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Relapse to cocaine seeking in an invertebrate
Accepted Manuscript Relapse to cocaine seeking in an invertebrate
Akua O. Amaning-Kwarteng, Aman Asif-Malik, Yue Pei, Juan J. Canales PII: DOI: Reference:
S0091-3057(17)30053-9 doi: 10.1016/j.pbb.2017.04.008 PBB 72465
To appear in:
Pharmacology, Biochemistry and Behavior
Received date: Revised date: Accepted date:
23 January 2017 7 April 2017 21 April 2017
Please cite this article as: Akua O. Amaning-Kwarteng, Aman Asif-Malik, Yue Pei, Juan J. Canales , Relapse to cocaine seeking in an invertebrate. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Pbb(2017), doi: 10.1016/j.pbb.2017.04.008
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ACCEPTED MANUSCRIPT Pharmacology, Biochemistry and Behavior, submitted 21/01/2016
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Revised
Relapse to Cocaine Seeking in an Invertebrate
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Akua O. Amaning-Kwarteng, Aman Asif-Malik, Yue Pei and Juan J. Canales Department of Neuroscience, Psychology and Behaviour University of Leicester, Lancaster Road, Leicester, LE1 9HN, United Kingdom
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Running head: Drug relapse in an invertebrate
Address for correspondence: Juan J. Canales, D.Phil. Department of Neuroscience, Psychology and Behaviour University of Leicester Leicester, LE1 9HN United Kingdom Phone: +44 (0)116 229 7125 Fax: +44 (0)116 229 7196 Email: [email protected] 1
ACCEPTED MANUSCRIPT Abstract Addiction is characterised by cycles of compulsive drug taking, periods of abstinence and episodes of relapse. The extinction/reinstatement paradigm has been extensively used in rodents to model human relapse and explore underlying mechanisms and therapeutics.
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However, relapse to drug seeking behaviour has not been previously demonstrated in
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invertebrates. Here, we used a cocaine conditioned place preference (CPP) paradigm in the
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flatworm, planarian, followed by extinction and reinstatement of drug seeking. Once baseline
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preference was established for one of two distinctly textured environments (i.e. compartments with a coarse or smooth surface), planarian received pairings of cocaine (5 M) in the non-
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preferred, and vehicle in the most preferred, environment, and were tested for conditioning
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thereafter. Cocaine produced robust CPP, measured as a significant increase in the time spent in the cocaine-paired compartment. Subsequently, planarian underwent extinction training,
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reverting back to their original preference within three sessions. Brief exposure to cocaine (5
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M) or methamphetamine (5 M) reinstated cocaine-seeking behaviour. By contrast, the high affinity dopamine transporter inhibitor, (N-(n-butyl)-3α-[bis (4-fluorophenyl) methoxy]-
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tropane) (JHW007), which in rodents exhibits a neurochemical and behavioural profile distinct
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from cocaine, was ineffective. The present findings demonstrate for the first time reinstatement of extinguished cocaine seeking in an invertebrate model and suggest that the long-term adaptations underlying drug conditioning and relapse are highly conserved through evolution.
Keywords: planarian, conditioned place preference, reinstatement, relapse, cocaine, methamphetamine
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ACCEPTED MANUSCRIPT Introduction One of the greatest challenges in the treatment of drug addiction is the prevention of relapse after successful detoxification and abstinence. Therefore the study of the psychological and physiological mechanisms underlying craving and relapse has become a prolific area of research. A variety of
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animal models have been developed in rodents and monkeys to study relapse to drug seeking,
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including drug-primed, cue- and drug-induced reinstatement, contextual relapse, and spontaneous
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recovery of drug seeking behaviour (Martin-Fardon and Weiss, 2013, Venniro et al. , 2016,
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Yahyavi-Firouz-Abadi and See, 2009). One of the best known properties of addictive drugs is their ability to promote Pavlovian associations between the drug’s effect and conditioned cues and/or
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specific environments, or contexts, where the drug is experienced, which are thought to underlie the
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phenomenon of conditioned place preference (Tzschentke, 1998). Following extinction training, an extinguished place preference can be readily reinstated by exposing the animals to stress or by
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administering a dose of the unconditioned stimulus (i.e. the drug used during conditioning)
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(Mantsch et al. , 2016, Tzschentke, 2007). Moreover, reinstatement can be elicited by drugs other than the conditioning drug. For example, methamphetamine and methylphenidate reinstated an
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extinguished cocaine place preference, suggesting that exposure to compounds acting similarly to,
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or sharing similar unconditioned stimulus properties as, the drug used during conditioning can induce renewed craving and relapse (Itzhak and Martin, 2002). Thus the place preference paradigm is a valuable tool used to assess in animal models both the rewarding properties of drugs of abuse and associated relapse-like behaviours. Invertebrates have become a popular model system to study various genetic, physiological and behavioural effects of drugs of abuse. One of such invertebrate models is planarian, one of the many flatworms of the Turbellaria class. The brain of planarian is a bi-lobed mass of nerve tissue with
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ACCEPTED MANUSCRIPT distinct structural and functional regions defined by the distinct expression of homeobox genes, and of neural networks, transmitters and neuromodulators akin to those found in the mammalian brain (Buttarelli et al. , 2008, Inoue et al. , 2015). Early evidence showing the usefulness of planarian in drug studies revealed that planarian display behaviours that resemble tolerance and dependence
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following repeated exposure to morphine (Needleman, 1967). More recently, planarian have been
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observed to display behavioural responses to drugs of abuse that are similar to that seen in
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mammals, including behavioural sensitization to cocaine (Rawls et al. , 2010) and withdrawal-like behaviours following cocaine and methamphetamine treatment (Raffa et al. , 2008, Rawls et al. ,
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2008, Sacavage et al. , 2008). Moreover, psychomotor stimulants such as methamphetamine,
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cocaine, mephedrone, and nicotine all exhibit rewarding-like properties in planarian in the place preference paradigm (Hutchinson et al. , 2015, Raffa et al., 2008, Ramoz et al. , 2012, Rawls et al. ,
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2011). These findings suggest that planarian constitute a suitable and translational model to study
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drug interactions, neurotoxicology and certain addiction-related behaviours (Barron et al. , 2010).
an invertebrate model.
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However, relapse-like behaviour, a defining feature of addiction, has not been previously shown in
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In the present experiments, we set out to study reinstatement of drug seeking behaviour following
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cocaine conditioning in planarian. We used a standard cocaine place conditioning paradigm followed by extinction and drug/context-induced reinstatement of cocaine seeking. To explore reinstatement mechanisms, primed exposure treatment to cocaine, methamphetamine or the benztropine analogue, JHW007, was given after extinction training. JHW007 is an atypical dopamine transporter inhibitor that exhibits high affinity for the dopamine transporter but behavioural effects in rodents that are distinct from cocaine (Desai et al. , 2014, Desai et al. , 2005, Hiranita et al. , 2014, Velazquez-Sanchez et al. , 2013). Therefore, we hypothesised that cocaine
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ACCEPTED MANUSCRIPT and methamphetamine, but not JHW007, would be effective at reinstating cocaine seeking behaviour in planarian. Methods
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Subjects and materials
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Large brown planarian of the genus Dugesia (n= 50) (Blades Biological Ltd., UK) comparable in
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size were used. Planarian were kept in a dimly lit room on a 10 h light cycle (light on at 08.00 AM) and maintained in aqua safe solution (Interpet Bioactive Tapsafe, UK) and fed raw chicken three
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times a week. Prior to experimenting, planarian were placed in individual plastic cups filled with
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aqua safe, which were labelled numerically. Planarians were also habituated to their environment and handling procedure before experimentation. All methods were performed in accordance with
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the guidelines and regulations of the University of Leicester.
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Cocaine HCl and methamphetamine HCl were purchased from Sigma- Aldrich (UK) and (N-(nbutyl)-3α-[bis (4-fluorophenyl) methoxy]-tropane) (JHW007) was synthesized by Dr. Juan Murga
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(University Jaime I, Spain). A final concentration 5 μM was used for each drug, diluted in aqua
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safe solution. This concentration was established as effective for reinstatement from previous doseresponse experiments in our laboratory. Singular plastic petri dishes (60 mm diameter; Corning Inc.,
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USA) were used for conditioning. Eight of these were covered internally with silicone (RS Components Ltd., UK) and sand to prepare a ‘rough’ surface in comparison to the ‘smooth’ empty petri dishes. Another eight petri dishes used for testing pre-conditioning, CPP and reinstatement were split internally so half of the dishes had a rough side and the other had a smooth side. Planarian were kept in numerically labelled plastic cups filled with aqua safe at all nonexperimental times and fine artists brushes were used to gently pick up planarian from cups to dishes and vice versa, to ensure minimal liquid transfer and contamination. Tracking of the 5
ACCEPTED MANUSCRIPT planarian was carried out using two CCD cameras. Viewpoint video tracking software (Lyon, France) was used to record both CPP and open field locomotor activity data. Procedure Phase 1: Pre-conditioning. Each planarian was placed at the centre of the two-textured, split petri
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dishes, filled with 8 ml of aqua safe, for 15 min to test for baseline preference. Planarian were
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video-tracked for 15 min and the amount of time spent in each compartment was recorded. Phase 2: Conditioning. Depending on the compartment least preferred during pre-conditioning,
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planarian were placed in a singular dish for 20 min that was either all ‘rough’ or ‘smooth’. The dish
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contained a final concentration of 5 μM cocaine, diluted in 8 ml of aqua safe. The following day, planarian were placed in the compartment they most preferred containing only aqua safe. This was
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repeated for 8 consecutive sessions, conducted once daily, during which the planarian locomotor
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activity (i.e. swimming) was also measured.
Phase 3: Test of conditioning. 24 h after the last conditioning session, each planarian was placed in
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the centre of a split compartment dish for 15 min and activity was recorded to test for CPP. The
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total time spent in each compartment in the pre-conditioning phase was compared with the total time spent in the test of conditioning to assess if the effect of cocaine produced a place preference to
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the environment that was least preferred in Phase 1. Phase 4: Extinction. Following cocaine-induced CPP, each planarian was placed in the split dish filled with aqua safe and the time spent in each compartment was measured, as in the test of conditioning (phase 3). This procedure was repeated until place preference for the compartment previously associated with cocaine was extinguished. Extinction was defined as the point where on
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ACCEPTED MANUSCRIPT average for all planarian the preference for the drug-paired environment during extinction sessions was not statistically different from the preference for the drug-paired environment at baseline. Phase 5: Reinstatement. Following extinction, each planarian was placed in a singular dish as in Phase 2 (conditioning) for 10 min. Planarian were split into three groups (n=10-12), and placed in a
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dish containing 5 μM cocaine, 5 μM methamphetamine or 5 μM JHW-007. Following the 10 min of
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drug exposure, each planarian was placed in a split compartment dish containing only aqua safe, as
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in phase 3 (test of conditioning). The time spent in each compartment was measured to establish
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whether preference for the environment associated with cocaine was reinstated following extinction. Control experiment. To exclude the possibility that brief 10 min exposure to the drug in the least
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preferred compartment could account for the effects observed in the reinstatement test (phase 5), an
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additional experiment was conducted. Baseline place preference was initially established in 16 naïve planarian (as in phase 1). The next day each planarian was exposed to a brief cocaine-
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conditioning period by placing them in the least preferred compartment filled with 5 μM cocaine for
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10 min (as in phase 2). Planarians were then placed in split compartment dishes and time spent in
Statistical analysis
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each compartment was measured to test for conditioning (as in phase 3).
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Data were analysed by analysis of variance (ANOVA) with repeated measures, followed by post hoc comparisons with the method of Fisher’s Protected Least Significance Difference (PLSD). Statistical significance was set at α= 0.05 for all conditions. All statistical analyses were performed using StatView 5.0 (SAS Institute, NC, USA).
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ACCEPTED MANUSCRIPT Results Induction of cocaine CPP and extinction Locomotor activity was measured during the conditioning phase. Two-way ANOVA with repeated measures was conducted with two within-subject factors, Drug (cocaine, vehicle) and Session (four
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conditioning sessions for each drug condition). ANOVA showed no significant effect of the Drug
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factor [F(1,33)= 0.857, p= 0.361], the Session factor [F(3,99)= 0.738, p= 0.533] or the interaction [F(1,33)= 1.866, p= 0.137]. Therefore cocaine exposure during conditioning did not produce
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significant effects on swimming activity (Fig.2a).
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[Fig. 2 about here]
For the test of conditioning, a two-way ANOVA with repeated measures was performed with two
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factor variables, Test (pre-conditioning, post-conditioning) and Compartment (drug-paired, DP, and
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non-drug-paired, NDP). The ANOVA showed a non-significant effect of the factor, Test [F(1,33)= 0.857, p= 0.361] and a significant effects of the factor, Compartment [F(1,33)= 7.837, p= 0.008].
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Furthermore, a significant interaction effect between Test and Compartment was found [F(1,33)=
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29.100, p< 0.001]. Post-hoc tests showed that the time spent by the planarian in the DP compartment in the post-conditioning test increased significantly compared with that spent in the
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same compartment during pre-conditioning (p < 0.01). Therefore, cocaine exposure produced robust CPP in planarian (Fig. 2b). Following conditioning, extinction sessions began. A one-way ANOVA for time spent in each compartment was conducted to test for significance during the extinction phase. On extinction day 1, a significant effect of compartment was observed [F(1, 33) = 26.974, p < 0.001] (Fig. 3a). In session 2, there was no longer a significant difference in the time spent in one compartment or the
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ACCEPTED MANUSCRIPT other [F(1, 33) = 0.032, p < 0.860] (Fig. 3b), and in session 3 planarian reverted back to the preference shown at baseline, effectively extinguishing preference for the DP environment [F(1, 33) = 30.391, p < 0.001] (Fig. 3c). [Fig. 3 about here]
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Reinstatement of cocaine seeking
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For the reinstatement test, planarians were exposed to cocaine, methamphetamine or JHW007. A three-way ANOVA was conducted with Drug (cocaine, methamphetamine or JHW-007) as a
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between-factor, and Test (third day of extinction and reinstatement test) and Compartment (DP and
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NDP), as within-factors. Planarians were matched in terms of relative preference for the DP and NDP compartments prior to the drug assignment. ANOVA revealed a significant high order
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interaction between the Drug, Test and Compartment variables [F(2, 31) = 8.166, p < 0.001] (Fig. 4a).
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Post hoc comparisons showed that while all three groups displayed similar preference for the NDP compartment on the last day of extinction, the cocaine (p< 0.01) and methamphetamine (p< 0.05)
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groups showed robust reinstatement drug seeking behaviour (i.e. comparing time spent in NDP and
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DP in the reinstatement test). By contrast, the group treated with JHW007 did not (Fig. 4a). [Fig. 4 about here]
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For the control experiment, which included a group of planarian receiving identical treatment except for the conditioning and extinction training, a two-way ANOVA with repeated measures showed that there was no significant effect of brief cocaine conditioning on time spent in the DP compartment [F(1, 15) = 0.003, p < 0.955] (Fig. 4b).
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ACCEPTED MANUSCRIPT Discussion The present study showed conditioning, extinction and reinstatement of cocaine place preference in planarian and provided the first evidence of relapse-like behaviour in an invertebrate. Following cocaine conditioning and extinction of a cocaine place preference, both cocaine and
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methamphetamine, but not the high affinity dopamine transport blocker, JHW007, reinstated drug
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seeking behaviour.
The Platyhelminthes, including the flatworm, planarian, are capable of exhibiting complex
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learning in classical conditioning and operant tasks despite their rudimentary cephalisation (Best and Rubinstein, 1962, Lee, 1963, Shafer and Corman, 1963). Planarian possess an
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intricate network of neural connections allowing both synaptic and paracrine signalling by way
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of a collection of neurotransmitter systems, including catecholamines, acetylcholine, serotonin
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and excitatory amino acids such as glutamate and its inhibitory derivative, -aminobutyric acid, amongst others (Ribeiro et al. , 2005). Although little is known about the contributions of these
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transmitter systems to the coordination of motor and complex behaviour in planarian, it is thought that dopamine plays a vital role in movement and reward-related learning in planarian.
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Previous studies led to the identification of the gene encoding tyrosine hydroxylase in
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the planarian, Dugesia japonica, whose knockdown renders planarian unable to synthesize dopamine (Nishimura et al. , 2007), and of the gene encoding the dopamine transporter (DAT), whose expression overlaps with that of the tyrosine hydroxylase (Tashiro et al. , 2014). DAT interference induced increased spontaneous locomotion in planarian that was effectively blocked by selective dopamine D1 and D2 receptor antagonists (Tashiro et al., 2014), suggesting the presence of a fully functional dopamine system in planarian that resembles that of mammalian counterparts. Indeed, methamphetamine-stimulated place learning in planarian
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ACCEPTED MANUSCRIPT was suppressed by dopamine D1 and D2 receptor antagonists (Kusayama and Watanabe, 2000), indicating that psychostimulant-induced place preference requires dopamine receptor activation for its induction in planarian. Our findings showed that repeated cocaine treatment during conditioning neither enhanced nor
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reduced locomotor activity in planarian. This is consistent with previous studies reporting
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cocaine induced hypomotility and C-like hyperkinesia but only at higher concentrations than
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that used in the current study (Hutchinson et al., 2015, Pagan et al. , 2008). Although we acknowledge that using only one dose of cocaine for the conditioning tests is an intrinsic
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limitation, the dose selected was within the range of doses that produce conditioning in
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planarian (1 μM and 10 μM) (Hutchinson et al., 2015) and therefore the interpretation of the findings is straightforward. Moreover, there was no evidence of sensitization to cocaine
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treatment, which also requires higher concentrations to be used in this species (Rawls et al.,
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2010).
The place preference paradigm is a well-established and widely used tool in behavioural
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pharmacology and addiction research to assess drug-induced reward and its underlying
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neurobiological and neuropharmacological substrates (Tzschentke, 2007). Our findings showed that cocaine produced robust place preference in planarian, evidenced by a clear shift in preference for
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the cocaine-paired environment after four cocaine-context pairings, corroborating previous observations (Raffa et al. , 2003, Tallarida et al. , 2014, Vouga et al. , 2015). We took advantage of these rewarding properties of cocaine to test relapse-like behaviour using the model of extinctionreinstatement. One of the major obstacles in the treatment of addiction is the susceptibility to relapse even after long periods of abstinence. Reinstatement of drug-seeking has been well documented in rodents exposed to psychostimulants and other addictive substances, and may be
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ACCEPTED MANUSCRIPT triggered by drug predictive stimuli such as environmental context, stressors, drug-related cues, as well as the previously administered drug itself, as may in humans (Bossert et al. , 2013, Shaham et al. , 2003). In the current experiments, extinction was achieved within three sessions, with planarian progressively and completely reversing their preference for the cocaine-paired context.
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Following extinction training, brief exposure to cocaine or methamphetamine immediately before a
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subsequent test of conditioning produced strong reinstatement of drug seeking behaviour. These
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findings provide the first evidence of reinstatement and cross-reinstatement of a drug-induced place preference in an invertebrate. A variety of abused drugs seem to exert potent rewarding and
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reinforcing effects via interaction with common brain substrates, one of which is the dopaminergic
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system and its affiliated basal ganglia thalamo-cortical systems in mammals (Ikemoto et al. , 2015, Volkow and Morales, 2015), and therefore it could be argued that such drugs can prime one another
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by reactivating drug-associated memory circuits. Locomotor cross-sensitization between cocaine
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and methamphetamine has been previously shown in rodent models (Hirabayashi et al. , 1991, Shanks et al. , 2015). Similarly, cocaine and methamphetamine cross-reinstated an extinguished
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morphine place preference in mice (Do Ribeiro Couto et al. , 2005), suggesting that following
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abstinence drug exposure may elicit renewed craving for the previously abused drug.
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The reinstatement model we used here, which involved re-exposing the planarian to the cocainepaired context in the presence of cocaine, has both ecological validity and experimental support. Humans relapse to drug seeking is most likely to occur in the same context in which the drugs were originally taken, and renewed drug taking in that context instigates further drug taking. Moreover, it is not at all uncommon in rodent models of reinstatement that animals are placed in the original context (i.e. self-administration chamber or CPP apparatus) immediately after the injection of the drug (Mueller et al. , 2002, Mueller and Stewart, 2000, Pei et al. , 2016, Pei et al. , 2014). In such
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ACCEPTED MANUSCRIPT cases, both the context and the re-experience of the drug (as a compound stimulus) produce reinstatement of drug seeking, as was the case here. Further, the fact that CPP can be easily reinstated when testing is immediately preceded by a priming injection of the conditioning drug suggests that drugs may induce relapse by renewing the incentive value of drug-associated cues
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(Mueller et al., 2002). We conducted an additional control experiment to rule out the possibility that
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reinstatement of cocaine seeking behaviour could be simply due to conditioning. Naïve planarian
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were exposed to the same priming cocaine treatment as that received by planarian which previously underwent conditioning and extinction training. In naïve planarian brief exposure to cocaine in the
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least preferred compartment was ineffective at inducing place preference. Therefore, this evidence
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strongly suggests that cocaine exposure after extinction re-activated memory traces of learned
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associations between cocaine and specific environmental cues. As predicted, unlike cocaine and methamphetamine, the high affinity dopamine uptake
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inhibitor, JHW007, failed to reinstate cocaine seeking following extinction. JHW007 belongs to
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a class of derivatives of the benztropine molecule and displays strong affinity for the DAT and >50-fold selectivity relative to other aminergic transporter sites (Desai et al., 2014, Desai et al.,
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2005, Katz et al. , 2001). Previous evidence showed that several of these analogues possess
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pharmacokinetic/dynamic properties that are distinct from cocaine (Li et al. , 2011). JHW007 exhibits two phases of association to the DAT: a rapid phase, with a half-life of seconds, followed by a slow phase, with a half-life of tens of seconds (Kopajtic et al. , 2010), and induces distinct conformational changes at the DAT that seem to be responsible for its reduced cocaine-like subjective effects (Kohut et al. , 2014). Further, unlike cocaine, JHW007 did not produce locomotor stimulation when administered alone and prevented the place preference and locomotor sensitization induced by repeated cocaine treatment in mice (Velazquez-Sanchez
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ACCEPTED MANUSCRIPT et al. , 2010), as well as the locomotor sensitization and structural synaptic rearrangement elicited by amphetamine exposure in the nucleus accumbens (Velazquez-Sanchez et al., 2013). The evidence that the effects of JHW007 were different from those of cocaine and methamphetamine in the reinstatement test suggest that the planarian DAT shares similar
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pharmacological features with its mammalian ortholog.
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Conclusions
In summary, the current findings provide a straightforward demonstration of reinstatement and
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cross-reinstatement of a previously extinguished drug-induced place preference in an invertebrate system, suggesting that the neuroplasticity and learning mechanisms involved in
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drug relapse may be highly conserved through evolution. Such invertebrate models can thereby
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provide a useful tool in the study of neurotoxicology and drug addiction, including vulnerability to relapse. The comparison among distantly related species permits the
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identification of putative genetic, neurophysiological and regulatory elements underpinning
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complex drug-related behaviours and uniquely bridges between the proximate (i.e. genetic,
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molecular and physiological) and distant (i.e. evolutionary) causes of addiction.
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ACCEPTED MANUSCRIPT Figures Figure 1. Schematics of the experimental procedure. The pre-conditioning phase was conducted in the two-textured, split petri dish on day 1. During the conditioning phase (days 2-9), planaria were placed in a singular dish with the texture of the least preferred environment containing 5 μM
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cocaine or vehicle in the most preferred compartment. Day 10 involved the test of conditioning,
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whereby planaria were placed in the two-textured petri-dishes, as in the pre-conditioning phase.
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Extinction was carried out on days 11-13 in the two-textured dishes and was found to occur fully by
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day 13, with planaria reverting to their original preference. Reinstatement was induced by placing the planaria in least preferred environment with cocaine, methamphetamine or JHW-007 and then
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the split-compartment phases) was measured.
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repeating the test of conditioning. Locomotor activity and time spent in each compartment (during
Figure 2. Effects of cocaine on locomotor activity and place preference. Swimming activity was not
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altered by cocaine treatment during the cocaine-conditioning sessions (a; n=34). Cocaine produced
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robust place conditioning, measured as a difference between the time spent in the non-drug-paired compartment (NDP) and the drug-paired compartment (DP) across pre-conditioning (pre-cond) and
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conditioning (pre-cond) sessions (b; n=34). **, p< 0.01, significantly different from NDP. Bars
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represent means ± SEM.
Figure 3. Extinction training following cocaine conditioning. Preference for the cocaine-paired compartment (DP) decreased during the three consecutive tests of extinction (a-c; n=34), compared with the preference for the non-drug-paired compartment (NDP), until baseline preference was fully re-established. **, p< 0.01, significantly different from NDP. Bars represent means ± SEM. Figure 4. Cocaine and methamphetamine-induced reinstatement of cocaine seeking. Both cocaine and methamphetamine, but not JHW007, priming effectively reinstated cocaine preference after 15
ACCEPTED MANUSCRIPT conditioning and extinction of cocaine preference (a; n=33). In naïve planarian primed exposure to cocaine was insufficient to produce cocaine place preference (b; n=16). **, p< 0.01, significantly
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different from NDP; *, p< 0.05, significantly different from NDP. Bars represent means ± SEM.
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ACCEPTED MANUSCRIPT Pagan OR, Rowlands AL, Azam M, Urban KR, Bidja AH, Roy DM, et al. Reversal of cocaine-induced planarian behavior by parthenolide and related sesquiterpene lactones. Pharmacol Biochem Behav. 2008;89:160-70. Pei Y, Asif-Malik A, Hoener M, Canales JJ. A partial trace amine-associated receptor 1 agonist exhibits properties consistent with a methamphetamine substitution treatment. Addict Biol. 2016.
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ACCEPTED MANUSCRIPT Shanks RA, Ross JM, Doyle HH, Helton AK, Picou BN, Schulz J, et al. Adolescent exposure to cocaine, amphetamine, and methylphenidate cross-sensitizes adults to methamphetamine with drug- and sex-specific effects. Behav Brain Res. 2015;281:11624. Tallarida CS, Egan E, Alejo GD, Raffa R, Tallarida RJ, Rawls SM. Levamisole and cocaine synergism: a prevalent adulterant enhances cocaine's action in vivo. Neuropharmacology. 2014;79:590-5.
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Velazquez-Sanchez C, Ferragud A, Murga J, Carda M, Canales JJ. The high affinity dopamine uptake inhibitor, JHW 007, blocks cocaine-induced reward, locomotor stimulation and sensitization. Eur Neuropsychopharmacol. 2010;20:501-8.
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Velazquez-Sanchez C, Garcia-Verdugo JM, Murga J, Canales JJ. The atypical dopamine transport inhibitor, JHW 007, prevents amphetamine-induced sensitization and synaptic reorganization within the nucleus accumbens. Prog Neuropsychopharmacol Biol Psychiatry. 2013;44:73-80.
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Venniro M, Caprioli D, Shaham Y. Animal models of drug relapse and craving: From drug priming-induced reinstatement to incubation of craving after voluntary abstinence. Prog Brain Res. 2016;224:25-52.
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Volkow ND, Morales M. The Brain on Drugs: From Reward to Addiction. Cell. 2015;162:712-25. Vouga A, Gregg RA, Haidery M, Ramnath A, Al-Hassani HK, Tallarida CS, et al. Stereochemistry and neuropharmacology of a 'bath salt' cathinone: S-enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates. Neuropharmacology. 2015;91:109-16. Yahyavi-Firouz-Abadi N, See RE. Anti-relapse medications: preclinical models for drug addiction treatment. Pharmacol Ther. 2009;124:235-47.
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ACCEPTED MANUSCRIPT Akua et al. submitted 23/01/2017 Highlights
Cocaine priming produced reinstatement of extinguished cocaine seeking in planarian Methamphetamine priming reinstated extinguished cocaine place preference
The DAT inhibitor, JHW007, was ineffective at reinstating cocaine place
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Akua O. Amaning-Kwarteng, Aman Asif-Malik, Yue Pei, Juan J. Canales PII: DOI: Reference:
S0091-3057(17)30053-9 doi: 10.1016/j.pbb.2017.04.008 PBB 72465
To appear in:
Pharmacology, Biochemistry and Behavior
Received date: Revised date: Accepted date:
23 January 2017 7 April 2017 21 April 2017
Please cite this article as: Akua O. Amaning-Kwarteng, Aman Asif-Malik, Yue Pei, Juan J. Canales , Relapse to cocaine seeking in an invertebrate. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Pbb(2017), doi: 10.1016/j.pbb.2017.04.008
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ACCEPTED MANUSCRIPT Pharmacology, Biochemistry and Behavior, submitted 21/01/2016
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Revised
Relapse to Cocaine Seeking in an Invertebrate
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Akua O. Amaning-Kwarteng, Aman Asif-Malik, Yue Pei and Juan J. Canales Department of Neuroscience, Psychology and Behaviour University of Leicester, Lancaster Road, Leicester, LE1 9HN, United Kingdom
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Running head: Drug relapse in an invertebrate
Address for correspondence: Juan J. Canales, D.Phil. Department of Neuroscience, Psychology and Behaviour University of Leicester Leicester, LE1 9HN United Kingdom Phone: +44 (0)116 229 7125 Fax: +44 (0)116 229 7196 Email: [email protected] 1
ACCEPTED MANUSCRIPT Abstract Addiction is characterised by cycles of compulsive drug taking, periods of abstinence and episodes of relapse. The extinction/reinstatement paradigm has been extensively used in rodents to model human relapse and explore underlying mechanisms and therapeutics.
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However, relapse to drug seeking behaviour has not been previously demonstrated in
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invertebrates. Here, we used a cocaine conditioned place preference (CPP) paradigm in the
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flatworm, planarian, followed by extinction and reinstatement of drug seeking. Once baseline
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preference was established for one of two distinctly textured environments (i.e. compartments with a coarse or smooth surface), planarian received pairings of cocaine (5 M) in the non-
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preferred, and vehicle in the most preferred, environment, and were tested for conditioning
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thereafter. Cocaine produced robust CPP, measured as a significant increase in the time spent in the cocaine-paired compartment. Subsequently, planarian underwent extinction training,
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reverting back to their original preference within three sessions. Brief exposure to cocaine (5
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M) or methamphetamine (5 M) reinstated cocaine-seeking behaviour. By contrast, the high affinity dopamine transporter inhibitor, (N-(n-butyl)-3α-[bis (4-fluorophenyl) methoxy]-
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tropane) (JHW007), which in rodents exhibits a neurochemical and behavioural profile distinct
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from cocaine, was ineffective. The present findings demonstrate for the first time reinstatement of extinguished cocaine seeking in an invertebrate model and suggest that the long-term adaptations underlying drug conditioning and relapse are highly conserved through evolution.
Keywords: planarian, conditioned place preference, reinstatement, relapse, cocaine, methamphetamine
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ACCEPTED MANUSCRIPT Introduction One of the greatest challenges in the treatment of drug addiction is the prevention of relapse after successful detoxification and abstinence. Therefore the study of the psychological and physiological mechanisms underlying craving and relapse has become a prolific area of research. A variety of
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animal models have been developed in rodents and monkeys to study relapse to drug seeking,
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including drug-primed, cue- and drug-induced reinstatement, contextual relapse, and spontaneous
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recovery of drug seeking behaviour (Martin-Fardon and Weiss, 2013, Venniro et al. , 2016,
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Yahyavi-Firouz-Abadi and See, 2009). One of the best known properties of addictive drugs is their ability to promote Pavlovian associations between the drug’s effect and conditioned cues and/or
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specific environments, or contexts, where the drug is experienced, which are thought to underlie the
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phenomenon of conditioned place preference (Tzschentke, 1998). Following extinction training, an extinguished place preference can be readily reinstated by exposing the animals to stress or by
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administering a dose of the unconditioned stimulus (i.e. the drug used during conditioning)
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(Mantsch et al. , 2016, Tzschentke, 2007). Moreover, reinstatement can be elicited by drugs other than the conditioning drug. For example, methamphetamine and methylphenidate reinstated an
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extinguished cocaine place preference, suggesting that exposure to compounds acting similarly to,
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or sharing similar unconditioned stimulus properties as, the drug used during conditioning can induce renewed craving and relapse (Itzhak and Martin, 2002). Thus the place preference paradigm is a valuable tool used to assess in animal models both the rewarding properties of drugs of abuse and associated relapse-like behaviours. Invertebrates have become a popular model system to study various genetic, physiological and behavioural effects of drugs of abuse. One of such invertebrate models is planarian, one of the many flatworms of the Turbellaria class. The brain of planarian is a bi-lobed mass of nerve tissue with
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ACCEPTED MANUSCRIPT distinct structural and functional regions defined by the distinct expression of homeobox genes, and of neural networks, transmitters and neuromodulators akin to those found in the mammalian brain (Buttarelli et al. , 2008, Inoue et al. , 2015). Early evidence showing the usefulness of planarian in drug studies revealed that planarian display behaviours that resemble tolerance and dependence
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following repeated exposure to morphine (Needleman, 1967). More recently, planarian have been
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observed to display behavioural responses to drugs of abuse that are similar to that seen in
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mammals, including behavioural sensitization to cocaine (Rawls et al. , 2010) and withdrawal-like behaviours following cocaine and methamphetamine treatment (Raffa et al. , 2008, Rawls et al. ,
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2008, Sacavage et al. , 2008). Moreover, psychomotor stimulants such as methamphetamine,
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cocaine, mephedrone, and nicotine all exhibit rewarding-like properties in planarian in the place preference paradigm (Hutchinson et al. , 2015, Raffa et al., 2008, Ramoz et al. , 2012, Rawls et al. ,
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2011). These findings suggest that planarian constitute a suitable and translational model to study
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drug interactions, neurotoxicology and certain addiction-related behaviours (Barron et al. , 2010).
an invertebrate model.
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However, relapse-like behaviour, a defining feature of addiction, has not been previously shown in
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In the present experiments, we set out to study reinstatement of drug seeking behaviour following
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cocaine conditioning in planarian. We used a standard cocaine place conditioning paradigm followed by extinction and drug/context-induced reinstatement of cocaine seeking. To explore reinstatement mechanisms, primed exposure treatment to cocaine, methamphetamine or the benztropine analogue, JHW007, was given after extinction training. JHW007 is an atypical dopamine transporter inhibitor that exhibits high affinity for the dopamine transporter but behavioural effects in rodents that are distinct from cocaine (Desai et al. , 2014, Desai et al. , 2005, Hiranita et al. , 2014, Velazquez-Sanchez et al. , 2013). Therefore, we hypothesised that cocaine
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ACCEPTED MANUSCRIPT and methamphetamine, but not JHW007, would be effective at reinstating cocaine seeking behaviour in planarian. Methods
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Subjects and materials
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Large brown planarian of the genus Dugesia (n= 50) (Blades Biological Ltd., UK) comparable in
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size were used. Planarian were kept in a dimly lit room on a 10 h light cycle (light on at 08.00 AM) and maintained in aqua safe solution (Interpet Bioactive Tapsafe, UK) and fed raw chicken three
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times a week. Prior to experimenting, planarian were placed in individual plastic cups filled with
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aqua safe, which were labelled numerically. Planarians were also habituated to their environment and handling procedure before experimentation. All methods were performed in accordance with
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the guidelines and regulations of the University of Leicester.
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Cocaine HCl and methamphetamine HCl were purchased from Sigma- Aldrich (UK) and (N-(nbutyl)-3α-[bis (4-fluorophenyl) methoxy]-tropane) (JHW007) was synthesized by Dr. Juan Murga
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(University Jaime I, Spain). A final concentration 5 μM was used for each drug, diluted in aqua
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safe solution. This concentration was established as effective for reinstatement from previous doseresponse experiments in our laboratory. Singular plastic petri dishes (60 mm diameter; Corning Inc.,
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USA) were used for conditioning. Eight of these were covered internally with silicone (RS Components Ltd., UK) and sand to prepare a ‘rough’ surface in comparison to the ‘smooth’ empty petri dishes. Another eight petri dishes used for testing pre-conditioning, CPP and reinstatement were split internally so half of the dishes had a rough side and the other had a smooth side. Planarian were kept in numerically labelled plastic cups filled with aqua safe at all nonexperimental times and fine artists brushes were used to gently pick up planarian from cups to dishes and vice versa, to ensure minimal liquid transfer and contamination. Tracking of the 5
ACCEPTED MANUSCRIPT planarian was carried out using two CCD cameras. Viewpoint video tracking software (Lyon, France) was used to record both CPP and open field locomotor activity data. Procedure Phase 1: Pre-conditioning. Each planarian was placed at the centre of the two-textured, split petri
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dishes, filled with 8 ml of aqua safe, for 15 min to test for baseline preference. Planarian were
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video-tracked for 15 min and the amount of time spent in each compartment was recorded. Phase 2: Conditioning. Depending on the compartment least preferred during pre-conditioning,
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planarian were placed in a singular dish for 20 min that was either all ‘rough’ or ‘smooth’. The dish
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contained a final concentration of 5 μM cocaine, diluted in 8 ml of aqua safe. The following day, planarian were placed in the compartment they most preferred containing only aqua safe. This was
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repeated for 8 consecutive sessions, conducted once daily, during which the planarian locomotor
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activity (i.e. swimming) was also measured.
Phase 3: Test of conditioning. 24 h after the last conditioning session, each planarian was placed in
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the centre of a split compartment dish for 15 min and activity was recorded to test for CPP. The
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total time spent in each compartment in the pre-conditioning phase was compared with the total time spent in the test of conditioning to assess if the effect of cocaine produced a place preference to
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the environment that was least preferred in Phase 1. Phase 4: Extinction. Following cocaine-induced CPP, each planarian was placed in the split dish filled with aqua safe and the time spent in each compartment was measured, as in the test of conditioning (phase 3). This procedure was repeated until place preference for the compartment previously associated with cocaine was extinguished. Extinction was defined as the point where on
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ACCEPTED MANUSCRIPT average for all planarian the preference for the drug-paired environment during extinction sessions was not statistically different from the preference for the drug-paired environment at baseline. Phase 5: Reinstatement. Following extinction, each planarian was placed in a singular dish as in Phase 2 (conditioning) for 10 min. Planarian were split into three groups (n=10-12), and placed in a
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dish containing 5 μM cocaine, 5 μM methamphetamine or 5 μM JHW-007. Following the 10 min of
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drug exposure, each planarian was placed in a split compartment dish containing only aqua safe, as
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in phase 3 (test of conditioning). The time spent in each compartment was measured to establish
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whether preference for the environment associated with cocaine was reinstated following extinction. Control experiment. To exclude the possibility that brief 10 min exposure to the drug in the least
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preferred compartment could account for the effects observed in the reinstatement test (phase 5), an
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additional experiment was conducted. Baseline place preference was initially established in 16 naïve planarian (as in phase 1). The next day each planarian was exposed to a brief cocaine-
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conditioning period by placing them in the least preferred compartment filled with 5 μM cocaine for
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10 min (as in phase 2). Planarians were then placed in split compartment dishes and time spent in
Statistical analysis
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each compartment was measured to test for conditioning (as in phase 3).
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Data were analysed by analysis of variance (ANOVA) with repeated measures, followed by post hoc comparisons with the method of Fisher’s Protected Least Significance Difference (PLSD). Statistical significance was set at α= 0.05 for all conditions. All statistical analyses were performed using StatView 5.0 (SAS Institute, NC, USA).
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ACCEPTED MANUSCRIPT Results Induction of cocaine CPP and extinction Locomotor activity was measured during the conditioning phase. Two-way ANOVA with repeated measures was conducted with two within-subject factors, Drug (cocaine, vehicle) and Session (four
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conditioning sessions for each drug condition). ANOVA showed no significant effect of the Drug
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factor [F(1,33)= 0.857, p= 0.361], the Session factor [F(3,99)= 0.738, p= 0.533] or the interaction [F(1,33)= 1.866, p= 0.137]. Therefore cocaine exposure during conditioning did not produce
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significant effects on swimming activity (Fig.2a).
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[Fig. 2 about here]
For the test of conditioning, a two-way ANOVA with repeated measures was performed with two
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factor variables, Test (pre-conditioning, post-conditioning) and Compartment (drug-paired, DP, and
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non-drug-paired, NDP). The ANOVA showed a non-significant effect of the factor, Test [F(1,33)= 0.857, p= 0.361] and a significant effects of the factor, Compartment [F(1,33)= 7.837, p= 0.008].
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Furthermore, a significant interaction effect between Test and Compartment was found [F(1,33)=
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29.100, p< 0.001]. Post-hoc tests showed that the time spent by the planarian in the DP compartment in the post-conditioning test increased significantly compared with that spent in the
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same compartment during pre-conditioning (p < 0.01). Therefore, cocaine exposure produced robust CPP in planarian (Fig. 2b). Following conditioning, extinction sessions began. A one-way ANOVA for time spent in each compartment was conducted to test for significance during the extinction phase. On extinction day 1, a significant effect of compartment was observed [F(1, 33) = 26.974, p < 0.001] (Fig. 3a). In session 2, there was no longer a significant difference in the time spent in one compartment or the
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ACCEPTED MANUSCRIPT other [F(1, 33) = 0.032, p < 0.860] (Fig. 3b), and in session 3 planarian reverted back to the preference shown at baseline, effectively extinguishing preference for the DP environment [F(1, 33) = 30.391, p < 0.001] (Fig. 3c). [Fig. 3 about here]
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Reinstatement of cocaine seeking
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For the reinstatement test, planarians were exposed to cocaine, methamphetamine or JHW007. A three-way ANOVA was conducted with Drug (cocaine, methamphetamine or JHW-007) as a
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between-factor, and Test (third day of extinction and reinstatement test) and Compartment (DP and
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NDP), as within-factors. Planarians were matched in terms of relative preference for the DP and NDP compartments prior to the drug assignment. ANOVA revealed a significant high order
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interaction between the Drug, Test and Compartment variables [F(2, 31) = 8.166, p < 0.001] (Fig. 4a).
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Post hoc comparisons showed that while all three groups displayed similar preference for the NDP compartment on the last day of extinction, the cocaine (p< 0.01) and methamphetamine (p< 0.05)
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groups showed robust reinstatement drug seeking behaviour (i.e. comparing time spent in NDP and
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DP in the reinstatement test). By contrast, the group treated with JHW007 did not (Fig. 4a). [Fig. 4 about here]
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For the control experiment, which included a group of planarian receiving identical treatment except for the conditioning and extinction training, a two-way ANOVA with repeated measures showed that there was no significant effect of brief cocaine conditioning on time spent in the DP compartment [F(1, 15) = 0.003, p < 0.955] (Fig. 4b).
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ACCEPTED MANUSCRIPT Discussion The present study showed conditioning, extinction and reinstatement of cocaine place preference in planarian and provided the first evidence of relapse-like behaviour in an invertebrate. Following cocaine conditioning and extinction of a cocaine place preference, both cocaine and
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methamphetamine, but not the high affinity dopamine transport blocker, JHW007, reinstated drug
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seeking behaviour.
The Platyhelminthes, including the flatworm, planarian, are capable of exhibiting complex
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learning in classical conditioning and operant tasks despite their rudimentary cephalisation (Best and Rubinstein, 1962, Lee, 1963, Shafer and Corman, 1963). Planarian possess an
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intricate network of neural connections allowing both synaptic and paracrine signalling by way
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of a collection of neurotransmitter systems, including catecholamines, acetylcholine, serotonin
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and excitatory amino acids such as glutamate and its inhibitory derivative, -aminobutyric acid, amongst others (Ribeiro et al. , 2005). Although little is known about the contributions of these
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transmitter systems to the coordination of motor and complex behaviour in planarian, it is thought that dopamine plays a vital role in movement and reward-related learning in planarian.
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Previous studies led to the identification of the gene encoding tyrosine hydroxylase in
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the planarian, Dugesia japonica, whose knockdown renders planarian unable to synthesize dopamine (Nishimura et al. , 2007), and of the gene encoding the dopamine transporter (DAT), whose expression overlaps with that of the tyrosine hydroxylase (Tashiro et al. , 2014). DAT interference induced increased spontaneous locomotion in planarian that was effectively blocked by selective dopamine D1 and D2 receptor antagonists (Tashiro et al., 2014), suggesting the presence of a fully functional dopamine system in planarian that resembles that of mammalian counterparts. Indeed, methamphetamine-stimulated place learning in planarian
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ACCEPTED MANUSCRIPT was suppressed by dopamine D1 and D2 receptor antagonists (Kusayama and Watanabe, 2000), indicating that psychostimulant-induced place preference requires dopamine receptor activation for its induction in planarian. Our findings showed that repeated cocaine treatment during conditioning neither enhanced nor
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reduced locomotor activity in planarian. This is consistent with previous studies reporting
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cocaine induced hypomotility and C-like hyperkinesia but only at higher concentrations than
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that used in the current study (Hutchinson et al., 2015, Pagan et al. , 2008). Although we acknowledge that using only one dose of cocaine for the conditioning tests is an intrinsic
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limitation, the dose selected was within the range of doses that produce conditioning in
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planarian (1 μM and 10 μM) (Hutchinson et al., 2015) and therefore the interpretation of the findings is straightforward. Moreover, there was no evidence of sensitization to cocaine
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treatment, which also requires higher concentrations to be used in this species (Rawls et al.,
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2010).
The place preference paradigm is a well-established and widely used tool in behavioural
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pharmacology and addiction research to assess drug-induced reward and its underlying
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neurobiological and neuropharmacological substrates (Tzschentke, 2007). Our findings showed that cocaine produced robust place preference in planarian, evidenced by a clear shift in preference for
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the cocaine-paired environment after four cocaine-context pairings, corroborating previous observations (Raffa et al. , 2003, Tallarida et al. , 2014, Vouga et al. , 2015). We took advantage of these rewarding properties of cocaine to test relapse-like behaviour using the model of extinctionreinstatement. One of the major obstacles in the treatment of addiction is the susceptibility to relapse even after long periods of abstinence. Reinstatement of drug-seeking has been well documented in rodents exposed to psychostimulants and other addictive substances, and may be
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ACCEPTED MANUSCRIPT triggered by drug predictive stimuli such as environmental context, stressors, drug-related cues, as well as the previously administered drug itself, as may in humans (Bossert et al. , 2013, Shaham et al. , 2003). In the current experiments, extinction was achieved within three sessions, with planarian progressively and completely reversing their preference for the cocaine-paired context.
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Following extinction training, brief exposure to cocaine or methamphetamine immediately before a
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subsequent test of conditioning produced strong reinstatement of drug seeking behaviour. These
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findings provide the first evidence of reinstatement and cross-reinstatement of a drug-induced place preference in an invertebrate. A variety of abused drugs seem to exert potent rewarding and
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reinforcing effects via interaction with common brain substrates, one of which is the dopaminergic
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system and its affiliated basal ganglia thalamo-cortical systems in mammals (Ikemoto et al. , 2015, Volkow and Morales, 2015), and therefore it could be argued that such drugs can prime one another
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by reactivating drug-associated memory circuits. Locomotor cross-sensitization between cocaine
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and methamphetamine has been previously shown in rodent models (Hirabayashi et al. , 1991, Shanks et al. , 2015). Similarly, cocaine and methamphetamine cross-reinstated an extinguished
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morphine place preference in mice (Do Ribeiro Couto et al. , 2005), suggesting that following
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abstinence drug exposure may elicit renewed craving for the previously abused drug.
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The reinstatement model we used here, which involved re-exposing the planarian to the cocainepaired context in the presence of cocaine, has both ecological validity and experimental support. Humans relapse to drug seeking is most likely to occur in the same context in which the drugs were originally taken, and renewed drug taking in that context instigates further drug taking. Moreover, it is not at all uncommon in rodent models of reinstatement that animals are placed in the original context (i.e. self-administration chamber or CPP apparatus) immediately after the injection of the drug (Mueller et al. , 2002, Mueller and Stewart, 2000, Pei et al. , 2016, Pei et al. , 2014). In such
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ACCEPTED MANUSCRIPT cases, both the context and the re-experience of the drug (as a compound stimulus) produce reinstatement of drug seeking, as was the case here. Further, the fact that CPP can be easily reinstated when testing is immediately preceded by a priming injection of the conditioning drug suggests that drugs may induce relapse by renewing the incentive value of drug-associated cues
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(Mueller et al., 2002). We conducted an additional control experiment to rule out the possibility that
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reinstatement of cocaine seeking behaviour could be simply due to conditioning. Naïve planarian
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were exposed to the same priming cocaine treatment as that received by planarian which previously underwent conditioning and extinction training. In naïve planarian brief exposure to cocaine in the
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least preferred compartment was ineffective at inducing place preference. Therefore, this evidence
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strongly suggests that cocaine exposure after extinction re-activated memory traces of learned
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associations between cocaine and specific environmental cues. As predicted, unlike cocaine and methamphetamine, the high affinity dopamine uptake
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inhibitor, JHW007, failed to reinstate cocaine seeking following extinction. JHW007 belongs to
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a class of derivatives of the benztropine molecule and displays strong affinity for the DAT and >50-fold selectivity relative to other aminergic transporter sites (Desai et al., 2014, Desai et al.,
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2005, Katz et al. , 2001). Previous evidence showed that several of these analogues possess
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pharmacokinetic/dynamic properties that are distinct from cocaine (Li et al. , 2011). JHW007 exhibits two phases of association to the DAT: a rapid phase, with a half-life of seconds, followed by a slow phase, with a half-life of tens of seconds (Kopajtic et al. , 2010), and induces distinct conformational changes at the DAT that seem to be responsible for its reduced cocaine-like subjective effects (Kohut et al. , 2014). Further, unlike cocaine, JHW007 did not produce locomotor stimulation when administered alone and prevented the place preference and locomotor sensitization induced by repeated cocaine treatment in mice (Velazquez-Sanchez
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ACCEPTED MANUSCRIPT et al. , 2010), as well as the locomotor sensitization and structural synaptic rearrangement elicited by amphetamine exposure in the nucleus accumbens (Velazquez-Sanchez et al., 2013). The evidence that the effects of JHW007 were different from those of cocaine and methamphetamine in the reinstatement test suggest that the planarian DAT shares similar
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pharmacological features with its mammalian ortholog.
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Conclusions
In summary, the current findings provide a straightforward demonstration of reinstatement and
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cross-reinstatement of a previously extinguished drug-induced place preference in an invertebrate system, suggesting that the neuroplasticity and learning mechanisms involved in
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drug relapse may be highly conserved through evolution. Such invertebrate models can thereby
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provide a useful tool in the study of neurotoxicology and drug addiction, including vulnerability to relapse. The comparison among distantly related species permits the
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identification of putative genetic, neurophysiological and regulatory elements underpinning
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complex drug-related behaviours and uniquely bridges between the proximate (i.e. genetic,
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molecular and physiological) and distant (i.e. evolutionary) causes of addiction.
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ACCEPTED MANUSCRIPT Figures Figure 1. Schematics of the experimental procedure. The pre-conditioning phase was conducted in the two-textured, split petri dish on day 1. During the conditioning phase (days 2-9), planaria were placed in a singular dish with the texture of the least preferred environment containing 5 μM
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cocaine or vehicle in the most preferred compartment. Day 10 involved the test of conditioning,
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whereby planaria were placed in the two-textured petri-dishes, as in the pre-conditioning phase.
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Extinction was carried out on days 11-13 in the two-textured dishes and was found to occur fully by
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day 13, with planaria reverting to their original preference. Reinstatement was induced by placing the planaria in least preferred environment with cocaine, methamphetamine or JHW-007 and then
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the split-compartment phases) was measured.
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repeating the test of conditioning. Locomotor activity and time spent in each compartment (during
Figure 2. Effects of cocaine on locomotor activity and place preference. Swimming activity was not
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altered by cocaine treatment during the cocaine-conditioning sessions (a; n=34). Cocaine produced
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robust place conditioning, measured as a difference between the time spent in the non-drug-paired compartment (NDP) and the drug-paired compartment (DP) across pre-conditioning (pre-cond) and
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conditioning (pre-cond) sessions (b; n=34). **, p< 0.01, significantly different from NDP. Bars
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represent means ± SEM.
Figure 3. Extinction training following cocaine conditioning. Preference for the cocaine-paired compartment (DP) decreased during the three consecutive tests of extinction (a-c; n=34), compared with the preference for the non-drug-paired compartment (NDP), until baseline preference was fully re-established. **, p< 0.01, significantly different from NDP. Bars represent means ± SEM. Figure 4. Cocaine and methamphetamine-induced reinstatement of cocaine seeking. Both cocaine and methamphetamine, but not JHW007, priming effectively reinstated cocaine preference after 15
ACCEPTED MANUSCRIPT conditioning and extinction of cocaine preference (a; n=33). In naïve planarian primed exposure to cocaine was insufficient to produce cocaine place preference (b; n=16). **, p< 0.01, significantly
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different from NDP; *, p< 0.05, significantly different from NDP. Bars represent means ± SEM.
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ACCEPTED MANUSCRIPT Akua et al. submitted 23/01/2017 Highlights
Cocaine priming produced reinstatement of extinguished cocaine seeking in planarian Methamphetamine priming reinstated extinguished cocaine place preference
The DAT inhibitor, JHW007, was ineffective at reinstating cocaine place
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