Behavioural Pharmacology

The endocannabinoid system and drug-associated contextual memories

--Manuscript Draft--

Manuscript Number: BP-20-91

Full Title: The endocannabinoid system and drug-associated contextual memories

Article Type: Review Article

Section/Category: Non-United States/Canada

Keywords: Cannabinoids; reward; addiction; drug abuse; memory; conditioned place

preference

Corresponding Author: Fabricio A. Moreira, PhD

University of Sao Paulo
Ribeirao Preto, BRAZIL

Corresponding Author Secondary

Information:

Corresponding Author's Institution: University of Sao Paulo

Corresponding Author's Secondary

Institution:

First Author: Laila Asth

First Author Secondary Information:

Order of Authors: Laila Asth

Aline C. Santos

Fabricio A. Moreira, PhD

Order of Authors Secondary Information:

Manuscript Region of Origin: BRAZIL

Abstract: Drug abuse and addiction can be initiated and reinstated by contextual stimuli
previously paired with the drug use. The influence exerted by the context on drug
seeking behaviour can be modelled in experimental animals with place conditioning
protocols. Here we review the effects of cannabinoids in place conditioning and the
therapeutic potential of the endocannabinoid system for interfering with drug-related
memories. The phytocannabinoid delta 9 -tetrahydrocannabinol (THC) tends induce
CPP at low doses and CPA at high doses; cannabidiol is devoid of any effect and yet it
can inhibit CPP induced by some drugs. Synthetic CB 1 receptor agonists tend to
recapitulate the biphasic profile observed with THC, whereas selective
antagonists/inverse agonists inhibit CPP induced by cocaine, nicotine, alcohol and
opioids. The CB 2 receptor has also attracted attention, as selective CB 2 agonists
inhibit cocaine-induced CPP. Inhibitors of endocannabinoid membrane transport and
hydrolysis yield mixed results. In targeting the endocannabinoid system for developing
new treatments for drug addiction, future research should focus on “neutral” CB 1
receptor antagonists and CB 2 receptor agonists. Such compounds may offer a safe
pharmacological profile and curb addiction by preventing drug seeking triggered by
conditioned contextual cues.

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Covering Letter

___________________________________________________________________________
Universidade Federal de Minas Gerais (UFMG)
Instituto de Ciências Biológicas | Departamento de Farmacologia

Belo Horizonte, Brazil

06th May 2020

Dear Dr. Louk Vanderschuren, Editor of Behavioural Pharmacology

We are submitting the manuscript

The endocannabinoid system and drug-associated contextual memories

To be considered for publication in the special issue Translational Research in Behavioural

Pharmacology.

All authors have contributed to this work and have agreed on the final version of the
manuscript. Also, this manuscript has not been submitted to any other journal. We have no
conflict of interest to declare.

Thank you and best regards,

Fabricio A. Moreira, PhD

Department of Pharmacology, ICB, Federal University of Minas Gerais
Av. Antonio Carlos 6627; 31270-901 Belo Horizonte, MG; Brazil
Tel.: +55 31 3409 2720; E-mail: fabriciomoreira@icb.ufmg.br
Manuscript (All Manuscript Text Pages, including Title Page,
References and Figure Legends)

Review paper

The endocannabinoid system and drug-associated contextual memories

Running head: Cannabinoids and drug memories

Laila ASTH#, Aline C. SANTOS#, Fabrício A. MOREIRA*

Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de

Minas Gerais, Belo Horizonte, MG, Brazil, 31270-901.

#
Shared first authorship

*Corresponding author:

Dr. Fabrício de Araújo Moreira, Department of Pharmacology, Institute of Biological

Sciences, Universidade Federal de Minas Gerais. Av. Antônio Carlos 6627, Belo

Horizonte, MG, 31270-901, Brazil. E-mail: fabriciomoreira@icb.ufmg.br

Conflicts of interest

None.

1
Acknowledgements

LA thanks “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil

(CAPES) – Finance Code 001” for the post-doctoral fellowship. ACS thanks “The

Brazilian National Council for Scientific and Technological Development (CNPq)” for

a master degree fellowship. FAM thanks “The Brazilian National Council for Scientific

and Technological Development (CNPq)” for the research grant 406122/2016-4 and for

the research productivity fellowship (level 2); “Fundação de Amparo à Pesquisa do

Estado de Minas Gerais” (FAPEMIG), for the research grant APQ-02064-15;

“Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP, Projeto Temático

2017/24304-0) and Instituto Nacional de Ciência e Tecnologia (INCT-Translational

Medicine).

2
Abstract

Drug abuse and addiction can be initiated and reinstated by contextual stimuli

previously paired with the drug use. The influence exerted by the context on drug

seeking behaviour can be modelled in experimental animals with place conditioning

protocols. Here we review the effects of cannabinoids in place conditioning and the

therapeutic potential of the endocannabinoid system for interfering with drug-related

memories. The phytocannabinoid 9-tetrahydrocannabinol (THC) tends induce CPP at

low doses and CPA at high doses; cannabidiol is devoid of any effect and yet it can

inhibit CPP induced by some drugs. Synthetic CB1 receptor agonists tend to recapitulate

the biphasic profile observed with THC, whereas selective antagonists/inverse agonists

inhibit CPP induced by cocaine, nicotine, alcohol and opioids. The CB2 receptor has

also attracted attention, as selective CB2 agonists inhibit cocaine-induced CPP.

Inhibitors of endocannabinoid membrane transport and hydrolysis yield mixed results.

In targeting the endocannabinoid system for developing new treatments for drug

addiction, future research should focus on “neutral” CB1 receptor antagonists and CB2

receptor agonists. Such compounds may offer a safe pharmacological profile and curb

addiction by preventing drug seeking triggered by conditioned contextual cues.

Key words: Cannabinoids; reward; addiction; drug abuse; memory; conditioned place

preference

3
INTRODUCTION

Several substances consumed by humans, including ethanol (alcohol), opioids, cocaine,

amphetamines, nicotine and caffeine, serve as rewarding and reinforcing stimuli leading

some individuals to compulsive abuse and addiction (Everitt and Robbins, 2005). Drug

addiction is considered as the most severe form of substance use disorder, which entails

several components, including craving, use of a substance despite all related problems

and negative consequences for everyday life, pharmacological tolerance, withdrawal

symptoms and relapse (DSM-5). Substance use disorder represents as a world health

problem whose prevalence has been increasing over the years (Collaborators, 2018).

Despite its huge impact on individual health and society, few effective treatments exist

(O'Brien, 2008,Welberg, 2011). A major challenge in developing addiction

pharmacotherapies can be attributed to the continuous change in brain circuitry after

chronic exposure to drugs of abuse. One conceptual framework divides drug addiction

in three phases: binge and intoxication, withdrawal and negative affect, and

preoccupation and anticipation (or craving) leading to relapse, which perpetuates drug

use (Koob and Le Moal, 2008,Koob and Volkow, 2016). Acute administration of drugs

of abuse facilitates dopamine neurotransmission in the reward mesolimbic pathway,

which projects from ventral tegmental area (VTA) to the nucleus accumbens (NAcc) in

the ventral striatum (Di Chiara and Imperato, 1988). As drug use progress toward

addiction, molecular and cellular modifications occur in the mesolimbic system, dorsal

aspects of the basal ganglia, extended amygdala, hippocampus and subregions of the

prefrontal cortex (Everitt and Robbins, 2005,Wise and Koob, 2014,Koob and Volkow,

2016).

4
The memory related to the context in which drug is consumed also constitutes a key

feature in the process from initial drug use towards addiction. A previously neutral

context and its related cues may become rewarding (conditioned stimuli) once they are

associated with drugs of abuse (interoceptive unconditioned stimuli), precipitating drug-

seeking behaviour and relapse. Therefore, drug addiction has been proposed as a

disorder whose investigation should take into account the subjacent memory systems

(Milton and Everitt, 2012). Specific sub-nucleus of the amygdala seem to be necessary

for processing emotional memories associated with drug use; the hippocampus, in turn,

is required for the acquisition and retrieval of contextual information previously paired

with the drug, which in turn can perpetuate drug seeking through neuronal connections

with the mesolimbic reward pathway (Milton and Everitt, 2012).

Several neurotransmitter systems are thought to be involved in addiction and substance

use disorders. Among them, the endocannabinoid system stands as a particularly

interesting neurochemical mechanism, controlling brain mechanisms underlying both

contextual memory and reward. This review focuses primarily on the cannabinoid link

between contextual memory and rewarding properties of drugs. First, we will discuss

the place conditioning protocol, emphasizing its translational value, strengths and

limitations. Next, we provide a brief overview on the role of the endocannabinoid

system in reward and addiction. The main session will focus on the relevance of place

conditioning to investigate the effects of cannabinoids and compounds targeting the

endocannabinoid system as potential interventions for drug-related contextual

memories. Finally, we will provide a summary of the main picture emerging from these

studies and suggest directions for further pharmacological research.

5
CONDITIONED PLACE PREFERENCE

Considering the need for better understanding the neurobiology drug addiction and

developing new pharmacotherapies, experimental studies with laboratory animals have

been extensively used (Kuhn et al., 2019). Of special interest for investigating drug-

related contextual memories is the conditioned place preference protocol (CPP), which

was first validated for studying opioids (Katz and Gormezano, 1979) and have been

extensively used for several drugs (Bardo and Bevins, 2000,Cunningham et al.,

2006,Huston et al., 2013). CPP is a simple and reliable procedure, which can be used

with mice and rats as experimental animals. A schematic overview of the apparatus and

the protocol is depicted in Figure 1. The apparatus consists of two chambers with

different visual and tactile cues, which can be connected by a small third compartment.

Although protocols may vary across studies, a typical CPP study consists in at least

three phases. First, animals are exposed to a pre-conditioning session, in which they are

allowed to explore the apparatus (two or three compartments). Then, animals undergo

the conditioned phase. The drug (unconditioned stimulus) is administered and the

animal is immediately confined to one of the chambers (which will serve as the

contextual conditioned stimulus). In alternate sessions, the vehicle is administered and

paired with the opposite chamber. During this phase, animals learn to associate the

environment with reward (or aversive) interoceptive effects induced by the drug.

Finally, in the test, drug-free animals are again able to explore the entire apparatus. If

they spend more time in the compartment previously paired to the drug than in unpaired

one, the drugs is considered as rewarding (CPP), while if the opposite occurs, the drug

effect is interpreted as aversive (conditioned place aversion, CPA) (Bardo and Bevins,

2000,Cunningham et al., 2006,Tzschentke, 2007,Huston et al., 2013). Place

conditioning (CPP or CPA) probably reflects the animals seeking (or avoiding) the drug

6
in the environment associated with its rewarding (or aversive) effects. In extended

protocols, reconsolidation, extinction and reinstatement can also be investigated (Parker

and Mcdonald, 2000). Reconsolidation can be evaluated when animals undergo a drug

memory reactivation phase followed by a retesting of drug-induced conditioned place

preference or aversion. Extinction is achieved by exposuring animals to paired

compartment without drug injection and reinstatement is induced by drug-priming

injections or exposure to stressors (Liu et al., 2008). In this way, it is also possible to

evaluate relapse to drug-seeking behaviour using CPP.

CPP has some important limitations (Bardo and Bevins, 2000). A pharmacological

limitation concerns the difficult in generating graded dose-effect curves due to both the

nature of the response and the requirement for a between-groups experimental design.

Another major problem is the broad variation in protocols used in different laboratories.

Some studies report no initial preference by the animal for any compartment of the

apparatus in the pre-conditioning session (unbiased protocol), while others report

preference for one of the sides (biased protocol). In case of a biased protocol, the reward

stimulus can be paired preferentially with the non-preferred compartment. Other source

of cross-studies variation are the duration and number of exposures to the apparatus.

However, the most obvious drawback of CPP concerns the drug administration by the

experimenter, rather than by the animal itself. In this way, the protocol fails to

recapitulate the contingency between the operant response (the drug self-administration)

and the positive reinforcing properties of the drug (leading to subsequent drug seeking),

an important feature of addiction.

In spite of these limitations, several advantages and applications are ascribed to place

conditioning protocols. The experiments can be easily performed with high

7
reproducibility, allowing investigation of both preference (CPP) and aversion (CPA).

Moreover, CPP exhibits robust face, construct and predictive validities (Bardo and

Bevins, 2000). CPP face validity relies on the possibility of analysing drug seeking

behaviour triggered by a conditioned reward, the context. Although this seems less

straightforward as compared to the face validity of self-administration protocols, CPP

can be induced also in humans by contextual cues associated with drugs (Childs and de

Wit, 2009,2013) as well as music (Molet et al., 2013), food (Astur et al., 2014), and

money (Childs et al., 2017). Furthermore, the exposure of animals to different types of

stressors induces reinstatement of CPP (Mantsch et al., 2016), modelling observations

in humans in which aversive events motivate relapse to drugs of abuse (Sinha et al.,

2006,Back et al., 2010,Higley et al., 2011,Sinha et al., 2011). The construct validity of

this protocol is supported by studies demonstrating the increase in dopamine released in

forebrain areas after the exposition to a drug-related cue in both animals and humans

(Milella et al., 2016,Shinohara et al., 2017). Moreover, brain structures and circuits

responsible for processing contextual information paired with drugs of abuse, such as

the hippocampus and its connections to the NAcc, have been demonstrated to underly

CPP (LeGates et al., 2018,Sjulson et al., 2018).

As for its pharmacological predictability, various drugs have been investigated in mice

and rats in CPP protocols. Among the drugs of abuse consistently reported to induce

conditioned reward are opioids, alcohol, amphetamines, cocaine, caffeine and nicotine

(Tzschentke, 1998,Liu et al., 2008). The protocol has also been demonstrated in humans

for amphetamine (Childs and de Wit, 2009,2013) and alcohol (Childs and de Wit,

2016). Apart from drugs, CPP can also be induced by food, sex and social interaction as

unconditioned stimuli (Agmo et al., 1995,Cunningham et al., 2006). Thus, CPP has a

robust predictive validity for detecting stimuli which are rewarding in humans. In

8
addition, CPP has been extensively used in the search for new pharmacological

treatments for addiction. The testing compound can be investigated for its effect upon

conditioning (acquisition), by administrations before each injection of the drug of abuse,

expression, by administering the compound before the test, as well as reconsolidation,

extinction and reinstatement (Tzschentke, 2007).

THE ENDOCANNABINOID SYSTEM AND DRUG ADDICTION

Among the compounds investigated in CPP/CPA protocols are the cannabinoids and

those targeting various molecular components of the endocannabinoid system. This

neuromodulatory system is named after the herb Cannabis sativa, whose main active

constituent is Δ9-tetrahydrocannabinol (THC), in addition to cannabidiol (CBD) and

other compounds called phytocannabinoids. The structure and functioning of this

system have been extensively reviewed (Pertwee et al., 2010,Mechoulam and Parker,

2013,Mechoulam et al., 2014). Endocannabinoid signalling comprises at least two

metabotropic, Gi protein-compled receptors, type 1 (CB1) and type 2 (CB2) cannabinoid

receptors (Matsuda et al., 1990,Munro et al., 1993); their endogenous ligands

(endocannabinoids), the lipids anandamide and 2-arachidonoylglycerol (2-AG) (Devane

et al., 1992,Sugiura et al., 1995); and their metabolizing enzymes. Endocannabinoids

are synthesized on-demand from postsynaptic membrane lipids after calcium influx and

subsequent activation of different enzymes, among them phospholipase D, for

anandamide, and diacylglycerol lipase α, for 2-AG (Bisogno et al., 2003,Okamoto et al.,

2004). As for their hydrolysis, anandamide and 2-AG hydrolysis are internalized in

neurons through a membrane transporter and serve as substrates primarily for fatty acid

amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively (Cravatt

9
et al., 1996,Dinh et al., 2002). A schematic view of the endocannabinoid system is

presented in Figure 2.

The CB1 receptor is the main responsible for the typical effects of THC (a

phytocannabinoid) and its derivatives (synthetic cannabinoids). It is densely expressed

throughout the encephalon, including regions related to the development of various

aspects of reward and addiction, such as ventral and dorsal striatum, hippocampus,

amygdala and the prefrontal cortex (Herkenham et al., 1991,Tsou et al., 1998). Based

morphological, neurochemical and electrophysiological studies, the CB1 receptor is

proposed be located mainly in presynaptic terminals, with endocannabinoids being

synthesized from cell membranes in postsynaptic neurons. Therefore the

endocannabinoid system is considered as a retrograde neuromodulatory system, which

inhibit neuronal activity, firing rates and neurotransmitter release (Elphick and

Egertová, 2001,Kreitzer and Regehr, 2001,Ohno-Shosaku et al., 2001,Wilson and

Nicoll, 2001). Contrary to the CB1 receptor, CB2 expression and distribution in brain

has been controversial. It was initially proposed as a “peripheral” cannabinoid receptor,

absent from neurons in the encephalon, where it would be expressed only in glial cell

(Munro et al., 1993). More recent studies, however, have been challenging this view,

providing evidence for the expression of the CB2 receptor in neurons, including in

brains circuits related to addiction (Gong et al., 2006,Onaivi et al., 2008b,Zhang et al.,

2014,Zhang et al., 2017).

The connection between cannabinoids and addiction emerges from the well-known

abuse potential of cannabis preparations. Cannabis effects are mediated mainly by THC,

a phytocannabinoid with moderate efficacy as a CB1 and CB2 receptor agonist. Various

synthetic cannabinoids mimic THC effects, some of which possessing higher

10
selectivity, affinity, efficacy for the CB1 receptor than THC itself (Pertwee et al., 2010).

In experimental animals, cannabinoids tend to induce anxiolytic-like effects, sustain

self-administration, serve as unconditioned stimuli for CPP and facilitate mesolimbic

dopaminergic activity through CB1 receptor activation, at least when administered at

low doses. As a corollary, CB1 receptor antagonists consistently reduce rewarding and

reinforcing effects induced by cannabinoid and non-cannabinoid drugs, including

cocaine, ethanol, opioids and nicotine, suggesting a modulatory role for the

endocannabinoid system in drug addiction (Moreira et al., 2015,Parsons and Hurd,

2015,Manzanares et al., 2018). Accordingly, certain drugs of abuse, namely cocaine,

nicotine and ethanol, require CB1 receptor activation to induce dopamine release (Cheer

et al., 2007). Moreover, endocannabinoid-synthesizing enzymes are expressed in

dopaminergic neurons (Mátyás et al., 2008) and cocaine triggers the synthesis of 2-AG

in the VTA, which in turn activates CB1 to inhibits gamma-aminobutyric acid (GABA)

afferences and disinhibit dopaminergic neurons (Wang et al., 2015). In addition to the

VTA, 2-AG-mediated activation of CB1 receptor may also indirectly modulate

dopaminergic terminals the striatum (Uchigashima et al., 2007). Finally, the CB2

receptor has also been implicated in drug addiction, although its function seems to be

opposite to those ascribed to the CB1 receptor, at least for cocaine. CB2 receptor

agonists inhibit the behavioural effects of cocaine in mice after systemic or intra-NAcc

administrations (Xi et al., 2011,Zhang et al., 2014,Delis et al., 2017,Gobira et al.,

2019,Lopes et al., 2020). The mechanisms might involve direct modulation of

dopaminergic activity, since the CB2 receptor is co-expressed with tyrosine hydroxylase

in the VTA, inhibiting the activity dopamine-producing neurons (Zhang et al., 2017)

The expression and functioning of the endocannabinoid system point to an important

role of this neuro-modulatory system in pathways and brain regions underlying

11
contextual memory formation. Thus, place conditioning protocols have been extensively

used to investigate the reward or aversive characteristics of cannabinoids as well as the

role of the endocannabinoid system in the drug addiction.

CANNABINOIDS AND PLACE CONDITIONING

This section will report on the effects of cannabinoids and compounds targeting the

endocannabinoid system on acquisition (conditioning), expression (test), extinction,

reinstatement, and reconsolidation of reward memory. Table 1 shows the main ligands

employed in these studies along with their molecular targets. Their detailed

pharmacology has been reviewed elsewhere (Pertwee et al., 2010).

THC and CBD

Notwithstanding the well-known rewarding properties of cannabis, it has been difficult

to recapitulate these effects with systemic THC administration on place conditioning

protocols. Rewarding effects (CPP) tend to occur at low doses (Braida et al., 2004,Le

Foll et al., 2006) and depend not only on the CB1 receptor activation, but also on

recruitment of endogenous opioid mechanisms (Braida et al., 2004). In contrast,

aversion (CPA) is more commonly observed by increasing THC doses (Sañudo-Peña et

al., 1997,Hutcheson et al., 1998,Cheer et al., 2000,Quinn et al., 2008,DeVuono et al.,

2017). Various parameter may interfere with THC effects, since the aversiveness

induced by this drug is not observed in adolescent rats (Quinn et al., 2008) or when a

stress stimulus precedes the conditioning phase (DeVuono et al., 2017). Moreover, THC

12
effects on CPP/CPA are dependent on the previous experience of the subject with the

drug, since CPP has been attained, even at doses otherwise aversive to naïve subjects,

when the animals receive a previous THC administration (Valjent and Maldonado,

2000). Other studies reported no effects regardless the doses tested (Parker et al.,

2004,Vlachou et al., 2007,Hempel et al., 2016). Thus, studies with systemic

administration of THC report CPP most commonly at low doses (1 mg/kg, in average)

and CPA at high doses (around 5 mg/kg) although, in fact, the most frequent result is no

effect at all (Kubilius et al., 2018). Apart from systemic injections, THC induces CPP

after local administration into either the VTA or the NAcc (Zangen et al., 2006).

Regarding the interactions between THC and other drugs, this phytocannabinoid

facilitates the extinction of cocaine- and amphetamine-induced CPP (Parker et al.,

2004). In nicotine pre-exposed rats, low doses of THC does not induce CPP, whereas

higher doses induce CPA (Le Foll et al., 2006). In addition, protracted treatment with

THC followed by short and long abstinence periods prevent morphine-induced CPP

(Valverde et al., 2001,Jardinaud et al., 2006). THC also influences the response to other

cannabinoids. Previous THC administration5 reduced CPA induced by the potent

cannabinoid JWH-018, suggesting protection against the aversive effects of synthetic

cannabinoids (Hyatt and Fantegrossi, 2014).

CBD, a major cannabis constituent, binds to multiple targets in brain and exert a myriad

of pharmacological effects, among them anxiolytic, antipsychotic and antiepileptic (Izzo

et al., 2009). It seems to lack rewarding properties and fails to induce CPP or CPA per

se (Parker et al., 2004,Vann et al., 2008,Markos et al., 2018). However, it attenuates

acquisition of CPP induced by cocaine (Luján et al., 2018) and morphine (Markos et al.,

2018), and facilitates the extinction of cocaine- and amphetamine-induced CPP (Parker

et al., 2004). Moreover, CBD prevents drug- and stress-induced reinstatement of CPP to

13
morphine and cocaine (de Carvalho and Takahashi, 2017), as well as reinstatement of

CPP precipitated by methamphetamine (DeVuono et al., 2017). Yet, CBD inhibits CPA

precipitated by naltrexone (de Carvalho and Takahashi, 2017). Thus, CBD prevents

different phases of drug-induced CPP or CPA. CBD and THC have also been evaluated

in combination. CBD was able to prevent THC-induced CPA (Vann et al., 2008), an

observation with potential therapeutic implication since some cannabis-based medicines

contain a combination of both compounds. Therefore, these results highlight the

therapeutic potential of CBD for the treatment of drug addiction. In agreement, a pilot

study performed in tobacco smokers found reduction in the number of cigarettes

smoked after inhalation of CBD for one-week (Morgan et al., 2013) and a study with

opioid users observed that CBD reduces drug craving induced by conditioned cues

(Hurd et al., 2019).

CB1 receptor

The CB1 receptor used to be considered as the only cannabinoid receptor expressed in

neurons in the encephalon, accounting for the typical behavioural effects of THC and

synthetic cannabinoids. Both agonists and antagonists have been evaluated for its

effects on CPP/CPA. They have also been investigated on CPP induced by drugs of

abuse. In the same fashion as THC, synthetic cannabinoids (with different degrees of

CB1 selectivity) yield complex effects. Some authors observed CPP after systemic CB1

receptor activation (Braida et al., 2001), others reported CPA (Chaperon et al.,

1998,López-Moreno et al., 2004,Scherma et al., 2008a) and others found no effect

(Polissidis et al., 2009). However, intra-cranial injections into the NAcc (Karimi et al.,

2013) or VTA (Rashidy-Pour et al., 2013,Ahmad and Laviolette, 2017) induced CPP.

14
Overall, experiments with synthetic cannabinoids yield a profile similar to THC, with

CPP observed mainly at low doses and CPA at high doses (Kubilius et al., 2018).

CB1 receptor agonists have also been investigated in relation to their effects on CPP

induced by other drugs. They facilitate stress-induced reinstatement of cocaine CPP

when combined with an α-2 adrenergic antagonist, suggesting involvement of

noradrenergic pathways (Vaughn et al., 2012). Indeed, noradrenaline is one of the

neurotransmitters involved in negative emotional states associated with addiction (Koob

and Le Moal, 2008). CB1 receptor agonists also faciliate MDMA-induced reinstatement

of CPP by potentiating the effects of subthreshold priming doses of the drug (Daza-

Losada et al., 2011). Moreover, cannabinoid exposure during adolescence faciliate

reinstatement of MDMA-induced CPP in adult animals (Rodríguez-Arias et al., 2010).

These animals were also more susceptible to acquisition and reinstatement of CPP

induced by cocaine (Rodríguez-Arias et al., 2016). These results build on the hypothesis

that previous exposure to cannabinoids may lead to the subsequent use of other drugs

(Lynskey et al., 2003).

In contrast to agonists, CB1 receptor antagonists/inverse agonists generally fail to

induce any place preference or aversion per se (Mas-Nieto et al., 2001,Manzanedo et

al., 2004,Scherma et al., 2008a,Fang et al., 2011), although some studies do detect CPP

(Cheer et al., 2000,Ramiro-Fuentes et al., 2010) or CPA (Karimi et al., 2013).

Although CB1 receptor antagonists generally are devoid of effects on CPP/CPA

protocols, they consistently impair CPP induced by different classes of drugs,

particularly psychostimulants, which inhibit the uptake and/or facilitate the release of

dopamine. On cocaine-induced CPP, CB1 antagonism prevented acquisition (Chaperon

et al., 1998,Yu et al., 2011,Delis et al., 2017,Gobira et al., 2019,Lopes et al., 2020),

15
expression (Delis et al., 2017,Lopes et al., 2020), and reinstatement of reward memory

(Yu et al., 2011,Vaughn et al., 2012). We observed that the inhibitory effect of CB1

antagonists on cocaine CPP are reversed by a CB2 antagonist, suggesting an interplay

between cannabinoid receptors in modulating cocaine reward memories (Gobira et al.,

2019). CB1 antagonists also decrease CPP elicited by methamphetamine (Yu et al.,

2011), although they facilitate MDMA reinstatement of extinguished CPP (Daza-

Losada et al., 2011).

In addition to psychostimulants, CB1 antagonists have been investigated in CPP induced

by drugs acting through other mechanisms. They decreased acquisition (Le Foll and

Goldberg, 2004,Forget et al., 2005,Merritt et al., 2008), expression (Le Foll and

Goldberg, 2004), reinstatement (Budzyńska et al., 2009,Fang et al., 2011) and

reconsolidation (Fang et al., 2011) of nicotine-induced CPP. These consistent result are

supported by clinical trials showing the ameliorating effects of the CB1 receptor

antagonist rimonabant in nicotine dependence (Le Foll et al., 2008). They also impaired

acquisition (Chaperon et al., 1998,Mas-Nieto et al., 2001,Manzanedo et al., 2004,Singh

et al., 2004) and reconsolidation of CPP induced by morphine (De Carvalho et al.,

2014). These effects were mimicked with specific administration into the VTA or the

NAcc (Karimi et al., 2013,Rashidy-Pour et al., 2013). CB1 blockage is also effective in

CPP induced by other types of reward, particularly palatable food (Chaperon et al.,

1998,Méndez-Díaz et al., 2012).

The role of the CB1 receptor has also been investigated in genetically modified animals

(i.e. with receptor deletion or overexpression). The majority of the results converge with

data obtained using pharmacological approaches. CB1 knockout animals display

decreased CPP for ethanol (Houchi et al., 2005,Thanos et al., 2005), morphine (Martin

16
et al., 2000), and nicotine (Castañé et al., 2002,Merritt et al., 2008), although not for

cocaine and quinpirole (Martin et al., 2000,Castañé et al., 2002,Houchi et al., 2005). In

contrast, drug reward is not significantly changed in mice with a hyper-sensitive form of

CB1, since they develop CPP for ethanol, morphine, and cocaine (Marcus et al., 2017).

These discrepant results may be related to developmental changes in knock-out animals

in reward pathways (Houchi et al., 2005), which are modulated by CB1 receptor (Cheer

et al., 2007).

Therefore, CB1 receptor agonists induce either reward or aversive effects and facilitate

or inhibit CPP induced by other drugs, depending on the dose, protocol and

experimental settings. In contrast, CB1 receptor antagonists/inverse agonists are inert

per se, yet they consistently prevent all phases of CPP induced not only by drugs of

abuse, but also by other types of reward. The results from CB1 knockout mice tend to

confirm the conclusion from studies employing selective antagonist.

CB2 receptor

Contrary to the CB1 receptor, which accounts for most of the typical effects of

cannabinoids, data on CB2 expression in neurons and its possible role in behaviour are

still scant. CB2 receptor ligands are mostly devoid of reward or aversive properties on

place conditioning protocols. As for their effects on drug-induced CPP, contrasting with

CB1, it is the agonism (not the antagonism) of CB2 that prevents acquisition and

expression of cocaine-induced CPP (Delis et al., 2017,Lopes et al., 2020). Either CB1

antagonism or CB2 agonism prevented the increase in cFos expression in the

hippocampus of animals exposed to the cocaine-paired compartment (Lopes et al.,

17
2020). Therefore, endocannabinoid signalling possibly modulates drug-associated

memories through inhibition of hippocampal activation in presence of contextual cues.

Evidence from opposite roles of cannabinoid receptors also came from results showing

CB2 antagonists preventing the effects of CB1 antagonist on cocaine-induced CPP

(Gobira et al., 2019). Moreover, CB2 agonists also prevented acquisition and expression

of ethanol-induced CPP (Al Mansouri et al., 2014,Powers et al., 2015,Martín-Sánchez et

al., 2019). Accordingly, there is a reduction on CB2 gene expression in the ventral

midbrain of mice after prolonged alcohol consumption (Onaivi et al., 2008a). Other

studies, however, reached the opposite conclusions, since CB2 antagonism was able to

block acquisition of nicotine-induced CPP (Ignatowska-Jankowska et al.,

2013,Navarrete et al., 2013) and expression of alcohol-induced CPP (Martín-Sánchez et

al., 2019). This impairment may be result of aversive proprieties of CB2 antagonists,

since they did induce CPA (Navarrete et al., 2013).

Results from genetically modified animals have been mixed. Ethanol-induced CPP was

increased in CB2 knockout mice, although this phenotype was not recapitulated by

pharmacological interventions in the same study (Powers et al., 2015). Another study

also found that not only CPP, but also other behavioural responses to ethanol, were

exacerbated in these animals (Ortega-Álvaro et al., 2015). The opposite was found in

studies with nicotine, in which CB2 receptor deletion abolished CPP (Ignatowska-

Jankowska et al., 2013,Navarrete et al., 2013). Selective deletion of CB2 in

dopaminergic neurons also prevented CPP for nicotine (Canseco-Alba et al., 2019) and

ethanol (Canseco-Alba et al., 2018), although the effects of psychostimulant drugs such

as cocaine, amphetamine, and methamphetamine, remained unchanged (Canseco-Alba

et al., 2019). This discrepancy may be attributed to different drug actions on

dopaminergic singling in the mesolimbic pathway. While psychostimulants induce

18
direct increase of dopamine levels, nicotine acts indirectly on the dopaminergic system.

Finally, ~transgenic mice overexpressing CB2 receptors showed CPA, rather than CPP,

after cocaine administration (Aracil-Fernández et al., 2012).

Therefore, it still premature to draw any firm conclusion on the role of the CB2 receptor

on CPP. Based on the few pharmacological and genetic studies available so far, the

functions of CB2 receptor tend to be the opposite to those ascribed for CB1, at least for

cocaine-induced CPP. It is the activation of CB2, rather than its antagonism, that tend to

inhibit cocaine effects, although this observation does not necessary apply to the effects

of other drugs.

Endocannabinoid uptake and hydrolysis

Inhibition of endocannabinoid uptake (membrane transport) and hydrolysis represents

an interesting pharmacological approach to investigate the biological functions of

endocannabinoids. The specific roles of anandamide and 2-AG can be investigated

through the inhibition of their metabolizing enzymes, FAAH and MAGL, respectively.

Since endocannabinoids are produced on-demand, it is possible to selectively increase

their endogenous levels with temporal and anatomical resolution in the brain.

Systemic anandamide administration did not induce CPP (Scherma et al.,

2008a,Méndez-Díaz et al., 2012) or CPA per se (Scherma et al., 2008a). The same was

observed after increase in endogenous anandamide levels by inhibition of its reuptake or

hydrolysis (Gobbi et al., 2005,Bortolato et al., 2006,Scherma et al., 2008a,Scherma et

al., 2008b,Gamaleddin et al., 2013,DeVuono et al., 2017). However, when animals were

pretreated with a FAAH inhibitor, anandamide induced CPA, which was prevented by

19
CB1 antagonism (Scherma et al., 2008a). In fact, intravenous administration of

anandamide produced an increase in extracellular dopamine levels in NAcc shell, which

was magnified by FAAH inhibition and blocked by CB1 antagonism (Solinas et al.,

2006). Therefore, anandamide modulation of brain dopaminergic pathway results in

biphasic behavioural responses.

Although pharmacological FAAH inhibition does not serve as an unconditioned

stimulus to induce CPP, increasing anandamide levels modulates the effects of addictive

drugs in different phases of CPP. The main evidence has been obtained from studies

with nicotine. Inhibition of anandamide transport or hydrolysis impaired acquisition and

reinstatement of nicotine-induced CPP as well as nicotine-induced increase in dopamine

levels in the NAcc (Scherma et al., 2008b,Scherma et al., 2012). In contrast, another

study observed enhanced expression of nicotine-induced CPP after FAAH inhibition

(Merritt et al., 2008). Regarding morphine-induced CPP, FAAH and MAGL inhibition

did not affect CPA induced by morphine withdrawal (Gamage et al., 2015). One study

found changes in expression of mRNA levels of FAAH and MAGL in dorsal

hippocampus of animals submitted to different phases of morphine-induced CPP,

indicating alterations in the mechanisms of clearance of endocannabinoids following

expression and reinstatement (Li et al., 2017).

Some studies have investigated the phenotypes of FAAH knockout mice in CPP

protocols. An otherwise inactive dose of nicotine was able to induce CPP in these

animals through CB1 receptor facilitation, suggesting an endocannabinoid-mediated

enhancement of nicotine reward (Merritt et al., 2008,Pavon et al., 2018). There was also

an increase in dopamine levels in NAcc of FAAH knockout animals or after FAAH

pharmacological inhibition in wildtype animals (Pavon et al., 2018). Genetic deletion of

20
FAAH, however, failed to modify ethanol-induced CPP (Blednov et al., 2007).

Collected brain samples from animals submitted to ethanol-induced CPP found

decreased anandamide levels in the medial prefrontal cortex and ventral midbrain

(Martín-Sánchez et al., 2019). Possibly, repeated alcohol exposure misbalances

endocannabinoid levels throughout the mesocorticolimbic pathway, resulting in

increased dopaminergic activity.

Thus, the lack of effect of inhibitors of endocannabinoid membrane transport or

hydrolysis in place conditioning protocols implies that these compounds can be

developed as therapeutic agents devoid of rewarding properties and therefore with low

abuse liability. This is an important observation, since these compounds have been

investigated for the treatment of pain and various neuro-psychiatric disorders (Fowler,

2015a,b). Their effects against drug-induced CPP, however, are inconsistent, possibly

due to the opposite roles of CB1 and CB2 receptors in modulating drug rewarding

effects.

GENERAL DISCUSSION

Place conditioning protocols (CPP/CPA) have been instrumental to investigate the

effects of cannabinoids and the role of the endocannabinoid system on reward memory

associated with drugs of abuse. These studies contribute to the understanding of

important questions and hypothesis about cannabinoid addiction, including their impact

on the effects of other drugs, their biphasic effects on reward/aversion and their variable

effects on the first drug experience. Moreover, CPP studies have shed light on how

21
specific components of the endocannabinoid system relate to the biological effects of

other drugs, impacting the developing of new treatments for addiction.

THC and selective CB1 agonists induce CPP or CPA, depending on the dose and

previous experiences. The pattern emerging from studies investigating cannabinoids on

CPP (that is, the inconsistent and biphasic profiles) is similar to those observed with

other types of behavioural responses. It has also been challenging to detect the

reinforcing properties of THC in self-administration protocols (Justinova et al., 2005).

Moreover, in animal models of anxiety/fear, these cannabinoids tend to induce

anxiolytic-like effects at low doses, whereas the opposite generally occurs at high doses

(Moreira and Wotjak, 2010). This complex pattern of effects may result from

cannabinoid modulation of diverse neurotransmitter systems and effects on specific

neural subpopulations in certain brain regions (Lutz et al., 2015,Patel et al., 2017).

Accordingly, in humans, the effects of cannabis also change depending on dose and

previous stress experiences (Viveros et al., 2005). Contrary to THC and synthetic

cannabinoids, CBD induces neither reward nor aversion, even though it inhibits the

effects of other drugs in different phases of CPP, an interesting profile for therapeutic

exploitation. Noteworthy, CBD reduces cue-induced craving in humans with heroin use

disorder (Hurd et al., 2019), further supporting its potential therapeutic use in drug

addiction.

The potential role of CB1 receptor in the rewarding and reinforcing effects of various

drugs has drawn much attention to selective antagonists/inverse agonists as potential

treatments for addiction. Therefore, these compounds have been investigated in drug-

induced CPP. Various studies converge showing CB1 receptor antagonists reducing the

acquisition and expression of CPP induced by cocaine, alcohol, opioids and nicotine.

22
These consistent results might be explained by antagonism of CB1 receptor located in

neuronal terminals projecting onto dopaminergic neurons in VTA and NAcc. In these

regions, drugs of abuse recruits endocannabinoids, whereas CB1 receptor blockade

restrains the mesolimbic reward pathway (Cheer et al., 2007,Hernandez et al.,

2014,Wang et al., 2015), as illustrated in Figure 3. In addition, CB1 receptor in

hippocampus, amygdala, and prefrontal cortex possibly participate in the mechanisms

through which conditioned contextual cues trigger compulsive drug seeking (Moreira et

al., 2015) as well as control extinction and reinstatement of reward memories (Stern et

al., 2018,Goode and Maren, 2019). As result, and further reinforcing the translational

value of CPP, the CB1 antagonists/inverse agonist, rimonabant, surinabant and

taranabant have been investigated in humans studies for alcohol, nicotine and opioid use

disorders (Sloan et al., 2017,Chye et al., 2019). The expectations, however, have been

dampened by the psychiatric side effects of these compounds, which can facilitate

anxiety, panic and depressive mood, as revealed by several preclinical studies and by

the short-living experience with rimonabant for the treatment of obesity (Moreira and

Crippa, 2009) Since drug addiction is frequently co-morbid with other psychiatric

disorders (Verheul, 2001,Morozova et al., 2015,Rahman, 2015,Hillemacher and

Frieling, 2019), alternative possibilities have been pursued. One of them is the use of

neutral antagonists, which block the receptor without inverse agonistic activity and are

less prone to induce aversive side effects (Gueye et al., 2016).

More recently, the focus has shifted to the CB2 receptor. Notwithstanding the

controversies concerning its expression and function in brain, CB2 selective agonists

impair CPP induced by cocaine or ethanol. Two hypotheses, not mutually exclusive,

could explain CB2 receptor-modulation of drug reward. The non-neuronal hypothesis

can be built based on evidence that microglia and astrocytes, which express CB2

23
receptors (Stella, 2010), modulate mesolimbic dopamine signalling and drug reward

(Neuhofer and Kalivas, 2018,Linker et al., 2019,Stellwagen et al., 2019). Thus, CB2

agonists could inhibit glial function, including cytokine signalling, and indirectly

modulate neuronal activity related to drug reward. An alternative, neuronal hypothesis,

can also be posited. Evidence has emerged suggesting CB2 receptor expression in

central neurons, although in different locations from CB1. While the latter is expressed

in neuronal terminals projecting to dopaminergic neurons, CB2 is proposed to be located

directly in dopaminergic cell bodies in VTA, where its activation inhibits the release of

dopamine on NAcc in mice (Zhang et al., 2017). These hypothesis are illustrate in

Figure 3. CB2 is also expressed in the hippocampus (Stempel et al., 2016), a key

structure involved in CPP expression due its implication on spatial memory recovery

(Sjulson et al., 2018). Indeed, CB2 activation prevented neuronal activation in the

hippocampus of animals exposed to cocaine-induced CPP (Lopes et al., 2020). A

potential advantage of exploiting the favourable effects of CB2 receptor agonists is the

possibility of reducing the reward actions of drug of abuse without inducing the

psychiatric side effects associated with CB1 antagonists. Actually, CB2 receptor agonists

even alleviate anxiety-like behaviour in rodents (Bahi et al., 2014). Therefore, CB2 is a

promising target for new addiction treatments. However, CB2 involvement in drug

reward warrants further investigation, including studies concerning its expression and

function across different species. An approach deserving more evaluation is the

simultaneous modulation of CB1 and CB2 receptors, since recent findings suggest they

work in concert, with opposite function, to counteract reward responses (Gobira et al.,

2019).

The opposite roles of CB1 and CB2 may at least partially explain the inconsistent effects

of endocannabinoids hydrolysis inhibitors on CPP induced by drugs of abuse. Once

24
endocannabinoids levels are increased, they can promote simultaneous activation of

CB1 and CB2, despite differences on affinity. In addition, given the interaction between

endocannabinoid system and both inhibitory and excitatory neurotransmitters (Figure

3), the contradictory actions of endocannabinoid hydrolysis inhibitors are not surprising.

CONCLUSIONS

CPP is a valuable protocol for investigating the effects of compounds on every stage of

memories related to drug reward, i.e. acquisition, evocation, extinction, reinstatement,

and reconsolidation. It has been instrumental for deepening our understanding on the

rewarding and aversive properties of cannabinoids as well as the effects of substances

targeting the endocannabinoid system on contextual conditioning induced by various

drugs of abuse. “Neutral” CB1 receptor antagonists, selective CB2 receptor agonists and

substances combining both mechanisms emerge as therapeutic possibilities. Along with

other protocols, particularly sensitization and drug self-administration, place

conditioning studies point to the endocannabinoid system as a “drugable” target to

interfere with contextual memories responsible for initiating and maintaining drug

addiction.

25
Figure legends

Figure 1. The place conditioning protocol. In the pre-conditioning phase, animals (mice

or rats) are exposed to the apparatus with two distinguishable compartments, which they

are free to explore (in an unbiased protocol, animals spend similar times in both sides of

the box). In the conditioning phase, the drug is paired to one side of the box, where the

animals remain confined, and vehicle is paired to the opposite side. Finally, in the test

phase, drug-free animals explore all the compartments of the apparatus, similarly to the

pre-conditioning phase. An increase exploratory activity in the drug-paired side

indicates conditioned place preference (CPP), whereas an increase in time spent in the

vehicle-paired side indicates conditioned place aversion (CPA).

Figure 2. Schematic representation of the endocannabinoid system. Anandamide

(arachidonoyl ethanolamide, AEA) synthesis depends initially on glycerophospholipid

and phosphatidylethanolamine, which form N-arachidonoyl phosphatidylethanolamine

(NArPE) through N-acyltransferase. NArPE, in turn, is cleaved to anandamide through

N-acylphosphatidylethanolamine specific phospholipase D (PLD). 2-AG is formed by

glycerophospholipid cleavage facilitated by phospholipase C (PLC), followed by

diacylglycerol lipase (DAGL). They bind do CB1 and CB2 receptor and are internalised

by an endocannabinoid membrane transporter (EMT). Endocannabinoid main

hydrolysis processes depend on fatty acid amid hydrolase (FAAH), for anandamide, and

monoacylgycerol lipase (MAGL), for 2-AG.

26
Figure 3. Endocannabinoid modulation of the rewarding dopaminergic pathway. CB1

receptor could facilitate or inhibit dopaminergic activity and reward memory by

exerting inhibitory activity on GABAergic and glutamatergic terminals, respectively, in

the VTA/NAcc pathway. CB2 receptors could inhibit dopaminergic activity directly at

cell bodies at the VTA or through surround glial cells.

27
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Table

Tables

Table 1: Selected cannabinoids and compounds targeting the endocannabinoid system.

Compound Mechanism

Phytocannabinoids

THC Partial agonist at CB1 and CB2

CBD Various mechanisms

Synthetic cannabinoids

CP-55940 CB1/CB2 agonist

HU210 CB1/CB2 agonist

WIN-55212-2 CB1/CB2 agonist

ACEA CB1 agonist

JWH133 CB2 agonist

Antagonists/inverse agonists

AM251 CB1 antagonist/inverse agonist

Rimonabant (SR 141716) CB1 antagonist/inverse agonist

AM4113 CB1 “neutral” antagonist

AM630 CB2 antagonist

Endocannabinoids

Anandamide CB1/CB2 agonist

2-arachidonoylglycerol (2-AG) CB1/CB2 agonist

Endocannabinoid hydrolysis and uptake inhibitors

URB597 FAAH inhibitor

JZL184 MAGL inhibitor

AM404 Endocannabinoid uptake inhibitor
Figure Click here to download Figure Figure CPP.tiff
Figure Click here to download Figure Figure eCBs.tiff
Figure Click here to download Figure Figure pathway SIMPLES.tiff