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Dissociation of conditioned taste avoidance from conditioned disgust reactions induced by wheel running in rats
Behavioural Processes 90 (2012) 223–228
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Dissociation of conditioned taste avoidance from conditioned disgust reactions induced by wheel running in rats Virginia L. Grant ∗ , Sarah V. McDonald, Robyn C. Sheppard, Catherine L. Caldwell, Thomas H. Heeley, Adam R. Brown, Gerard M. Martin Department of Psychology, Memorial University of Newfoundland, St. John’s, NL A1B 3X9, Canada
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Article history: Received 8 September 2011 Received in revised form 10 January 2012 Accepted 30 January 2012 Keywords: Conditioned disgust reactions Conditioned taste aversion Conditioned taste avoidance Rats Taste reactivity test Wheel running
a b s t r a c t It is well established that wheel running in rats produces conditioned taste avoidance; that is, rats that run in wheels after consuming a novel-tasting solution later consume less of that solution than rats that do not run. In experiment 1, we found that wheel running also produces conditioned disgust reactions, indicated by gapes elicited by both the taste and context that were experienced before running. Experiment 2 showed that the conditioned disgust reactions were likely not due to running itself but to a by-product of running, the rocking of the wheel that occurs when the running stops. When rocking was reduced, the disgust reactions were also reduced, but consumption of the taste solution was not changed, showing dissociation of conditioned taste avoidance and disgust. These findings indicate that the taste avoidance induced by wheel running itself is more like the taste avoidance produced by rewarding drugs than that produced by nausea-inducing drugs. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Wheel running produces conditioned taste avoidance in rats; that is, when rats drink a novel-tasting solution and then run in running wheels they subsequently drink less of that taste solution than rats treated equivalently but which do not run (e.g., Baysari and Boakes, 2004; Heth et al., 2001; Lett and Grant, 1996; Nakajima et al., 2000). Reduced consumption produced by wheel running does not necessarily reflect conditioned disgust. Parker (2003) distinguishes between treatments (usually drugs) that are truly aversive and clearly induce nausea and sickness (e.g., lithium chloride) and those that are rewarding and do not appear to induce nausea (e.g., amphetamine). When administered after consumption of a novel tasting solution, both types of drug can produce strong conditioned taste avoidance, indicated by reduced consumption, but only nausea-inducing drugs produce conditioned disgust reactions, indicated by such behaviors as gaping, paw pushing, and chin rubbing, which are elicited by the associated taste solution in a taste reactivity test (Grill and Norgren, 1978). Wheel running is similar to rewarding drugs, in that it also has rewarding effects. It can serve as a reinforcer for bar pressing (e.g., Belke and Wagner, 2005; Iversen, 1993) and it can condition a place preference (e.g., Belke and Wagner, 2005; Lett et al., 2000). Wheel running is also similar to rewarding drugs in terms of
∗ Corresponding author. Tel.: +1 709 864 8020; fax: +1 709 864 4000. E-mail addresses: [email protected], [email protected] (V.L. Grant).
its taste conditioning effects; like them, it produces conditioned taste avoidance. To complete the parallel between wheel running and rewarding drugs, there should be no evidence of conditioned disgust reactions; that is, like rewarding drugs (Parker, 1995), wheel running should produce conditioned taste avoidance, but not conditioned disgust reactions. This seems likely because Parker and her colleagues have not found any treatment that produces both a place preference and conditioned disgust reactions (e.g., see Table 6.1 in Parker et al., 2009). However there is some evidence that wheel running may be an exception. Dwyer et al. (2008), using analysis of the rats’ pattern of licking to evaluate palatability, found that wheel running produced a pattern of licks similar to that produced by lithium chloride and unlike that produced by amphetamine. This observation suggests that wheel running is more like lithium chloride, and thus may also produce disgust reactions as measured by a taste reactivity test. Such tests have shown disgust reactions (e.g., gapes) in response to novel tastes (e.g., Kent et al., 2000; Parker, 1984) and contexts (e.g., Brown et al., 2011; Limebeer et al., 2008) that have been paired with lithium chloride. The current experiments determined whether wheel running produces conditioned disgust reactions as well as conditioned taste avoidance. In experiment 1, we used a modified version of the taste reactivity test in which rats were allowed to drink the taste solution in a distinctive context while being observed for disgust reactions (Brown et al., 2011). In experiment 2 we tested whether disgust reactions were conditioned by a by-product of running, the rocking of the wheel when the rat stops running, rather than wheel running itself.
0376-6357/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2012.01.011
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2. Experiment 1
the remainder of the pretraining period and during the conditioning and test period.
2.1. Introduction On four conditioning trials, the rats consumed a novel tasting solution in a drinking box, following which they were either placed in running wheels (experimental group) or returned to their home cages (control group). The fluid consumption measure was used to assess conditioned taste avoidance. On the day following the last conditioning trial, the rats were given a test trial during which, in addition to the fluid consumption measure, the rats were also video-recorded and observed for any evidence of disgust reactions. Parker and her colleagues (e.g., Parker, 2003; Parker et al., 2008, 2009) have repeatedly noted that only emetic or nausea-inducing treatments produce conditioned disgust reactions, like gaping, in rats. Thus, if rats show the gaping reaction in response to a context and taste that have been paired with wheel running, it implies that wheel running produces nausea effects similar to those of emetic drugs. 2.2. Method 2.2.1. Subjects We obtained 24 male Sprague-Dawley rats (Rattus norvegicus) as weanlings from the breeding colony at Memorial University of Newfoundland. On the first day of the pretraining procedure the rats were approximately 40 days old and weighed between 167 and 249 g. The rats were housed, trained, and tested in a room maintained at approximately 20 ◦ C. The 12-h light/dark cycle had lights on at 7:30 a.m. The rats were individually housed in clear plastic cages (45 cm × 25 cm × 21 cm) with lids of metal bars that were indented to hold food and a drinking bottle. Food was always available in the home cages, as was water, except during the water deprivation period as outlined below. 2.2.2. Apparatus and materials Twelve freely rotating running wheels were used in the conditioning procedure. The circumference of each wheel was approximately 113 cm and the wire mesh running surface inside the wheel was between 11 and 12 cm in width. Each wheel had a magnet on the rotating part which passed two sensors situated 180◦ apart; the sensors were connected to a computer which recorded complete revolutions. Floorless drinking boxes with removable lids were placed on a square glass table top (85.1 cm × 85.1 cm) that was 73.7 cm above the floor, so that the rats confined in the boxes could be video recorded with a Canon high-definition camera on a small tripod positioned on the floor beneath the glass table top. The inside dimensions of the drinking boxes were 25.4 cm × 15.2 cm × 38.1 cm. They were made of wood; two were painted black and two white. Each rat was assigned to a box for the duration of the experiment. The table top under each box was cleaned with a wet paper towel and then wiped dry before each rat was confined in the box. A small hole drilled in one of the short sides of the box allowed insertion of a bottle spout for drinking. There was a second hole in one of the long sides which was not used. The novel-tasting solution was a 0.1% saccharin solution (1 g sodium saccharin per 1000 ml tap water). 2.2.3. Procedure Water was removed at 4 p.m. the day before the second pretraining session (see Section 2.2.3.1). The rats were allowed access to water in their home cages between 2 p.m. and 4 p.m. each day for
2.2.3.1. Pretraining. Three pretraining sessions familiarized each rat with its drinking box and with the water deprivation schedule. The rats spent the first 20-min session in their drinking boxes when not thirsty, followed by two 20-min sessions when thirsty. Sessions occurred in the morning on 3 consecutive days. After 10 min in the box, the spout of a water bottle was inserted to provide access to water for the last 10 min.
2.2.3.2. Conditioning. Conditioning trials were between 10:30 a.m. and 1:50 p.m. on 4 consecutive days immediately following the final day of the pretraining procedure. Six squads of 4 rats were conditioned on each day. In each squad, one rat in a white and one rat in a black drinking box were randomly assigned to run in the wheels after drinking (the wheel group); the other two rats were returned to their home cages (the control group). Each rat in the wheel group had its own wheel which was the same for each conditioning trial. On each conditioning day, the four rats in the first squad were confined in their drinking boxes for 20 min. During the last 10 min in the box, the rats were given access to the saccharin solution. Then the 2 rats in the wheel group were placed into their running wheels where they remained for 30 min before returning to the home cage; the 2 rats in the control group were returned to their home cages. Then the next squad was given a conditioning trial and so on for each of the six squads.
2.2.3.3. Test. Each rat was tested once for taste reactivity. On the day following the final conditioning trial, 6 rats in the wheel group and 6 in the control group were tested, one at a time, starting at 10:30 a.m. The remaining rats were similarly tested on the following day. As during conditioning, each rat was confined in its box for 20 min, with access to the saccharin solution during the last 10 min in the box. The video camera situated beneath the drinking box recorded each rat for the entire 20-min duration of the test trial.
2.2.4. Data analysis To evaluate the measure of conditioned taste avoidance, the mean intake of saccharin solution over the conditioning trials was analyzed by a 2 × 4 (Groups by Trials) mixed analysis of variance (ANOVA), with repeated measures on the trial factor. Linear trend analyses across conditioning trials assessed progressive changes in drinking for both groups as well as wheel running scores (number of revolutions) for the wheel group. Independent t-tests were conducted between groups for the saccharin solution consumption on the test. For all analyses the alpha level was set at .05, two-tailed for t-tests. The video record for each rat’s test was observed for the rejection reaction of gaping (the only rejection reaction observed). The scoring was validated by having two additional observers each score five of the records. Correlation coefficients were above +.90 (ps < .05) indicating that inter-rater reliability was high. All observers were blind to the observed rat’s experimental condition. To determine if the wheel and control groups differed in the number of subjects showing gapes, a chi square analysis was used. A 2 (Group) × 2 (Time in Session: first versus second 10 min) mixed ANOVA with repeated measures on the Time in Session factor assessed the extent to which gapes occurred before and after the introduction of the saccharin solution.
Saccharin Solution Consumed (gm)
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Fig. 1. Mean (±SE) amount of saccharin solution consumed on each trial and on the test for rats in the control and wheel groups in experiment 1.
2.3. Results 2.3.1. Saccharin solution consumption on conditioning and test trials Our findings on consumption replicate the many studies that show that wheel running induces conditioned taste avoidance (see Boakes and Nakajima, 2009, for a review). Mean saccharin solution intake over the four conditioning trials and the test trial is shown for wheel and control groups in Fig. 1. Analysis of the four conditioning trials revealed a significant group by trial interaction, F (3, 66) = 25.94, p < .001, reflecting the diverging scores as the wheel group consumed progressively less of the saccharin fluid over trials than the control group. Moreover, a significant group main effect, F (1, 22) = 27.36, p < .001, shows that overall, the wheel group consumed less than the control group. A linear trend analysis indicated a significant decrease in consumption by the wheel group over trials and an increase in consumption by the controls (ps < .001). On the test trial, significantly less saccharin was consumed by the wheel group than the control group, t (22) = 7.42, p < .001. 2.3.2. Disgust reactions on the test trial The only aversive measure observed was gaping. For some unexplained reason, one rat in the control group gaped once. Despite that anomaly, the number of rats that showed gapes was significantly greater in the wheel group (7 out of 12) than the control group (1 out of 12), 2 (1, N = 24) = 6.75, p = .009. Rats in the wheel group gaped more (M = 1.92, SE = 0.85) than rats in the control group (M = 0.08, SE = 0.08), F(1, 22) = 4.63, p = .04. Although there were more gapes in the second 10 min (M = 0.83, SE = 0.41), when saccharin solution was available, than in the first (M = 0.17, SE = 0.09), the difference was not significant, p = .09. 2.3.3. Wheel running The rats ran more each successive day; that is, the number of wheel revolutions increased across the four conditioning trials (Trial 1: M = 142.4, SE = 15.6; Trial 2: M = 210.1, SE = 34.4; Trial 3: M = 287.2, SE = 39.8; Trial 4: M = 302.7, SE = 39.7). The increase was significant, F (3, 33) = 20.84, p = .0001, and was confirmed by a significant linear trend, p < .001. 2.4. Discussion Wheel running produced both conditioned taste avoidance and conditioned disgust reactions. The rats that ran in the wheels after drinking the saccharin solution subsequently drank less of the solution and gaped more than the control rats that did not wheel run. Thus wheel running appears to be more like sickness-inducing
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drugs than rewarding drugs in conditioning both taste avoidance and disgust. The finding that gaping occurred before the introduction of the saccharin solution indicates that there was likely conditioning to the context. Such contextual conditioning of gaping has been reported with lithium chloride as the unconditioned stimulus (e.g., Brown et al., 2011; Limebeer et al., 2008). Moreover, although backward conditioning of a place with wheel running produces a conditioned place preference (Lett et al., 2000), forward conditioning produces a conditioned place aversion (Masaki and Nakajima, 2008), also indicating that aversive aspects of wheel running can be associated with a context experienced before the running. The context here was a distinctive one, solid wooden walls painted black or white, compared to the transparent plastic walls of the home cages, and thus could support contextual conditioning. Additionally, this test was after four conditioning trials which might account for the strong contextual conditioning effect. In contrast to the usual taste reactivity test (e.g., Grill and Norgren, 1978), our rats were not forced to taste the saccharin solution. The absence of forced exposure to the taste solution may have contributed to the lack of difference between the first and second 10 min of the test session.
3. Experiment 2 3.1. Introduction Forristall et al. (2007) compared free wheel running and forced wheel running in a motorized wheel as agents to produce conditioned taste avoidance. They found that both types of wheel running produced conditioned taste avoidance, however, the avoidance was greater when the taste solution was followed by free wheel running. The authors speculated on possible reasons for this difference; one hypothesis was that the free wheels rocked back and forth when the rats stopped running, whereas the motorized wheels just turned at a constant rate and never rocked. It could be that the rocking of the free wheels caused motion sickness, and this in turn might cause the stronger conditioned taste avoidance in the free wheel running group. Rotation-induced motion sickness produces both conditioned taste avoidance and disgust (Cordick et al., 1999). Rocking of the wheel could also induce motion sickness and thus produce the conditioned disgust reactions of experiment 1. In experiment 2 we tested whether rocking is responsible for the conditioned taste avoidance (reduction in consumption of the taste solution) as well as the disgust reactions (gapes) noted in experiment 1. We replicated experiment 1 but with an additional group – a group that ran in the wheels and had rocking of the wheels reduced by a researcher who placed a finger on the wheel any time the rat stopped running. Thus there were three groups of rats: a Rock group that ran freely in wheels that rocked; a LoRock group that ran freely in wheels that rocked less; and a control group that did not go in the wheels.
3.2. Method 3.2.1. Subjects The Memorial University of Newfoundland breeding colony provided 39 male, Sprague-Dawley rats. There were five sets of rats that arrived, as weanlings, at approximately 2-week intervals. There were 3 rats in the first set and 9 in each of the subsequent sets. They were group-housed for 7 days and then moved to individual housing 8 days before the start of water deprivation, when they were between 35 and 40 days old. Housing conditions were as in experiment 1 (Section 2.2.1.).
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3.2.2. Materials and apparatus The materials and apparatuses were as described in experiment 1 (Section 2.2.2), except that only six of the wheels were used and all drinking boxes were painted white. 3.2.3. Procedure In unspecified details the procedure was as in experiment 1 (Section 2.2.3). Within each of the five sets of rats, except the first, there were three squads. A squad was composed of 3 rats which were trained at the same time; each rat in a squad was randomly assigned to one of three experimental groups, described below, so that each squad contained one rat from each of the three groups. 3.2.3.1. Pretraining. The pretraining procedure was the same as that in experiment 1 (Section 2.2.3.1), except that the pretraining sessions were in the afternoon, and water was made available in the home cage for an hour after the sessions in the drinking boxes were completed. 3.2.3.2. Conditioning. There were four daily conditioning trials starting the day after the last pretraining trial. The conditioning procedure was as described for experiment 1 (Section 2.2.3.2), except that there were three experimental treatments: placement in a running wheel which was allowed to rock (Group Rock); placement in a running wheel which was not allowed to rock (Group LoRock); and return to the home cage (Group Control). Each rat in Group Rock and LoRock was always placed in the same wheel, where they remained for 30 min. The wheels for the two members of a squad that were in Group Rock and Group LoRock were always side by side. A researcher sat between the two wheels and prevented the wheel for the rat in the LoRock condition from rocking. Whenever the rat stopped running and the wheel was about to rock, the researcher gently put a finger on the wheel and prevented it from rocking. In the last set of 9 rats, the three rats in Group Rock and the three in Group LoRock were video-recorded while they ran in the wheels. The video records were analyzed for the number of times each wheel rocked, as a check on the normal amount of rocking and on how successful the researcher was in preventing rocking. 3.2.3.3. Test. The day after the last training trial the test trial was conducted; as in experiment 1 (Section 2.2.3.3.) the rats were video-recorded during the 20 min in their drinking boxes. The tests were started at approximately 11 a.m. with the last rat finishing at approximately 5:30 p.m. 3.2.4. Data analysis As before, video records of the test session were scored for gaping during the 10 min before and the 10 min while they had access to saccharin solution. The video record for one rat in Group LoRock was lost due to a camera malfunction. The total number of gapes for that rat was acquired through direct observation, but the timing of the gapes was not noted, so this rat’s data were included in the Chi Square analysis, but not in the Group by Time in Session ANOVA (see Section 3.3.2). The scoring of the videos was again validated by having two additional observers, blind to each rat’s condition, score the records for gaping. Video records that were made of a wheel running session for 6 rats were scored for instances of rocking. These were also evaluated by two additional observers. Inter-rater correlation coefficients for gapes and rocks were all above +.90 (ps < .01) indicating high reliability of the scoring procedure. To evaluate the measure of conditioned taste avoidance, the mean intake of saccharin solution over the conditioning trials was analyzed by a 3 × 4 (Groups by Trials) mixed ANOVA, with repeated measures on the trial factor. Wheel turns were similarly analyzed
Saccharin Solution Consumed (gm)
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20
Control
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12 8 4 0 1
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Trials Fig. 2. Mean (±SE) amount of saccharin solution consumed on each trial and on the test for rats in Group Rock, LoRock and Control in experiment 2.
with a 2 × 4 ANOVA. Other analyses were as in experiment 1 (Section 2.2.4). 3.3. Results 3.3.1. Saccharin solution consumption on conditioning and test trials As can be seen in Fig. 2, over the first four conditioning trials, Groups Rock and LoRock drank progressively less while Group Control drank progressively more. The reliability of these effects is supported by the 3 (Groups) × 4 (Trials) ANOVA. There were significant effects of Group, F(2, 36) = 33.62, p < .001, and Trial, F(3, 108) = 14.96, p < .001, and a Group by Trial interaction, F(6, 108) = 32.38, p < .001. A linear trend analysis indicated that the saccharin-solution consumption of the two wheel groups decreased over trials, while that of the control group increased (ps < .001). There were no group differences between Group Rock and LoRock, F(1, 24) = 0.83, p = .37; but both groups differed significantly from the control group (ps < .001). A one-way ANOVA of saccharin solution on the test trial showed that the groups continued to differ in saccharin consumption, F(2, 36) = 80.91, p < .001 (see Fig. 2). Both Group Rock and LoRock drank significantly less saccharin solution than Group Control, t(24) = 11.56 and t(24) = 10.15, respectively, ps < .001, and they did not differ from each other, t(24) = 0.50, p = .62. 3.3.2. Disgust reactions Parametric analysis of disgust reactions was limited to Groups Rock and LoRock because no rat in Group Control emitted any disgust reaction. The two sets of bars in Fig. 3 show the mean number of gapes emitted by Group Rock and LoRock in the first 10 min (before the solution was introduced), in the second 10 min (when the saccharin solution was available), and in total. Although Groups Rock and LoRock did not differ in consumption of saccharin solution on the test, they did differ in the number of gapes as assessed in the Group × Time in Session ANOVA, F(1, 23) = 5.48, p = .03. As in experiment 1, there were more gapes in the second 10 min of the session than the first, but the difference was not reliable, p = .06. The present findings indicate that rocking produces conditioned disgust reactions, but that running without rocking may only produce conditioned taste avoidance. For Group Rock, the number of rats showing disgust responses was 10 (n = 13; 77%) whereas for Group LoRock, the number was 5 (n = 13; 38%). As mentioned above, no disgust responses were observed for any rat in Group Control (n = 13; 0%). A Chi Square analysis indicated differences overall, 2 (2, N = 39) = 16.25, p < .001. Compared to Group Control, both Group Rock and Group LoRock had greater
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6.3 (SE = 2.03), t(4) = 20.29, p = .001. Thus rocking was reduced for Group LoRock, but not eliminated. This lower level of rocking may have resulted in the gaping observed in some rats in Group LoRock. 3.4. Discussion
Fig. 3. Mean (+SE) number of gapes shown by the Rock and LoRock Groups in experiment 2. Gapes during the first 10 min of the test, before the saccharin solution was introduced, are shown in the left-hand bar and for the second 10 min, during access to the saccharin solution, in the middle bar, and the total number of gapes during the 20-min test in the right-hand bar.
numbers of rats showing disgust, 2 (1, N = 26) = 16.25, p < .001 and 2 (1, N = 26) = 6.19, p = .013, respectively. More rats in Group Rock showed disgust than in Group LoRock, 2 (1, N = 26) = 3.94, p = .047. The disgust reactions data indicate that a greater aversion to the taste is found when rocking is allowed. When rocking is reduced, the disgust responses are also reduced. As indicated in Section 3.3.1, the amount of rocking does not appear to affect consumption. Wheel running with rocking and without rocking produced equivalent conditioned taste avoidance as measured by the amount of the taste solution consumed.
Mean Number of Revolutions
3.3.3. Wheel turns for Group Rock and LoRock Our actions to prevent rocking did not affect the rats’ running behavior. Fig. 4 shows the mean number of wheel revolutions made by Group Rock and LoRock on each of the four conditioning trials. A 2 (Groups) × 4 (Trials) ANOVA showed that the groups did not differ in wheel turns, F(1, 24) = 1.36, p = .25, nor was there an interaction between Groups and Trials, F(3,72) = 0.09, p = .97. There was, however, a large trials effect, F(3,72) = 15.52, p < .001. A linear trend analysis indicated that both groups ran more over trials (ps < .001). As a manipulation check, 3 rats in Group Rock and 3 in Group LoRock in the last squad were video-recorded while they were running in the wheels and the videos were analyzed for the number of times the wheel rocked. Compared to Group Rock, our prevention of rocking reduced the number of rocks for Group LoRock significantly. The mean number of rocks for the three rats in Group Rock was 130.7 (SE = 5.78) rocks, whereas for Group LoRock it was
300
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200 150 100 50 0 1
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Trials Fig. 4. Mean (±SE) number of wheel revolutions on each trial for rats in Groups Rock and LoRock in experiment 2.
Wheel running produced conditioned taste avoidance whether the wheel rocked or was prevented from rocking; that is, both Group Rock and LoRock drank less of the saccharin solution than the controls. Moreover, both Group Rock and LoRock drank equivalent amounts of the saccharin solution, so the level of rocking had no effect on conditioned taste avoidance. However it did have an effect on conditioned disgust responses; rats in Group Rock made more gapes than rats in Group LoRock – that is, conditioned disgust was stronger in Group Rock than LoRock. As in experiment 1, disgust responses were evident both before and after the saccharin solution was made available, suggesting an association of the context with the running. There remains a question as to whether the lower level of gaping observed in the rats in Group LoRock was due to wheel running or to the rocks that the researcher was unable to prevent. An alternative interpretation of our findings is that wheel rocking produces sensitization, in that rats are more likely to gape and avoid flavored solutions after running and being rocked in the wheel. Early work with lithium chloride revealed that sensitization did not contribute to taste avoidance when unpaired control groups were included (Domjan, 1975, 1977), and thus probably does not contribute to the effects observed in our experiments. Moreover, there have been a number of studies of the effect of wheel running on the consumption measure which used differential conditioning (e.g., Heth et al., 2001; Lett & Grant, 1996; Nakajima et al., 2000). None report a sensitization effect, and it seems unlikely that gaping would be sensitized when consumption is not. 4. General discussion In experiment 1, we found that wheel running not only induced conditioned taste avoidance, but also conditioned disgust reactions to the taste and context. There was conditioned taste avoidance because rats that ran in the wheels after consuming saccharin subsequently drank less saccharin than rats that did not run and there was conditioned disgust because rats that ran in the wheels after consuming saccharin made more conditioned gapes indicative of disgust on the test than rats that did not run. This finding is consistent with that of Dwyer et al. (2008), who, using the pattern of licking as a measure, found reduced palatability of a taste solution that had been paired with wheel running. Experiment 2 revealed that the number of conditioned gapes was significantly reduced when wheel rocking was greatly reduced. Thus, it is possible that the wheel-running reduced palatability of the taste solution reported by Dwyer et al. (2008) is due, at least in part, to rocking. Reducing the rocking should not only reduce the number of conditioned disgust responses but it should also increase palatability as assessed by the pattern of licking. Parker et al. (2009) have suggested that there are two systems producing conditioned taste avoidance – only one of which is mediated by nausea. Drugs, like lithium chloride, that produce nausea not only produce avoidance but also produce conditioned disgust responses, whereas drugs, like amphetamine, that do not produce nausea only produce conditioned taste avoidance. Boakes and Nakajima (2009) outline five theories of the mechanisms by which wheel-running induces conditioned taste avoidance: activation of the mesolimbic-dopamine system; gastrointestinal discomfort; general stress; motion sickness; and energy expenditure. The current findings indicate that wheel running induces conditioned
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taste avoidance (possibly through the mesolimbic-dopamine system) but that conditioned disgust (gaping) results at least partly from rocking (possibly through gastrointestinal discomfort resulting from rocking-induced motion sickness). Group Rock and LoRock showed equivalent levels of avoidance and equivalent levels of wheel running. This finding indicates that absence of rocking in the motorized wheel did not account for the weak conditioned taste avoidance reported in the Forristall et al. (2007) study. More likely, the weaker conditioned taste avoidance produced by motorized wheel running compared to free wheel running (with rocking) was due to the pattern of wheel running. Rats in the motorized wheel were made to run at a constant rate so that they ran the same average distance as the rats in the free wheel. In contrast, rats in the free wheel do not run at a constant rate; they typically run in bursts and pauses (e.g., Premack and Schaeffer, 1962) with more running at the beginning of the session and less at the end (e.g., Aoyama and McSweeney, 2001). Thus there was more intense running immediately after drinking the taste solution for the rats in the free wheel group than in the motorized wheel group. This would mean that compared to the motorized wheel group, the free wheel group would get a more intense unconditioned stimulus (greater speed of running) soon after the conditioned stimulus (the taste), which should facilitate conditioned taste avoidance by the free wheel. It should be noted that others have found stronger conditioned taste avoidance induced by running in a motorized wheel than Forristall et al. (e.g., Masaki and Nakajima, 2006). However, others have not matched the number of wheel revolutions in the motorized wheel to rats running in free wheels. For example, the rats in the motorized wheel in the Forristall et al. study never reached the speed or distance run by the rats in the motorized wheel in the Masaki and Nakajima study. So the stronger aversion observed by Masaki and Nakajima may have been due to the increased speed/distance run. There has long been a question as to whether illness-inducing and rewarding drugs produce conditioned taste avoidance through similar mechanisms (e.g., Gamzu, 1977; Goudie, 1979; Grant, 1987). As mentioned earlier, Parker and her colleagues (e.g., Parker et al., 2009) have suggested that there are different mechanisms because differences are revealed in the taste reactivity test; nauseainducing drugs condition disgust reactions whereas rewarding drugs do not. Nausea-inducing drugs that condition disgust reactions do not serve as reinforcers and all such drugs condition place avoidance. Moreover, Parker et al., in a table comparing drugs that do and do not produce conditioned disgust reactions, found no rewarding drugs (as measured by conditioned place preference) that also produced disgust reactions (see Table 6.1 in Parker et al., 2009). Our findings in experiment 1 initially suggested that wheel running might be an exception in this regard. However the findings of experiment 2 suggest this empty category may remain empty – wheel running alone produces conditioned place preference and conditioned taste avoidance, but perhaps not conditioned disgust reactions. Conditioned disgust reactions may only result from the rocking of the wheel when the rat stops running. Acknowledgments The research was funded by grants to VLG and GMM from the Natural Sciences and Engineering Research Council of Canada and was conducted in compliance with the regulations of the Canadian Council on Animal Care with approval of the Institutional Animal Care Committee.
References Aoyama, K., McSweeney, F.K., 2001. Habituation contributes to within-session changes in free wheel running. J. Exp. Anal. Behav. 76, 289–302. Baysari, M.T., Boakes, R.A., 2004. Flavour aversion produced by running and attenuated by prior exposure to wheels. Q. J. Exp. Psychol. B 57, 273–286. Belke, T.W., Wagner, J.P., 2005. The reinforcing property and the rewarding aftereffect of wheel running in rats: a combination of two paradigms. Behav. Process. 68, 165–172. Boakes, R.A., Nakajima, S., 2009. Conditioned taste aversions based on running or swimming. In: Reilly, S., Schachtman, T.R. (Eds.), Conditioned Taste Aversion: Behavioral and Neural Processes. Oxford University Press, Oxford, UK, pp. 159–178. Brown, A.R., Penney, A.M., Skinner, D.M., Martin, G.M., 2011. Aversive, appetitive and flavour avoidance responses in the presence of contextual cues. Learn. Behav. 39, 95–103. Cordick, N., Parker, L.A., Ossenkopp, K.-P., 1999. Rotation-induced conditioned rejection in the taste reactivity test. NeuroReport 10, 1557–1559. Domjan, M., 1975. Poison-induced neophobia: role of stimulus generalization of conditioned taste aversions. Anim. Learn. Behav. 3, 205–211. Domjan, M., 1977. Attenuation and enhancement of neophobia for edible substances. In: Barker, L.M., Best, M.R., Domjan, M. (Eds.), Learning Mechanisms in Food Selection. Baylor University Press, Waco, TX, pp. 151–179. Dwyer, D.M., Boakes, R.A., Hayward, A.J., 2008. Reduced palatability in lithiumand activity-based, but not amphetamine-based, taste aversion learning. Behav. Neurosci. 122, 1051–1060. Forristall, J.R., Hookey, B.L., Grant, V.L., 2007. Conditioned taste avoidance induced by forced and voluntary wheel running in rats. Behav. Process. 74, 326–333. Gamzu, E., 1977. The multifaceted nature of taste-aversion-inducing agents: is there a single common factor? In: Barker, L.M., Best, M.R., Domjan, M. (Eds.), Learning Mechanisms in Food Selection. Baylor University Press, Waco, TX, pp. 477–509. Goudie, A.J., 1979. Aversive stimulus properties of drugs. Neuropharmacology 18, 971–979. Grant, V.L., 1987. Do conditioned taste aversions result from activation of emetic mechanisms? Psychopharmacology 93, 405–415. Grill, H.J., Norgren, R., 1978. The taste reactivity test: I. Mimetic responses to gustatory stimuli in neurologically normal rats. Brain Res. 143, 263–279. Heth, D.C., 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. Iversen, I.H., 1993. Techniques for establishing schedules with wheel running as reinforcement in rats. J. Exp. Anal. Behav. 60, 219–238. Kent, W.D.T., Cross-Mellor, S.K., Kavaliers, M., Ossenkopp, K.-P., 2000. Acute effects of corticosterone on LiCl-induced rapid gustatory conditioning in rats: a taste reactivity analysis. NeuroReport 11, 3903–3908. 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., Byrne, M.J., Koh, M.T., 2000. Pairings of a distinctive chamber with the aftereffect of wheel running produce conditioned place preference. Appetite 34, 87–94. Limebeer, C.L., Krohn, J.P., Cross-Mellor, S., Litt, D.E., Ossenkopp, K.-P., Parker, L.A., 2008. Exposure to a context previously associated with nausea elicits conditioned gaping in rats: a model of anticipatory nausea. Behav. Brain Res. 187, 33–40. Masaki, T., Nakajima, S., 2006. Taste aversion in rats induced by forced swimming, voluntary running, forced running, and lithium chloride injection treatments. Physiol. Behav. 88, 411–416. Masaki, T., Nakajima, S., 2008. Forward conditioning with wheel running causes place aversion in rats. Behav. Process. 79, 43–47. Nakajima, S., Hayashi, H., Kato, T., 2000. Taste aversion induced by confinement in a running wheel. Behav. Process. 49, 35–42. Parker, L.A., 1984. Behavioral conditioned responses across multiple conditioning/testing trials elicited by lithium- and amphetamine-paired flavors. Behav. Neural Biol. 41, 190–199. Parker, L.A., 1995. Rewarding drugs produce taste avoidance, but not taste aversion. Neurosci. Biobehav. Rev. 19, 143–151. Parker, L.A., 2003. Taste avoidance and taste aversion: evidence for two different processes. Learn. Behav. 31, 165–172. Parker, L.A., Limebeer, C.L., Rana, S.A., 2009. Conditioned disgust, but not conditioned taste avoidance, may reflect conditioned nausea in rats. In: Reilly, S., Schachtman, T.R. (Eds.), Conditioned Taste Aversion: Behavioral and Neural Processes. Oxford University Press, Oxford, UK, pp. 92–113. Parker, L.A., Rana, S.A., Limebeer, C.L., 2008. Conditioned nausea in rats: assessment by conditioned disgust reactions, rather than conditioned taste avoidance. Can. J. Exp. Psychol. 62, 198–209. Premack, D., Schaeffer, R.W., 1962. Distributional properties of operant-level locomotion in the rat. J. Exp. Anal. Behav. 5, 89–95.
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Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Dissociation of conditioned taste avoidance from conditioned disgust reactions induced by wheel running in rats Virginia L. Grant ∗ , Sarah V. McDonald, Robyn C. Sheppard, Catherine L. Caldwell, Thomas H. Heeley, Adam R. Brown, Gerard M. Martin Department of Psychology, Memorial University of Newfoundland, St. John’s, NL A1B 3X9, Canada
a r t i c l e
i n f o
Article history: Received 8 September 2011 Received in revised form 10 January 2012 Accepted 30 January 2012 Keywords: Conditioned disgust reactions Conditioned taste aversion Conditioned taste avoidance Rats Taste reactivity test Wheel running
a b s t r a c t It is well established that wheel running in rats produces conditioned taste avoidance; that is, rats that run in wheels after consuming a novel-tasting solution later consume less of that solution than rats that do not run. In experiment 1, we found that wheel running also produces conditioned disgust reactions, indicated by gapes elicited by both the taste and context that were experienced before running. Experiment 2 showed that the conditioned disgust reactions were likely not due to running itself but to a by-product of running, the rocking of the wheel that occurs when the running stops. When rocking was reduced, the disgust reactions were also reduced, but consumption of the taste solution was not changed, showing dissociation of conditioned taste avoidance and disgust. These findings indicate that the taste avoidance induced by wheel running itself is more like the taste avoidance produced by rewarding drugs than that produced by nausea-inducing drugs. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Wheel running produces conditioned taste avoidance in rats; that is, when rats drink a novel-tasting solution and then run in running wheels they subsequently drink less of that taste solution than rats treated equivalently but which do not run (e.g., Baysari and Boakes, 2004; Heth et al., 2001; Lett and Grant, 1996; Nakajima et al., 2000). Reduced consumption produced by wheel running does not necessarily reflect conditioned disgust. Parker (2003) distinguishes between treatments (usually drugs) that are truly aversive and clearly induce nausea and sickness (e.g., lithium chloride) and those that are rewarding and do not appear to induce nausea (e.g., amphetamine). When administered after consumption of a novel tasting solution, both types of drug can produce strong conditioned taste avoidance, indicated by reduced consumption, but only nausea-inducing drugs produce conditioned disgust reactions, indicated by such behaviors as gaping, paw pushing, and chin rubbing, which are elicited by the associated taste solution in a taste reactivity test (Grill and Norgren, 1978). Wheel running is similar to rewarding drugs, in that it also has rewarding effects. It can serve as a reinforcer for bar pressing (e.g., Belke and Wagner, 2005; Iversen, 1993) and it can condition a place preference (e.g., Belke and Wagner, 2005; Lett et al., 2000). Wheel running is also similar to rewarding drugs in terms of
∗ Corresponding author. Tel.: +1 709 864 8020; fax: +1 709 864 4000. E-mail addresses: [email protected], [email protected] (V.L. Grant).
its taste conditioning effects; like them, it produces conditioned taste avoidance. To complete the parallel between wheel running and rewarding drugs, there should be no evidence of conditioned disgust reactions; that is, like rewarding drugs (Parker, 1995), wheel running should produce conditioned taste avoidance, but not conditioned disgust reactions. This seems likely because Parker and her colleagues have not found any treatment that produces both a place preference and conditioned disgust reactions (e.g., see Table 6.1 in Parker et al., 2009). However there is some evidence that wheel running may be an exception. Dwyer et al. (2008), using analysis of the rats’ pattern of licking to evaluate palatability, found that wheel running produced a pattern of licks similar to that produced by lithium chloride and unlike that produced by amphetamine. This observation suggests that wheel running is more like lithium chloride, and thus may also produce disgust reactions as measured by a taste reactivity test. Such tests have shown disgust reactions (e.g., gapes) in response to novel tastes (e.g., Kent et al., 2000; Parker, 1984) and contexts (e.g., Brown et al., 2011; Limebeer et al., 2008) that have been paired with lithium chloride. The current experiments determined whether wheel running produces conditioned disgust reactions as well as conditioned taste avoidance. In experiment 1, we used a modified version of the taste reactivity test in which rats were allowed to drink the taste solution in a distinctive context while being observed for disgust reactions (Brown et al., 2011). In experiment 2 we tested whether disgust reactions were conditioned by a by-product of running, the rocking of the wheel when the rat stops running, rather than wheel running itself.
0376-6357/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2012.01.011
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2. Experiment 1
the remainder of the pretraining period and during the conditioning and test period.
2.1. Introduction On four conditioning trials, the rats consumed a novel tasting solution in a drinking box, following which they were either placed in running wheels (experimental group) or returned to their home cages (control group). The fluid consumption measure was used to assess conditioned taste avoidance. On the day following the last conditioning trial, the rats were given a test trial during which, in addition to the fluid consumption measure, the rats were also video-recorded and observed for any evidence of disgust reactions. Parker and her colleagues (e.g., Parker, 2003; Parker et al., 2008, 2009) have repeatedly noted that only emetic or nausea-inducing treatments produce conditioned disgust reactions, like gaping, in rats. Thus, if rats show the gaping reaction in response to a context and taste that have been paired with wheel running, it implies that wheel running produces nausea effects similar to those of emetic drugs. 2.2. Method 2.2.1. Subjects We obtained 24 male Sprague-Dawley rats (Rattus norvegicus) as weanlings from the breeding colony at Memorial University of Newfoundland. On the first day of the pretraining procedure the rats were approximately 40 days old and weighed between 167 and 249 g. The rats were housed, trained, and tested in a room maintained at approximately 20 ◦ C. The 12-h light/dark cycle had lights on at 7:30 a.m. The rats were individually housed in clear plastic cages (45 cm × 25 cm × 21 cm) with lids of metal bars that were indented to hold food and a drinking bottle. Food was always available in the home cages, as was water, except during the water deprivation period as outlined below. 2.2.2. Apparatus and materials Twelve freely rotating running wheels were used in the conditioning procedure. The circumference of each wheel was approximately 113 cm and the wire mesh running surface inside the wheel was between 11 and 12 cm in width. Each wheel had a magnet on the rotating part which passed two sensors situated 180◦ apart; the sensors were connected to a computer which recorded complete revolutions. Floorless drinking boxes with removable lids were placed on a square glass table top (85.1 cm × 85.1 cm) that was 73.7 cm above the floor, so that the rats confined in the boxes could be video recorded with a Canon high-definition camera on a small tripod positioned on the floor beneath the glass table top. The inside dimensions of the drinking boxes were 25.4 cm × 15.2 cm × 38.1 cm. They were made of wood; two were painted black and two white. Each rat was assigned to a box for the duration of the experiment. The table top under each box was cleaned with a wet paper towel and then wiped dry before each rat was confined in the box. A small hole drilled in one of the short sides of the box allowed insertion of a bottle spout for drinking. There was a second hole in one of the long sides which was not used. The novel-tasting solution was a 0.1% saccharin solution (1 g sodium saccharin per 1000 ml tap water). 2.2.3. Procedure Water was removed at 4 p.m. the day before the second pretraining session (see Section 2.2.3.1). The rats were allowed access to water in their home cages between 2 p.m. and 4 p.m. each day for
2.2.3.1. Pretraining. Three pretraining sessions familiarized each rat with its drinking box and with the water deprivation schedule. The rats spent the first 20-min session in their drinking boxes when not thirsty, followed by two 20-min sessions when thirsty. Sessions occurred in the morning on 3 consecutive days. After 10 min in the box, the spout of a water bottle was inserted to provide access to water for the last 10 min.
2.2.3.2. Conditioning. Conditioning trials were between 10:30 a.m. and 1:50 p.m. on 4 consecutive days immediately following the final day of the pretraining procedure. Six squads of 4 rats were conditioned on each day. In each squad, one rat in a white and one rat in a black drinking box were randomly assigned to run in the wheels after drinking (the wheel group); the other two rats were returned to their home cages (the control group). Each rat in the wheel group had its own wheel which was the same for each conditioning trial. On each conditioning day, the four rats in the first squad were confined in their drinking boxes for 20 min. During the last 10 min in the box, the rats were given access to the saccharin solution. Then the 2 rats in the wheel group were placed into their running wheels where they remained for 30 min before returning to the home cage; the 2 rats in the control group were returned to their home cages. Then the next squad was given a conditioning trial and so on for each of the six squads.
2.2.3.3. Test. Each rat was tested once for taste reactivity. On the day following the final conditioning trial, 6 rats in the wheel group and 6 in the control group were tested, one at a time, starting at 10:30 a.m. The remaining rats were similarly tested on the following day. As during conditioning, each rat was confined in its box for 20 min, with access to the saccharin solution during the last 10 min in the box. The video camera situated beneath the drinking box recorded each rat for the entire 20-min duration of the test trial.
2.2.4. Data analysis To evaluate the measure of conditioned taste avoidance, the mean intake of saccharin solution over the conditioning trials was analyzed by a 2 × 4 (Groups by Trials) mixed analysis of variance (ANOVA), with repeated measures on the trial factor. Linear trend analyses across conditioning trials assessed progressive changes in drinking for both groups as well as wheel running scores (number of revolutions) for the wheel group. Independent t-tests were conducted between groups for the saccharin solution consumption on the test. For all analyses the alpha level was set at .05, two-tailed for t-tests. The video record for each rat’s test was observed for the rejection reaction of gaping (the only rejection reaction observed). The scoring was validated by having two additional observers each score five of the records. Correlation coefficients were above +.90 (ps < .05) indicating that inter-rater reliability was high. All observers were blind to the observed rat’s experimental condition. To determine if the wheel and control groups differed in the number of subjects showing gapes, a chi square analysis was used. A 2 (Group) × 2 (Time in Session: first versus second 10 min) mixed ANOVA with repeated measures on the Time in Session factor assessed the extent to which gapes occurred before and after the introduction of the saccharin solution.
Saccharin Solution Consumed (gm)
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Fig. 1. Mean (±SE) amount of saccharin solution consumed on each trial and on the test for rats in the control and wheel groups in experiment 1.
2.3. Results 2.3.1. Saccharin solution consumption on conditioning and test trials Our findings on consumption replicate the many studies that show that wheel running induces conditioned taste avoidance (see Boakes and Nakajima, 2009, for a review). Mean saccharin solution intake over the four conditioning trials and the test trial is shown for wheel and control groups in Fig. 1. Analysis of the four conditioning trials revealed a significant group by trial interaction, F (3, 66) = 25.94, p < .001, reflecting the diverging scores as the wheel group consumed progressively less of the saccharin fluid over trials than the control group. Moreover, a significant group main effect, F (1, 22) = 27.36, p < .001, shows that overall, the wheel group consumed less than the control group. A linear trend analysis indicated a significant decrease in consumption by the wheel group over trials and an increase in consumption by the controls (ps < .001). On the test trial, significantly less saccharin was consumed by the wheel group than the control group, t (22) = 7.42, p < .001. 2.3.2. Disgust reactions on the test trial The only aversive measure observed was gaping. For some unexplained reason, one rat in the control group gaped once. Despite that anomaly, the number of rats that showed gapes was significantly greater in the wheel group (7 out of 12) than the control group (1 out of 12), 2 (1, N = 24) = 6.75, p = .009. Rats in the wheel group gaped more (M = 1.92, SE = 0.85) than rats in the control group (M = 0.08, SE = 0.08), F(1, 22) = 4.63, p = .04. Although there were more gapes in the second 10 min (M = 0.83, SE = 0.41), when saccharin solution was available, than in the first (M = 0.17, SE = 0.09), the difference was not significant, p = .09. 2.3.3. Wheel running The rats ran more each successive day; that is, the number of wheel revolutions increased across the four conditioning trials (Trial 1: M = 142.4, SE = 15.6; Trial 2: M = 210.1, SE = 34.4; Trial 3: M = 287.2, SE = 39.8; Trial 4: M = 302.7, SE = 39.7). The increase was significant, F (3, 33) = 20.84, p = .0001, and was confirmed by a significant linear trend, p < .001. 2.4. Discussion Wheel running produced both conditioned taste avoidance and conditioned disgust reactions. The rats that ran in the wheels after drinking the saccharin solution subsequently drank less of the solution and gaped more than the control rats that did not wheel run. Thus wheel running appears to be more like sickness-inducing
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drugs than rewarding drugs in conditioning both taste avoidance and disgust. The finding that gaping occurred before the introduction of the saccharin solution indicates that there was likely conditioning to the context. Such contextual conditioning of gaping has been reported with lithium chloride as the unconditioned stimulus (e.g., Brown et al., 2011; Limebeer et al., 2008). Moreover, although backward conditioning of a place with wheel running produces a conditioned place preference (Lett et al., 2000), forward conditioning produces a conditioned place aversion (Masaki and Nakajima, 2008), also indicating that aversive aspects of wheel running can be associated with a context experienced before the running. The context here was a distinctive one, solid wooden walls painted black or white, compared to the transparent plastic walls of the home cages, and thus could support contextual conditioning. Additionally, this test was after four conditioning trials which might account for the strong contextual conditioning effect. In contrast to the usual taste reactivity test (e.g., Grill and Norgren, 1978), our rats were not forced to taste the saccharin solution. The absence of forced exposure to the taste solution may have contributed to the lack of difference between the first and second 10 min of the test session.
3. Experiment 2 3.1. Introduction Forristall et al. (2007) compared free wheel running and forced wheel running in a motorized wheel as agents to produce conditioned taste avoidance. They found that both types of wheel running produced conditioned taste avoidance, however, the avoidance was greater when the taste solution was followed by free wheel running. The authors speculated on possible reasons for this difference; one hypothesis was that the free wheels rocked back and forth when the rats stopped running, whereas the motorized wheels just turned at a constant rate and never rocked. It could be that the rocking of the free wheels caused motion sickness, and this in turn might cause the stronger conditioned taste avoidance in the free wheel running group. Rotation-induced motion sickness produces both conditioned taste avoidance and disgust (Cordick et al., 1999). Rocking of the wheel could also induce motion sickness and thus produce the conditioned disgust reactions of experiment 1. In experiment 2 we tested whether rocking is responsible for the conditioned taste avoidance (reduction in consumption of the taste solution) as well as the disgust reactions (gapes) noted in experiment 1. We replicated experiment 1 but with an additional group – a group that ran in the wheels and had rocking of the wheels reduced by a researcher who placed a finger on the wheel any time the rat stopped running. Thus there were three groups of rats: a Rock group that ran freely in wheels that rocked; a LoRock group that ran freely in wheels that rocked less; and a control group that did not go in the wheels.
3.2. Method 3.2.1. Subjects The Memorial University of Newfoundland breeding colony provided 39 male, Sprague-Dawley rats. There were five sets of rats that arrived, as weanlings, at approximately 2-week intervals. There were 3 rats in the first set and 9 in each of the subsequent sets. They were group-housed for 7 days and then moved to individual housing 8 days before the start of water deprivation, when they were between 35 and 40 days old. Housing conditions were as in experiment 1 (Section 2.2.1.).
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3.2.2. Materials and apparatus The materials and apparatuses were as described in experiment 1 (Section 2.2.2), except that only six of the wheels were used and all drinking boxes were painted white. 3.2.3. Procedure In unspecified details the procedure was as in experiment 1 (Section 2.2.3). Within each of the five sets of rats, except the first, there were three squads. A squad was composed of 3 rats which were trained at the same time; each rat in a squad was randomly assigned to one of three experimental groups, described below, so that each squad contained one rat from each of the three groups. 3.2.3.1. Pretraining. The pretraining procedure was the same as that in experiment 1 (Section 2.2.3.1), except that the pretraining sessions were in the afternoon, and water was made available in the home cage for an hour after the sessions in the drinking boxes were completed. 3.2.3.2. Conditioning. There were four daily conditioning trials starting the day after the last pretraining trial. The conditioning procedure was as described for experiment 1 (Section 2.2.3.2), except that there were three experimental treatments: placement in a running wheel which was allowed to rock (Group Rock); placement in a running wheel which was not allowed to rock (Group LoRock); and return to the home cage (Group Control). Each rat in Group Rock and LoRock was always placed in the same wheel, where they remained for 30 min. The wheels for the two members of a squad that were in Group Rock and Group LoRock were always side by side. A researcher sat between the two wheels and prevented the wheel for the rat in the LoRock condition from rocking. Whenever the rat stopped running and the wheel was about to rock, the researcher gently put a finger on the wheel and prevented it from rocking. In the last set of 9 rats, the three rats in Group Rock and the three in Group LoRock were video-recorded while they ran in the wheels. The video records were analyzed for the number of times each wheel rocked, as a check on the normal amount of rocking and on how successful the researcher was in preventing rocking. 3.2.3.3. Test. The day after the last training trial the test trial was conducted; as in experiment 1 (Section 2.2.3.3.) the rats were video-recorded during the 20 min in their drinking boxes. The tests were started at approximately 11 a.m. with the last rat finishing at approximately 5:30 p.m. 3.2.4. Data analysis As before, video records of the test session were scored for gaping during the 10 min before and the 10 min while they had access to saccharin solution. The video record for one rat in Group LoRock was lost due to a camera malfunction. The total number of gapes for that rat was acquired through direct observation, but the timing of the gapes was not noted, so this rat’s data were included in the Chi Square analysis, but not in the Group by Time in Session ANOVA (see Section 3.3.2). The scoring of the videos was again validated by having two additional observers, blind to each rat’s condition, score the records for gaping. Video records that were made of a wheel running session for 6 rats were scored for instances of rocking. These were also evaluated by two additional observers. Inter-rater correlation coefficients for gapes and rocks were all above +.90 (ps < .01) indicating high reliability of the scoring procedure. To evaluate the measure of conditioned taste avoidance, the mean intake of saccharin solution over the conditioning trials was analyzed by a 3 × 4 (Groups by Trials) mixed ANOVA, with repeated measures on the trial factor. Wheel turns were similarly analyzed
Saccharin Solution Consumed (gm)
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20
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16
Rock LoRock
12 8 4 0 1
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Trials Fig. 2. Mean (±SE) amount of saccharin solution consumed on each trial and on the test for rats in Group Rock, LoRock and Control in experiment 2.
with a 2 × 4 ANOVA. Other analyses were as in experiment 1 (Section 2.2.4). 3.3. Results 3.3.1. Saccharin solution consumption on conditioning and test trials As can be seen in Fig. 2, over the first four conditioning trials, Groups Rock and LoRock drank progressively less while Group Control drank progressively more. The reliability of these effects is supported by the 3 (Groups) × 4 (Trials) ANOVA. There were significant effects of Group, F(2, 36) = 33.62, p < .001, and Trial, F(3, 108) = 14.96, p < .001, and a Group by Trial interaction, F(6, 108) = 32.38, p < .001. A linear trend analysis indicated that the saccharin-solution consumption of the two wheel groups decreased over trials, while that of the control group increased (ps < .001). There were no group differences between Group Rock and LoRock, F(1, 24) = 0.83, p = .37; but both groups differed significantly from the control group (ps < .001). A one-way ANOVA of saccharin solution on the test trial showed that the groups continued to differ in saccharin consumption, F(2, 36) = 80.91, p < .001 (see Fig. 2). Both Group Rock and LoRock drank significantly less saccharin solution than Group Control, t(24) = 11.56 and t(24) = 10.15, respectively, ps < .001, and they did not differ from each other, t(24) = 0.50, p = .62. 3.3.2. Disgust reactions Parametric analysis of disgust reactions was limited to Groups Rock and LoRock because no rat in Group Control emitted any disgust reaction. The two sets of bars in Fig. 3 show the mean number of gapes emitted by Group Rock and LoRock in the first 10 min (before the solution was introduced), in the second 10 min (when the saccharin solution was available), and in total. Although Groups Rock and LoRock did not differ in consumption of saccharin solution on the test, they did differ in the number of gapes as assessed in the Group × Time in Session ANOVA, F(1, 23) = 5.48, p = .03. As in experiment 1, there were more gapes in the second 10 min of the session than the first, but the difference was not reliable, p = .06. The present findings indicate that rocking produces conditioned disgust reactions, but that running without rocking may only produce conditioned taste avoidance. For Group Rock, the number of rats showing disgust responses was 10 (n = 13; 77%) whereas for Group LoRock, the number was 5 (n = 13; 38%). As mentioned above, no disgust responses were observed for any rat in Group Control (n = 13; 0%). A Chi Square analysis indicated differences overall, 2 (2, N = 39) = 16.25, p < .001. Compared to Group Control, both Group Rock and Group LoRock had greater
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6.3 (SE = 2.03), t(4) = 20.29, p = .001. Thus rocking was reduced for Group LoRock, but not eliminated. This lower level of rocking may have resulted in the gaping observed in some rats in Group LoRock. 3.4. Discussion
Fig. 3. Mean (+SE) number of gapes shown by the Rock and LoRock Groups in experiment 2. Gapes during the first 10 min of the test, before the saccharin solution was introduced, are shown in the left-hand bar and for the second 10 min, during access to the saccharin solution, in the middle bar, and the total number of gapes during the 20-min test in the right-hand bar.
numbers of rats showing disgust, 2 (1, N = 26) = 16.25, p < .001 and 2 (1, N = 26) = 6.19, p = .013, respectively. More rats in Group Rock showed disgust than in Group LoRock, 2 (1, N = 26) = 3.94, p = .047. The disgust reactions data indicate that a greater aversion to the taste is found when rocking is allowed. When rocking is reduced, the disgust responses are also reduced. As indicated in Section 3.3.1, the amount of rocking does not appear to affect consumption. Wheel running with rocking and without rocking produced equivalent conditioned taste avoidance as measured by the amount of the taste solution consumed.
Mean Number of Revolutions
3.3.3. Wheel turns for Group Rock and LoRock Our actions to prevent rocking did not affect the rats’ running behavior. Fig. 4 shows the mean number of wheel revolutions made by Group Rock and LoRock on each of the four conditioning trials. A 2 (Groups) × 4 (Trials) ANOVA showed that the groups did not differ in wheel turns, F(1, 24) = 1.36, p = .25, nor was there an interaction between Groups and Trials, F(3,72) = 0.09, p = .97. There was, however, a large trials effect, F(3,72) = 15.52, p < .001. A linear trend analysis indicated that both groups ran more over trials (ps < .001). As a manipulation check, 3 rats in Group Rock and 3 in Group LoRock in the last squad were video-recorded while they were running in the wheels and the videos were analyzed for the number of times the wheel rocked. Compared to Group Rock, our prevention of rocking reduced the number of rocks for Group LoRock significantly. The mean number of rocks for the three rats in Group Rock was 130.7 (SE = 5.78) rocks, whereas for Group LoRock it was
300
Rock
250
LoRock
200 150 100 50 0 1
2
3
4
Trials Fig. 4. Mean (±SE) number of wheel revolutions on each trial for rats in Groups Rock and LoRock in experiment 2.
Wheel running produced conditioned taste avoidance whether the wheel rocked or was prevented from rocking; that is, both Group Rock and LoRock drank less of the saccharin solution than the controls. Moreover, both Group Rock and LoRock drank equivalent amounts of the saccharin solution, so the level of rocking had no effect on conditioned taste avoidance. However it did have an effect on conditioned disgust responses; rats in Group Rock made more gapes than rats in Group LoRock – that is, conditioned disgust was stronger in Group Rock than LoRock. As in experiment 1, disgust responses were evident both before and after the saccharin solution was made available, suggesting an association of the context with the running. There remains a question as to whether the lower level of gaping observed in the rats in Group LoRock was due to wheel running or to the rocks that the researcher was unable to prevent. An alternative interpretation of our findings is that wheel rocking produces sensitization, in that rats are more likely to gape and avoid flavored solutions after running and being rocked in the wheel. Early work with lithium chloride revealed that sensitization did not contribute to taste avoidance when unpaired control groups were included (Domjan, 1975, 1977), and thus probably does not contribute to the effects observed in our experiments. Moreover, there have been a number of studies of the effect of wheel running on the consumption measure which used differential conditioning (e.g., Heth et al., 2001; Lett & Grant, 1996; Nakajima et al., 2000). None report a sensitization effect, and it seems unlikely that gaping would be sensitized when consumption is not. 4. General discussion In experiment 1, we found that wheel running not only induced conditioned taste avoidance, but also conditioned disgust reactions to the taste and context. There was conditioned taste avoidance because rats that ran in the wheels after consuming saccharin subsequently drank less saccharin than rats that did not run and there was conditioned disgust because rats that ran in the wheels after consuming saccharin made more conditioned gapes indicative of disgust on the test than rats that did not run. This finding is consistent with that of Dwyer et al. (2008), who, using the pattern of licking as a measure, found reduced palatability of a taste solution that had been paired with wheel running. Experiment 2 revealed that the number of conditioned gapes was significantly reduced when wheel rocking was greatly reduced. Thus, it is possible that the wheel-running reduced palatability of the taste solution reported by Dwyer et al. (2008) is due, at least in part, to rocking. Reducing the rocking should not only reduce the number of conditioned disgust responses but it should also increase palatability as assessed by the pattern of licking. Parker et al. (2009) have suggested that there are two systems producing conditioned taste avoidance – only one of which is mediated by nausea. Drugs, like lithium chloride, that produce nausea not only produce avoidance but also produce conditioned disgust responses, whereas drugs, like amphetamine, that do not produce nausea only produce conditioned taste avoidance. Boakes and Nakajima (2009) outline five theories of the mechanisms by which wheel-running induces conditioned taste avoidance: activation of the mesolimbic-dopamine system; gastrointestinal discomfort; general stress; motion sickness; and energy expenditure. The current findings indicate that wheel running induces conditioned
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taste avoidance (possibly through the mesolimbic-dopamine system) but that conditioned disgust (gaping) results at least partly from rocking (possibly through gastrointestinal discomfort resulting from rocking-induced motion sickness). Group Rock and LoRock showed equivalent levels of avoidance and equivalent levels of wheel running. This finding indicates that absence of rocking in the motorized wheel did not account for the weak conditioned taste avoidance reported in the Forristall et al. (2007) study. More likely, the weaker conditioned taste avoidance produced by motorized wheel running compared to free wheel running (with rocking) was due to the pattern of wheel running. Rats in the motorized wheel were made to run at a constant rate so that they ran the same average distance as the rats in the free wheel. In contrast, rats in the free wheel do not run at a constant rate; they typically run in bursts and pauses (e.g., Premack and Schaeffer, 1962) with more running at the beginning of the session and less at the end (e.g., Aoyama and McSweeney, 2001). Thus there was more intense running immediately after drinking the taste solution for the rats in the free wheel group than in the motorized wheel group. This would mean that compared to the motorized wheel group, the free wheel group would get a more intense unconditioned stimulus (greater speed of running) soon after the conditioned stimulus (the taste), which should facilitate conditioned taste avoidance by the free wheel. It should be noted that others have found stronger conditioned taste avoidance induced by running in a motorized wheel than Forristall et al. (e.g., Masaki and Nakajima, 2006). However, others have not matched the number of wheel revolutions in the motorized wheel to rats running in free wheels. For example, the rats in the motorized wheel in the Forristall et al. study never reached the speed or distance run by the rats in the motorized wheel in the Masaki and Nakajima study. So the stronger aversion observed by Masaki and Nakajima may have been due to the increased speed/distance run. There has long been a question as to whether illness-inducing and rewarding drugs produce conditioned taste avoidance through similar mechanisms (e.g., Gamzu, 1977; Goudie, 1979; Grant, 1987). As mentioned earlier, Parker and her colleagues (e.g., Parker et al., 2009) have suggested that there are different mechanisms because differences are revealed in the taste reactivity test; nauseainducing drugs condition disgust reactions whereas rewarding drugs do not. Nausea-inducing drugs that condition disgust reactions do not serve as reinforcers and all such drugs condition place avoidance. Moreover, Parker et al., in a table comparing drugs that do and do not produce conditioned disgust reactions, found no rewarding drugs (as measured by conditioned place preference) that also produced disgust reactions (see Table 6.1 in Parker et al., 2009). Our findings in experiment 1 initially suggested that wheel running might be an exception in this regard. However the findings of experiment 2 suggest this empty category may remain empty – wheel running alone produces conditioned place preference and conditioned taste avoidance, but perhaps not conditioned disgust reactions. Conditioned disgust reactions may only result from the rocking of the wheel when the rat stops running. Acknowledgments The research was funded by grants to VLG and GMM from the Natural Sciences and Engineering Research Council of Canada and was conducted in compliance with the regulations of the Canadian Council on Animal Care with approval of the Institutional Animal Care Committee.
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