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Consumption of a highly palatable food induces a lasting place-conditioning memory in marmoset monkeys
Behavioural Processes 107 (2014) 163–166
Contents lists available at ScienceDirect
Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Short report
Consumption of a highly palatable food induces a lasting place-conditioning memory in marmoset monkeys R.B.M. Duarte a , E. Patrono b,c , A.C. Borges e , A.A.S. César a , C. Tomaz a , R. Ventura c,d , A. Gasbarri b,c , S. Puglisi-Allegra c,d , M. Barros e,∗ a
Primate Center and Department of Physiological Sciences, Institute of Biology, University of Brasilia, CEP 70910-900 Brasilia, DF, Brazil Department of Applied Clinical and Biotechnological Sciences, University of L’Aquila, 67100 L’Aquila, Italy c Santa Lucia Foundation IRCCS, via del Fosso di Fiorano 65, 00143 Rome, Italy d Department of Psychology and Research Center “Daniel Bovet”, Sapienza University, via dei Marsi 78, 00185 Rome, Italy e Department of Pharmaceutical Sciences, School of Health Sciences, University of Brasilia, CEP 70910-900 Brasilia, DF, Brazil b
a r t i c l e
i n f o
Article history: Received 26 May 2014 Received in revised form 8 July 2014 Accepted 21 August 2014 Available online 28 August 2014 Keywords: Chocolate Food reward Long-term memory Marmoset Place-conditioning
a b s t r a c t Highly palatible foods may induce addiction-related behaviors. However, this has yet to be established in non-human primates. Therefore, we evaluated whether marmoset monkeys (Calllithrix penicillata) acquire a conditioned-place-preference (CPP) for chocolate and if this response is detectable after a 24h and 15-day period. Subjects were first habituated to a two-compartment CPP box and then randomly assigned to a chocolate or control group. Thereafter, they were given access to only one compartment during daily 15-min conditionings, held on six consecutive days. On each trial, the chocolate group received pieces of chocolate (50 g) in this context, whereas controls were not given a food reward. Marmosets were subsequently tested for preferring this (food) paired context after a 24-h and 15-day interval. During conditioning, individual foraging and the amount of chocolate ingested by each pair of the chocolate group remained constant. However, compared to pre-CPP levels, the time spent inside/in contact with the conditioned compartment increased significantly, while the latency to first entry decreased on both post-CPP intervals. For controls, the parameters remained unaltered. Thus, chocolate induced a persistent CPP response—an aspect usually associated with drug-related rewards. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The overconsumption of highly caloric palatable foods is a nonhomeostatic feeding pattern. It usually results in an energy intake that exceeds normal daily requirements and occurs when an individual is in a physiologically sated state (Berthoud, 2006). It is also commonly associated with the development of addictionrelated behaviors, eating disorders and obesity (Avena, 2010), with both learned and motivated processes being involved—including contextual cue-learning, mnemonic representation of different food items and their intrinsic reinforcing properties (Johnson, 2013). Chocolate, in particular, has an elevated hedonic rating (Bruinsma and Taren, 1999; Parker et al., 2006) and a prominent craving-inducing property (Rozin et al., 1991). In fact, similar to
∗ Corresponding author. Tel.: +55 61 31072002; fax: +55 61 31072002. E-mail address: [email protected] (M. Barros). http://dx.doi.org/10.1016/j.beproc.2014.08.021 0376-6357/© 2014 Elsevier B.V. All rights reserved.
reports using drugs of abuse (reviewed in Volkow et al., 2013), reward-processing neural circuits are activated when humans (Small et al., 2001) and rodents voluntarily consume chocolate (Latagliata et al., 2010; Ventura et al., 2008, 2007). In these animals, chocolate also induced a conditioned-place-preference (CPP) (Dickson et al., 2012; La Mela et al., 2010; Latagliata et al., 2010; Orsini et al., 2013; Schroeder et al., 2001; Ventura et al., 2008, 2007). This effect is an associatively learned preference for certain locations that is acquired in response to experiencing a rewarding agent at that same location (Huston et al., 2013; Tzschentke, 2007). The CPP paradigm has only recently been demonstrated in non-human primates using both drug- (Barros et al., 2013; Wang et al., 2012) and food-related stimuli (Monclaro et al., 2014; Valentinuzzi et al., 2008). However, the persistence of a food-induced CPP response has not been assessed, regardless of the species and food type. Here we evaluated whether marmoset monkeys acquire a conditioned preference for where they consumed a highly palatable food (i.e., chocolate), and if this response is detectable after a 24-h and 15-day period.
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2. Materials and methods 2.1. Subjects and housing conditions Fourteen adult captive black tufted-ear marmosets (C. penicillata; 7 males, 7 females) were used, weighing 250–450 g at the beginning of the study. They were stable pairs (n = 7) housed at the Primate Center of the University of Brasilia in cages (2 m × 1 m × 2 m each) of a same colony room. This room consisted of a semi-outdoor/indoor housing system with two parallel rows of 12 cages each, separated by a common wire-mesh enclosed central corridor. The animals were thus exposed to natural light, temperature and humidity conditions. Fresh food, consisting of a mixture of pieces of fruits and vegetables, was provided at 07:00 h and removed at 17:00 h. Boiled eggs, nuts and/or cooked chicken breast were given several times a week. Water and monkey chow were available ad libitum. Animals were not food or water deprived, except during specific trials (see Section 2.3). For further housing conditions see (Monclaro et al., 2014). These complied with the regulations of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA). 2.2. Apparatus Testing was conducted in a CPP box (60 cm × 30 cm × 30 cm) set on a 1.5 m high pedestal. One wall (60 cm × 30 cm) was made of 4 mm transparent glass and the remaining sides of aluminum. It was divided into two compartments (30 cm × 30 cm × 30 cm): one had smooth interior and exterior surfaces and was painted white, whereas the other had rough inner and outer surfaces and was painted black. The side wall of each compartment was a sliding door, acting as its entry/exit point. Thus, the white and black compartments could only be accessed from the left and right side of the box, respectively. Also, a stainless-steel bowl (9 cm in diameter) was placed in a hole on the floor of each compartment, located as far away as possible from its access door. Seven identical CPP boxes were used, each being placed in the middle of a home-cage as not to come in contact with anything. The glass wall faced the central corridor from where behaviors were observed and recorded. 2.3. Procedure The apparatus was placed in the home-cage and the pair was given 24-h access to both compartments during two consecutive days. On day 3, a 15-min observation trial (pre-CPP) was held and then both access doors were closed. During this phase a food reward was not provided. Each pair was then randomly assigned to the control (3 males, 3 females) or chocolate group (4 males, 4 females). Thereafter, subjects were only given access to one of the compartments during daily 15-min conditioning sessions held on six consecutive days (C1–C6). Trials were not performed in the opposite compartment. This is typically done to control for possible novelty-based preferences and (aversive) emotional effects of a drug-reward application procedure (Huston et al., 2013). As subjects were tested in their home-cages and had been priorly habituated to the CPP box, the latter had become part of the ‘background’ environment. An unbiased counterbalanced design was used, in which half of the pairs were arbitrarily conditioned to the white context and half to the black. Subjects from a same home-cage were conditioned simultaneously to the same compartment and their regular diet was removed 2h prior to each session. Before each trial, an experienced observer entered the home-cage and opened the door of the pre-established conditioned compartment. The door of the non-conditioned side remained closed at all times. For the chocolate group, 50 g of pieces of milk chocolate was placed in the food bowl. For the controls,
the same procedure was held, yet nothing was placed in the food bowl. This design aimed at isolating the influence of the chocolate consumption on the marmosets’ future behavior (chocolate group), while controlling for any possible changes due to repeated exposures to the test environment (control group). When the observer exited the home-cage, a 15-min trial began. At the end of this interval, the observer re-entered the home-cage, retrieved any leftover food item and closed the door of the conditioned compartment. Between trials, marmosets did not have access to either compartment. A test trial was held 24-h (T24h) and 15-days after (T15d) the last conditioning, with subjects once again being given 15-min access to both compartments in the absence of the food reward. Subjects were tested in random order and all sessions held between 13:30 and 17:00 h. Even though pairs were tested simultaneously, all behaviors were scored individually by one of two observers positioned in front of their home-cage. Observers had 95% inter-rater reliability and used the Anymaze software (Stoelting Co., USA) to score the following parameters during the pre-CPP, C1, C6, T24h and T15d trials: (1) frequency and duration inside/in contact with the top surface of each compartment; (2) latency to first entry in the conditioned compartment; and (3) individual foraging frequency and duration (search, manipulation and/or ingestion of the chocolate). The first parameter was scored as a single variable since, based on a similar study (Monclaro et al., 2014), all subjects consistently use the top of the open compartment as a continuum of its inside. Several aspects may contribute to this response, including having one of the highest rates of predation among primates (Cheney and Wrangham, 1987), an impaired visibility of the surroundings from within the compartment, and free transit throughout the home-cage. In addition, the amount of chocolate consumed by each pairmate (kcal) on trials C1 and C6 was calculated, based on the amount ingested in grams. Data from males and females in each group were pooled together as no significant differences in gender were observed. Data were analyzed using a mixed-design two-way ANOVA. For the habituation trials, ‘group’ was used as the independent factor and ‘compartment’ as the repeated measure variable, while for the remaining analyses ‘group’ and ‘trial’ were the independent and dependent variables, respectively. Whenever significant, subsequent comparisons were held using Tukey’s test for multiple pair-wise comparisons. All latency data were log transformed prior to analyses due to a non-normal distribution, yet results were reported as the median and interquartile range (IQR). For the chocolate group, individual foraging frequency and duration, as well as the amount of chocolate consumed (kcal/pair), were analyzed via paired t-test (C1 vs. C6). In addition, partial eta squared (2p ) and Cohen’s d for correlated samples (dz ) were used to determine effect size for the ANOVAs and t-tests, respectively. Significance level was set at p ≤ 0.05. All procedures were approved by the Animal Ethics Committee of the University of Brasilia and complied with the Brazilian National Council for Control of Animal Experimentation (CONCEA) and NIH guidelines for care and use of laboratory animals.
3. Results Pre-CPP, marmosets had no initial preference for either side of the apparatus (see supplementary data—Table S1). During conditioning, the time spent inside/in contact with the ‘conditioned’ compartment differed significantly between groups (F1,13 = 13.59, p = 0.003, 2p = 0.53), yet with no significant trial effect (C1 vs. C6) or group vs. trial interaction (F1,13 < 1.0, p = n.s.; trial: 2p = 0.06; interaction: 2p = 0.05; Table 1). For the chocolate group, individual foraging rates and the amount of food consumed by the pair (in kcal) did not differ between C1 and C6 (frequency:
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parameters remained unaltered throughout the study (Fig. 1). Furthermore, significant gender differences within each group were not observed for the parameters described above (data not shown).
4. Discussion
Fig. 1. Mean (+SEM) number of entries (top) and time spent (in seconds; middle) inside and/or in contact with the conditioned compartment of the CPP box, as well as the median (and interquartile range—IQR) for the latency to first entry (in seconds; bottom) in this same context by marmosets of the control (n = 6) and chocolate groups (n = 8) during the 15-min session after the initial habituation (pre-CPP) and on each of the two 15-min test trials held 24-h (T24h) and 15-days (T15d) after conditioning. *p < 0.05 control vs. chocolate; **p < 0.05 vs. pre-CPP trial of the chocolate group.
t7 = 1.68, p = 0.14, dz = 0.39; duration: t7 = 1.90, p = 0.10, dz = 0.68; consumption: t3 < 1.0, p = n.s., dz = 0.16; Table 1). Incidents of pairmate aggression or cooperative feeding behavior were rare. However, on both test trials held after conditioning (i.e., T24h and T15d), the time spent inside/in contact with the conditioned compartment increased significantly, yet only in the chocolate group compared to pre-CPP levels (trial effect: F2,13 = 4.62, p = 0.03, 2p = 0.28; group effect: F1,13 = 4.31, p = 0.06, 2p = 0.26; group × trial interaction: F2,13 = 5.55, p = 0.02, 2p = 0.32; Fig. 1). The latency to enter the conditioned compartment for the first time, on both post-CPP trials, also decreased in the chocolate group compared to pre-CPP levels, but not significantly (trial effect: F2,13 = 1.40, p = 0.27; 2p = 0.10; group effect: F1,13 < 1.0, p = n.s., 2p = 0.63; group × trial interaction: F2,13 = 1.32, p = 0.29, 2p = 0.10; Fig. 1). This same group had a significantly higher frequency inside/in contact with the conditioned compartment than controls (F1,13 = 10.92, p = 0.006, 2p = 0.48; Fig. 1). However, the latter was seen on all trials, with no significant group vs. trial interaction (F2,13 < 1.0, p = n.s., trial: 2p = 0.02, interaction: 2p = 0.01). For controls, who had not received food during conditioning, these
Table 1 Response (mean ± SEM) of each experimental group in the conditioned compartment of the CPP box during the first and last conditioning trials. Parameter
Group
Time spent (s) Time spent (s) Individual foraging frequency Individual foraging duration (s) Chocolate consumption (kcal/pair)
Control Chocolate Chocolate Chocolate Chocolate
a
p < 0.05 vs. the time spent by the control group.
Conditioning trial 1
6
70 ± 23 299 ± 63 a 8±2 206 ± 60 10 ± 2
67 ± 19 245 ± 40 a 5±1 118 ± 20 11 ± 4
This study showed that chocolate, repeatedly consumed by marmosets in a specific environment, induced a CPP response for this location. Only in the chocolate group did the time spent inside/in contact with the conditioned compartment increase postCPP, compared to pre-CPP levels. Also, after conditioning, the latency to enter the chocolate-paired context decreased compared to pre-CPP rates, although a significance level was not attained. The latter parameter has been viewed as a measure of an individual’s immediate response to this experimental setup (Monclaro et al., 2014; Valentinuzzi et al., 2008). This increase in dwell time was not accompanied by changes in frequency, which further corroborates the CPP response observed. However, the chocolate group entered/contacted this location more often than controls. Prior to the group assignment and chocolate conditioning, a general preference for either compartment was not detected. Following these events, group differences were seen throughout the study, even pre-CPP. Thus, a higher entry frequency may be inherent to the animals that were randomly assigned to the chocolate group, regardless of the CPP training. Throughout the chocolate conditioning, marmosets remained motivated for this food reward. Incidences of aggression or cooperative feeding behavior between pairmates were rare, and individual foraging rates and the amount of chocolate consumed by each pair remained unaltered (C1 vs. C6). The pairs tested formed stable groups and were not food-deprived prior to conditioning. Also, the amount of chocolate provided exceeded the estimated individual consumption, based on a pilot study using different subjects. It is also important to emphasize that feeding occurred voluntarily and in a familiar (physical and social) environment, and thus the animals were in a minimally stressful condition (Monclaro et al., 2014). Furthermore, for both groups, dwell time in the ‘conditioned’ compartment was the same in C1 vs. C6, which minimizes the possibility that changes in exposure over time and novelty may have confounded the observed CPP effect. The chocolate group did remain significantly longer at this location compared to controls, yet this may reflect the time they dedicated to forage for the food reward. In fact, pre-CPP rates did not differ between groups and the levels seen in the control animals remained constant throughout the study. Although chocolate-related rewards have induced a CPP response in rodents (Dickson et al., 2012; La Mela et al., 2010; Latagliata et al., 2010; Orsini et al., 2013; Schroeder et al., 2001; Ventura et al., 2008, 2007), this is to our knowledge the first report in a non-human primate. Marmosets have, nevertheless, been place-conditioned using other types of food rewards (Monclaro et al., 2014; Valentinuzzi et al., 2008). Chocolate’s orosensory properties (i.e., flavor, aroma and texture; reviewed in Parker et al., 2006), fat-sugar content and/or psychoactive constituents (Bruinsma and Taren, 1999; Parker et al., 2006) seem to contribute directly or via post-ingestive learning to its hedonic property. In fact, both ingestive and reward-associated neural circuits are activated (Bruinsma and Taren, 1999; Parker et al., 2006), the latter being via mechanisms that are putatively attributed to drug addiction (e.g., Volkow et al., 2013). Of particular interest was the persistence of the present CPP behavior. Even in the absence of additional training, the time spent inside/in contact with the chocolate-paired compartment 15 days after conditioning was not only significantly higher than pre-CPP rates, but also equivalent to the duration seen after only 24 h. A
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preference for specific stimuli, such as a location, may be acquired as a consequence of events that became associatively learned with those stimuli, influencing future behaviors. As these rewardassociated learned behaviors endure, they are commonly linked to episodes of craving and relapse (Tzschentke, 2007), particularly in the context of drug addiction (e.g., Mueller et al., 2002). However, data on the persistence of maladaptive memories for food-related stimuli are still scarce. In summary, this is the first study to report that chocolate induces a CPP response in a non-human primate. Interestingly, the effect persisted for at least 15 days, an aspect that to date has only been consistently demonstrated for drug-related rewards. Considering that the CPP paradigm in now being established in non-human primates (Barros et al., 2013; Monclaro et al., 2014; Valentinuzzi et al., 2008; Wang et al., 2012) and humans (Childs and de Wit, 2009), future studies comparing different food items and evaluating possible long-term effects may play a significant role in elucidating the neural mechanisms underlying the overconsumption of highly palatable foods items (like chocolate). Acknowledgements This study was supported by CNPq (478930/2012-7) and FAP-DF (193.000.408/2010). R.B.M.D. received a doctoral fellowship from CNPq and M.B. a researcher fellowship from CNPq (303331/20127). These funding sources had no involvement in the study design, data collection, analysis or interpretation, writing the manuscript or decision to submit it for publication. The authors would like to thank A.P. Souto Maior for constructing the marmoset CPP boxes and Drs. C. Dias and R. de Oliveira, as well as G.V. da Silva and A.G. de Araújo for their dedicated care of the animals. References Avena, N.M., 2010. The study of food addiction using animal models of binge eating. Appetite 55, 734–737. Barros, M., Dempster, E.L., Illott, N., Chabrawi, S., Maior, R.S., Tomaz, C., de Souza Silva, M.A., Huston, J.P., Mill, J., Müller, C.P., 2013. Decreased methylation of the NK3 receptor coding gene (TACR3) after cocaine-induced place preference in marmoset monkeys. Addict. Biol. 18, 452–454. Berthoud, H.R., 2006. Homeostatic and non-homeostatic pathways involved in the control of food intake and energy balance. Obesity 14, 197–200. Bruinsma, K., Taren, D.L., 1999. Chocolate: food or drug? J. Am. Diet Assoc. 99, 1249–1256.
Cheney, D.L., Wrangham, R.W., 1987. Predation. In: Smutts, B.B., Cheney, D.L., Seyfarth, R.M., Wrangham, R.W., Struhsaker, T.T. (Eds.), Primate Societies. University of Chicago Press, Chicago, pp. 227–239. Childs, E., de Wit, H., 2009. Amphetamine-induced place preference in humans. Biol. Psychiatry 65, 900–904. Dickson, S.L., Shirazi, R.H., Hansson, C., Bergquist, F., Nissbrandt, H., Skibicka, K.P., 2012. The glucagon-like peptide 1 (GLP-1) analogue, exendin-4, decreases the rewarding value of food: a new role for mesolimbic GLP-1 receptors. J. Neurosci. 32, 4812–4820. Huston, J.P., de Souza Silva, M.A., Topic, B., Müller, C.P., 2013. What’s conditioned in conditioned in place preference? Trends Pharmacol. Sci. 34, 162–166. Johnson, A.W., 2013. Eating beyond metabolic need: how environmental cues influence feeding behavior. Trends Neurosci. 36, 101–109. La Mela, I., Latagliata, E.C., Patrono, E., Puglisi-Allegra, S., Ventura, R., 2010. Olfactory priming reinstates extinguished chocolate-induced conditioned place preference. Appetite 54, 237–240. Latagliata, E.C., Patrono, E., Puglisi-Allegra, S., Ventura, R., 2010. Food seeking in spite of harmful consequences is under prefrontal cortical noradrenergic control. BMC Neurosci. 11, 1–15. Monclaro, A.V., Sampaio, A.C., Ribeiro, N.B., Barros, M., 2014. Time-of-day effect on a food-induced conditioned place preference task in monkeys. Behav. Brain Res. 259, 336–341. Mueller, D., Perdikaris, D., Stewart, J., 2002. Persistence and drug induced reinstatement of a morphine-induced conditioned place preference. Behav. Brain Res. 136, 389–397. Orsini, C., Bonito-Oliva, A., Montanari, C., Conversi, D., Cabib, S., 2013. Partial extinction of a conditioned context enhances preference for elements previously associated with cocaine but not with chocolate. Physiol. Behav. 120, 1–10. Parker, G., Parker, I., Brotchie, H., 2006. Mood state effects of chocolate. J. Affect. Disord. 92, 149–159. Rozin, P., Levine, E., Stoess, C., 1991. Chocolate craving and liking. Appetite 17, 199–212. Schroeder, B.E., Binzak, J.M., Kelley, A.E., 2001. A common profile of prefrontal cortical activation following exposure to nicotine- or chocolate associated contextual cues. Neuroscience 10, 535–545. Small, D.M., Zatorre, R.J., Dagher, A., Evans, A.C., Jones-Gotman, M., 2001. Changes in brain activity related to eating chocolate: from pleasure to aversion. Brain 124, 1720–1733. Tzschentke, T.M., 2007. Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade. Addict. Biol. 12, 227–462. Valentinuzzi, V.S., Neto, S.P., Carneiro, B.T., Santana, K.S., Araújo, J.F., Ralph, M.R., 2008. Memory for time of training modulates performance on a place conditioning task in marmosets. Neurobiol. Learn. Mem. 89, 604–607. Ventura, R., Morrone, C., Puglisi-Allegra, S., 2007. Prefrontal/accumbal catecholamine system determines motivational salience attribution to both rewardand aversion-related stimuli. Proc. Natl. Acad. Sci. U.S.A. 104, 5181–5186. Ventura, R., Latagliata, E.C., Morrone, C., La Mela, I., Puglisi-Allegra, S., 2008. Prefrontal norepinephrine determines attribution of high motivational salience. PLoS One 3, 1–10. Volkow, N.D., Wang, G.J., Tomasi, D., Baler, R.D., 2013. Obesity and addiction: neurobiological overlaps. Obes. Rev. 14, 2–18. Wang, J.H., Wu, X.J., Li, C.Y., Wei, J.K., Jiang, H.H., Liu, C.R., Yu, C., Carlson, S., Hu, X., Duan, W., Ma, Y., 2012. Effect of morphine on conditioned place preference in rhesus monkeys. Addict. Biol. 17, 539–546.
Contents lists available at ScienceDirect
Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Short report
Consumption of a highly palatable food induces a lasting place-conditioning memory in marmoset monkeys R.B.M. Duarte a , E. Patrono b,c , A.C. Borges e , A.A.S. César a , C. Tomaz a , R. Ventura c,d , A. Gasbarri b,c , S. Puglisi-Allegra c,d , M. Barros e,∗ a
Primate Center and Department of Physiological Sciences, Institute of Biology, University of Brasilia, CEP 70910-900 Brasilia, DF, Brazil Department of Applied Clinical and Biotechnological Sciences, University of L’Aquila, 67100 L’Aquila, Italy c Santa Lucia Foundation IRCCS, via del Fosso di Fiorano 65, 00143 Rome, Italy d Department of Psychology and Research Center “Daniel Bovet”, Sapienza University, via dei Marsi 78, 00185 Rome, Italy e Department of Pharmaceutical Sciences, School of Health Sciences, University of Brasilia, CEP 70910-900 Brasilia, DF, Brazil b
a r t i c l e
i n f o
Article history: Received 26 May 2014 Received in revised form 8 July 2014 Accepted 21 August 2014 Available online 28 August 2014 Keywords: Chocolate Food reward Long-term memory Marmoset Place-conditioning
a b s t r a c t Highly palatible foods may induce addiction-related behaviors. However, this has yet to be established in non-human primates. Therefore, we evaluated whether marmoset monkeys (Calllithrix penicillata) acquire a conditioned-place-preference (CPP) for chocolate and if this response is detectable after a 24h and 15-day period. Subjects were first habituated to a two-compartment CPP box and then randomly assigned to a chocolate or control group. Thereafter, they were given access to only one compartment during daily 15-min conditionings, held on six consecutive days. On each trial, the chocolate group received pieces of chocolate (50 g) in this context, whereas controls were not given a food reward. Marmosets were subsequently tested for preferring this (food) paired context after a 24-h and 15-day interval. During conditioning, individual foraging and the amount of chocolate ingested by each pair of the chocolate group remained constant. However, compared to pre-CPP levels, the time spent inside/in contact with the conditioned compartment increased significantly, while the latency to first entry decreased on both post-CPP intervals. For controls, the parameters remained unaltered. Thus, chocolate induced a persistent CPP response—an aspect usually associated with drug-related rewards. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The overconsumption of highly caloric palatable foods is a nonhomeostatic feeding pattern. It usually results in an energy intake that exceeds normal daily requirements and occurs when an individual is in a physiologically sated state (Berthoud, 2006). It is also commonly associated with the development of addictionrelated behaviors, eating disorders and obesity (Avena, 2010), with both learned and motivated processes being involved—including contextual cue-learning, mnemonic representation of different food items and their intrinsic reinforcing properties (Johnson, 2013). Chocolate, in particular, has an elevated hedonic rating (Bruinsma and Taren, 1999; Parker et al., 2006) and a prominent craving-inducing property (Rozin et al., 1991). In fact, similar to
∗ Corresponding author. Tel.: +55 61 31072002; fax: +55 61 31072002. E-mail address: [email protected] (M. Barros). http://dx.doi.org/10.1016/j.beproc.2014.08.021 0376-6357/© 2014 Elsevier B.V. All rights reserved.
reports using drugs of abuse (reviewed in Volkow et al., 2013), reward-processing neural circuits are activated when humans (Small et al., 2001) and rodents voluntarily consume chocolate (Latagliata et al., 2010; Ventura et al., 2008, 2007). In these animals, chocolate also induced a conditioned-place-preference (CPP) (Dickson et al., 2012; La Mela et al., 2010; Latagliata et al., 2010; Orsini et al., 2013; Schroeder et al., 2001; Ventura et al., 2008, 2007). This effect is an associatively learned preference for certain locations that is acquired in response to experiencing a rewarding agent at that same location (Huston et al., 2013; Tzschentke, 2007). The CPP paradigm has only recently been demonstrated in non-human primates using both drug- (Barros et al., 2013; Wang et al., 2012) and food-related stimuli (Monclaro et al., 2014; Valentinuzzi et al., 2008). However, the persistence of a food-induced CPP response has not been assessed, regardless of the species and food type. Here we evaluated whether marmoset monkeys acquire a conditioned preference for where they consumed a highly palatable food (i.e., chocolate), and if this response is detectable after a 24-h and 15-day period.
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2. Materials and methods 2.1. Subjects and housing conditions Fourteen adult captive black tufted-ear marmosets (C. penicillata; 7 males, 7 females) were used, weighing 250–450 g at the beginning of the study. They were stable pairs (n = 7) housed at the Primate Center of the University of Brasilia in cages (2 m × 1 m × 2 m each) of a same colony room. This room consisted of a semi-outdoor/indoor housing system with two parallel rows of 12 cages each, separated by a common wire-mesh enclosed central corridor. The animals were thus exposed to natural light, temperature and humidity conditions. Fresh food, consisting of a mixture of pieces of fruits and vegetables, was provided at 07:00 h and removed at 17:00 h. Boiled eggs, nuts and/or cooked chicken breast were given several times a week. Water and monkey chow were available ad libitum. Animals were not food or water deprived, except during specific trials (see Section 2.3). For further housing conditions see (Monclaro et al., 2014). These complied with the regulations of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA). 2.2. Apparatus Testing was conducted in a CPP box (60 cm × 30 cm × 30 cm) set on a 1.5 m high pedestal. One wall (60 cm × 30 cm) was made of 4 mm transparent glass and the remaining sides of aluminum. It was divided into two compartments (30 cm × 30 cm × 30 cm): one had smooth interior and exterior surfaces and was painted white, whereas the other had rough inner and outer surfaces and was painted black. The side wall of each compartment was a sliding door, acting as its entry/exit point. Thus, the white and black compartments could only be accessed from the left and right side of the box, respectively. Also, a stainless-steel bowl (9 cm in diameter) was placed in a hole on the floor of each compartment, located as far away as possible from its access door. Seven identical CPP boxes were used, each being placed in the middle of a home-cage as not to come in contact with anything. The glass wall faced the central corridor from where behaviors were observed and recorded. 2.3. Procedure The apparatus was placed in the home-cage and the pair was given 24-h access to both compartments during two consecutive days. On day 3, a 15-min observation trial (pre-CPP) was held and then both access doors were closed. During this phase a food reward was not provided. Each pair was then randomly assigned to the control (3 males, 3 females) or chocolate group (4 males, 4 females). Thereafter, subjects were only given access to one of the compartments during daily 15-min conditioning sessions held on six consecutive days (C1–C6). Trials were not performed in the opposite compartment. This is typically done to control for possible novelty-based preferences and (aversive) emotional effects of a drug-reward application procedure (Huston et al., 2013). As subjects were tested in their home-cages and had been priorly habituated to the CPP box, the latter had become part of the ‘background’ environment. An unbiased counterbalanced design was used, in which half of the pairs were arbitrarily conditioned to the white context and half to the black. Subjects from a same home-cage were conditioned simultaneously to the same compartment and their regular diet was removed 2h prior to each session. Before each trial, an experienced observer entered the home-cage and opened the door of the pre-established conditioned compartment. The door of the non-conditioned side remained closed at all times. For the chocolate group, 50 g of pieces of milk chocolate was placed in the food bowl. For the controls,
the same procedure was held, yet nothing was placed in the food bowl. This design aimed at isolating the influence of the chocolate consumption on the marmosets’ future behavior (chocolate group), while controlling for any possible changes due to repeated exposures to the test environment (control group). When the observer exited the home-cage, a 15-min trial began. At the end of this interval, the observer re-entered the home-cage, retrieved any leftover food item and closed the door of the conditioned compartment. Between trials, marmosets did not have access to either compartment. A test trial was held 24-h (T24h) and 15-days after (T15d) the last conditioning, with subjects once again being given 15-min access to both compartments in the absence of the food reward. Subjects were tested in random order and all sessions held between 13:30 and 17:00 h. Even though pairs were tested simultaneously, all behaviors were scored individually by one of two observers positioned in front of their home-cage. Observers had 95% inter-rater reliability and used the Anymaze software (Stoelting Co., USA) to score the following parameters during the pre-CPP, C1, C6, T24h and T15d trials: (1) frequency and duration inside/in contact with the top surface of each compartment; (2) latency to first entry in the conditioned compartment; and (3) individual foraging frequency and duration (search, manipulation and/or ingestion of the chocolate). The first parameter was scored as a single variable since, based on a similar study (Monclaro et al., 2014), all subjects consistently use the top of the open compartment as a continuum of its inside. Several aspects may contribute to this response, including having one of the highest rates of predation among primates (Cheney and Wrangham, 1987), an impaired visibility of the surroundings from within the compartment, and free transit throughout the home-cage. In addition, the amount of chocolate consumed by each pairmate (kcal) on trials C1 and C6 was calculated, based on the amount ingested in grams. Data from males and females in each group were pooled together as no significant differences in gender were observed. Data were analyzed using a mixed-design two-way ANOVA. For the habituation trials, ‘group’ was used as the independent factor and ‘compartment’ as the repeated measure variable, while for the remaining analyses ‘group’ and ‘trial’ were the independent and dependent variables, respectively. Whenever significant, subsequent comparisons were held using Tukey’s test for multiple pair-wise comparisons. All latency data were log transformed prior to analyses due to a non-normal distribution, yet results were reported as the median and interquartile range (IQR). For the chocolate group, individual foraging frequency and duration, as well as the amount of chocolate consumed (kcal/pair), were analyzed via paired t-test (C1 vs. C6). In addition, partial eta squared (2p ) and Cohen’s d for correlated samples (dz ) were used to determine effect size for the ANOVAs and t-tests, respectively. Significance level was set at p ≤ 0.05. All procedures were approved by the Animal Ethics Committee of the University of Brasilia and complied with the Brazilian National Council for Control of Animal Experimentation (CONCEA) and NIH guidelines for care and use of laboratory animals.
3. Results Pre-CPP, marmosets had no initial preference for either side of the apparatus (see supplementary data—Table S1). During conditioning, the time spent inside/in contact with the ‘conditioned’ compartment differed significantly between groups (F1,13 = 13.59, p = 0.003, 2p = 0.53), yet with no significant trial effect (C1 vs. C6) or group vs. trial interaction (F1,13 < 1.0, p = n.s.; trial: 2p = 0.06; interaction: 2p = 0.05; Table 1). For the chocolate group, individual foraging rates and the amount of food consumed by the pair (in kcal) did not differ between C1 and C6 (frequency:
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parameters remained unaltered throughout the study (Fig. 1). Furthermore, significant gender differences within each group were not observed for the parameters described above (data not shown).
4. Discussion
Fig. 1. Mean (+SEM) number of entries (top) and time spent (in seconds; middle) inside and/or in contact with the conditioned compartment of the CPP box, as well as the median (and interquartile range—IQR) for the latency to first entry (in seconds; bottom) in this same context by marmosets of the control (n = 6) and chocolate groups (n = 8) during the 15-min session after the initial habituation (pre-CPP) and on each of the two 15-min test trials held 24-h (T24h) and 15-days (T15d) after conditioning. *p < 0.05 control vs. chocolate; **p < 0.05 vs. pre-CPP trial of the chocolate group.
t7 = 1.68, p = 0.14, dz = 0.39; duration: t7 = 1.90, p = 0.10, dz = 0.68; consumption: t3 < 1.0, p = n.s., dz = 0.16; Table 1). Incidents of pairmate aggression or cooperative feeding behavior were rare. However, on both test trials held after conditioning (i.e., T24h and T15d), the time spent inside/in contact with the conditioned compartment increased significantly, yet only in the chocolate group compared to pre-CPP levels (trial effect: F2,13 = 4.62, p = 0.03, 2p = 0.28; group effect: F1,13 = 4.31, p = 0.06, 2p = 0.26; group × trial interaction: F2,13 = 5.55, p = 0.02, 2p = 0.32; Fig. 1). The latency to enter the conditioned compartment for the first time, on both post-CPP trials, also decreased in the chocolate group compared to pre-CPP levels, but not significantly (trial effect: F2,13 = 1.40, p = 0.27; 2p = 0.10; group effect: F1,13 < 1.0, p = n.s., 2p = 0.63; group × trial interaction: F2,13 = 1.32, p = 0.29, 2p = 0.10; Fig. 1). This same group had a significantly higher frequency inside/in contact with the conditioned compartment than controls (F1,13 = 10.92, p = 0.006, 2p = 0.48; Fig. 1). However, the latter was seen on all trials, with no significant group vs. trial interaction (F2,13 < 1.0, p = n.s., trial: 2p = 0.02, interaction: 2p = 0.01). For controls, who had not received food during conditioning, these
Table 1 Response (mean ± SEM) of each experimental group in the conditioned compartment of the CPP box during the first and last conditioning trials. Parameter
Group
Time spent (s) Time spent (s) Individual foraging frequency Individual foraging duration (s) Chocolate consumption (kcal/pair)
Control Chocolate Chocolate Chocolate Chocolate
a
p < 0.05 vs. the time spent by the control group.
Conditioning trial 1
6
70 ± 23 299 ± 63 a 8±2 206 ± 60 10 ± 2
67 ± 19 245 ± 40 a 5±1 118 ± 20 11 ± 4
This study showed that chocolate, repeatedly consumed by marmosets in a specific environment, induced a CPP response for this location. Only in the chocolate group did the time spent inside/in contact with the conditioned compartment increase postCPP, compared to pre-CPP levels. Also, after conditioning, the latency to enter the chocolate-paired context decreased compared to pre-CPP rates, although a significance level was not attained. The latter parameter has been viewed as a measure of an individual’s immediate response to this experimental setup (Monclaro et al., 2014; Valentinuzzi et al., 2008). This increase in dwell time was not accompanied by changes in frequency, which further corroborates the CPP response observed. However, the chocolate group entered/contacted this location more often than controls. Prior to the group assignment and chocolate conditioning, a general preference for either compartment was not detected. Following these events, group differences were seen throughout the study, even pre-CPP. Thus, a higher entry frequency may be inherent to the animals that were randomly assigned to the chocolate group, regardless of the CPP training. Throughout the chocolate conditioning, marmosets remained motivated for this food reward. Incidences of aggression or cooperative feeding behavior between pairmates were rare, and individual foraging rates and the amount of chocolate consumed by each pair remained unaltered (C1 vs. C6). The pairs tested formed stable groups and were not food-deprived prior to conditioning. Also, the amount of chocolate provided exceeded the estimated individual consumption, based on a pilot study using different subjects. It is also important to emphasize that feeding occurred voluntarily and in a familiar (physical and social) environment, and thus the animals were in a minimally stressful condition (Monclaro et al., 2014). Furthermore, for both groups, dwell time in the ‘conditioned’ compartment was the same in C1 vs. C6, which minimizes the possibility that changes in exposure over time and novelty may have confounded the observed CPP effect. The chocolate group did remain significantly longer at this location compared to controls, yet this may reflect the time they dedicated to forage for the food reward. In fact, pre-CPP rates did not differ between groups and the levels seen in the control animals remained constant throughout the study. Although chocolate-related rewards have induced a CPP response in rodents (Dickson et al., 2012; La Mela et al., 2010; Latagliata et al., 2010; Orsini et al., 2013; Schroeder et al., 2001; Ventura et al., 2008, 2007), this is to our knowledge the first report in a non-human primate. Marmosets have, nevertheless, been place-conditioned using other types of food rewards (Monclaro et al., 2014; Valentinuzzi et al., 2008). Chocolate’s orosensory properties (i.e., flavor, aroma and texture; reviewed in Parker et al., 2006), fat-sugar content and/or psychoactive constituents (Bruinsma and Taren, 1999; Parker et al., 2006) seem to contribute directly or via post-ingestive learning to its hedonic property. In fact, both ingestive and reward-associated neural circuits are activated (Bruinsma and Taren, 1999; Parker et al., 2006), the latter being via mechanisms that are putatively attributed to drug addiction (e.g., Volkow et al., 2013). Of particular interest was the persistence of the present CPP behavior. Even in the absence of additional training, the time spent inside/in contact with the chocolate-paired compartment 15 days after conditioning was not only significantly higher than pre-CPP rates, but also equivalent to the duration seen after only 24 h. A
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preference for specific stimuli, such as a location, may be acquired as a consequence of events that became associatively learned with those stimuli, influencing future behaviors. As these rewardassociated learned behaviors endure, they are commonly linked to episodes of craving and relapse (Tzschentke, 2007), particularly in the context of drug addiction (e.g., Mueller et al., 2002). However, data on the persistence of maladaptive memories for food-related stimuli are still scarce. In summary, this is the first study to report that chocolate induces a CPP response in a non-human primate. Interestingly, the effect persisted for at least 15 days, an aspect that to date has only been consistently demonstrated for drug-related rewards. Considering that the CPP paradigm in now being established in non-human primates (Barros et al., 2013; Monclaro et al., 2014; Valentinuzzi et al., 2008; Wang et al., 2012) and humans (Childs and de Wit, 2009), future studies comparing different food items and evaluating possible long-term effects may play a significant role in elucidating the neural mechanisms underlying the overconsumption of highly palatable foods items (like chocolate). Acknowledgements This study was supported by CNPq (478930/2012-7) and FAP-DF (193.000.408/2010). R.B.M.D. received a doctoral fellowship from CNPq and M.B. a researcher fellowship from CNPq (303331/20127). These funding sources had no involvement in the study design, data collection, analysis or interpretation, writing the manuscript or decision to submit it for publication. The authors would like to thank A.P. Souto Maior for constructing the marmoset CPP boxes and Drs. C. Dias and R. de Oliveira, as well as G.V. da Silva and A.G. de Araújo for their dedicated care of the animals. References Avena, N.M., 2010. The study of food addiction using animal models of binge eating. Appetite 55, 734–737. Barros, M., Dempster, E.L., Illott, N., Chabrawi, S., Maior, R.S., Tomaz, C., de Souza Silva, M.A., Huston, J.P., Mill, J., Müller, C.P., 2013. Decreased methylation of the NK3 receptor coding gene (TACR3) after cocaine-induced place preference in marmoset monkeys. Addict. Biol. 18, 452–454. Berthoud, H.R., 2006. Homeostatic and non-homeostatic pathways involved in the control of food intake and energy balance. Obesity 14, 197–200. Bruinsma, K., Taren, D.L., 1999. Chocolate: food or drug? J. Am. Diet Assoc. 99, 1249–1256.
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