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Persistent conditioned place preference to cocaine and withdrawal hypo-locomotion to mephedrone in the flatworm planaria
Neuroscience Letters 593 (2015) 19–23
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Persistent conditioned place preference to cocaine and withdrawal hypo-locomotion to mephedrone in the flatworm planaria Claire V Hutchinson a , Jose Prados a , Colin Davidson b,∗ a b
School of Psychology, University of Leicester, Leicester LE1 9HN, UK Basic Medical Sciences, St George’s University of London, London SW17 0RE, UK
h i g h l i g h t s • We show conditioned place preference in planaria to cocaine. • We find long-lasting hypo-motility to mephedrone. • We have used clinically relevant concentrations.
a r t i c l e
i n f o
Article history: Received 9 November 2014 Received in revised form 26 February 2015 Accepted 11 March 2015 Available online 13 March 2015 Keywords: Planaria Cocaine Mephedrone Conditioned place preference Locomotion Withdrawal
a b s t r a c t The purpose of the present study was to determine the effects of exposure to cocaine and mephedrone on conditioned place preference (CPP) and locomotion in the flatworm planaria. Planaria were treated with either cocaine or mephedrone at 1 or 10 M. Planaria were exposed to 15 min of drug in their nonpreferred place (either a rough- or smooth-floored petri dish) on alternate days, and were exposed to normal water in their preferred place on the following day. There were 5 days of conditioning to drug. Planaria were then tested for CPP on day 2, 6 and 13 after withdrawal. We found that animals exhibited CPP to cocaine at both 1 and 10 M, but not to mephedrone. When examining locomotor activity we found that neither cocaine nor mephedrone treatment showed any evidence of evoking increased motility or locomotor sensitisation. Hypo-motility was seen on the first day of conditioning at concentrations of 10 M for both cocaine and mephedrone, but had disappeared by the last day of conditioning. Examining chronic withdrawal, only 10 M mephedrone had a significant effect on motility, decreasing locomotion on day 2 of withdrawal. Taken together we have shown that cocaine evoked CPP in planaria. We have also shown withdrawal depressing effects of mephedrone on motility. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction There is an increasing pressure on researchers to justify their use of vertebrate animals in pre-clinical studies. Over the last decade, the principles of the 3Rs (reduction, refinement and replacement) have gained more prominence and researchers are actively looking for non-vertebrate species in which to undertake their research. One such invertebrate species that has become more popular in pre-clinical research is the flatworm planaria. There are two main advantages of undertaking research in planaria; first its entire genome is known [1] and second it is cheap and easy to maintain.
Abbreviations: CPP, conditioned place preference; NPS, novel psychoactive substance; ANOVA, analysis of variance. ∗ Corresponding author. Tel.: +44 208725 5659. E-mail address: [email protected] (C. Davidson). http://dx.doi.org/10.1016/j.neulet.2015.03.021 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
In the field of neuroscience, further advantages to using planaria include its ability to regenerate its nervous system and the belief that it is the simplest organism to have a ‘brain’ [2]. In the field of neuropharmacology, the planaria may be of use because it possesses most of the main neurotransmitters that are found in rodents and humans [3]. It has been used recently in studying drugs of abuse and has been found to show withdrawal signs, most notably reduced locomotion, after being taken out of various drugs of abuse including cocaine, opioids, methamphetamine, cannabinoids and mephedrone [4–8]. Hypomotility is a core symptom of stimulant withdrawal in humans and rodents. In addition, behaviours such as head-nodding and head-swinging, squirming, clinging, tail-twising and corkscrew spiral motions along the longaxis have been seen [4]. More recently conditioned place preference (CPP) has been shown in planaria for cocaine, mephedrone and MDMA [8,9] and we have shown more subtle responses such as blocking [10] suggesting that planaria show complex information
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processing. It has also been shown [11] that planaria exhibit environmental familiarisation that endures for up to 14 days. This raises the interesting possibility that drug-induced CPP effects in planaria might be long-lasting, in a manner similar to the effects observed in rodent models [12] where rodents, after conditioning with mephedrone for a number of days, demonstrated significant CPP that lasted for at least 3 weeks. If similar effects were evident in planaria, their utility modeling drug-related changes in mammalian behaviour would be extremely promising. One possible limitation of previous studies concerns whether the concentrations tested have clinical relevance, where studies use drug concentrations of up to 1000 M. In the present study we aimed to further show the validity of using planaria in drug abuse research by using drugs at lower, perhaps more clinically relevant, concentrations and by examining the persistence of the conditioning effect. Having a simple model organism that allows high throughput screening of drugs of abuse would be particularly useful in the testing of legal highs (novel psychoactive substances, NPS) where around 100 NPS are found each year, with virtually nothing known of their pharmacology.
2. Methods Planaria. Brown planaria (Dugesia Tigrina) were purchased from Blades Biological Ltd (UK) and were fed raw chicken every 2 days for 1 h. Their water was changed after feeding. We used AquaSafe© (Tetra, Germany) to treat normal tap water. Planaria were kept about 20 worms to 500 ml treated water in Tupperware boxes. They were kept in a dimly lit room with a dark cycle from 6PM to 9AM. Drugs and conditioning. We examined cocaine, one of the most addictive drugs, and mephedrone for which much less is known about its addictive liability. Cocaine is primarily a DAT inhibitor whereas we have previously shown that mephedrone causes reverse transport of dopamine (Opacka-Juffry et al., 2014). In a pre-test, planaria were allowed to move freely in a plastic petri dish (9 cm diameter) which had one half smooth (white shiny card) and one half rough (brown, wet–dry sandpaper, grade 100 grit). The floor coverings were fitted up the sides of the dishes to avoid planaria getting trapped under paper and also so that if they swam around the edge, they were still in contact with a smooth or rough
3. Results In our pre-conditioning test of side preference we found no statistically significant difference in the amount of time spent in each side in all our planaria combined. In the 15 min (900 s) test, planaria spent 411.1 ± 31 s in the rough side and 488.9 ± 31 s in the smooth side, t(42) = 1.25, p = 0.22. Thus, our CPP design was non-biased. Conditioned place preference. The main finding was that CPP was seen with cocaine at both 1 and 10 M, but no effect was seen with mephedrone. Fig. 1A shows CPP findings for planaria conditioned with 1 M cocaine (n = 9), 1 M mephedrone (n = 13) and a control group (n = 7) conditioned in treated water. Fig 1B shows CPP findings for planaria conditioned with 10 M cocaine (n = 6) or 10 M mephedrone (n = 9). The control group is also
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surface. The dish was filled with 3 ml of treated tap water. This meant that the fluid level was just above the floor of the dish, forcing the worms to contact the different surfaces. The time spent in each half of the dish was measured after 15 min. During conditioning the worms were placed in a dish, which only contained a floor of their non-preferred side (sandpaper or shiny white card) in either 1 or 10 M of cocaine or mephedrone, made up in treated tap water, for 15 min. The following day worms were placed in a petri dish with their preferred floor, again for 15 min. This was repeated for 10 consecutive days (5 days drug, 5 days water), as is standard in CPP experiments [16]. Planaria were then tested on days 2, 6 and 13 after withdrawal in the original petri dishes, which were ½ sandpaper and ½ shiny white card. The amount of time spent in each half was measured after video analysis. We were also able to measure locomotor activity using video analysis with a suitable sized grid placed in the video monitor, allowing us to estimate the number gridlines crossed. If more than 50% of the planaria’s body crossed a line we counted this as a crossing. See Fig. 1 for an overview of the testing and drug administration schedule. Worms were transferred from dish to dish by gently picking them up with a fine artists brush, occasionally a worm would be damaged during this process and data from these worms were excluded from analysis, (i.e. planaria that did not move). Cocaine was purchased from Sigma–Aldrich, mephedrone was a gift from John Ramsay (TICTAC Communications Ltd., UK).
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Withdrawal day Fig. 1. Overview of testing and drug administration schedule and conditioned place preference after cocaine. Top panel: we always started the planaria with drug on day 1 of conditioning, on the non-preferred side. This meant that the last day of conditioning was with water on the preferred side. Thus the CPP tests were done on days 2, 6 and 13 of drug withdrawal (WD). Time spent on the non-preferred side before conditioning (pre) and 2, 6, and 13 days after withdrawal. CPP was seen with cocaine at both 1 and 10 M, but not with mephedrone, see text for details. A. 1 M cocaine (closed circles) and 1 M mephedrone (open circles) and for the control group (open triangles). B. 10 M cocaine (closed circles) and 10 M mephedrone (open circles). Control group data (open triangles) are replotted from A. Values are means ± 1 SEM. N = 6–13.
C.V. Hutchinson et al. / Neuroscience Letters 593 (2015) 19–23
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shown (replotted from Fig. 1A). These data suggest that cocainetreated worms spent more time in their conditioned side than mephedrone-treated worms. A 3 (drug type: treated water, cocaine, mephedrone) by 3 (drug concentration: no drug, 1, 10 M) by 4 (test day: pre-conditioning, +2, +6, +13 days after withdrawal) analysis of variance (ANOVA) revealed significant effects of drug type [F(1,39) = 17.845; p < .001] and withdrawal day [F(3117) = 3.389; p < .05]. There was no significant effect of drug concentration and no significant interaction. To confirm these findings, we performed separate ANOVAs for cocaine and mephedrone. For cocaine, a 3 (drug concentration: no drug, 1, 10 M) by 4 (test day: pre-conditioning, +2, +6, +13 days after withdrawal) ANOVA yielded significant effects of drug concentration [F(2,19) = 3.922; p < .05] and withdrawal day [F(3,57) = 4.104; p < .005]. A comparable ANOVA revealed no significant effects of mephedrone. Followup within group t-tests (one-tailed) compared the time spent on the conditioned side on each post-cocaine withdrawal day (2, 6 and 13) with the time spent on the conditioned side before conditioning began. After being conditioned at a concentration of 1 M cocaine, planaria spent significantly longer of the conditioned side (compared to pre-conditioning) when tested 6 days after withdrawal [t(8) = 3.173; p < .01]. After being conditioned at a concentration of 10 M cocaine, planaria spent significantly longer on the conditioned side on all 3 withdrawal days: 2 days [t(5) = 2.131; p < .05], 6 days and 13 days [t(5) = 4.203; p < .005] after withdrawal [t(5) = 3.128; p < .05]. Locomotor activity. The main finding was that mephedrone, at 10 M, reduced locomotor activity during chronic withdrawal, this was most evident on day 2 of withdrawal). Fig. 2 A shows locomotor activity (number of 1 cm gridlines crossed in 15 min) for planaria conditioned with 1 M cocaine (n = 9), 1 M mephedrone (n = 8)
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and a control group (n = 7) conditioned in treated water. Fig 2B shows locomotor activity for planaria conditioned with 10 M cocaine (n = 6) or 10 M mephedrone (n = 8). Control group data are replotted from Fig 2A. A three (drug type: treated water, cocaine, mephedrone) by 3 (drug concentration: no drug, 1, 10 M) by 3 (test day: pre-conditioning, conditioning day 1, conditioning day 5) ANOVA revealed a significant effect of testing day [F(2,66) = 3.489; p < .05]. We followed this up with individual ANOVAs for each group. As expected, there was no significant effect of day in the control group (no drug). Although at higher drug concentrations (10 M) locomotion appeared to decrease activity relative to baseline on conditioning days 1 and 5, this effect was not significant for either cocaine or mephedrone. At 1 M concentrations, there was a significant effect of day for both cocaine [F(2,16) = 3.944; p < .05] and mephedrone [F(2,14) = 10.963; p < .05]. Follow-up t-tests confirmed that this reflected a reduction in motility on conditioning day 1 in the drug compared to pre-conditioning (cocaine: t(8) = 2.293; p = .051; mephedrone: t(7) = 2.569; p < .05). There was no significant change in locomotion relative to preconditioning by conditioning day 5. This suggests that the planaria did not show an increase in motility when placed in 1 M cocaine or mephedrone, but showed hypo-motility. There was no evidence of locomotor sensitisation or tolerance by day 5 of drug treatment, but the planaria appeared to be tolerant to the hypolocomotor effects of both cocaine and mephedrone (Fig 2A). We next examined motility during chronic withdrawal by testing the numbers of lines crossed during the post-test days and days 2, 6 and 13 after withdrawal. Fig. 2C shows locomotor activity (number of 1 cm gridlines crossed in 15 min) before and after conditioning for planaria conditioned with 1 M cocaine (n = 8), 1 M mephedrone (n = 8) and a control group (n = 7) conditioned in treated water. Fig 2D shows locomotor activity before and after conditioning for planaria conditioned with 10 M cocaine (n = 6) or 10 M mephedrone (n = 8). At 10 M, the mephedrone treated planaria appeared to display hypo-motility compared to cocaine treated planaria. A 3 (drug type: treated water, cocaine, mephedrone) by 3 (drug concentration: no drug, 1, 10 M) by 3 (test day: pre-conditioning, + 2 days, 6 days and 13 days after withdrawal) ANOVA revealed a significant effect of testing day [F(3,96) = 2.83; p < .05] and drug type [F(1,32) = 4.997; p < .05]. Individual ANOVAs for each individual group confirmed a significant effect of chronic withdrawal for mephedrone at 10 M [F(3,21) = 3.884; p < .05]. Follow-up t-tests showed that planaria exhibited significantly decreased locomotion, relative to preconditioning, 2 days after withdrawal [t(7) = 2.683; p < .05]. This decrease in locomotion appeared to persist at 6 and 13 days after withdrawal. However, these effects only approached significance [+6 days: t(7) = 2.135; p = .07, +13 days: t(7) = 2.194; p = .064].
4. Discussion 0
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Fig. 2. Locomotion during drug treatment and during chronic withdrawal. Number of 1 cm gridlines crossed in 15 min. A and B. Locomotion on pre-conditioning day and days 1 and 5 (the first and last days) of drug conditioning. Both cocaine and mephedrone at 1 M reduced locomotor activity on day 1 of conditioning, an effect that had disappeared by day 5. There was no significant effect at 10 M with either drug. A. 1 M cocaine (closed circles) and mephedrone (open circles) and control group (open triangles). B. 10 M cocaine (closed circles) and mephedrone (open circles). Control group (open triangles) are replotted from A, C and D. Locomotion on pre-conditioning day and days 2, 6 and 13 after withdrawal (WD). C. Mephedrone at 10 M reduced locomotor activity on day 2 of withdrawal. 10 M cocaine (closed circles) and mephedrone (open circles) and control group (open triangles). D. 10 M cocaine (closed circles) and mephedrone (open circles). Control group (open triangles) are replotted from C. Values are means ± 1 SEM. N = 6–9.
Rodent models of psychostimulant abuse such as locomotor sensitisation, CPP and drug self-administration have shown complex responses dependant upon the drug tested, dose, route of administration and day of withdrawal [17,18]. In addition to these reinforcing effects, one might also expect a psychostimulant to increase locomotion, but in-place stereotypies, common with amphetamine-like drugs or high doses of stimulants, can lead to reduced locomotion [19,20]. We have compared cocaine, which exerts its addictive effects primarily through dopamine transporter inhibition, with the novel psychoactive substance mephedrone, which has been shown to cause reverse transport of dopamine [30]. We have tested for locomotor sensitisation (or tolerance), CPP and withdrawal effects.
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In the current study we have shown CPP to cocaine on day 2, 6 and 13 after withdrawal but we found no evidence of locomotor sensitisation. Previous studies have shown CPP to mephedrone [8] in planaria, but only at much higher concentrations (100–500 M). To our knowledge, no group have shown locomotor sensitisation to cocaine or mephedrone in planaria at clinically relevant concentrations, although Rawls and colleagues have shown sensitization to cocaine at 100 M [22]. Indeed, a simple explanation might be that the concentrations tested have been toxic, and mephedrone has been described as evoking toxicity in humans [21], and thus reduces locomotion (as we have seen). An alternative explanation is that the planaria move around less due to stereotypies [8]. However, cocaine at high concentrations increases planarian activity [22] defined as an increase in ‘C-like hyperkinesias’ rather than increased motility. Another explanation might be that the planarian locomotor system is not driven by the dopamine system to the same extent as it is in mammals. However, numerous studies have found dopamine D2 antagonists to inhibit the effects of stimulants in planaria [8,23] suggesting a reasonable similarity to the mammalian system. The lack of a consistent effect of stimulants on planarian motility has been previously discussed [8]. Previous studies have shown hypo-locomotion after withdrawal from drugs of abuse, with various distinct acute withdrawal behaviours, such as ‘corkscrew’ movements and ‘inverted C-shapes’ [12,24]. We tested cocaine and mephedrone at lower concentrations to determine if the lack of hyper-motility was due to the concentrations of the drug being too great, leading to toxicity and/or stereotypies, but even at the lower concentrations we used (1 and 10 M) there was no evidence of increased locomotion, nor did we see stereotypies. Ramoz et al. found that 60 min treatment with 1 or 10 M mephedrone caused a reduction in motility in the first 5 min of withdrawal [8]. Differences between studies in planarian CPP and motility might be due to different species of planaria used or different dosing regimens. For example, Rawls’ group used a biased CPP design, 30 min contact time during mephedrone conditioning, 5 min post-conditioning test time and Dugesia dorotocephala [8], whereas we have used an unbiased design, 15 min drug contact time, a 15 min post conditioning test time and D. Tigrina. A second goal of our study was to test how long CPP lasts in planaria. Previous studies in rodents suggest that CPP can last for days, and drug craving can last for weeks or months [25]. In general, rodent studies show that the more conditioning days and the higher the drug dose, the more persistent the CPP. With 5 days of drug pairing (in the present study) one might expect to see CPP for 10 days or more in a rodent [16]. In a study by Brabant et al. [27] CPP to cocaine persisted for up to 35 days in mice, but only at the highest dose tested (12 mg/kg IP). Our data show that CPP is still evident on day 13 after withdrawal. These data extend previous studies, which have found CPP in planaria with methamphetamine [26], amphetamine [28] and mephedrone [8] only immediately after conditioning. The finding that mephedrone (10 M) treatment caused an extended hypo-motility in planaria lasting up to 13 days after withdrawal, suggests a chronic withdrawal syndrome similar to that which one might expect to find in a stimulant-dependant human [29]. The lack of chronic withdrawal hypo-motility in cocaine treated planaria suggests pharmacological differences between these 2 drugs. We have shown in rat brain slices, that mephedrone has a neurochemical effects similar to amphetamine in that it causes reverse transport of dopamine [30], whereas cocaine acts primarily as a dopamine transporter blocker and mobiliser of dopamine storage pools [31,32]. Thus these simple locomotor tests in planaria could have the potential to tease out different types of stimulants. One limitation of these studies is whether the concentrations tested have clinical relevance. Using data from human subjects
taking a relatively low dose of cocaine, Zheng and Zhan [13] modelled the extracellular cocaine concentration in the brain and found the peak to be 2.6 M. Nicolaysen et al. [14] using microdialysis in rats found that a 30 mg/kg i.p. cocaine injection (considered a large dose) caused a peak striatal cocaine concentration of 10.1 M. In addition, rats chronically treated with cocaine had almost double the plasma cocaine concentration found in acutely treated rats Pettit et al. [15]. Thus 10 M may be a clinically relevant dose of cocaine. Taken together, these data further confirm the utility of planaria in drug abuse research. They may allow us to predict the behavioural pharmacology of drugs of abuse and hint at neurochemical mechanisms. NPS or legal highs, for which very little is known, are currently flooding European markets through internet vendors. A simple and cheap high throughput screen, such as described here, might be able to offer important pharmacological data on potential abuse liability and toxicity of these many new drugs of abuse. Acknowledgements This work was supported by an NC3Rs small grant to CD and the European Commission-funded EU-MADNESS project (JUST2013/DPIP/AG/4823) resources were used to assist with the preparation of this research document. Some of the work was carried out during a period of study leave awarded by the University of Leicester to CH. References [1] E. Saló, J.F. Abril, T. Adell, F. Cebrià, K. Eckelt, E. Fernandez-Taboada, M. Handberg-Thorsager, M. Iglesias, M.D. Molina, G. Rodríguez-Esteban, Planarian regeneration: achievements and future directions after 20 years of research, Int. J. Dev. Biol. 53 (2009) 1317–1327. [2] T. Sandmann, M.C. Vogg, S. Owlarn, M. Boutros, K. Bartscherer, The head-regeneration transcriptome of the planarian Schmidtea mediterranea, Genome Biol. 12 (2011) R76. [3] F.R. Buttarelli, C. Pellicano, F.E. Pontieri, Neuropharmacology and behavior in planarians: translations to mammals, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 147 (2008) 399–408. [4] R.B. Raffa, P. Desai, Description and quantification of cocaine withdrawal signs in planaria, Brain Res. 1032 (2005) 200–202. [5] R.B. Raffa, G.W. Stagliano, S. Umeda, Kappa-opioid withdrawal in planaria, Neurosci. Lett. 349 (2003) 139–142. [6] S.M. Rawls, H. Shah, G. Ayoub, R.B. Raffa, 5-HT1A-like receptor activation inhibits abstinence-induced methamphetamine withdrawal in planarians, Neurosci. Lett. 484 (2010) 113–117. [7] S.M. Rawls, K. Gerber, Z. Ding, C. Roth, R.B. Raffa, Agmatine: identification and inhibition of methamphetamine, kappa opioid, and cannabinoid withdrawal in planarians, Synapse 62 (2008) 927–934. [8] L. Ramoz, S. Lodi, P. Bhatt, A.B. Reitz, C. Tallarida, R.B. Raffa, S.M. Rawls, Mephedrone (bath salt) pharmacology: insights from invertebrates, Neuroscience 208 (2012) 79–84. [9] T. Kusayama, S. Watanabe, Reinforcing effects of methamphetamine in planarians, Neuroreport 11 (2000) 2511–2513. [10] J. Prados, B. Alvarez, J. Howarth, K. Stewart, C.L. Gibson, C.V. Hutchinson, A.M. Young, C. Davidson, Cue competition effects in the planarian, Animal Cogn. 16 (2012) 177–186. [11] T. Shomrat, M. Levin, An automated training paradigm reveals long-term memory in planaria and its persistence through head regeneration, J. Exp. Biol. 216 (2013) 3799–3810. [12] R. Lisek, W. Xu, E. Yuvasheva, Y.T. Chiu, A.B. Reitz, L.Y. Liu-Chen, S.M. Rawls, Mephedrone (‘bath salt’) elicits conditioned place preference and dopamine-sensitive motor activation, Drug Alcohol Depend. 126 (2012) 257–262. [13] F. Zheng, C.G. Zhan, Modeling of pharmacokinetics of cocaine in human reveals the feasibility for development of enzyme therapies for drugs of abuse, PLoS Computat. Biol. 8 (2012) e1002610. [14] L.C. Nicolaysen, H.T. Pan, J.B. Justice Jr., Extracellular cocaine and dopamine concentrations are linearly related in rat striatum, Brain Res. 456 (1988) 317–323. [15] H.O. Pettit, H.T. Pan, L.H. Parsons, J.B. Justice Jr., Extracellular concentrations of cocaine and dopamine are enhanced during chronic cocaine administration, J. Neurochem. 55 (1990) 798–804. [16] T.M. Tzschentke, Review on CPP: measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade, Addict. Biol. 12 (2007) 227–462.
C.V. Hutchinson et al. / Neuroscience Letters 593 (2015) 19–23 [17] C. Davidson, A.J. Gow, T.H. Lee, E.H. Ellinwood, Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms: relevance to human abuse and treatments, Brain Res. Rev. 36 (2001) 1–22. [18] T.E. Robinson, K.E. Browman, H.S. Crombag, A. Badiani, Modulation of the induction or expression of psychostimulant sensitization by the circumstances surrounding drug administration, Neurosci. Biobehav. Rev. 22 (1998) 347–354. [19] J. Shoblock, E. Sullivan, I. Maisonneuve, S. Glick, Neurochemical and behavioral differences between d-methamphetamine and d-amphetamine in rats, Psychopharmacology 165 (2003) 359–369. [20] D.A. Hall, J.J. Stanis, H.M. Avila, J.M. Gulley, A comparison of amphetamineand methamphetamine-induced locomotor activity in rats: evidence for qualitative differences in behavior, Psychopharmacology 195 (2008) 469–478. [21] A.R. Winstock, L.R. Mitcheson, P. Deluca, Z. Davey, O. Corazza, F. Schifano, Mephedrone, new kid for the chop? Addiction 106 (2011) 154–161. [22] S.M. Rawls, T. Patil, E. Yuvasheva, R.B. Raffa, First evidence that drugs of abuse produce behavioral sensitization and cross-tolerance in planarians, Behav. Pharmacol. 21 (2010) 301–313. [23] G. Palladini, S. Ruggeri, F. Stocchi, M.F. De Pandis, G. Venturini, V. Margotta, A pharmacological study of cocaine activity in planaria, Comp. Biochem. Physiol. 115C (1996) 41–45. [24] R.B. Raffa, P. Desai, Description and quantification of cocaine withdrawal signs in planaria, Brain Res. 1032 (2005) 200–202. [25] L. Ju, J.W. Grimm, B.T. Hope, Y. Shaham, Incubation of cocaine craving after withdrawal: a review of preclinical data, Neuropharmacology 47 (2004) 214–216.
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[26] T. Kusayama, S. Watanabe, Reinforcing effects of methamphetamine in planarians, Neuroreport 11 (2000) 2511–2513. [27] C. Brabant, E. Quertemont, E. Tirelli, Influence of the dose and the number of drug – context pairings on the magnitude and the long-lasting retention of cocaine-induced conditioned place preference in C57BL/6J mice, Psychopharmacology 180 (2005) 33–40. [28] R.B. Raffa, S. Shah, C.S. Tallarida, S.M. Rawls, Amphetamine conditioned place preference in planarians, J. Behav. Brain Sci. 3 (2013) 131–136. [29] F.H. Gawin, H.D. Kleber, Abstinence symptomatology and psychiatric diagnosis in cocaine abusers, Arch. Gen. Psychiatry 43 (1986) 107–113. [30] J. Opacka-Juffry, T. Pinnel, N. Patel, M. Bevan, M. Meintel, C. Davidson, Stimulant mechanisms of cathinones – effects of mephedrone and other cathinones on basal and electrically evoked dopamine efflux in rat accumbens brain slices, Prog. Neuro-Psychopharmcol. Biol. Psychiatry 54 (2014) 122–130. [31] T.H. Lee, R. Balu, C. Davidson, E.H. Ellinwood, Differential time-course profiles of dopamine release and uptake changes induced by three dopamine uptake inhibitors, Synapse 41 (2001) 301–310. [32] B.J. Venton, A.T. Seipel, P.E.M. Phillips, W.C. Wetsel, D. Gitler, P. Greengard, G.J. Augustine, R.M. Wightman, Cocaine increases dopamine release by mobilization of a synapsin-dependent reserve pool, J. Neurosci. 26 (2006) 3206–3209.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Persistent conditioned place preference to cocaine and withdrawal hypo-locomotion to mephedrone in the flatworm planaria Claire V Hutchinson a , Jose Prados a , Colin Davidson b,∗ a b
School of Psychology, University of Leicester, Leicester LE1 9HN, UK Basic Medical Sciences, St George’s University of London, London SW17 0RE, UK
h i g h l i g h t s • We show conditioned place preference in planaria to cocaine. • We find long-lasting hypo-motility to mephedrone. • We have used clinically relevant concentrations.
a r t i c l e
i n f o
Article history: Received 9 November 2014 Received in revised form 26 February 2015 Accepted 11 March 2015 Available online 13 March 2015 Keywords: Planaria Cocaine Mephedrone Conditioned place preference Locomotion Withdrawal
a b s t r a c t The purpose of the present study was to determine the effects of exposure to cocaine and mephedrone on conditioned place preference (CPP) and locomotion in the flatworm planaria. Planaria were treated with either cocaine or mephedrone at 1 or 10 M. Planaria were exposed to 15 min of drug in their nonpreferred place (either a rough- or smooth-floored petri dish) on alternate days, and were exposed to normal water in their preferred place on the following day. There were 5 days of conditioning to drug. Planaria were then tested for CPP on day 2, 6 and 13 after withdrawal. We found that animals exhibited CPP to cocaine at both 1 and 10 M, but not to mephedrone. When examining locomotor activity we found that neither cocaine nor mephedrone treatment showed any evidence of evoking increased motility or locomotor sensitisation. Hypo-motility was seen on the first day of conditioning at concentrations of 10 M for both cocaine and mephedrone, but had disappeared by the last day of conditioning. Examining chronic withdrawal, only 10 M mephedrone had a significant effect on motility, decreasing locomotion on day 2 of withdrawal. Taken together we have shown that cocaine evoked CPP in planaria. We have also shown withdrawal depressing effects of mephedrone on motility. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction There is an increasing pressure on researchers to justify their use of vertebrate animals in pre-clinical studies. Over the last decade, the principles of the 3Rs (reduction, refinement and replacement) have gained more prominence and researchers are actively looking for non-vertebrate species in which to undertake their research. One such invertebrate species that has become more popular in pre-clinical research is the flatworm planaria. There are two main advantages of undertaking research in planaria; first its entire genome is known [1] and second it is cheap and easy to maintain.
Abbreviations: CPP, conditioned place preference; NPS, novel psychoactive substance; ANOVA, analysis of variance. ∗ Corresponding author. Tel.: +44 208725 5659. E-mail address: [email protected] (C. Davidson). http://dx.doi.org/10.1016/j.neulet.2015.03.021 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
In the field of neuroscience, further advantages to using planaria include its ability to regenerate its nervous system and the belief that it is the simplest organism to have a ‘brain’ [2]. In the field of neuropharmacology, the planaria may be of use because it possesses most of the main neurotransmitters that are found in rodents and humans [3]. It has been used recently in studying drugs of abuse and has been found to show withdrawal signs, most notably reduced locomotion, after being taken out of various drugs of abuse including cocaine, opioids, methamphetamine, cannabinoids and mephedrone [4–8]. Hypomotility is a core symptom of stimulant withdrawal in humans and rodents. In addition, behaviours such as head-nodding and head-swinging, squirming, clinging, tail-twising and corkscrew spiral motions along the longaxis have been seen [4]. More recently conditioned place preference (CPP) has been shown in planaria for cocaine, mephedrone and MDMA [8,9] and we have shown more subtle responses such as blocking [10] suggesting that planaria show complex information
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processing. It has also been shown [11] that planaria exhibit environmental familiarisation that endures for up to 14 days. This raises the interesting possibility that drug-induced CPP effects in planaria might be long-lasting, in a manner similar to the effects observed in rodent models [12] where rodents, after conditioning with mephedrone for a number of days, demonstrated significant CPP that lasted for at least 3 weeks. If similar effects were evident in planaria, their utility modeling drug-related changes in mammalian behaviour would be extremely promising. One possible limitation of previous studies concerns whether the concentrations tested have clinical relevance, where studies use drug concentrations of up to 1000 M. In the present study we aimed to further show the validity of using planaria in drug abuse research by using drugs at lower, perhaps more clinically relevant, concentrations and by examining the persistence of the conditioning effect. Having a simple model organism that allows high throughput screening of drugs of abuse would be particularly useful in the testing of legal highs (novel psychoactive substances, NPS) where around 100 NPS are found each year, with virtually nothing known of their pharmacology.
2. Methods Planaria. Brown planaria (Dugesia Tigrina) were purchased from Blades Biological Ltd (UK) and were fed raw chicken every 2 days for 1 h. Their water was changed after feeding. We used AquaSafe© (Tetra, Germany) to treat normal tap water. Planaria were kept about 20 worms to 500 ml treated water in Tupperware boxes. They were kept in a dimly lit room with a dark cycle from 6PM to 9AM. Drugs and conditioning. We examined cocaine, one of the most addictive drugs, and mephedrone for which much less is known about its addictive liability. Cocaine is primarily a DAT inhibitor whereas we have previously shown that mephedrone causes reverse transport of dopamine (Opacka-Juffry et al., 2014). In a pre-test, planaria were allowed to move freely in a plastic petri dish (9 cm diameter) which had one half smooth (white shiny card) and one half rough (brown, wet–dry sandpaper, grade 100 grit). The floor coverings were fitted up the sides of the dishes to avoid planaria getting trapped under paper and also so that if they swam around the edge, they were still in contact with a smooth or rough
3. Results In our pre-conditioning test of side preference we found no statistically significant difference in the amount of time spent in each side in all our planaria combined. In the 15 min (900 s) test, planaria spent 411.1 ± 31 s in the rough side and 488.9 ± 31 s in the smooth side, t(42) = 1.25, p = 0.22. Thus, our CPP design was non-biased. Conditioned place preference. The main finding was that CPP was seen with cocaine at both 1 and 10 M, but no effect was seen with mephedrone. Fig. 1A shows CPP findings for planaria conditioned with 1 M cocaine (n = 9), 1 M mephedrone (n = 13) and a control group (n = 7) conditioned in treated water. Fig 1B shows CPP findings for planaria conditioned with 10 M cocaine (n = 6) or 10 M mephedrone (n = 9). The control group is also
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surface. The dish was filled with 3 ml of treated tap water. This meant that the fluid level was just above the floor of the dish, forcing the worms to contact the different surfaces. The time spent in each half of the dish was measured after 15 min. During conditioning the worms were placed in a dish, which only contained a floor of their non-preferred side (sandpaper or shiny white card) in either 1 or 10 M of cocaine or mephedrone, made up in treated tap water, for 15 min. The following day worms were placed in a petri dish with their preferred floor, again for 15 min. This was repeated for 10 consecutive days (5 days drug, 5 days water), as is standard in CPP experiments [16]. Planaria were then tested on days 2, 6 and 13 after withdrawal in the original petri dishes, which were ½ sandpaper and ½ shiny white card. The amount of time spent in each half was measured after video analysis. We were also able to measure locomotor activity using video analysis with a suitable sized grid placed in the video monitor, allowing us to estimate the number gridlines crossed. If more than 50% of the planaria’s body crossed a line we counted this as a crossing. See Fig. 1 for an overview of the testing and drug administration schedule. Worms were transferred from dish to dish by gently picking them up with a fine artists brush, occasionally a worm would be damaged during this process and data from these worms were excluded from analysis, (i.e. planaria that did not move). Cocaine was purchased from Sigma–Aldrich, mephedrone was a gift from John Ramsay (TICTAC Communications Ltd., UK).
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Withdrawal day Fig. 1. Overview of testing and drug administration schedule and conditioned place preference after cocaine. Top panel: we always started the planaria with drug on day 1 of conditioning, on the non-preferred side. This meant that the last day of conditioning was with water on the preferred side. Thus the CPP tests were done on days 2, 6 and 13 of drug withdrawal (WD). Time spent on the non-preferred side before conditioning (pre) and 2, 6, and 13 days after withdrawal. CPP was seen with cocaine at both 1 and 10 M, but not with mephedrone, see text for details. A. 1 M cocaine (closed circles) and 1 M mephedrone (open circles) and for the control group (open triangles). B. 10 M cocaine (closed circles) and 10 M mephedrone (open circles). Control group data (open triangles) are replotted from A. Values are means ± 1 SEM. N = 6–13.
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shown (replotted from Fig. 1A). These data suggest that cocainetreated worms spent more time in their conditioned side than mephedrone-treated worms. A 3 (drug type: treated water, cocaine, mephedrone) by 3 (drug concentration: no drug, 1, 10 M) by 4 (test day: pre-conditioning, +2, +6, +13 days after withdrawal) analysis of variance (ANOVA) revealed significant effects of drug type [F(1,39) = 17.845; p < .001] and withdrawal day [F(3117) = 3.389; p < .05]. There was no significant effect of drug concentration and no significant interaction. To confirm these findings, we performed separate ANOVAs for cocaine and mephedrone. For cocaine, a 3 (drug concentration: no drug, 1, 10 M) by 4 (test day: pre-conditioning, +2, +6, +13 days after withdrawal) ANOVA yielded significant effects of drug concentration [F(2,19) = 3.922; p < .05] and withdrawal day [F(3,57) = 4.104; p < .005]. A comparable ANOVA revealed no significant effects of mephedrone. Followup within group t-tests (one-tailed) compared the time spent on the conditioned side on each post-cocaine withdrawal day (2, 6 and 13) with the time spent on the conditioned side before conditioning began. After being conditioned at a concentration of 1 M cocaine, planaria spent significantly longer of the conditioned side (compared to pre-conditioning) when tested 6 days after withdrawal [t(8) = 3.173; p < .01]. After being conditioned at a concentration of 10 M cocaine, planaria spent significantly longer on the conditioned side on all 3 withdrawal days: 2 days [t(5) = 2.131; p < .05], 6 days and 13 days [t(5) = 4.203; p < .005] after withdrawal [t(5) = 3.128; p < .05]. Locomotor activity. The main finding was that mephedrone, at 10 M, reduced locomotor activity during chronic withdrawal, this was most evident on day 2 of withdrawal). Fig. 2 A shows locomotor activity (number of 1 cm gridlines crossed in 15 min) for planaria conditioned with 1 M cocaine (n = 9), 1 M mephedrone (n = 8)
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and a control group (n = 7) conditioned in treated water. Fig 2B shows locomotor activity for planaria conditioned with 10 M cocaine (n = 6) or 10 M mephedrone (n = 8). Control group data are replotted from Fig 2A. A three (drug type: treated water, cocaine, mephedrone) by 3 (drug concentration: no drug, 1, 10 M) by 3 (test day: pre-conditioning, conditioning day 1, conditioning day 5) ANOVA revealed a significant effect of testing day [F(2,66) = 3.489; p < .05]. We followed this up with individual ANOVAs for each group. As expected, there was no significant effect of day in the control group (no drug). Although at higher drug concentrations (10 M) locomotion appeared to decrease activity relative to baseline on conditioning days 1 and 5, this effect was not significant for either cocaine or mephedrone. At 1 M concentrations, there was a significant effect of day for both cocaine [F(2,16) = 3.944; p < .05] and mephedrone [F(2,14) = 10.963; p < .05]. Follow-up t-tests confirmed that this reflected a reduction in motility on conditioning day 1 in the drug compared to pre-conditioning (cocaine: t(8) = 2.293; p = .051; mephedrone: t(7) = 2.569; p < .05). There was no significant change in locomotion relative to preconditioning by conditioning day 5. This suggests that the planaria did not show an increase in motility when placed in 1 M cocaine or mephedrone, but showed hypo-motility. There was no evidence of locomotor sensitisation or tolerance by day 5 of drug treatment, but the planaria appeared to be tolerant to the hypolocomotor effects of both cocaine and mephedrone (Fig 2A). We next examined motility during chronic withdrawal by testing the numbers of lines crossed during the post-test days and days 2, 6 and 13 after withdrawal. Fig. 2C shows locomotor activity (number of 1 cm gridlines crossed in 15 min) before and after conditioning for planaria conditioned with 1 M cocaine (n = 8), 1 M mephedrone (n = 8) and a control group (n = 7) conditioned in treated water. Fig 2D shows locomotor activity before and after conditioning for planaria conditioned with 10 M cocaine (n = 6) or 10 M mephedrone (n = 8). At 10 M, the mephedrone treated planaria appeared to display hypo-motility compared to cocaine treated planaria. A 3 (drug type: treated water, cocaine, mephedrone) by 3 (drug concentration: no drug, 1, 10 M) by 3 (test day: pre-conditioning, + 2 days, 6 days and 13 days after withdrawal) ANOVA revealed a significant effect of testing day [F(3,96) = 2.83; p < .05] and drug type [F(1,32) = 4.997; p < .05]. Individual ANOVAs for each individual group confirmed a significant effect of chronic withdrawal for mephedrone at 10 M [F(3,21) = 3.884; p < .05]. Follow-up t-tests showed that planaria exhibited significantly decreased locomotion, relative to preconditioning, 2 days after withdrawal [t(7) = 2.683; p < .05]. This decrease in locomotion appeared to persist at 6 and 13 days after withdrawal. However, these effects only approached significance [+6 days: t(7) = 2.135; p = .07, +13 days: t(7) = 2.194; p = .064].
4. Discussion 0
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Fig. 2. Locomotion during drug treatment and during chronic withdrawal. Number of 1 cm gridlines crossed in 15 min. A and B. Locomotion on pre-conditioning day and days 1 and 5 (the first and last days) of drug conditioning. Both cocaine and mephedrone at 1 M reduced locomotor activity on day 1 of conditioning, an effect that had disappeared by day 5. There was no significant effect at 10 M with either drug. A. 1 M cocaine (closed circles) and mephedrone (open circles) and control group (open triangles). B. 10 M cocaine (closed circles) and mephedrone (open circles). Control group (open triangles) are replotted from A, C and D. Locomotion on pre-conditioning day and days 2, 6 and 13 after withdrawal (WD). C. Mephedrone at 10 M reduced locomotor activity on day 2 of withdrawal. 10 M cocaine (closed circles) and mephedrone (open circles) and control group (open triangles). D. 10 M cocaine (closed circles) and mephedrone (open circles). Control group (open triangles) are replotted from C. Values are means ± 1 SEM. N = 6–9.
Rodent models of psychostimulant abuse such as locomotor sensitisation, CPP and drug self-administration have shown complex responses dependant upon the drug tested, dose, route of administration and day of withdrawal [17,18]. In addition to these reinforcing effects, one might also expect a psychostimulant to increase locomotion, but in-place stereotypies, common with amphetamine-like drugs or high doses of stimulants, can lead to reduced locomotion [19,20]. We have compared cocaine, which exerts its addictive effects primarily through dopamine transporter inhibition, with the novel psychoactive substance mephedrone, which has been shown to cause reverse transport of dopamine [30]. We have tested for locomotor sensitisation (or tolerance), CPP and withdrawal effects.
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In the current study we have shown CPP to cocaine on day 2, 6 and 13 after withdrawal but we found no evidence of locomotor sensitisation. Previous studies have shown CPP to mephedrone [8] in planaria, but only at much higher concentrations (100–500 M). To our knowledge, no group have shown locomotor sensitisation to cocaine or mephedrone in planaria at clinically relevant concentrations, although Rawls and colleagues have shown sensitization to cocaine at 100 M [22]. Indeed, a simple explanation might be that the concentrations tested have been toxic, and mephedrone has been described as evoking toxicity in humans [21], and thus reduces locomotion (as we have seen). An alternative explanation is that the planaria move around less due to stereotypies [8]. However, cocaine at high concentrations increases planarian activity [22] defined as an increase in ‘C-like hyperkinesias’ rather than increased motility. Another explanation might be that the planarian locomotor system is not driven by the dopamine system to the same extent as it is in mammals. However, numerous studies have found dopamine D2 antagonists to inhibit the effects of stimulants in planaria [8,23] suggesting a reasonable similarity to the mammalian system. The lack of a consistent effect of stimulants on planarian motility has been previously discussed [8]. Previous studies have shown hypo-locomotion after withdrawal from drugs of abuse, with various distinct acute withdrawal behaviours, such as ‘corkscrew’ movements and ‘inverted C-shapes’ [12,24]. We tested cocaine and mephedrone at lower concentrations to determine if the lack of hyper-motility was due to the concentrations of the drug being too great, leading to toxicity and/or stereotypies, but even at the lower concentrations we used (1 and 10 M) there was no evidence of increased locomotion, nor did we see stereotypies. Ramoz et al. found that 60 min treatment with 1 or 10 M mephedrone caused a reduction in motility in the first 5 min of withdrawal [8]. Differences between studies in planarian CPP and motility might be due to different species of planaria used or different dosing regimens. For example, Rawls’ group used a biased CPP design, 30 min contact time during mephedrone conditioning, 5 min post-conditioning test time and Dugesia dorotocephala [8], whereas we have used an unbiased design, 15 min drug contact time, a 15 min post conditioning test time and D. Tigrina. A second goal of our study was to test how long CPP lasts in planaria. Previous studies in rodents suggest that CPP can last for days, and drug craving can last for weeks or months [25]. In general, rodent studies show that the more conditioning days and the higher the drug dose, the more persistent the CPP. With 5 days of drug pairing (in the present study) one might expect to see CPP for 10 days or more in a rodent [16]. In a study by Brabant et al. [27] CPP to cocaine persisted for up to 35 days in mice, but only at the highest dose tested (12 mg/kg IP). Our data show that CPP is still evident on day 13 after withdrawal. These data extend previous studies, which have found CPP in planaria with methamphetamine [26], amphetamine [28] and mephedrone [8] only immediately after conditioning. The finding that mephedrone (10 M) treatment caused an extended hypo-motility in planaria lasting up to 13 days after withdrawal, suggests a chronic withdrawal syndrome similar to that which one might expect to find in a stimulant-dependant human [29]. The lack of chronic withdrawal hypo-motility in cocaine treated planaria suggests pharmacological differences between these 2 drugs. We have shown in rat brain slices, that mephedrone has a neurochemical effects similar to amphetamine in that it causes reverse transport of dopamine [30], whereas cocaine acts primarily as a dopamine transporter blocker and mobiliser of dopamine storage pools [31,32]. Thus these simple locomotor tests in planaria could have the potential to tease out different types of stimulants. One limitation of these studies is whether the concentrations tested have clinical relevance. Using data from human subjects
taking a relatively low dose of cocaine, Zheng and Zhan [13] modelled the extracellular cocaine concentration in the brain and found the peak to be 2.6 M. Nicolaysen et al. [14] using microdialysis in rats found that a 30 mg/kg i.p. cocaine injection (considered a large dose) caused a peak striatal cocaine concentration of 10.1 M. In addition, rats chronically treated with cocaine had almost double the plasma cocaine concentration found in acutely treated rats Pettit et al. [15]. Thus 10 M may be a clinically relevant dose of cocaine. Taken together, these data further confirm the utility of planaria in drug abuse research. They may allow us to predict the behavioural pharmacology of drugs of abuse and hint at neurochemical mechanisms. NPS or legal highs, for which very little is known, are currently flooding European markets through internet vendors. A simple and cheap high throughput screen, such as described here, might be able to offer important pharmacological data on potential abuse liability and toxicity of these many new drugs of abuse. Acknowledgements This work was supported by an NC3Rs small grant to CD and the European Commission-funded EU-MADNESS project (JUST2013/DPIP/AG/4823) resources were used to assist with the preparation of this research document. Some of the work was carried out during a period of study leave awarded by the University of Leicester to CH. References [1] E. Saló, J.F. Abril, T. Adell, F. Cebrià, K. Eckelt, E. Fernandez-Taboada, M. Handberg-Thorsager, M. Iglesias, M.D. Molina, G. Rodríguez-Esteban, Planarian regeneration: achievements and future directions after 20 years of research, Int. J. Dev. Biol. 53 (2009) 1317–1327. [2] T. Sandmann, M.C. Vogg, S. Owlarn, M. Boutros, K. Bartscherer, The head-regeneration transcriptome of the planarian Schmidtea mediterranea, Genome Biol. 12 (2011) R76. [3] F.R. Buttarelli, C. Pellicano, F.E. Pontieri, Neuropharmacology and behavior in planarians: translations to mammals, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 147 (2008) 399–408. [4] R.B. Raffa, P. Desai, Description and quantification of cocaine withdrawal signs in planaria, Brain Res. 1032 (2005) 200–202. [5] R.B. Raffa, G.W. Stagliano, S. Umeda, Kappa-opioid withdrawal in planaria, Neurosci. Lett. 349 (2003) 139–142. [6] S.M. Rawls, H. Shah, G. Ayoub, R.B. Raffa, 5-HT1A-like receptor activation inhibits abstinence-induced methamphetamine withdrawal in planarians, Neurosci. Lett. 484 (2010) 113–117. [7] S.M. Rawls, K. Gerber, Z. Ding, C. Roth, R.B. Raffa, Agmatine: identification and inhibition of methamphetamine, kappa opioid, and cannabinoid withdrawal in planarians, Synapse 62 (2008) 927–934. [8] L. Ramoz, S. Lodi, P. Bhatt, A.B. Reitz, C. Tallarida, R.B. Raffa, S.M. Rawls, Mephedrone (bath salt) pharmacology: insights from invertebrates, Neuroscience 208 (2012) 79–84. [9] T. Kusayama, S. Watanabe, Reinforcing effects of methamphetamine in planarians, Neuroreport 11 (2000) 2511–2513. [10] J. Prados, B. Alvarez, J. Howarth, K. Stewart, C.L. Gibson, C.V. Hutchinson, A.M. Young, C. Davidson, Cue competition effects in the planarian, Animal Cogn. 16 (2012) 177–186. [11] T. Shomrat, M. Levin, An automated training paradigm reveals long-term memory in planaria and its persistence through head regeneration, J. Exp. Biol. 216 (2013) 3799–3810. [12] R. Lisek, W. Xu, E. Yuvasheva, Y.T. Chiu, A.B. Reitz, L.Y. Liu-Chen, S.M. Rawls, Mephedrone (‘bath salt’) elicits conditioned place preference and dopamine-sensitive motor activation, Drug Alcohol Depend. 126 (2012) 257–262. [13] F. Zheng, C.G. Zhan, Modeling of pharmacokinetics of cocaine in human reveals the feasibility for development of enzyme therapies for drugs of abuse, PLoS Computat. Biol. 8 (2012) e1002610. [14] L.C. Nicolaysen, H.T. Pan, J.B. Justice Jr., Extracellular cocaine and dopamine concentrations are linearly related in rat striatum, Brain Res. 456 (1988) 317–323. [15] H.O. Pettit, H.T. Pan, L.H. Parsons, J.B. Justice Jr., Extracellular concentrations of cocaine and dopamine are enhanced during chronic cocaine administration, J. Neurochem. 55 (1990) 798–804. [16] T.M. Tzschentke, Review on CPP: measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade, Addict. Biol. 12 (2007) 227–462.
C.V. Hutchinson et al. / Neuroscience Letters 593 (2015) 19–23 [17] C. Davidson, A.J. Gow, T.H. Lee, E.H. Ellinwood, Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms: relevance to human abuse and treatments, Brain Res. Rev. 36 (2001) 1–22. [18] T.E. Robinson, K.E. Browman, H.S. Crombag, A. Badiani, Modulation of the induction or expression of psychostimulant sensitization by the circumstances surrounding drug administration, Neurosci. Biobehav. Rev. 22 (1998) 347–354. [19] J. Shoblock, E. Sullivan, I. Maisonneuve, S. Glick, Neurochemical and behavioral differences between d-methamphetamine and d-amphetamine in rats, Psychopharmacology 165 (2003) 359–369. [20] D.A. Hall, J.J. Stanis, H.M. Avila, J.M. Gulley, A comparison of amphetamineand methamphetamine-induced locomotor activity in rats: evidence for qualitative differences in behavior, Psychopharmacology 195 (2008) 469–478. [21] A.R. Winstock, L.R. Mitcheson, P. Deluca, Z. Davey, O. Corazza, F. Schifano, Mephedrone, new kid for the chop? Addiction 106 (2011) 154–161. [22] S.M. Rawls, T. Patil, E. Yuvasheva, R.B. Raffa, First evidence that drugs of abuse produce behavioral sensitization and cross-tolerance in planarians, Behav. Pharmacol. 21 (2010) 301–313. [23] G. Palladini, S. Ruggeri, F. Stocchi, M.F. De Pandis, G. Venturini, V. Margotta, A pharmacological study of cocaine activity in planaria, Comp. Biochem. Physiol. 115C (1996) 41–45. [24] R.B. Raffa, P. Desai, Description and quantification of cocaine withdrawal signs in planaria, Brain Res. 1032 (2005) 200–202. [25] L. Ju, J.W. Grimm, B.T. Hope, Y. Shaham, Incubation of cocaine craving after withdrawal: a review of preclinical data, Neuropharmacology 47 (2004) 214–216.
23
[26] T. Kusayama, S. Watanabe, Reinforcing effects of methamphetamine in planarians, Neuroreport 11 (2000) 2511–2513. [27] C. Brabant, E. Quertemont, E. Tirelli, Influence of the dose and the number of drug – context pairings on the magnitude and the long-lasting retention of cocaine-induced conditioned place preference in C57BL/6J mice, Psychopharmacology 180 (2005) 33–40. [28] R.B. Raffa, S. Shah, C.S. Tallarida, S.M. Rawls, Amphetamine conditioned place preference in planarians, J. Behav. Brain Sci. 3 (2013) 131–136. [29] F.H. Gawin, H.D. Kleber, Abstinence symptomatology and psychiatric diagnosis in cocaine abusers, Arch. Gen. Psychiatry 43 (1986) 107–113. [30] J. Opacka-Juffry, T. Pinnel, N. Patel, M. Bevan, M. Meintel, C. Davidson, Stimulant mechanisms of cathinones – effects of mephedrone and other cathinones on basal and electrically evoked dopamine efflux in rat accumbens brain slices, Prog. Neuro-Psychopharmcol. Biol. Psychiatry 54 (2014) 122–130. [31] T.H. Lee, R. Balu, C. Davidson, E.H. Ellinwood, Differential time-course profiles of dopamine release and uptake changes induced by three dopamine uptake inhibitors, Synapse 41 (2001) 301–310. [32] B.J. Venton, A.T. Seipel, P.E.M. Phillips, W.C. Wetsel, D. Gitler, P. Greengard, G.J. Augustine, R.M. Wightman, Cocaine increases dopamine release by mobilization of a synapsin-dependent reserve pool, J. Neurosci. 26 (2006) 3206–3209.