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Taste aversion learning induced by delayed swimming activity
Behavioural Processes 67 (2004) 357–362
Taste aversion learning induced by delayed swimming activity Takahisa Masaki, Sadahiko Nakajima∗ Department of Integrated Psychological Science, Kwansei Gakuin University, Nishinomiya 662-8501, Japan Received 16 January 2004; accepted 22 June 2004
Abstract The experiment reported here demonstrated that forced swimming endows rats with aversion to a taste solution consumed 30 min before the swimming. The experimental rats were allowed to drink 0.2% sodium saccharin solution, which was followed by a 30-min empty interval, and then a 20-min swimming opportunity in water. Compared with the control rats, which were returned to their home cages after drinking the saccharin, the experimental rats drank a small amount of saccharin solution both in the later sessions of one-bottle training and in the subsequent two-bottle choice (saccharin versus tap water) testing. The delayed swimming procedure was as effective as an immediate swimming procedure, extending the generality of the swimming-induced taste aversion, which we recently discovered with the immediate swimming procedure. © 2004 Elsevier B.V. All rights reserved. Keywords: Taste aversion learning; Conditioned taste aversion; Delayed learning; Swimming; Exercise; Rat
1. Introduction Lett and Grant (1996) discovered that voluntary running in an activity wheel endows rats with aversion to a taste solution consumed before the running. This finding has been successfully replicated not only in their laboratory (Lett et al., 1998, 2001; Sparkes et al., 2003) but also by other researchers (Hayashi et al., 2002; Heth et al., 2001; Nakajima et al., 2000; Salvy et al., 2002, 2003). Procedural details of these studies (e.g., rat strains, taste stimuli, durations of running, numbers of taste-running pairing trials, deprivation levels) differed ∗ Corresponding author. Tel.: +81 798 546076; fax: +81 798 546076. E-mail address: [email protected] (S. Nakajima).
0376-6357/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2004.06.005
from one another, indicating the robustness of the phenomenon. Because a correlation of taste and running is necessary for this phenomenon, it has been discussed in the framework of Pavlovian conditioning. In other words, associative or conditioned taste aversion learning is established with taste as the conditioned stimulus (CS) and running as the unconditioned stimulus (US). In a recent series of experiments we found that forced swimming also works as a US for conditioned taste aversion (Masaki and Nakajima, submitted for publication; Masaki and Nakajima, in press; Nakajima and Masaki, 2004). Immersion in water pools immediately after drinking a taste solution endowed a group of rats with aversion to that solution. The aversion was associative, because these rats drank the
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T. Masaki, S. Nakajima / Behavioural Processes 67 (2004) 357–362
solution less than did the control rats, which had received a taste–swimming unpaired treatment. Furthermore, a within-subject comparison of paired and unpaired taste solutions demonstrated clear differential intakes: rats began to drink less of the former than of the latter. Another experiment also revealed that swimming activity in water rather than being wet is critical in obtaining the taste aversion. The swimming-induced taste aversion also follows the Pavlovian law of US strength, because the aversion was a positive function of the length of water immersion. These experiments strongly demonstrated that forced swimming after drinking of a target taste induces aversion to that taste, but the finding is novel and calls for further replication with modified procedures to extend the generality of the finding. In all of the experiments we reported, water immersion immediately followed taste drinking. However, it is widely known that taste aversion is established in rats with a delayed US when the US is any of a variety of chemical substances (e.g., Andrews and Braveman, 1975; Barker and Smith, 1974; Etscorn and Stephens, 1973; Garcia et al., 1966; Nachman, 1970; Schafe et al., 1995; Wright et al., 1971), irradiation (e.g., Barker and Smith, 1974; McLaurin, 1964; Smith and Roll, 1967), motion sickness (Green and Rachlin, 1976; Haroutunian and Riccio, 1975), a poisoned conspecific (Coombes et al., 1980), and voluntary wheel running (Hayashi et al., 2002). In the experiment reported below, we explored whether forced swimming could be established with a 30-min empty interval between the end of taste drinking and the onset of water immersion.
2. Methods 2.1. Subjects The subjects were 24 experimentally na¨ıve male Wistar rats of 60 days old with a mean weight of 321 g (range: 297–338 g) on the day before the water deprivation schedule began. The animals were housed in individual hanging home cages on a 12 h light:12 h dark cycle (lights on at 08:00 h) at about 23 ◦ C and maintained on an ad-lib food schedule. Watering in the home cages was limited to 30 min/day 2 days before the beginning of the adaptation training. Thereafter, the rats were able to drink water for 15 min in their home cages
90 min after experimental sessions of 15-min fluid intake. 2.2. Apparatus The experimental sessions were carried out in a conventionally illuminated room where eight drinking cages and eight swimming pools were located. The drinking cages were copies of the home cages and the inner dimension of each was 20 cm wide, 25 cm long, and 18.7 cm high, and fluid was provided via a plastic bottle with a metal spout inserted from the cage ceiling. The end of the spout was 16.5 cm above the cage floor. When two bottles were used, they were separated 8 cm apart. The swim pools were blue–gray plastic garbage containers, and the inner dimension of each was 48 cm high and 34 cm in diameter on the bottom and 43 cm in diameter at the top. Tap water of room temperature was filled up to a height of 36 cm. The pools were cleaned after each session, so that each rat swam in fresh water without being affected by any fecal or urinary materials of other rats. 2.3. Training All experimental sessions were conducted at the same time (around the middle of the lighted condition) of successive days. Each rat was initially adapted to drinking tap water from a bottle for 15 min/day. After 4-day adaptation training, the rats were assigned to one of three groups of equal number (each n = 8) matched for their amount of water intake and bodyweights. Conditioning was then conducted for 8 days. Rats of Group 0-min were allowed to drink 0.2% sodium saccharin solution, immediately followed by a 20-min water immersion. Rats of Group 30-min were given identical treatment with the exception that they spent 30 min in the drinking cages without bottles between the end of saccharin drinking and the onset of water immersion. Rats of Group Control were returned to their home cages immediately after the saccharin drinking (no immersion). 2.4. Testing All rats were tested on the following 20 days. On each day, each rat was offered two bottles, one containing saccharin solution and the other containing tap water, concurrently for 15 min. The left–right po-
T. Masaki, S. Nakajima / Behavioural Processes 67 (2004) 357–362
sitions of bottles were interchanged across rats and days. 2.5. Analysis The amount of fluid intake was measured by weighing each bottle before and after the drinking period using an electric balance to the nearest 0.1 g. All statistical decisions were based on the alpha level of 0.05 or less.
3. Results Fig. 1 presents the mean saccharin intake during the conditioning phase. Although Group Control increased their saccharin intake over the 8 days, the other groups kept the initial intake levels. A 3 (group) × 8 (day) analysis of variance (ANOVA) applied to the data illustrated in Fig. 1 yielded significant main effects of group, F(2,21) = 4.98, P < 0.05, and day, F(7,147) = 6.15, P < 0.001, and their interaction, F(14,147) = 2.01, P < 0.05. Subsequent Newman–Keuls tests applied to the group effect revealed that Group Control drank the solution more than Groups 0-min and 30-min (P’s < 0.05). Furthermore, the tests for the group × day interaction revealed that Group Control drank more than
Fig. 1. Mean amount of saccharin (0.2% in water) intake during onebottle training. On each day, an access to the saccharin solution was followed by returning to their home cages for Group Control, an immediate swimming for Group 0-min, or a delayed swimming for Group 30-min. Error bars indicate the standard errors.
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both Groups 0-min and 30-min on the 4th, 6th, and 7th days (P’s < 0.05). Group Control also drank more than Group 0-min on the 5th and 8th days (P’s < 0.05). Groups 0-min and 30-min did not differ from each other on any day. These results suggest that moderate aversion induced by immediate or delayed swimming prevents normal development of saccharin preference by repeated exposure. Fig. 2 shows the results of the choice preference test over 20 days. The top panel depicts the mean saccharin and water intakes, and the bottom panel presents the mean saccharin preference ratios in the form of x/(x + y), where x is saccharin intake and y is water intake. The lower the ratio value, the stronger the saccharin aversion estimated. As in the training phase, Group Control drank more saccharin than Groups 0-min and 30-min in the testing, indicating conditioned saccharin aversion induced by immediate or delayed swimming. A 3 (group) × 2 (fluid) × 20 (day) ANOVA applied to the data illustrated in the top panel of Fig. 2 yielded significant main effects of group, F(2,21) = 4.22, P < 0.05, and day, F(19,399) = 7.80, P < 0.001, and first-order interactions of group × day, F(38,399) = 2.12, P < 0.001, and fluid × day, F(19,399) = 2.38, P < 0.01. Most importantly, the interaction of group × fluid was significant, F(2,21) = 8.12, P < 0.01, supporting conditioned saccharin aversion in Groups 0-min and 30-min but not in Group Control. The main effect of fluid, F < 1, and the second-order interaction of group × fluid × day, F(38,399) = 1.12, were nonsignificant. Post hoc analyses of the group × fluid interaction with the Newman–Keuls method revealed that Group Control consumed the saccharin solution significantly more than Groups 0-min and 30-min (P’s < 0.05), which did not differ from each other. On the other hand, water consumption was significantly less in Group Control than in Groups 0-min and 30-min (P’s < 0.05), which did not differ from each other. A 3 (group) × 20 (day) ANOVA applied to the data illustrated in the bottom panel of Fig. 2 yielded significant main effects of group, F(2,21) = 7.00, P < 0.01, and day, F(19,399) = 3.00, P < 0.001, but their interaction was nonsignificant, F < 1. Subsequent Newman–Keuls tests revealed that Group Control scored higher than Groups 0-min and 30-min (P’s < 0.05), which did not differ from each other. The statistical equivalence in the saccharin aversion of the latter groups suggests that the trace interval of 30 min between the end of drinking
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Fig. 2. Performance of rats during two-bottle testing. Top panel: mean amount of saccharin (0.2% in water) and tap water intake during two-bottle testing. Error bars were omitted for simplicity. Bottom panel: mean saccharin preference ratio, in the form of x/(x + y), where x is the intake of saccharin solution (0.2% in water) and y is that of tap water. Bars of standard errors are shown on either side for simplicity.
and the onset of water immersion had little effect on conditioning.
4. Discussion The experiment reported here demonstrated a successful replication of our previous finding (Masaki
and Nakajima, submitted for publication; Masaki and Nakajima, in press; Nakajima and Masaki, 2004) that forced swimming caused aversion to a taste solution consumed immediately before swimming. Group 0min drank less of a target taste (saccharin) solution than Group Control both in the one-bottle conditioning phase and the two-bottle choice testing phase. Although we employed a CS-only control in the present
T. Masaki, S. Nakajima / Behavioural Processes 67 (2004) 357–362
experiment, it is highly unlikely that the obtained taste aversion was caused by any nonassociative process, because we have already demonstrated elsewhere that correlation of a target taste solution and swimming is necessary to establish taste aversion (Masaki and Nakajima, submitted for publication; Masaki and Nakajima, in press; Nakajima and Masaki, 2004). A new finding of this experiment is that delayed administration of forced swimming treatment is also effective in establishing taste aversion when the delay between the end of drinking and the onset of swimming is 30 min. Although the group averages shown in Figs. 1 and 2 suggest that taste aversion was slightly weaker for Group 30-min than for Group 0-min, the group differences were not statistically significant in the conditioning and testing phases. In other words, saccharin intakes of these groups were equivalent and less than that of Group Control in both phases. The demonstration of taste aversion induced by delayed swimming extends the generality of the phenomenon and broadens the degree of freedom in conducting experiments of this training technique: we do not have to put a rat into the water pool in a hurry to establish taste aversion. In the experiment reported here, the interval between the end of drinking and the onset of water immersion was 30 min. Further research is required in order to determine the effective length of the interval needed to establish taste aversion with a swimming US. It is notable that the swimming-induced taste aversion demonstrated in this article was long lasting or highly resistant to extinction. Groups 0-min and 30-min both showed substantial saccharin aversion throughout 20 days of testing. Such a strong effect was partially due to our test procedure. A two-bottle choice preference technique is generally more sensitive than a one-bottle intake technique to detect taste aversion (e.g., Dragoin et al., 1971; Grote and Brown, 1971; but see Batsell and Best, 1993), and taste aversion was resistant to extinction especially when a two-bottle test was used to measure the aversion (e.g., Elkins, 1973; Dragoin et al., 1971). For example, Elkins reports that saccharin aversion conditioned with a cyclophosphamide injection of 25 mg/kg was extinguished by the 2nd day of one-bottle testing, but that the aversion was substantial throughout 50 days of two-bottle testing. Thus, the long-lasting taste aversion in the two-bottle testing is not unique to our experiment with a forced swimming US. Another possible reason of the long-lasting effect
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in our experiment is that we paired saccharin solution and swimming over 8 days. The mechanism of swimming-induced taste aversion is not yet clarified. Our tentative hypothesis is that energy expenditure caused by swimming endows rats with aversion to the paired taste (Masaki and Nakajima, submitted for publication; Masaki and Nakajima, in press; Nakajima and Masaki, 2004; see also Nakajima et al., 2000 for the same argument for running-induced taste aversion). Benthem et al. (1994) show that swimming causes a large increase in energy expenditure calculated by online measurement of oxygen consumption and carbon dioxide production. Forced swimming, however, also leads to many physiological changes in rats including increases in serum corticosterone, glucose, lactate, prolactin, cholesterol, alkaline phosphatase, lipase, and phosphorus levels, and decreases in serum sodium, potassium, chloride, carbon dioxide, and pH levels (Abel, 1993, 1994a,b). One, some, or all of these changes might be critical in establishing taste aversion. Furthermore, swimming exercise might yield activation of the mesolimbic dopamine system or cause gastrointestinal discomfort to result in taste aversion (see Lett et al., 1998, 2001; Lett et al., 1999, for the argument of these possibilities in running-induced taste aversion). Further research is required to elucidate the mechanism of swimming-induced taste aversion.
Acknowledgements This research was supported by a Grant-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to S.N. (14710055) and a Grant-in-Aid for Scientific Research in Academic Frontier Promotion Project provided by MEXT to Research Center for Applied Psychological Science of KGU.
References Abel, E.L., 1993. Physiological correlates of the forced swim test in rats. Physiol. Behav. 54, 309–317. Abel, E.L., 1994a. A further analysis of physiological changes in rats in the forced swim test. Physiol. Behav. 56, 795–800. Abel, E.L., 1994b. Behavioral and physiological effects of different water depths in the forced swim test. Physiol. Behav. 56, 411–414.
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Andrews, E.A., Braveman, N.S., 1975. The combined effects of dosage level and interstimulus interval on the formation of onetrial poison-based aversions in rats. Anim. Learn. Behav. 3, 287–289. Barker, L.M., Smith, J.C., 1974. A comparison of taste aversions induced by radiation and lithium chloride in CS–US and US–CS paradigm. J. Comp. Physiol. Psychol. 87, 644–654. Batsell Jr., W.R., Best, M.R., 1993. One bottle too many? Method of testing determines the detection of overshadowing and retention of taste aversions. Anim. Learn. Behav. 21, 154–158. Benthem, L., Bolhuis, J.W., Van Der Leest, J., Steffens, A.B., Zock, J.P., Zijlstra, W.G., 1994. Methods for measurement of energy expenditure and substrate concentrations in swimming rats. Physiol. Behav. 56, 151–159. Coombes, S., Revusky, S., Lett, B.T., 1980. Long-delay taste aversion learning in an unpoisoned rat: exposure to a poisoned rat as the unconditioned stimulus. Learn. Motiv. 11, 256–266. Dragoin, W., McCleary, G.E., McCleary, P., 1971. A comparison of two methods of measuring conditioned taste aversions. Behav. Res. Methods Instrum. 3, 309–310. Elkins, R.L., 1973. Individual differences in bait shyness: effects of drug dose and measurement technique. Psychol. Rec. 23, 349–358. Etscorn, F., Stephens, R., 1973. Establishment of conditioned taste aversions with a 24-hours CS–US interval. Physiol. Psychol. 1, 251–253. Garcia, J., Ervin, F.R., Koelling, R.A., 1966. Learning with prolonged delay of reinforcement. Psychon. Sci. 5, 121–122. Green, L., Rachlin, H., 1976. Learned taste aversions in rats as a function of delay, speed, and duration of rotation. Learn. Motiv. 7, 283–289. Grote Jr., F.W., Brown, R.T., 1971. Conditioned taste aversions: two stimulus tests are more sensitive than one-stimulus tests. Behav. Res. Methods Instrum. 3, 311–312. Haroutunian, V., Riccio, D.C., 1975. Acquisition of rotation-induced taste aversion as a function of drinking-treatment delay. Physiol. Psychol. 3, 273–277. Hayashi, H., Nakajima, S., Urushihara, K., Imada, H., 2002. Taste avoidance caused by spontaneous wheel running: effects of duration and delay of wheel confinement. Learn. Motiv. 33, 390–409. Heth, C.D., Inglis, P., Russell, J.C., Pierce, W.D., 2001. Conditioned taste aversion induced by wheel running is not due to novelty of the wheel. Physiol. Behav. 74, 53–56.
Lett, B.T., Grant, V.L., 1996. Wheel running induces conditioned taste aversion in rats trained while hungry and thirsty. Physiol. Behav. 59, 699–702. Lett, B.T., Grant, V.L., Gaborko, L.L., 1998. Wheel running simultaneously induces CTA and facilitates feeding in non-deprived rats. Appetite 31, 351–360. Lett, B.T., Grant, V.L., Koh, M.T., Smith, J.F., 2001. Wheel running simultaneously produces conditioned taste aversion and conditioned place preference in rats. Learn. Motiv. 32, 129– 136. Masaki, T., Nakajima, S., submitted for publication. Further evidence for conditioned taste aversion induced by forced swimming. Masaki, T., Nakajima, S., in press. Swimming-induced taste aversion and its prevention by a prior history of swimming. Learn. Motiv. McLaurin, W.A., 1964. Postirradiation saccharin avoidance in rats as a function of the interval between ingestion and exposure. J. Comp. Physiol. Psychol. 57, 316–317. Nachman, M., 1970. Learned taste and temperature aversions due to lithium chloride sickness after temporal delays. J. Comp. Physiol. Psychol. 73, 22–30. Nakajima, S., Hayashi, H., Kato, T., 2000. Taste aversion induced by confinement in a running wheel. Behav. Proc. 49, 35– 42. Nakajima, S., Masaki, T., 2004. Taste aversion learning induced by forced swimming in rats. Physiol. Behav. 80, 623– 628. Salvy, S.-J., Pierce, W.D., Heth, D.C., Russell, J.C., 2002. Preexposure to wheel running disrupts taste aversion conditioning. Physiol. Behav. 76, 51–56. Salvy, S.-J., Pierce, W.D., Heth, D.C., Russell, J.C., 2003. Wheel running produces conditioned food aversion. Physiol. Behav. 80, 89–94. Schafe, G.E., Sollars, S.I., Bernstein, I.L., 1995. The CS–US interval and taste aversion learning: a brief look. Behav. Neurosci. 109, 799–802. Smith, J.C., Roll, D.L., 1967. Trace conditioning with X-ray as an aversive stimulus. Psychon. Sci. 9, 11–12. Sparkes, S., Grant, V.L., Lett, B.L., 2003. Role of conditioned taste aversion in the development of activity anorexia. Appetite 41, 161–165. Wright, W.E., Foshee, D.P., McCleary, G.E., 1971. Comparison of taste aversion with various delays and cyclophosphamide dose levels. Psychon. Sci. 22, 55–56.
Taste aversion learning induced by delayed swimming activity Takahisa Masaki, Sadahiko Nakajima∗ Department of Integrated Psychological Science, Kwansei Gakuin University, Nishinomiya 662-8501, Japan Received 16 January 2004; accepted 22 June 2004
Abstract The experiment reported here demonstrated that forced swimming endows rats with aversion to a taste solution consumed 30 min before the swimming. The experimental rats were allowed to drink 0.2% sodium saccharin solution, which was followed by a 30-min empty interval, and then a 20-min swimming opportunity in water. Compared with the control rats, which were returned to their home cages after drinking the saccharin, the experimental rats drank a small amount of saccharin solution both in the later sessions of one-bottle training and in the subsequent two-bottle choice (saccharin versus tap water) testing. The delayed swimming procedure was as effective as an immediate swimming procedure, extending the generality of the swimming-induced taste aversion, which we recently discovered with the immediate swimming procedure. © 2004 Elsevier B.V. All rights reserved. Keywords: Taste aversion learning; Conditioned taste aversion; Delayed learning; Swimming; Exercise; Rat
1. Introduction Lett and Grant (1996) discovered that voluntary running in an activity wheel endows rats with aversion to a taste solution consumed before the running. This finding has been successfully replicated not only in their laboratory (Lett et al., 1998, 2001; Sparkes et al., 2003) but also by other researchers (Hayashi et al., 2002; Heth et al., 2001; Nakajima et al., 2000; Salvy et al., 2002, 2003). Procedural details of these studies (e.g., rat strains, taste stimuli, durations of running, numbers of taste-running pairing trials, deprivation levels) differed ∗ Corresponding author. Tel.: +81 798 546076; fax: +81 798 546076. E-mail address: [email protected] (S. Nakajima).
0376-6357/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2004.06.005
from one another, indicating the robustness of the phenomenon. Because a correlation of taste and running is necessary for this phenomenon, it has been discussed in the framework of Pavlovian conditioning. In other words, associative or conditioned taste aversion learning is established with taste as the conditioned stimulus (CS) and running as the unconditioned stimulus (US). In a recent series of experiments we found that forced swimming also works as a US for conditioned taste aversion (Masaki and Nakajima, submitted for publication; Masaki and Nakajima, in press; Nakajima and Masaki, 2004). Immersion in water pools immediately after drinking a taste solution endowed a group of rats with aversion to that solution. The aversion was associative, because these rats drank the
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solution less than did the control rats, which had received a taste–swimming unpaired treatment. Furthermore, a within-subject comparison of paired and unpaired taste solutions demonstrated clear differential intakes: rats began to drink less of the former than of the latter. Another experiment also revealed that swimming activity in water rather than being wet is critical in obtaining the taste aversion. The swimming-induced taste aversion also follows the Pavlovian law of US strength, because the aversion was a positive function of the length of water immersion. These experiments strongly demonstrated that forced swimming after drinking of a target taste induces aversion to that taste, but the finding is novel and calls for further replication with modified procedures to extend the generality of the finding. In all of the experiments we reported, water immersion immediately followed taste drinking. However, it is widely known that taste aversion is established in rats with a delayed US when the US is any of a variety of chemical substances (e.g., Andrews and Braveman, 1975; Barker and Smith, 1974; Etscorn and Stephens, 1973; Garcia et al., 1966; Nachman, 1970; Schafe et al., 1995; Wright et al., 1971), irradiation (e.g., Barker and Smith, 1974; McLaurin, 1964; Smith and Roll, 1967), motion sickness (Green and Rachlin, 1976; Haroutunian and Riccio, 1975), a poisoned conspecific (Coombes et al., 1980), and voluntary wheel running (Hayashi et al., 2002). In the experiment reported below, we explored whether forced swimming could be established with a 30-min empty interval between the end of taste drinking and the onset of water immersion.
2. Methods 2.1. Subjects The subjects were 24 experimentally na¨ıve male Wistar rats of 60 days old with a mean weight of 321 g (range: 297–338 g) on the day before the water deprivation schedule began. The animals were housed in individual hanging home cages on a 12 h light:12 h dark cycle (lights on at 08:00 h) at about 23 ◦ C and maintained on an ad-lib food schedule. Watering in the home cages was limited to 30 min/day 2 days before the beginning of the adaptation training. Thereafter, the rats were able to drink water for 15 min in their home cages
90 min after experimental sessions of 15-min fluid intake. 2.2. Apparatus The experimental sessions were carried out in a conventionally illuminated room where eight drinking cages and eight swimming pools were located. The drinking cages were copies of the home cages and the inner dimension of each was 20 cm wide, 25 cm long, and 18.7 cm high, and fluid was provided via a plastic bottle with a metal spout inserted from the cage ceiling. The end of the spout was 16.5 cm above the cage floor. When two bottles were used, they were separated 8 cm apart. The swim pools were blue–gray plastic garbage containers, and the inner dimension of each was 48 cm high and 34 cm in diameter on the bottom and 43 cm in diameter at the top. Tap water of room temperature was filled up to a height of 36 cm. The pools were cleaned after each session, so that each rat swam in fresh water without being affected by any fecal or urinary materials of other rats. 2.3. Training All experimental sessions were conducted at the same time (around the middle of the lighted condition) of successive days. Each rat was initially adapted to drinking tap water from a bottle for 15 min/day. After 4-day adaptation training, the rats were assigned to one of three groups of equal number (each n = 8) matched for their amount of water intake and bodyweights. Conditioning was then conducted for 8 days. Rats of Group 0-min were allowed to drink 0.2% sodium saccharin solution, immediately followed by a 20-min water immersion. Rats of Group 30-min were given identical treatment with the exception that they spent 30 min in the drinking cages without bottles between the end of saccharin drinking and the onset of water immersion. Rats of Group Control were returned to their home cages immediately after the saccharin drinking (no immersion). 2.4. Testing All rats were tested on the following 20 days. On each day, each rat was offered two bottles, one containing saccharin solution and the other containing tap water, concurrently for 15 min. The left–right po-
T. Masaki, S. Nakajima / Behavioural Processes 67 (2004) 357–362
sitions of bottles were interchanged across rats and days. 2.5. Analysis The amount of fluid intake was measured by weighing each bottle before and after the drinking period using an electric balance to the nearest 0.1 g. All statistical decisions were based on the alpha level of 0.05 or less.
3. Results Fig. 1 presents the mean saccharin intake during the conditioning phase. Although Group Control increased their saccharin intake over the 8 days, the other groups kept the initial intake levels. A 3 (group) × 8 (day) analysis of variance (ANOVA) applied to the data illustrated in Fig. 1 yielded significant main effects of group, F(2,21) = 4.98, P < 0.05, and day, F(7,147) = 6.15, P < 0.001, and their interaction, F(14,147) = 2.01, P < 0.05. Subsequent Newman–Keuls tests applied to the group effect revealed that Group Control drank the solution more than Groups 0-min and 30-min (P’s < 0.05). Furthermore, the tests for the group × day interaction revealed that Group Control drank more than
Fig. 1. Mean amount of saccharin (0.2% in water) intake during onebottle training. On each day, an access to the saccharin solution was followed by returning to their home cages for Group Control, an immediate swimming for Group 0-min, or a delayed swimming for Group 30-min. Error bars indicate the standard errors.
359
both Groups 0-min and 30-min on the 4th, 6th, and 7th days (P’s < 0.05). Group Control also drank more than Group 0-min on the 5th and 8th days (P’s < 0.05). Groups 0-min and 30-min did not differ from each other on any day. These results suggest that moderate aversion induced by immediate or delayed swimming prevents normal development of saccharin preference by repeated exposure. Fig. 2 shows the results of the choice preference test over 20 days. The top panel depicts the mean saccharin and water intakes, and the bottom panel presents the mean saccharin preference ratios in the form of x/(x + y), where x is saccharin intake and y is water intake. The lower the ratio value, the stronger the saccharin aversion estimated. As in the training phase, Group Control drank more saccharin than Groups 0-min and 30-min in the testing, indicating conditioned saccharin aversion induced by immediate or delayed swimming. A 3 (group) × 2 (fluid) × 20 (day) ANOVA applied to the data illustrated in the top panel of Fig. 2 yielded significant main effects of group, F(2,21) = 4.22, P < 0.05, and day, F(19,399) = 7.80, P < 0.001, and first-order interactions of group × day, F(38,399) = 2.12, P < 0.001, and fluid × day, F(19,399) = 2.38, P < 0.01. Most importantly, the interaction of group × fluid was significant, F(2,21) = 8.12, P < 0.01, supporting conditioned saccharin aversion in Groups 0-min and 30-min but not in Group Control. The main effect of fluid, F < 1, and the second-order interaction of group × fluid × day, F(38,399) = 1.12, were nonsignificant. Post hoc analyses of the group × fluid interaction with the Newman–Keuls method revealed that Group Control consumed the saccharin solution significantly more than Groups 0-min and 30-min (P’s < 0.05), which did not differ from each other. On the other hand, water consumption was significantly less in Group Control than in Groups 0-min and 30-min (P’s < 0.05), which did not differ from each other. A 3 (group) × 20 (day) ANOVA applied to the data illustrated in the bottom panel of Fig. 2 yielded significant main effects of group, F(2,21) = 7.00, P < 0.01, and day, F(19,399) = 3.00, P < 0.001, but their interaction was nonsignificant, F < 1. Subsequent Newman–Keuls tests revealed that Group Control scored higher than Groups 0-min and 30-min (P’s < 0.05), which did not differ from each other. The statistical equivalence in the saccharin aversion of the latter groups suggests that the trace interval of 30 min between the end of drinking
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Fig. 2. Performance of rats during two-bottle testing. Top panel: mean amount of saccharin (0.2% in water) and tap water intake during two-bottle testing. Error bars were omitted for simplicity. Bottom panel: mean saccharin preference ratio, in the form of x/(x + y), where x is the intake of saccharin solution (0.2% in water) and y is that of tap water. Bars of standard errors are shown on either side for simplicity.
and the onset of water immersion had little effect on conditioning.
4. Discussion The experiment reported here demonstrated a successful replication of our previous finding (Masaki
and Nakajima, submitted for publication; Masaki and Nakajima, in press; Nakajima and Masaki, 2004) that forced swimming caused aversion to a taste solution consumed immediately before swimming. Group 0min drank less of a target taste (saccharin) solution than Group Control both in the one-bottle conditioning phase and the two-bottle choice testing phase. Although we employed a CS-only control in the present
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experiment, it is highly unlikely that the obtained taste aversion was caused by any nonassociative process, because we have already demonstrated elsewhere that correlation of a target taste solution and swimming is necessary to establish taste aversion (Masaki and Nakajima, submitted for publication; Masaki and Nakajima, in press; Nakajima and Masaki, 2004). A new finding of this experiment is that delayed administration of forced swimming treatment is also effective in establishing taste aversion when the delay between the end of drinking and the onset of swimming is 30 min. Although the group averages shown in Figs. 1 and 2 suggest that taste aversion was slightly weaker for Group 30-min than for Group 0-min, the group differences were not statistically significant in the conditioning and testing phases. In other words, saccharin intakes of these groups were equivalent and less than that of Group Control in both phases. The demonstration of taste aversion induced by delayed swimming extends the generality of the phenomenon and broadens the degree of freedom in conducting experiments of this training technique: we do not have to put a rat into the water pool in a hurry to establish taste aversion. In the experiment reported here, the interval between the end of drinking and the onset of water immersion was 30 min. Further research is required in order to determine the effective length of the interval needed to establish taste aversion with a swimming US. It is notable that the swimming-induced taste aversion demonstrated in this article was long lasting or highly resistant to extinction. Groups 0-min and 30-min both showed substantial saccharin aversion throughout 20 days of testing. Such a strong effect was partially due to our test procedure. A two-bottle choice preference technique is generally more sensitive than a one-bottle intake technique to detect taste aversion (e.g., Dragoin et al., 1971; Grote and Brown, 1971; but see Batsell and Best, 1993), and taste aversion was resistant to extinction especially when a two-bottle test was used to measure the aversion (e.g., Elkins, 1973; Dragoin et al., 1971). For example, Elkins reports that saccharin aversion conditioned with a cyclophosphamide injection of 25 mg/kg was extinguished by the 2nd day of one-bottle testing, but that the aversion was substantial throughout 50 days of two-bottle testing. Thus, the long-lasting taste aversion in the two-bottle testing is not unique to our experiment with a forced swimming US. Another possible reason of the long-lasting effect
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in our experiment is that we paired saccharin solution and swimming over 8 days. The mechanism of swimming-induced taste aversion is not yet clarified. Our tentative hypothesis is that energy expenditure caused by swimming endows rats with aversion to the paired taste (Masaki and Nakajima, submitted for publication; Masaki and Nakajima, in press; Nakajima and Masaki, 2004; see also Nakajima et al., 2000 for the same argument for running-induced taste aversion). Benthem et al. (1994) show that swimming causes a large increase in energy expenditure calculated by online measurement of oxygen consumption and carbon dioxide production. Forced swimming, however, also leads to many physiological changes in rats including increases in serum corticosterone, glucose, lactate, prolactin, cholesterol, alkaline phosphatase, lipase, and phosphorus levels, and decreases in serum sodium, potassium, chloride, carbon dioxide, and pH levels (Abel, 1993, 1994a,b). One, some, or all of these changes might be critical in establishing taste aversion. Furthermore, swimming exercise might yield activation of the mesolimbic dopamine system or cause gastrointestinal discomfort to result in taste aversion (see Lett et al., 1998, 2001; Lett et al., 1999, for the argument of these possibilities in running-induced taste aversion). Further research is required to elucidate the mechanism of swimming-induced taste aversion.
Acknowledgements This research was supported by a Grant-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to S.N. (14710055) and a Grant-in-Aid for Scientific Research in Academic Frontier Promotion Project provided by MEXT to Research Center for Applied Psychological Science of KGU.
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