABAergic interneurons possessed increased CB1 receptor number, active-zone complexity and receptor/effector ratio compared with dendritically projecting interneurons, consistent with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ(9)-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked marked CB1 downregulation in a dose-dependent manner. Full receptor recovery required several weeks after the cessation of Δ(9)-tetrahydrocannabinol treatment. These findings indicate that cell type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits and identify previously unknown molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.
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Among the many signalling lipids, endocannabinoids are increasingly recognized for their important roles in neuronal and glial development. Recent experimental evidence suggests that, during neuronal differentiation, endocannabinoid signalling undergoes a fundamental switch from the prenatal determination of cell fate to the homeostatic regulation of synaptic neurotransmission and bioenergetics in the mature nervous system. These studies also offer novel insights into neuropsychiatric disease mechanisms and contribute to the public debate about the benefits and the risks of cannabis use during pregnancy and in adolescence.
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The discovery of functional cannabinoid receptor 2 (CB2R) in brain suggests a potential new therapeutic target for neurological and psychiatric disorders. However, recent findings in experimental animals appear controversial. Here we report that there are significant species differences in CB2R mRNA splicing and expression, protein sequences, and the receptor responses to CB2R ligands in mice and rats. Systemic administration of JWH133, a highly-selective CB2R agonist, significantly and dose-dependently inhibited intravenous cocaine self-administration under a fixed-ratio (FR) schedule of reinforcement in mice, but not in rats. However, under a progressive-ratio (PR) schedule of reinforcement, JWH133 significantly increased break-point for cocaine self-administration in rats, but decreased it in mice. To explore the possible reasons for these conflicting findings, we examined CB2R gene expression and receptor structure in the brain. We found novel rat-specific CB2C and CB2D mRNA isoforms in addition to CB2 A and CB2B mRNA isoforms of mice. In situ hybridization RNAscope assays found higher levels of CB2R mRNA in different brain regions and cell types in mice than in rats. By comparing CB2R-encoding regions, we observed a premature stop codon in the mouse CB2R gene, which truncated 13 amino acid residues including a functional autophosphorylation site in the intracellular C-terminus. These findings suggest that species differences in the splicing and expression of CB2R genes and receptor structures may in part explain the different effects of CB2R-selective ligands on cocaine self-administration in mice and rats.Neuropsychopharmacology accepted article preview online, 06 November 2014. doi:10.1038/npp.2014.297.
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Isolation and structure elucidation of most of the major cannabinoid constituents - including Δ(9)-tetrahydrocannabinol (Δ(9)-THC), which is the principal psychoactive molecule in Cannabis sativa - was achieved in the 1960s and 1970s. It was followed by the identification of two cannabinoid receptors in the 1980s and the early 1990s and by the identification of the endocannabinoids shortly thereafter. There have since been considerable advances in our understanding of the endocannabinoid system and its function in the brain, which reveal potential therapeutic targets for a wide range of brain disorders.
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Over the last decades, the endocannabinoid system has been implicated in a large variety of functions, including a crucial modulation of brain-reward circuits and the regulation of motivational processes. Importantly, behavioral studies have shown that cannabinoid compounds activate brain reward mechanisms and circuits in a similar manner to other drugs of abuse, such as nicotine, alcohol, cocaine, and heroin, although the conditions under which cannabinoids exert their rewarding effects may be more limited. Furthermore, there is evidence on the involvement of the endocannabinoid system in the regulation of cue- and drug-induced relapsing phenomena in animal models. The aim of this review is to briefly present the available data obtained using diverse behavioral experimental approaches in experimental animals, namely, the intracranial self-stimulation paradigm, the self-administration procedure, the conditioned place preference procedure, and the reinstatement of drug-seeking behavior procedure, to provide a comprehensive picture of the current status of what is known about the endocannabinoid system mechanisms that underlie modification of brain-reward processes. Emphasis is placed on the effects of cannabinoid 1 (CB1) receptor agonists, antagonists, and endocannabinoid modulators. Further, the role of CB1 receptors in reward processes is investigated through presentation of respective genetic ablation studies in mice. The vast majority of studies in the existing literature suggest that the endocannabinoid system plays a major role in modulating motivation and reward processes. However, much remains to be done before we fully understand these interactions. Further research in the future will shed more light on these processes and, thus, could lead to the development of potential pharmacotherapies designed to treat reward-dysfunction-related disorders.
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We hypothesize that drug addiction can be viewed as the endpoint of a series of transitions from initial voluntary drug use through the loss of control over this behaviour, such that it becomes habitual and ultimately compulsive. We describe evidence that the switch from controlled to compulsive drug seeking represents a transition at the neural level from prefrontal cortical to striatal control over drug-seeking and drug-taking behaviours as well as a progression from ventral to more dorsal domains of the striatum, mediated by its serially interconnecting dopaminergic circuitry. These neural transitions depend upon the neuroplasticity induced by chronic self-administration of drugs in both cortical and striatal structures, including long-lasting changes that are the consequence of toxic drug effects. We further summarize evidence showing that impulsivity, a spontaneously occurring behavioural tendency in outbred rats that is associated with low dopamine D 2/3 receptors in the nucleus accumbens, predicts both the propensity to escalate cocaine intake and the switch to compulsive drug seeking and addiction.
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Stress affects a constellation of physiological systems in the body and evokes a rapid shift in many neurobehavioral processes. A growing body of work indicates that the endocannabinoid (eCB) system is an integral regulator of the stress response. In the current review, we discuss the evidence to date that demonstrates stress-induced regulation of eCB signaling and the consequential role changes in eCB signaling have with respect to many of the effects of stress. Across a wide array of stress paradigms, studies have generally shown that stress evokes bidirectional changes in the two eCB molecules, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), with stress exposure reducing AEA levels and increasing 2-AG levels. Additionally, in almost every brain region examined, exposure to chronic stress reliably causes a downregulation or loss of cannabinoid type 1 (CB1) receptors. With respect to the functional role of changes in eCB signaling during stress, studies have demonstrated that the decline in AEA appears to contribute to the manifestation of the stress response, including activation of the hypothalamic–pituitary–adrenal (HPA) axis and increases in anxiety behavior, while the increased 2-AG signaling contributes to termination and adaptation of the HPA axis, as well as potentially contributing to changes in pain perception, memory and synaptic plasticity. More so, translational studies have shown that eCB signaling in humans regulates many of the same domains and appears to be a critical component of stress regulation, and impairments in this system may be involved in the vulnerability to stress-related psychiatric conditions, such as depression and posttraumatic stress disorder. Collectively, these data create a compelling argument that eCB signaling is an important regulatory system in the brain that largely functions to buffer against many of the effects of stress and that dynamic changes in this system contribute to different aspects of the stress response.