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Does conspecific fighting yield conditioned taste aversion in rats?

The initially surprising finding of Lett and Grant (1996) that voluntary running in an activity wheel works as an effective agent to establish conditioned taste aversion (CTA) has now been well confirmed by subsequent studies (see Boakes & Nakajima, 2009, for a review). Because the correlation of a target taste and running is necessary to establish taste aversion, it has been considered a type of Pavlovian conditioning, with the taste as a conditioned stimulus (CS) and the running as an unconditioned stimulus (US). In addition, many major features of Pavlovian conditioning can be seen in running-based CTA. For example, running-based CTA follows the laws of US strength (Hayashi, Nakajima, Urushihara, & Imada, 2002; Masaki & Nakajima, 2006) and CS-US temporal contiguity (Hayashi et al., 2002). Furthermore, as in other Pavlovian conditioning preparations, running-based CTA is attenuated by CS preexposures (i.e., latent inhibition; Heth & Pierce, 2007; Satvat & Eikelboom, 2006; Sparkes, Grant, & Lett, 2003), US preexposures (Baysari & Boakes, 2004; Hughes & Boakes, 2008; Nakajima, Urata, & Ogawa, 2006; Salvy, Pierce, Heth, & Russell, 2002), degraded contingency (Nakajima, 2008), stimulus overshadowing (Nagaishi & Nakajima, 2010), and associative blocking (Pierce & Heth, 2010). Finally, backward inhibitory conditioning (i.e., conditioned taste preference) is possible by US-then-CS pairings (Hughes & Boakes, 2008; Salvy, Pierce, Heth, & Russell, 2004).

Despite a considerable literature on this relatively new phenomenon, its underlying physiological mechanism is still controversial. Among the several hypotheses that have been proposed for this running-based CTA, Nakajima, Hayashi, and Kato (2000) argued that energy expenditure caused by physical exercise is a critical factor. Nakajima et al. (2006) have also suggested that stress-induced physiological changes such as the elevation in plasma corticosterone levels may yield this running-based CTA. On the basis of these hypotheses, we speculated that not only running but also other stressful physical exercises should effectively function as USs for CTA. It has already been shown that a swimming US is capable of yielding CTA (e.g., Masaki & Nakajima, 2005, 2006; Nakajima & Masaki, 2004).

The goal of the present study was to behaviorally examine this hypothesis by using another stressful exercise to yield CTA. We chose conspecific fighting as a putative US, because confrontation of adult male rats is known to evoke a series of aggressive behaviors (Grant & Mackintosh, 1963; Koolhaas, Schuuman, & Wiepkema, 1980; Miczek & De Boer, 2005). Such antagonistic encounters also cause physiological stress, especially to the defeated or subordinate rats (Albonetti & Farabollini, 1994; Blanchard, McKittrick, & Blanchard, 2001; Caldwell, 2006; Koolhaas, Meerlo, De Boer, Strubbe, & Bohus, 1997; Martinez, Calvo-Torrent, & Pico-Alfonso, 1998). Thus, according to the aforementioned hypotheses, conspecific fighting should yield CTA in rats.

Experiment 1

In this experiment, we trained male rats in a simple conditioning paradigm with a single taste cue. After consuming saccharin solution, a group of rats was allowed to run for 30 min; rats of a second group encountered larger resident rats for the same duration. By comparing the saccharin intakes of these groups over the training period with those of rats in the third group, which received no special treatment after the daily saccharin consumption, we assessed the efficacy of running and fighting as USs for establishing CTA.


Subjects. The target subjects were 23 experimentally naive male Wistar rats that were purchased from a local supplier (Jbc:Wistar, Keari Co. Ltd., Osaka, Japan) at 8 weeks old and kept in our vivarium for 2 weeks before the experiment. Their mean weight was 340 g (range: 310-358 g) on the first conditioning day. The animals were individually housed in hanging wire-made home cages (20 x 25 x 19 cm) and maintained on an ad-lib food schedule. Water was removed on the day before the adaptation training. The vivarium was on a 12-hr light/12-hr dark cycle (lights on at 08:00) at 23 [degrees]C and 55% humidity. Each session started at 14:00. All rats were allowed to drink tap water for 15 min in the home cages 3.5 hr before the daily sessions throughout the experiment. This treatment properly set the rats' thirst level for monitoring the acquisition of CTA established by physical exercise.

An additional eight male Jbc:Wistar rats were individually housed in white polycarbonate tubs (30 x 36 x 18 cm, with pine-shaving bedding) on the shelves in the vivarium. These rats were 19 weeks old with a mean weight of 579 g (range: 535-626 g) on the first conditioning day. They were retired animals from an unrelated behavior study conducted two months prior to these studies. They lived in the tubs with free access to food and water in the wire tops for a week prior to the present experiment. The wire tops were replaced with perforated clear acrylic plates during confrontation with intruder subjects (see the following "Procedure" section).

Apparatus. A total of 23 replicas of the home cages served as drinking cages, which were placed on a table in a conventionally illuminated experimental room. Traveling time between the vivarium and the experimental room was around one minute. Fluid (tap water or 0.2% sodium saccharin solution) was provided from the cage ceiling via a bottle with a metal spout. The end of the spout was positioned 16.5 cm above the cage floor. On a wall of the same experimental room were eight activity wheels (15 cm wide, 30 cm in diameter). Each wheel had two side walls made of perforated metal sheets, and the running surface was made of 2-mm metal rods spaced 1 cm apart. A full turn of each wheel was counted automatically by a handcrafted system.

Procedure. On the First three clays of the experiment, all rats were trained to drink tap water for 15 min in the experimental room. They were then assigned to one of the following groups: run (n = 8), fight (n = 8), or control (n = 7), after matching them for their average water intake and body weight on the third day. On the next five days, each rat had access to the saccharin solution, which was provided in a bottle in the drinking cage, for 15 min. This was immediately followed by either a 30-min confinement to the wheel, a 30-min confrontation with a resident rat in one of the tubs, or a return to the home cage, depending on the assigned group. Each rat was trained in its own apparatus on all days; thus, the intruder--resident pairs were unchanged in the case of the fight group. Strong fighting occurred in all cases, but there was no bleeding or serious physical damage to the rats.

Although we did not measure physiological changes induced by fighting nor formally analyze the amount of fighting in this study due to equipment limitations, a pilot study with four resident--intruder pairs of Jbc:Wistar rats of age and weight similar to those of Experiment 1 illustrated that the mean ([+ or -] standard error) relative duration of summed major aggressive behaviors (threat, attack, and chase) was 7.1 [+ or -] 2.3% on average over five 30-min daily sessions. This value is comparable to the previously reported aggressive scores of rats showing physiological stress signs (e.g., Fokkema & Koolhaas, 1985; Fokkema, Smit, Van der Gugten, & Koolhaas, 1988), thus supporting the adequacy of our preparation for evoking conspecific fighting.

Results and Discussion

The amount of fluid intake was measured by weighing each bottle using an electric balance to the nearest 0.1 g, and results are significant at p < .050. As shown in Figure 1, the saccharin intake increased over the conditioning days in the control group, suggesting gradual release from neophobic reaction to the novel saccharin solution. Compared with this control group, saccharin intake was depressed in the run group, indicating the acquisition of conditioned saccharin aversion. It is unlikely that the obtained depression was caused by nonassociative processes, because our previous study that used the same saccharin CS and running US as employed here (Hayashi et al., 2002) showed that a CS-US correlation is necessary to yield this effect.


The data from the fight group were comparable to those of the control group. A 3 (group) x 5 (day) analysis of variance (ANOVA) yielded significant main effects of group, F(2, 20) = 7.61, p = .003, day, F(4, 80) = 17.61, p < .001, and, most importantly, their interaction, F(8, 80) = 7.07, p < .001. Subsequent analyses of the interaction revealed that the three groups significantly differed from the second day onward, Fs(2, 100) > 3.27, ps < .042. According to multiple-group comparisons with Ryan's procedure, the run group drank less than the control group on the second, fourth, and fifth days, ts(100) > 2.50, ps < .014, but there was no significant difference between the fight group and the control group on any conditioning days. In addition, the comparison between the run group and the fight group was significant on the third, fourth, and fifth conditioning days, ts(100) > 3.10, ps < .003. These results strongly imply that running, but not fighting, yielded CTA.

The mean numbers of wheel turns ([+ or -] standard error) of the run group were 141.6 [+ or -] 23.0, 174.0 [+ or -] 25.5, 162.3 [+ or -] 20.7, 162.1 [+ or -] 27.4, and 176.8 [+ or -] 16.4, respectively, for the five conditioning days.

Experiment 2

The possibility of fighting-based CTA was further explored in this study by employing a differential conditioning procedure with two taste cues: One was paired with confrontation with a conspecific, while the other was not. Another aim of this experiment was to use not only intruders but also resident rats as subjects for seeking evidence of fighting-based CTA.


Eight male subjects were chosen from among 48 Jbc: Wistar rats aged 12 weeks and individually kept in the vivarium. They were retired animals from an unrelated behavior study conducted one month prior to the present experiment. Half of them were selected as intruders, with a mean weight of 395 g (range: 381-408 g), on the first conditioning day, while the remaining were selected as residents, with a mean weight of 474 g (range: 465-485 g), on the corresponding day. The intruder--resident pairs were fixed throughout the experiment. The intruder rats were housed in the hanging cages, while the resident rats were moved to the white polycarbonate tubs with pine-shaving bedding three days before the experiment. These cages and tubs were identical to those employed in Experiment 1.

All experimental sessions, which started at 13:30, were administered in the vivarium. Rats were allowed a 15-min access to tap water 3.5 hr before the daily sessions. Food was freely available in the containers on the cage front or in the tub top, but it was removed during the periods of drinking and confrontation.

Water was removed on the day before the adaptation training. All rats were initially accustomed to drinking tap water from the bottles for 15 min for the first two days. Differential conditioning was initiated on the next day. Training began with a 15-min access to either a strawberry- or melon-flavored solution, which was prepared daily by mixing 24 g of artificial fruit juice powder (Matsuyama Confectionery, Nagoya, Japan) and 1 L of tap water. The sequence of solutions employed for 14 days of training was SMSMSMSMMSSMSM (S = strawberry, M = melon) for all rats. The target solution was strawberry flavored and the nontarget solution was melon flavored for two intruder--resident pairs, while the combinations were reversed for the remaining two pairs. Immediately after drinking the target solution, the intruders were individually introduced into the tubs of their partner residents, and they remained there for 30 min for the fighting exercise; the wire tops of the tubs were replaced with the acrylic plates during the confrontation. On the nontarget days, the rats were kept quietly in their own housing after the drinking period.

Notably, rats could easily distinguish between the flavored solutions employed here. A pilot experiment with another eight Wistar rats of similar age and background showed clear demonstration of running-based CTA with these solutions: The mean intakes ([+ or -] standard error) of target and nontarget solutions were 9.5 [+ or -] 1.6 g and 2.4 [+ or -] 0.4 g, respectively, at the end of the 10-day differential conditioning training (five blocks of two days each). This difference was statistically significant with a paired test, t(7) = 4.73, two-tailed p = .002.

Results and Discussion

The intake of solution by rats is shown in Figure 2 as seven blocks of two days each, with each block having one day each for target and nontarget training. There were no signs of differentiation of solution intake in the intruder rats or in the resident rats. A 2 (group) x 2 (target) x 7 (block) ANOVA yielded only a significant main effect of block, F(6, 36) = 20.71, p < .001, suggesting a gradual release from the neophobic reaction to the flavored solutions. All other main or interactive effects were not significant.


General Discussion

In the present study conspecific fighting did not yield an aversion to the taste consumed before the fighting, although wheel running successfully caused CTA as in the previous research (see Boakes & Nakajima, 2009, for a review of running-based CTA). Our attempts to demonstrate fighting-based CTA were unsuccessful both with a simple conditioning procedure (Experiment 1) and with a differential conditioning procedure (Experiment 2). These results question the validity of the two hypotheses proposed by Nakajima et al. (2000, 2006) regarding the mechanism underlying running-based CTA. According to these hypotheses, general physiological stress and/or energy expenditure caused by physical exercise should have yielded CTA. We therefore expected successful demonstration of fighting-based CTA in rats on the basis of these hypotheses. This was not the case.

Our failure to obtain fighting-based CTA can be explained by the associative overshadowing effect (e.g., Nagaishi & Nakajima, 2010), because the positions and movements of a rival rat are a much better and proximal predictor of stressful exercise than is the flavored solution consumed before confrontation. Therefore, a strong enemy-fight association may interfere with an otherwise possible taste-fight association in our preparation. If this were the case, the energy-expenditure and stress hypotheses would still be valid. However, it seems impossible to exclude all reliable predictors for fighting exercise from the confrontation situation. In any case, this study showed that conspecific fighting is not an efficient US for establishing CTA, at least with the conventional procedures.

One may argue that conspecific fighting is not suitable for yielding-CTA in rats because it is a part of the skin-defense system rather than the gut-defense system (Garcia, Lasiter, Bermudez-Rattoni, & Deems, 1985). Although such an argument seems plausible, it does not give any persuasive answers to the question of why rats have CTA based on running, which is also unlikely to be a part of the gut-defense system. This type of explanation will be difficult to evaluate until more is known about the physiological processes of running and fighting. At the moment, this explanation is little more than a restatement of the fact that the two types of physical exercises employed here are different in their ability to yield CTA.

It is noteworthy that Nakajima (2011) has also questioned the validity of the energy-expenditure hypothesis, because caloric energy supply did not alleviate running-based CTA in a series of experiments. Taken together with our failure to demonstrate fighting-based CTA, these results suggest that energy expenditure caused by exercise is neither necessary nor sufficient for yielding CTA. Thus, we must consider much better explanations of running-based CTA. For example, in a personal communication to Lett, Grant, Koh, and Parsons (1999), John Garcia claimed that the cause of running-based CTA is gastrointestinal discomfort induced by inhibition of stomach emptying by running. Despite the general observation that rats show no clear sign of illness after wheel running, Eccles, Kim, and O'Hare (2005) have demonstrated alleviation of running-based CTA by injecting granisetron (an anti-emetic drug) before the taste-running pairing. Furthermore, although wheel running can work as a positive reinforcer for instrumental behavior such as lever pressing (e.g., Kagan & Berkun, 1954; Iversen, 1993), it also functions as an unpleasant US to yield conditioned place aversion (Masaki & Nakajima, 2008). These results seem to support Garcia's gastrointestinal-discomfort hypothesis for running-based CTA, but further investigation is required before strong claims can be made.

This study was supported by the Japan Society for the Promotion of Science KAKENHI Grant C-21530779 to the first author.

We thank the undergraduate students of the first author's research seminar for helping with data collection in Experiment 1. We also appreciate the reviewers' helpful comments on the earlier version of this article.

This research project and the animal facility were approved by the Animal Care and Use Committee of KGU, based on the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences of the United States in 1996, as well as on Japanese law (the Act on Welfare and Management of Animals) and the guidelines published by the Science Council of Japan (Guidelines for Proper Conduct of Animal Experiments) in 2006.

Correspondence concerning this article should be addressed to Sadahiko Nakajima, Department of Psychological Science, Kwansei Gakuin University, Nishinomiya, 662-8501, Japan. E-mail:


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Sadahiko Nakajima, Gaku Kumazawa, Hayato Ieki, and Aya Hashimoto

Kwansei Gakuin University
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Author:Nakajima, Sadahiko; Kumazawa, Gaku; Ieki, Hayato; Hashimoto, Aya
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Date:Jan 1, 2012
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