Appetite 83 (2014) 178–184

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Research report

Running-based pica in rats. Evidence for the gastrointestinal discomfort hypothesis of running-based taste aversion ☆ Sadahiko Nakajima *, Tomomi Katayama Department of Psychological Science, Kwansei Gakuin University, Nishinomiya, 662-8501, Japan

A R T I C L E

I N F O

Article history: Received 18 July 2014 Received in revised form 18 August 2014 Accepted 23 August 2014 Available online 27 August 2014 Keywords: Conditioned taste aversion Wheel running Pica Kaolin Lithium chloride Rats

A B S T R A C T

Voluntary running in an activity wheel establishes aversion to paired taste in rats. A proposed mechanism underlying this taste aversion learning is gastrointestinal discomfort caused by running. We tested this hypothesis by measuring the pica behavior (kaolin clay intake) of rats, because it is known that rats engage in pica behavior after various nausea-inducing treatments including irradiation, motion sickness, and injection of emetic drugs such as lithium chloride (LiCl). Following a demonstration of the alreadyknown phenomenon of LiCl-based pica in Experiment 1, we successfully showed running-based pica behavior in Experiment 2 where the running treatment was compared with a non-running control treatment (i.e., confinement in a locked wheel). These results suggest that not only LiCl but also running induces nausea in rats, supporting the gastrointestinal discomfort hypothesis of running-based taste aversion learning. © 2014 Elsevier Ltd. All rights reserved.

Introduction Lett and Grant (1996) reported that voluntary running in an activity wheel establishes Pavlovian conditioned taste aversion (CTA) in laboratory rats to a substance consumed shortly before running. Running-based CTA has been replicated in their own and other researchers’ laboratories (e.g., Heth, Inglis, Russell, & Pierce, 2001; Lett, Grant, & Gaborko, 1998; Nakajima, Hayashi, & Kato, 2000), and subsequent studies have revealed many features of this relatively new Pavlovian conditioning phenomenon (see Boakes & Nakajima, 2009, for a review). One of the major hypotheses that addresses the underlying physiological mechanism of running-based CTA was proposed by John Garcia in a personal communication to Lett, Grant, Koh, and Parsons (1999), where he ascribed the physiological cause of this phenom-

☆ Acknowledgements: We thank Takefumi Kikusui for directing our attention to nausea-based pica behavior in rats. This study was supported by JSPS KAKENHI (24530931) to the first author, who designed the experiments and prepared the manuscript. The second author administered the experiment and collected the data in partial fulfillment of requirements for the BA degree in Psychology under the close supervision of the first author. The data were analyzed by the first author. This research project and the animal facility were approved by the Animal Care and Use Committee of KGU, based on a Japanese law (the Act on Welfare and Management of Animals) and the guideline published by the Science Council of Japan (the Guidelines for Proper Conduct of Animals Experiments) in 2006, which is shown in the following web site: http://www.scj.go.jp/ja/info/kohyo/pdf/kohyo-20-k16-2e.pdf * Corresponding author. E-mail address: [email protected] (S. Nakajima).

http://dx.doi.org/10.1016/j.appet.2014.08.031 0195-6663/© 2014 Elsevier Ltd. All rights reserved.

enon to gastrointestinal discomfort (e.g., nausea) induced by running. Now, we have at least three pieces of evidence for this hypothesis. First, Eccles, Kim, and O’Hare (2005) reported prevention of running-based CTA by an anti-emetic drug (granisetron) injection, suggesting that nausea plays a major role in establishing running-based taste aversion. Second, Nakajima, Urata, and Ogawa (2006) demonstrated that running-based CTA is alleviated not only by preexposure to running but also by prior injection of lithium chloride (LiCl), which is the most popular nausea-inducing drug in rat CTA studies (Riley & Freeman, 2004). This finding implies that a common process (presumably nausea) is physiologically habituated by preexposure. Third, by analyzing the microstructure of rats’ licking, Dwyer, Boakes, and Hayward (2008) measured the change in the palatability of the taste solution paired with running, LiCl, or a rewarding drug (amphetamine), and they found that a reduction in taste palatability accompanies running- and LiCl-based CTAs but not amphetamine-based CTA. They concluded that running- and LiCl-based CTAs are commonly caused by nausea, while the dopamine system plays a major role in amphetamine-based CTA. In the present study, we provide another piece of evidence for the gastrointestinal discomfort hypothesis of running-based CTA by measuring the pica behavior of rats. Almost 40 years ago, Mitchell and his colleagues found that geophagia (consumption of soil or kaolin clay) is generated in a majority of rats by a nausea-inducing drug (injection of LiCl or cyclophosphamide, or intragastric intubation of a rodenticide: Mitchell, Beatty, & Cox, 1977; Mitchell et al., 1976), or by rotation-induced motion sickness (Mitchell, Krusemark, & Hafner, 1977; Mitchell, Laycock, & Stephens, 1977). According to

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Mitchell et al. (1976), quantification of gastrointestinal discomfort in the rat is easily achieved when we use this aberrant pica behavior. Nausea-based pica behavior has been replicated by other researchers with a variety of emetogenic drugs including LiCl (e.g., McCutcheon, Ballard, & McCaffrey, 1992; Watson & Leitner, 1988), cyclophosphamide (e.g., Tohei, Kojima, Ikeda, Hokao, & Shinoda, 2011; Yamamoto, Nakai, Nohara, & Yamatodani, 2007), morphine (e.g., Aung, Mehendale, Xie, Moss, & Yuan, 2004), apomorphine (e.g., Takeda, Hasegawa, Morita, & Matsunaga, 1993; Takeda et al., 1995a), nicotine (Yamamoto, Ngan, Takeda, Yamatodani, & Rudd, 2004), copper sulfate (e.g., Hasegawa et al., 1992; Takeda et al., 1993), cisplatin (e.g., Liu, Malik, Sanger, Friedman, & Andrews, 2005; Takeda et al., 1993, 1995b), 2-deoxy-D-glucose (2DG: e.g., Watson & Leitner, 1988; Watson et al., 1987), ritonavir (e.g., Aung et al., 2005), cholecystokinin octapeptide (CCK-8: McCutcheon et al., 1992), actinomycin D (Yamamoto et al., 2007), 5-fluorouracil (Yamamoto et al., 2007), and ethanol (in a case of gavage administration: Constancio, Pereira-Derderian, Menani, & De Luca, 2011), as well as irradiation (e.g., Yamamoto, Takeda, & Yamatodani, 2002) and motion sickness (e.g., McCaffrey, 1985; Morita, Takeda, Kubo, & Matsunaga, 1988). Furthermore, pica caused by these agents is attenuated by antiemetic drugs. For example, Takeda et al. (1993) demonstrated that domperidone and ondansetron, respectively, inhibit apomorphineand cisplatin-based pica in rats. Notably, prevention of cisplatinbased pica has been extensively explored, because cisplatin is one of the most frequently used drugs in cancer chemotherapy. The list of drugs that have been shown to attenuate cisplatin-based pica includes ondansetron (Malik, Liu, Cole, Sanger, & Andrews, 2007; Takeda et al., 1993, 1995b), granisetron (Han et al., 2014; Yamamoto et al., 2014), diphenidol (Takeda et al., 1995b), thalidomide (Han et al., 2014), dexamethasone (Malik et al., 2007; Rudd, Yamamoto, Yamatodani, & Takeda, 2002), tachykinin NK1-receptor antagonists (HSP-117: Saeki et al., 2001; GR205171: Malik et al., 2007), several antioxidants (Sherma, Gupta, Kochupillai, Seth, & Gupta, 1997), and herbal medicines (Aung et al., 2003, 2005; Mehendale et al., 2004, 2005; Qian et al., 2011; Raghavendran et al., 2011; Wang et al., 2005). Pica caused by cyclophosphamide, morphine, apomorphine, ritonavir, irradiation, and motion sickness has also been attenuated by anti-emetics (e.g., Aung et al., 2004; Morita et al., 1988; Takeda et al., 1995a; Tohei et al., 2011; Yamamoto et al., 2002; Yuan et al., 2009). Therefore, we now have ample evidence to consider that pica behavior is a good tool for measuring nausea in rats. The present study, thus, uses pica as an index of nausea to test the hypothesis that running-based taste aversion learning is mediated by nausea. Because we had not conducted any research on pica behavior in rats, the first experiment of the present study aimed to replicate rats’ pica with a nausea-inducing drug (LiCl), before conducting the second experiment, which would explore whether running produces pica in rats. If pica is caused not only by LiCl injection but also by wheel running in similar settings in the same laboratory, we may then have a strong piece of evidence that LiCl and running share a similar physiological process (seemingly nausea) in taste aversion learning.

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ior in our laboratory, by emulating the techniques of previous reports on nausea-based kaolin intake in rats. As rats chew pellets into small pieces, we needed a couple of days to refine the procedure of collecting the kaolin splinters for measuring precisely the amount of kaolin intake. Method Subjects and apparatus Eight experimentally naïve male rats (Slc: Wistar/ST) were housed in individual hanging wire home cages (20 cm wide, 25 cm long, and 18.7 cm high) in a vivarium on a 16:8-h light–dark cycle (lights on at 0800 h) at 23 °C and 55% humidity. The animals were 9–10 weeks old on the first day of this experiment, and they were maintained with food pellets, tap water, and kaolin pellets available ad libitum throughout the experiment. The food pellets (MF diet; Oriental Yeast Co., Tokyo, Japan) were placed in a stainless container positioned inwards with its end apertures 3.5 cm above the cage floor. The tap water was accessible from a stainless needle-pin nozzle protruding through a hole in the center of the back wall of each cage. The kaolin pellets were made of kaolin powder (Shin Nihon Zokei Co., Tokyo, Japan) and gum arabic (Holbein Works, Ltd., Osaka, Japan) at a 99:1 (w/w) ratio; they were mixed with tap water to form cylindrical pellets and were completely dried at room temperature. Each day, three or four kaolin pellets (about 20–25 g in total) were presented to each rat in a stainless steel bowl (8 cm in diameter and 3.5 cm deep) clipped to the cage wall at floor level with an iron hoop holder. A plastic tray (22.5 cm wide, 32 cm long, and 5.5 cm deep) with paper bedding was positioned 10 cm below each cage to collect excreta, food shatters, and, most importantly, kaolin splinters. Split kaolin and crushed food were collected with a spoon and chopsticks, dried for a day, segregated, and weighed to obtain correct amounts of kaolin and food intake. Procedure Each day, the food and kaolin containers were removed at 1200 h, weighed with an electric balance (BJ-1500, Sartorius, K.K., Tokyo, Japan) to the nearest 0.1, refilled, and replaced at 1300 h. In short, we recorded the amounts of food and kaolin consumed in the preceding 23-h period every day. The rats were also weighed in the vivarium with an electric balance (KS-251, Dretec Co., Koshigaya, Japan) to the nearest 1 g between 1200 and 1300 h. After a six-day baseline phase, half of the rats were given an intraperitoneal (i.p.) injection of 0.15 M LiCl at 1% body weight (i.e., 63.6 mg/kg) while the remaining rats received physiological saline of the same amount. Three days later, the roles of the experimental and control treatments were exchanged: the formerly LiClinjected rats were now given an i.p. injection of saline, and vice versa. After an additional three baseline days, the group roles were changed again, but the dose of LiCl and saline was doubled to 2% body weight. Finally, three days later, the treatments were switched using the same dose, followed by two non-treatment days. All statistical analyses in this and the following experiments are based on an alpha level set at p < .050.

Experiment 1 Results and discussion The aim of Experiment 1 was to ensure nausea-based pica in our laboratory. We used LiCl as the nausea-inducing drug in this experiment, not only because it has been used in nausea-based pica studies (McCutcheon et al., 1992; Mitchell et al., 1976; Watson & Leitner, 1988; Yamamoto et al., 2004), but also because it is the most conventional emetogenic drug in rat taste aversion studies (see Riley & Freeman, 2004, for a database). A collateral aim of Experiment 1 was to set up a proper procedure for measuring rats’ pica behav-

Figure 1 illustrates the kaolin intake of the two groups of rats across days. The group names stand for the order of treatment: Group LiCl-Sal received LiCl and then saline, while Group Sal-LiCl was given saline and then LiCl. The data are shown from the fourth day of the initial baseline phase, because we needed three days to establish a procedure for precisely measuring the rats’ kaolin intake in our laboratory.

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Fig. 1. Mean amount of kaolin intake across the days of Experiment 1. Group LiClSal (n = 4) was given injections of 0.15 M LiCl at 1% body weight (bw), saline at 1% bw, 0.15 M LiCl at 2% bw, and saline at 2% bw, respectively, on the 6th, 9th, 12th, and 15th days. The order of the LiCl and saline injections was exchanged for Group Sal-LiCl (n = 4). The effect of the injection was expected to be reflected in the measurement of the next, hash-tagged, day. The error bars indicate the standard errors.

Figure 2 summarizes the kaolin intake on the critical days for assessing the effects of injection of 0.15 M LiCl or saline at 1% body weight (the 7th and 10th days) and at 2% body weight (the 13th and 16th days). A 2 (drug: LiCl vs. saline) × 2 (dose: 1% vs. 2%) analysis of variance (ANOVA) yielded significant main effects of the drug, F (1, 7) = 15.76, p = .005, dose, F (1, 7) = 14.63, p = .006, and their interaction, F (1, 7) = 7.21, p = .031. Subsequent simple main effect analyses of the interaction using separate error terms revealed that the effect of the drug was marginally, F (1, 7) = 5.49, p = .051, and highly, F (1, 7) = 18.68, p = .003, significant at 1% and 2% body weight, respectively. Furthermore, the effect of dose was significant in LiCl, F (1, 7) = 13.22, p = .008, and in saline, F (1, 7) = 10.39, p = .015. Notably, LiCl injection had no carryover effect on kaolin intake; a similar 2 × 2 ANOVA applied to the next days (i.e., the 8th, 11th, 14th, and 17th days) yielded no significant main or interactive effects, Fs (1, 7) < 3.73, ps > .094. Food intake was reduced by the LiCl injection. The average (±standard error, SE) food intakes on the critical days were as follows: 22.3 (±1.2), 24.7 (±0.9), 20.1 (±1.0), 25.1 (±0.6) g, respectively, for LiCl at 1% body weight, saline at 1% body weight, LiCl at 2% body weight, and saline at 2% body weight. Although a 2 (drug) × 2 (dose) ANOVA yielded a significant main effect of drug, F (1, 7) = 33.97, p < .001, the main effect of dose and drug × dose interaction failed to reach a level of significance, Fs (1, 7) < 2.97, ps > .128. The LiCl injection had no carryover effect on food intake over the next days; a similar 2 × 2

ANOVA yielded no significant main or interactive effects, Fs < 1, with an overall average (±SE) of 24.7 (±0.4) g. The rats’ body weights were unaffected by LiCl injection. The average (±SE) weights on the critical days were as follows: 349.9 (±4.5), 352.9 (±5.4), 371.3 (±5.3), and 372.5 (±5.0) g, respectively, for LiCl at 1% body weight, saline at 1% body weight, LiCl at 2% body weight, and saline at 2% body weight. A 2 (drug) × 2 (dose) ANOVA yielded a significant main effect of dose, F (1, 7) = 114.2, p < .001, but there was no significant main effect of drug or interaction, Fs < 1, indicating that the significant dose effect merely shows a developmental increase in the rats’ body weight over the test days. This was also the case for the body weights on the next days; the corresponding values were 356.1 (±4.8), 355.0 (±5.6), 374.5 (±5.0), and 374.3 (±5.5) g. A similar 2 × 2 ANOVA yielded a significant main effect of dose, F (1, 7) = 298.2, p < .001, and there was no significant main effect of drug or interaction, Fs < 1. This experiment successfully replicated previous studies demonstrating LiCl-based pica behavior in rats (McCutcheon et al., 1992; Mitchell et al., 1976; Watson & Leitner, 1988; Yamamoto et al., 2004). Furthermore, the effect was dose-dependent; the first LiCl administration (63.6 mg/kg) resulted in a marginally significant increase in kaolin intake, while the second administration (127.2 mg/kg) caused a statistically reliable increase. Although the order of the two dose administrations was not counterbalanced in the present experiment, we consider the observed dose-dependency to be genuine, because Yamamoto et al. (2004) reported using a between-group design that pica behavior was caused by an LiCl injection of 120 mg/ kg but not of 60 mg/kg. Watson and Leitner (1988) failed to demonstrate LiCl-based pica behavior with doses of 7.5–60 mg/kg in their first experiment, while a strong effect was observed with 120 mg/kg LiCl in the second experiment. Notably, McCutcheon et al. (1992) and Mitchell et al. (1976) demonstrated LiCl-based pica with a single dose of 127 mg/kg. Experiment 2 Having successfully demonstrated pica behavior with a typical nausea-inducing drug (LiCl) in Experiment 1, we are now ready to assess the gastrointestinal discomfort hypothesis of runningbased CTA by testing whether voluntary running in an activity wheel also generates pica behavior. In this experiment, the administration of 1-h wheel running was repeated for four successive days rather than a single episode of running, because running-based CTA is not substantial as compared to LiCl-based CTA (Masaki & Nakajima, 2006), suggesting nausea induced by running to be weaker than LiClinduced nausea. Method Subjects and apparatus A new set of eight experimentally naïve male rats (Slc:Wistar/ ST) was housed and maintained with ad libitum food, water, and kaolin, as in Experiment 1. The animals were 8–9 weeks old on the first day of this experiment. The rats were transferred, by a carrying cart having individual compartments, to a conventionally illuminated experimental room, which had eight holding cages (copies of the home cages) and eight hand-made activity wheels (see Nakajima, 2011, 2014, for details). A full turn of each wheel was counted automatically by a handcrafted system. Half of the wheels were freely mobile, while the remaining wheels were fixed to the wall net (hereafter referred to as “locked wheels”).

Fig. 2. Mean amount of kaolin intake on the critical days of Experiment 1, shown as functions of the drug (0.15 M LiCl vs. saline) and dose (1% vs. 2% body weight, bw). The data of the hash-tagged days shown in Fig. 1 are summarized here, by collapsing the counterbalancing group factor. The error bars indicate the standard errors.

Procedure Due to the experimenter’s schedule, the daily procedure was administered 2 h earlier than that in Experiment 1. At 1000 h of each

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Fig. 3. Mean amount of kaolin intake across the days of Experiment 2. Group Run-noRun (n = 4) was allowed to run in activity wheels for 1 h in the first treatment phase and confined in locked wheels for 1 h in the second treatment phase. The order of these treatment phases was exchanged for Group noRun-Run (n = 4). The effect of the treatment was expected to be reflected in the measurement of the following, hash-tagged, days. The error bars indicate the standard errors.

day, all rats were moved to the experimental room, where they were weighed with an electric balance (KS-251, Dretec Co., Koshigaya, Japan) to the nearest 1 g and then given the following treatment. On the initial five baseline days, the rats were confined in the individual holding cages for 1 h. On the next four days, half of the rats were allowed to run in the activity wheels for 1 h while the remaining rats were confined in the locked wheels for the same duration. After returning to the baseline treatment for four days, the running treatment was reinstated for four days with the roles of the two groups exchanged: the formerly running rats were now confined in the locked wheels, and vice versa. The experiment ended with additional four-day baseline treatment. As in Experiment 1, the amounts of food and kaolin consumed in the 23-h period were recorded every day in this experiment. This was conducted by removing the food and kaolin containers at 1000 h, when the rats were moved to the experimental room. The containers were weighed with an electric balance (BJ-1500, Sartorius, K.K., Tokyo, Japan) to the nearest 0.1, refilled, and replaced at 1100 h, when the rats were returned to the home cages. As in Experiment 1, split kaolin and crushed food were collected, dried, segregated, and weighed to obtain the correct amounts of kaolin and food intakes. Results and discussion Figure 3 illustrates the kaolin intake of the two groups of rats across the days. The group names stand for the order of experimental conditions; namely, Group Run-noRun was allowed to run in the first phase of wheel treatment and confined to the locked wheels in the second phase of wheel treatment, while the order of these treatments was reversed for Group noRun-Run. Figure 4 summarizes the kaolin intake generated by four wheel days for each experimental condition. The data of the preceding day and the two following days are also shown. The rats consumed equivalently low amounts of kaolin before the differential wheel treatments (the “pre” data in Fig. 4), paired t < 1. However, a 2 (condition: running vs. no-running) × 4 (day) ANOVA applied to the data on the wheel treatment days yielded significant main effects of condition, F (1, 28) = 15.81, p = .005, day, F (3, 21) = 6.16, p = .004, and their interaction, F (3, 21) = 6.91, p = .002. Subsequent simple main effect analyses of the interaction using separate error terms showed that the effect of condition was statistically significant on all days: F (1, 7) = 6.94, p = .034, F (1, 7) = 6.16, p = .042, F (1, 7) = 14.92, p = .006, and (1, 7) = 11.37, p = .012, respectively, for the 1st, 2nd, 3rd, and 4th test days. Furthermore, the effect of day was reliable only in the running condition, F (3, 42) = 13.0, p < .001, but not in the norunning condition, F < 1. The effect of running on kaolin intake was carried over after the running treatment (the “post-1” data in Fig. 4),

as supported by a statistically reliable difference between the two conditions, paired t (7) = 3.10, two-tailed p = .017; namely, the identical baseline treatment after the wheel phase resulted in differential amounts of kaolin intake, depending on the preceding running or no-running treatment. This difference, however, vanished on the second day (the “post-2” data in Fig. 4), paired t < 1. A similar 2 × 4 ANOVA applied to food intake revealed that the rats consumed the food pellets slightly, but significantly, less in the running condition than in the no-running condition, F (1, 7) = 14.7, p = .006, and food intake significantly decreased over the four days, F (3, 21) = 6.35, p = .003, from 23.1 (±0.8) to 22.0 (±0.7) g for the running condition and from 24.6 (±0.7) to 22.9 (±0.5) g for the norunning condition. The condition × day interaction was not significant, F < 1. There were no significant differences between the conditions on the preceding one and following two baseline days, paired ts (7) < 2.09, two-tailed ps > .075. The average (±SE) body weights of the rats on the four critical treatment days were as follows: 340.0 (±7.0), 342.4 (±6.9), 345.4 (±6.5), and 349.0 (±6.4) g, respectively, for the running rats and 336.9 (±8.7), 341.8 (±8.6), 345.8 (±8.1), and 351.1 (±8.5) g, respectively, for the no-running rats. A 2 × 4 ANOVA suggested that the body weights were unaffected by the two conditions, F < 1, but that they increased over the days, F (3, 21) = 64.7, p < .001. The condition × day interaction was not significant, F < 1. There were no significant

Fig. 4. Mean amount of kaolin intake on the critical days of Experiment 2, shown as functions of the treatment (running vs. no running) and successive days. The data of the hash-tagged days shown in Fig. 2 are summarized here, by collapsing the counterbalancing group factor. The digits (1–4) indicate the days after the beginning of the treatment, and the data of the preceding day and the two following days are also shown as pre, post-1, and post-2, respectively. The error bars indicate the standard errors. The two data points are overlapped at pre.

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differences between the conditions on the preceding one and following two baseline days, paired ts < 1. The average (±SE) number of wheel turns of the four running days was 157.8 (±22.0), 184.8 (±21.1), 168.5 (±26.2), and 194.0 (±37.0) from the first to the fourth days. A one-way ANOVA yielded no significant day effect, F < 1. There was no significant correlation between the number of wheel turns and the amount of kaolin intake on the critical days. For example, the product-moment correlation coefficient (Pearson’s r) between the cumulative number of wheel turns and the cumulative amount of kaolin intake of the critical days was –.12. General discussion The present study clearly showed that voluntary wheel running in an activity wheel, as well as LiCl injection, generates pica behavior in laboratory rats. LiCl-based pica, which was demonstrated in our Experiment 1, has already been reported in the literature where it is assumed that nausea mediates between LiCl injection and pica (McCutcheon et al., 1992; Mitchell et al., 1976; Watson & Leitner, 1988; Yamamoto et al., 2004). Furthermore, our rats engaged in pica behavior in proportion to the amount of LiCl administration as noted in the previous reports (Watson & Leitner, 1988; Yamamoto et al., 2004), although we have to qualify this point because our procedure was not ideal for arguing for it strongly. (See the Results and discussion section of Experiment 1 for further discussion on this issue.) A novel contribution of the present study is the demonstration of running-based pica behavior in rats (Experiment 2). Although running had only a small, but statistically reliable, effect on kaolin intake on the initial two running days, we observed a large increase in kaolin intake on the following two days. Given the ample literature indicating that pica is a reliable behavioral index of rats’ nausea (as reviewed in the introduction of this article), a straightforward conclusion to draw from the results of Experiment 2 is that wheel running induces nausea in rats. This is a novel piece of evidence supporting the hypothesis proposed by Garcia (Lett et al., 1999) that gastrointestinal discomfort is the underlying physiological cause of running-based CTA. A few features of the results of Experiment 2 merit special mention. First, as aforementioned, the intake of kaolin increased gradually over the four-day running phase, implying that the observed pica is a product of the cumulative effect of wheel running. Second, pica behavior was weakly but significantly carried over to the next day (the post-1 data in Fig. 4); namely, the baseline treatment of holding rats in the experimental room evoked some kaolin intake during the next 23-h period in the home cages if the baseline treatment had been preceded by the four-day running phase. These two features might indicate that at least a part of pica behavior is a conditioned response to the experimental room. Masaki and Nakajima (2008) have reported that wheel running endows conditioned aversion to the place of exposure shortly before running, and Grant et al. (2012) have shown that running also produces conditioned disgust reactions (gapes) to the paired chamber. Thus, our rats might have felt malaise when re-entering the experimental room, resulting in the enhanced kaolin intake during the next 23-h period in the home cages. This conditioned malaise would have been added to the unconditioned nausea to produce the cumulative running effect on the later days of the running phase, and it would also have evoked some kaolin intake without running on the day after the wheel treatment phase. In this scenario, the null effect on kaolin intake after the second post-running day suggests extinction of conditioned malaise by exposing the rats to the experimental room without running on the first post-running day. Although this account of the cumulative and carryover effects of running on kaolin intake seems plausible, one shortcoming is that

our rats had been exposed to the experimental room for several days without any event before the first running treatment. Because preexposure to a to-be-conditioned stimulus conventionally retards subsequent establishment of conditioned responses in general (Lubow, 1989) as well as in running-based CTA preparations (Heth & Pierce, 2007; Satvat & Eikelboom, 2006; Sparkes, Grant, & Lett, 2003), repeated preexposure to the experimental room would retard establishment of conditioned malaise in rats. Further research is needed to address this shortcoming of the conditioned malaise account of the cumulative and carryover effects of running on kaolin intake. In Experiment 2, wheel running induced not only kaolin intake but also a slight reduction in food intake. This was also the case in Experiment 1 with a LiCl injection. The null effect of LiCl on food intake has been reported by Watson and Leitner (1988) and Yamamoto et al. (2004), but McCutcheon et al. (1992) found a sizable reduction with liquid food. In addition, with solid food pellets, cyclophosphamide (Mitchell et al., 1976; Tohei et al., 2011; Yamamoto et al., 2007), cisplatin (De Jonghe & Horn, 2008; De Jonghe, Lawler, Horn, & Tordoff, 2009; Horn, De Jonghe, Matyas, & Norgren, 2009; Liu et al., 2005; Yamamoto et al., 2004, 2007), nicotine (Yamamoto et al., 2004), irradiation (e.g., Yamamoto et al., 2002), and motion sickness (e.g., Mitchell, Laycock et al., 1977; but see Mitchell, Krusemark et al., 1977) result in reduction in food intake with a dose that causes pica behavior. These studies, taken together, suggest that “pica and anorexia are complementary but independent behaviors” (Mitchell et al., 1976, p. 696) in rats. Another possible account of the reduction in food intake by running is activity-based anorexia (Boakes, 2007; Epling & Pierce, 1996; Pierce & Epling, 1994), although the duration of daily running employed in the present research (i.e., 1 h) was much shorter than the conventional procedure of activity-based anorexia, where rats are confined in a wheel for 23 h per day. Notably, the amount of wheel running had no significant correlation with the amount of kaolin intake in the present research. This might be partly due to the small sample size employed here (n = 8), but readers should be aware of a recent study from our laboratory (Nakajima, 2014) that revealed that the strength of runningbased CTA is also not easily predicted by the amount of running, unless the latter is subject to experimental manipulation as conducted by Masaki and Nakajima (2006). Presumably, the degree of nausea is not a simple linear function of wheel running, or individual differences in sensitivity to the effect of running are so large as to mask a simple linear running–nausea relationship. In any case, the running-based pica demonstrated in this research provides a new support for the gastrointestinal discomfort hypothesis proposed by Garcia (Lett et al., 1999) in explaining running-based CTA. It is worth briefly discussing here other hypotheses proposed for running-based CTA. Nakajima et al. (2000) suggested the possibility that energy expenditure caused by running is a critical factor in running-based CTA, and according to this hypothesis, Nakajima and Masaki (2004) successfully demonstrated CTA based on swimming, which is another energy-exhausting behavior (also see Nakajima, 2004; Masaki & Nakajima, 2004a, 2004b, 2005, 2006, 2010, for replications and extensions). However, recent two pieces of evidence do not support this hypothesis. First, conspecific fighting, which is also highly energy-exhausting, is not effective in establishing CTA (Nakajima, Kumazawa, Ieki, & Hashimoto, 2012). Second, energy supply (calories from sugar), which would compensate for the energy expended by running, does not alleviate running-based CTA (Nakajima, 2011). The present research also illuminates weaknesses of the energy expenditure hypothesis, because it cannot explicitly predict running-based pica in rats. Lett and Grant (1996) speculated, in the original research on running-based CTA, that the mesolimbic dopamine system plays a

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major role in this type of CTA. It is well documented that physical exercise influences the central dopaminergic as well as the noradrenergic and serotonergic systems (see Meeusen & Piacentini, 2001, for a review). The mesolimbic dopamine hypothesis argues that running acts, like rewarding drugs such as amphetamine and morphine, on this brain area. As noted in the introduction of this article, however, microstructure analysis of taste-licking behavior conducted by Dwyer et al. (2008) has revealed that runningbased CTA differs from amphetamine-based CTA in the palatability of conditioned taste, implying that these aversions do not share a common underlying physiological mechanism. Forristall, Hookey, and Grant (2007) argued that motion sickness induced by the collateral back-and-forth “rocking” movements of a wheel results in nausea in rats, and their argument is corroborated by a recent study that carefully manipulated the rocking movements (Grant et al., 2012). Wheel running with rocking movements produced not only conditioned avoidance (i.e., reduction in intake of a target taste solution) but also conditioned disgust (i.e., rejective reactions of “gaping”), while running without rocking movements produced only conditioned taste avoidance. This study, thus, implies that conventional running-based CTA has two underlying physiological processes: (1) gastrointestinal discomfort induced by the rocking movements of the wheel, and (2) mesolimbic dopamine activation induced by running itself. The former process is akin to the mechanism working in CTA (avoidance and disgust) based on nausea-inducing drugs, while the latter is similar to that of CTA (avoidance only) established by rewarding drugs (Parker, 1995; Parker, Limebeer, & Rana, 2009). Although further evidence is needed to ensure these conclusions, this two-process approach merits scrutiny. It is worth remembering here that effective emetogenic agents causing pica behavior include rewarding drugs such as morphine (e.g., Aung et al., 2004; Hasegawa et al., 1992), apomorphine (e.g., Takeda et al., 1993, 1995a), nicotine (Yamamoto et al., 2004), and ethanol (Constancio et al., 2011). Hasegawa et al. (1992) have argued that rewarding and non-rewarding emetogenic agents act on a common locus of nausea (presumably the emetic center in the brain stem), because they found significant positive correlations in individual susceptibility to pica behavior based on these agents. However, the finding of Horn et al. (2009) that lesion of the lateral parabrachial nucleus has no effect on apomorphine-based pica but enhances cisplatin-based pica suggests disparate neural mechanisms for pica caused by rewarding and non-rewarding emetogenic agents. According to De Jonghe et al. (2009), pica is an adaptive response to dietary toxin, because kaolin clay absorbs toxin (see Dominy, Davoust, & Minekus, 2004, for support of this idea in an artificial human intestinal model) and prevents diarrhea (see Beck, Jenkins, Thurber, & Ambrus, 1977, for support in monkey subjects). What then is the function (or adaptive role) of pica in running rats? Our tentative answer to this question is that it has no actual function: intestinal discomfort induced by running is fortuitously similar to poison-induced nausea, and it thus evokes innate behavior of kaolin clay intake. Further research on running-based pica, however, might reveal its adaptive function. For example, a finer time-course analysis of pica using an automatic monitoring system (Yamamoto, Asano, Matsukawa, Imaizumi, & Yamatodani, 2011) might be helpful for tracking the internal state of rats. As noted in the introduction of this article, many studies have shown that pica behavior is attenuated by anti-emetic drugs. Thus, the effect of anti-emetic drugs on running-based pica will be a target of future investigation. Future research should also include testing of whether other types of activity such as swimming produce pica behavior in rats. Another research question to be investigated is whether forced running in motorized wheels yields pica behavior in rats. According to Forristall et al. (2007), forced running

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produces taste avoidance but not taste disgust, because it lacks rocking movements to induce motion sickness (nausea). If this is the case, forced running should not yield pica behavior. The present research serves as an initial step in examining these intriguing topics.

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Running-based pica in rats. Evidence for the gastrointestinal discomfort hypothesis of running-based taste aversion.

Voluntary running in an activity wheel establishes aversion to paired taste in rats. A proposed mechanism underlying this taste aversion learning is g...
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