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Dissociation of conditioned and unconditioned factors in the running-induced feeding suppression
Physiology & Behavior 89 (2006) 428 – 437
Dissociation of conditioned and unconditioned factors in the running-induced feeding suppression Elham Satvat, Roelof Eikelboom ⁎ Department of Psychology, Wilfrid Laurier University, 75 University Ave West, Waterloo ON, Canada N2L 3C5 Received 12 October 2005; received in revised form 6 June 2006; accepted 5 July 2006
Abstract In adult rats, running wheel introduction induces a 7 to 10 day feeding suppression, either due to a learned conditioned taste avoidance or to the direct unconditioned effects of wheel running. The three experiments investigated the effects of wheel introduction on familiar (rat chow) and novel (24% sucrose solution) food consumption (Experiment 1), and then explored how alternate-day wheel access affected sucrose consumption when it was novel (Experiment 2) or familiar (Experiment 3). When paired with wheel introduction the consumption of a novel sucrose solution was completely suppressed for an extended period, whether the rats had continuous or alternate-day wheel access. In contrast, familiar food consumption was suppressed, for a limited period, only on wheel days. When rats were pre-exposed to the sucrose, consumption was suppressed only on wheel days. The results suggest that in addition to the direct unconditioned effects of wheel running on feeding, learning factors may influence the feeding suppression observed and thus wheel introduction supports a learned conditioned taste avoidance. © 2006 Elsevier Inc. All rights reserved. Keywords: Wheel running; Feeding; Novel sucrose; Associative learning; Taste avoidance; Male rats; Anorexia; Latent inhibition
1. Introduction Rats will voluntarily and spontaneously run soon after being given access to running wheels [1–3]. A substantial body of evidence demonstrates that wheel running influences energy balance in adult male and female rats [4–7]. The voluntary running would be expected to increase energy expenditure and an elevation in food intake might be expected. Paradoxically, when a wheel is first made available to rats, a noticeable suppression in food intake is evident [4–9]. With ad lib wheel and food access, feeding is suppressed by up to 30% when rats are first introduced to wheels. This feeding suppression disappears after about 10 days, and the rats gradually increase their food intake to a level similar to, or more than, those of nonrunning rats [4,6,9]. It is not yet clear why running induces this temporary feeding suppression. In a number of studies, the food suppression induced by wheel running has been suggested to be the result of a conditioned taste avoidance [10–16]. In these studies the ⁎ Corresponding author. Tel.: +1 519 884 1970x3465; fax: +1 519 746 7605. E-mail address: [email protected] (R. Eikelboom). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.07.005
traditional procedure for demonstrating taste avoidance conditioning has been employed, involving limited access to the novel CS (flavoured solution) followed by short term exposure to the US (wheel running). It was proposed that wheel running may produce a “sickness” state [11]. If wheel running induces a “sickness” state that supports a conditioned taste avoidance, then wheel running should have two effects on feeding, first the direct unconditioned suppression induced by the running, and second running should support a learned suppression. This learned suppression could add to the direct suppression, making the reduction in feeding with novel foods stronger than that seen with familiar foods (where learning would be reduced). The learned suppression should also be evident in feeding at times when animals do not have wheel access. Novelty is important in taste avoidance learning; it has been shown that prior exposure to the wheel running disrupts a conditioned taste avoidance [15,16] and food novelty is important for developing a strong taste avoidance. Given that familiar food consumption is suppressed by wheel access it becomes important to consider the effects of ad lib wheel access on novel food consumption. Two such studies exist [17,18] both using 32% sucrose solution as their novel food.
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However, these earlier experiments were complex and neither experiment was designed to compare the effects of initial wheel exposure on feeding of both familiar and novel foods and so lacked controls to address this issue. One study used only 4 rats and had no control animals [17]. The other study lacked animals with only familiar food access, given wheel or no wheel experience [18]. Moreover, while only intake of the novel sucrose was reported, the rats also had access to familiar rat chow and it is not clear what effect running had on consumption of this familiar food. In other words, from these studies the relative degree of wheel induced suppression of novel and familiar foods was not clear. This comparison would permit the exploration of the learned and unconditioned (direct) effects of wheel access. Our first experiment therefore, investigated the effects of wheel running on consumption of both familiar and novel food. This experiment was continued until the consumption of the novel and familiar food had stabilized. At this point it was possible to see what effect novel food introduction would have in animals with extensive wheel experience. 2. Experiment 1—the effect of continuous wheel access on intake of novel and familiar food 2.1. Animals Twenty-four male Sprague–Dawley rats (200–225 g, 47– 49 days old) from Charles River, Canada were used in this study. Upon arrival, rats were housed in pairs in standard plastic shoebox cages (48 × 27 × 20 cm) in a colony room, maintained at 21°–22 °C with a 12 h light–dark cycle (lights on at 0700 h). During this habituation period and throughout the study rats had ad lib access to food (Harlan Teklad, 8640, 22/5 Rodent Diet, 3.11 Kcal/g metabolizable energy) and tap water. They were weighed daily between 1000 h and 1200 h to become accustomed to handling. At the daily weighing of rats, food, sucrose, and water consumption were recorded by measuring the difference between the weight of the food, sucrose (24% w/w, 0.9 Kcal/g of solution), and water when introduced to the rats, and the weight remaining after 24 h. Small visible pieces of food spillage were included in the food intake measures but food powder was left on the tray as previous work had suggested such spillage was similar for all rats and weighed less than a gram [9]. All the procedures (in all these experiments) were approved by the Wilfrid Laurier University Animal Care Committee following the Canadian Council of Animal Care guidelines.
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(25 × 17 × 20 cm) were used as home cages for the animals without wheel access. 2.3. Procedure After 1 week of habituation, the rats (n = 6) were assigned to one of four conditions in a 2 × 2 design: Wheel–Sucrose, Wheel–No Sucrose, No Wheel–Sucrose, and No Wheel–No Sucrose, and were transferred to their assigned cages. For three baseline days (Days—2 to 0), the wheels were locked. On Day 0, sucrose solution, wheel or both became available ad lib to the animals, which were assigned to receive sucrose, wheel or both. The rats given sucrose access had one bottle of water and one bottle of sucrose solution, whereas the rats with no sucrose access had two bottles of water. To avoid having any sucrose spill on the rat chow, the sucrose bottles were always placed on the side away from the rat chow and the water bottles were always placed on top of the food hopper on the other side. However, such arrangements of the bottles did not allow controlling for bottle position preferences. On day 25 rats that had not had sucrose access (in Wheel–No Sucrose and No Wheel–No Sucrose Groups) were also introduced to the sucrose solution and consumption measured for another 3 days (till day 28). Therefore, during the last 3 days of the experiment, all the animals had ad lib access to sucrose and water. 2.4. Analysis Consumption was analyzed based on the caloric values of food (3.11 Kcal/g) and sucrose (0.9 Kcal/g of solution) for this and subsequent experiments, but in the figures we provide the actual consumption in grams on the right hand axis. Rat chow consumption was analyzed for the days around the environmental changes, using a 2 (wheel vs. no wheel) × 2 (sucrose vs. no sucrose) × 3 (days) mixed analysis of variance (ANOVA), for the baseline (days—2 to 0), days 1 to 3, days 23 to 25 and the last 3 days (days 26 to 28). The sucrose intake was analyzed using a 2 × 3 (days) mixed ANOVA for day 1 to 3 and day 23 to 25. For the last 3 days (day 26 to 28) when all the rats were given sucrose access, sucrose intake was analyzed, using a 2 × 2 × 3 mixed ANOVA. 2.5. Results and discussion One of the rats in the No Wheel–Sucrose condition broke its front teeth and its data were eliminated from analysis, resulting in five animals for this group.
2.2. Apparatus Twelve standard wire cages (25 × 17 × 20 cm), each with a custom-built wheel (diameter 30 cm, width 11 cm) attached, were used for the animals with wheel access in the first and subsequent studies. The number of wheel turns was recorded using a magnetic contact closure system, in 5-s bins by Dataquest III, a Mini-Mitter Co. data collection system. The cages were mounted on a three-tiered rack. Identical wire cages
2.5.1. Familiar food intake Fig. 1A shows the mean food calorie intake of the four groups over the entire study. The ANOVA of the 3 days of baseline found only a significant days effect, F(2, 38) = 4.81, p < 0.05. It is evident that the rats in the four groups all increased their feeding over these days. For the first 3 days of the experiment, the ANOVA of the food intake revealed a significant main effect for sucrose,
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p < 0.05, with wheel access animals eating more than animals without wheel access. After 23 days of wheel and sucrose availability, wheel access no longer had a major effect on food consumption, but sucrose access caused a pronounced suppression in food consumption. ANOVA of food intake for the last 3 days when all animals had sucrose access revealed a significant interaction between days and wheel, F(2, 38) = 5.23, p < 0.05, and a three way interaction approaching significance, F(2, 38) = 3.00, p = 0.06. Fig. 1A shows a sharp drop in the mean food consumption of the two groups that had no prior experience with sucrose. However, the interactions suggest that it took a few days for animals, introduced to sucrose, to completely readjust their feeding. 2.5.2. Unfamiliar sucrose intake The ANOVA of sucrose intake for days 1 to 3 showed a significant effect of wheel F(1, 10) = 170.50, p < 0.001, and a significant interaction, F(2, 20) = 12.02, p < 0.001. Fig. 1B demonstrates that whereas the rats with wheel access hardly consumed any sucrose at the start of the experiment, actually decreasing their consumption over these days, the rats with no wheel access consumed a large amount of sucrose with an increase from day 1 to 2. The ANOVA of sucrose intake for day 23 to 25 found no significant effects indicating, as is evident from Fig. 1B, that by this time sucrose consumption had increased and stabilized for animals with wheel access at levels similar to sucrose consumption in no wheel rats. For the last 3 days of the study, ANOVA for the sucrose calorie intake found only a significant main effect of wheel, F(1, 19) = 12.73, p < 0.01, with wheel access rats consuming more sucrose. Fig. 1. A. Mean (±SEM) daily food intake (Kcal left axis, grams right axis) of the four groups of rats in Experiment 1. The first vertical line indicates the point of wheel and sucrose solution introduction, the second line the point at which the no sucrose groups started to receive sucrose. B. Mean (± SEM) daily 24% sucrose solution intake (Kcal left axis, grams solution right axis) of the Wheel– Sucrose and No Wheel–Sucrose group in Experiment 1. On day 26 the no sucrose groups started to receive sucrose solution. C. Mean (±SEM) daily percentage of calories consumed as sucrose of rats that had access to this solution.
F(1, 19) = 14.53, p < 0.01, and a significant wheel × sucrose interaction, F(1, 19) = 24.71, p < 0.001. It is evident from Fig. 1A that the interaction is due to the fact that while both wheel groups showed a similar feeding suppression over these 3 days, for the rats in the no wheel groups, those with sucrose availability showed an even more marked suppression of the food intake relative to No Wheel-No Sucrose rats. The ANOVA of the food intake for days 23 to 25 found a significant effect for sucrose, F(1, 19) = 138.17, p < 0.001 and a days × wheel × sucrose interaction, F(2, 38) = 4.91, p < 0.05. As is evident in Fig. 1A, at this point in the experiment both groups given sucrose access consumed significantly less food than did the groups without sucrose access. Separate ANOVAs for each of these 3 days revealed significant sucrose effect for each day, and significant wheel effect only for day 23, F(1, 19) = 4.64,
2.5.3. Percentage of calories consumed as sucrose Fig. 1C represents the percentage of calories consumed as sucrose (100 × sucrose Kcal / total Kcal intake) in the four groups over the study. A ratio of 100 indicates consumption of only sucrose, a ratio of 0 indicates consumption of only rat chow. It is evident that about 50% of the no wheel group rats' calorie intake was from the sucrose consumption, but that the calorie intake of the rats with wheel access was initially almost completely from rat chow but they eventually increased their sucrose intake to the level of rats without wheel access. These results parallel almost exactly the results of the actual sucrose consumption (compare Fig. 1B and C). 2.5.4. Wheel running and body weight Table 1 presents the average wheel running for the first and last 3 days (days 23 to 25) of this experiment. The wheel running data were analyzed and revealed that while, as expected, the running increased over the experiment the running of the rats with and without sucrose access was similar, both F s < 1, suggesting that sucrose availability did not influence wheel running. The average body weights of the rats in the four groups for the 3 days of baseline and for days 23 to 25 are also presented in
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Table 1 Experimental groups
Average wheel running First 3 days
Body weight Last 3 days
3-day baseline
Last 3 days
1344.8 ± 106.01 1303.2 ± 161.63
4449.7 ± 950.04 4561.3 ± 1782.77
301.0 ± 7.14 295.4 ± 5.21 294.4 ± 6.34 299.9 ± 9.06
450.5 ± 9.34 473.7 ± 16.88 420.9 ± 14.16 434.8 ± 19.39
Experiment 2 Home Wheel Alternate-W
1582.8 ± 152.00 1473.9 ± 136.18
4570.7 ± 136.18 4000.5 ± 885.35
312.1 ± 3.06 315.0 ± 4.50 309.8 ± 2.58
424.5 ± 7.32 402.5 ± 9.92 369.1 ± 6.08
Experiment 3 Home Wheel Alternate-W
1426.3 ± 233.00 1843.9 ± 366.88
4751.7 ± 1241.16 5501.0 ± 1527.67
304.6 ± 9.95 302.6 ± 6.84 300.5 ± 4.71
405.2 ± 24.80 373.8 ± 13.93 340.5 ± 5.75
Experiment 1 No Wheel–No Sucrose No Wheel–Sucrose Wheel–No Sucrose Wheel–Sucrose
Table 1. The ANOVA for the baseline days revealed only a significant days effect, F(2, 38) = 5.64, p < 0.01, with rats gaining weight over these days. The ANOVA of days 23 to 25 revealed a significant days, F(2, 38) = 19.61, p < 0.001, and wheel, F(1, 19) = 4.61, p < 0.05, effects. While wheel access reduced body weight, the availability of sucrose did not cause a significant increase in body weight.
avoidance [11], then introducing rats to a novel food, in combination with alternate-day wheel access, should induce a suppression of the novel food, regardless of wheel availability. Thus, this experiment aims to examine the effect of the alternate-day wheel access on the consumption of novel and familiar food by comparing rats with alternate-day wheel access to rats with either no or continuous wheel access.
3. Experiment 2—the effect of alternate-day wheel access on intake of novel and familiar food
3.1. Procedure
The effects of alternate-day wheel access on consumption of a familiar food have been explored [9]. If learning has a role in the feeding suppression one might expect the suppression to be evident on both wheel and non-wheel days as learned effects should also be evident on the non-wheel days. In this study, however, for rats with alternate-day wheel exposure there was a significant difference in the food consumption on wheel and non-wheel days. These rats suppressed their food intake on the wheel days, but on the intervening non-wheel days they initially consumed as much as, and gradually more than, no wheel control rats. Thus, with a familiar food while wheel access had direct effects on feeding, it had no carry-over or learned effects on the non-wheel days. Rats with alternate-day wheel exposure had a total of 16 days of wheel running over the 32 days of the study. In contrast to animals with continuous wheel access where the feeding suppression only lasted 10 days, the feeding of the alternate day rats was suppressed each time these rats were given wheel access, with little change in the scale of the suppression. Note there was no difference in the amount of running between the two groups on the days when both groups had wheel access. The alternate-day wheel access study suggests that wheel running induces a direct anorexia lasting for about a day [9]. If wheel access only has direct effects on feeding then giving rats a novel food in combination with alternate-day wheel exposure, should induce a suppression of the novel food on wheel days and increased consumption of this food on non-wheel days. However, if the running can also support a conditioned taste
Thirty-six rats, similar to those in the first experiment, were tested in two replications for this study. Unspecified procedures and equipment were similar to those in Experiment 1. Following a week of habituation, rats were randomly assigned to one of three conditions, Home, Wheel, and Alternate-W. Rats were transferred from pair-housing to their assigned cages; Home group rats into the home cages; Wheel group rats into the wheel cages; Alternate-W group rats spent the first and consecutive odd days in the wheel cages and the second and consecutive even days of the experiment in the home cages. In the first replication there were 8 rats in Home group, 8 rats in Wheel group, and 4 rats in Alternate-W group. In the second replication there were 4 rats in the Home and Wheel groups and 8 rats in the Alternate-W group (sharing the wheel cages on alternate days, with the four lightest animals 1 day behind in the experiment), resulting in 12 rats in each group. After the four baseline days during which the wheels were locked, wheels were unlocked and all the animals were given ad lib access to a 24% sucrose solution, in addition to ad lib food and water. Wheel turns, body weight, and consumptions were measured daily for 18 days. 3.2. Analysis Because feeding suppression induced by wheel running is only evident for about a week [4,6,9], it was decided to focus our analysis on the effects of wheel running on consumptions over the first 6 days (3 cycles). Food calorie data for the baseline were analyzed using a 3 (groups) × 4 (days) mixed ANOVA. For
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the main part of the experiment, food calorie and sucrose calorie data were analyzed separately by a 3 (groups) × 2 (conditions, odd vs. even days) × 3 (blocks of 2 days) mixed ANOVAs for the first 6 days of the experiment. This was followed by three planned 2 (conditions) × 3 (blocks) ANOVAs looking at each group of rats individually. 3.3. Results and discussion 3.3.1. Familiar food intake Fig. 2A shows the mean food intake of the three groups over the complete experiment. The ANOVA of baseline food intake found a significant days effect, F(3, 99) = 26.61, p < 0.01 and a days × group interaction, F(6, 99) = 2.22, p < 0.05, suggesting that consumption differed from day to day. The food intake data for day 1 to 6 of the experiment (shown in Fig. 2A) showed, in a mixed ANOVA, a significant effects of group, F(2, 33) = 30.67, p < 0.01, and condition, F (1, 33) = 19.07, p < 0.001. The block × group, F(4, 66) = 4.66,
Fig. 2. A. Mean (±SEM) daily food intake (Kcal left axis, grams right axis) of the three groups of rats in Experiment 2. The vertical line indicates the point of wheel and sucrose solution introduction. B. Mean (±SEM) daily 24% sucrose solution intake (Kcal left axis, grams solution right axis) of the three groups of rats in Experiment 2. C. Mean (± SEM) daily percentage of calories consumed as sucrose of rats in all three groups.
p < 0.01, condition × group, F(2, 33) = 16.02, p < 0.01, and triple interactions, F(4, 66) = 3.91, p < 0.01 were all significant. The planned ANOVA of the Home group rats revealed no significant main effects or interactions, indicating that their food intake did not change over these 6 days of the experiment. It is also evident in Fig. 2A that the rats in the Wheel group decreased (relative to baseline), and then gradually increased their food consumption over the first part of the experiment. An ANOVA of these Wheel group rats found only a significant block effect, F(2, 22) = 7.18, p < 0.01. The ANOVA of the Alternate-W group rats revealed a significant effect of condition F(1,11) = 22.33, p < 0.01, and a significant block × condition interaction, F(2, 22) = 9.1, p < 0.01. Fig. 2A shows that when rats were given alternate-day wheel access, familiar food consumption was suppressed on wheel days and increased on non-wheel, home cage days. This difference became more pronounced over the initial few blocks. 3.3.2. Unfamiliar sucrose intake Fig. 2B shows the mean sucrose intake of the three groups of rats for the entire study. Sucrose consumption for the first 6 days of sucrose access was analysed as for the food, and showed a significant main effect of group, F(2,33) = 149.56, p < 0.001, and a significant block × condition interaction, F(2, 66) = 6.57, p < 0.01. It is clear that the rats without wheel access consumed significantly more sucrose in these 6 days of the experiment than did the rats in the other two groups, and that the drop in sucrose consumption over the first few days for the rats in the two wheel groups may have resulted in the significant interaction. The planned ANOVA for the Home group and the AlternateW group rats revealed no significant effects. As is evident in Fig. 2B, rats in both groups showed constant sucrose consumption over these 6 days. The Home group rats consumed about 60 Kcal/day while the Alternate-W group only 6 Kcal reflecting the almost complete suppression in this group on both wheel and no wheel days. Only in the latter part of this experiment did they start to show a zigzag pattern but with a low level of consumption. The ANOVA of the Wheel group rats revealed a significant effect of condition, F(1, 22) = 7.54, p < 0.05, and block × condition interaction, F(2, 22) = 11.15, p < 0.01. The rats in the Wheel group consumed an average of 18.4 ± 4.9 Kcal of sucrose on the first day of the experiment and their mean sucrose consumption decreased to 3.7 ± .8 Kcal by the second day and stayed low for day 3 to day 6. This decrease in consumption from day 1 to day 2 is most likely responsible for both significant effects. Only towards the end of this experiment did sucrose consumption in this group start to increase (at about the same rate as in Experiment 1). 3.3.3. Percentage of calories consumed as sucrose Fig. 2C shows the percentage of calories consumed as sucrose in the three groups over the entire study. It is evident that about 50% (slowly increasing) of the Home group rats' calorie intake was from the sucrose consumption, but that the calorie intake of the rats with continuous wheel access was mainly from the rat chow only gradually increasing their percentage sucrose consumption towards the end of this
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experiment. The Alternate-W rats' calorie intake is also initially almost completely from the rat chow but by the end of the study they are showing a zigzag pattern of sucrose preference. The rats come to show an increased preference for sucrose on home cage days and decreased preference on wheel days. As in the first experiment the percentage sucrose consumption parallels almost exactly the absolute sucrose consumption. 3.3.4. Wheel running and body weight As seen in previous work [9], the middle section of Table 1 shows that animals with alternate-day wheel access increase their running over days in a similar manner to that of animals with continuous wheel access, F < 1. The average body weights of the rats in the three groups for the 3 baseline days and for last 3 days of the experiment are presented in the middle of Table 1. The ANOVA for the baseline days revealed only a significant days effect, F(2, 66) = 221.12, p < 0.001, with rats gaining weight over these days. The ANOVA of days 16 to 18 revealed significant days, F(2, 66) = 35.09, p < 0.001, wheel, F(2, 33) = 12.37, p < 0.001, and interaction, F(4, 66) = 4.01, p < 0.01, effects. Interestingly while wheel access reduced body weight, the weight reduction was only significant in comparing the Alternate-W rats to animals in the other two groups. Alternate-day wheel access seemed to have a more profound suppressant effect on body weight than did continuous wheel access (Tukey p < 0.05).
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in this group that were lightest in weight, at the start of habituation, started the experiment 1 day later than the other animals permitting the 8 rats to share 4 wheel cages. Body weight, sucrose and food intakes were recorded daily for the 6 days of baseline. After 10 days of sucrose pre-exposure (4 days of habituation and 6 days of baseline), wheels were unlocked. Body weight, food, water and sucrose consumption of each rat was measured as before for 18 days. The results of this third experiment were analyzed in the same way as the results of the second experiment (using the last 4 days of the baseline period). 4.2. Results and discussion 4.2.1. Familiar food intake Fig. 3A shows the mean food intake for the three groups over the experiment. In this experiment the last 4 days of baseline food intake revealed only a significant day effect, F(3, 63) = 6.13, p < 0.01.
4. Experiment 3—the effect of alternate-day wheel access on intake of familiar sucrose and food Conditioned stimulus pre-exposure has been shown to reduce the strength of a conditioned taste avoidance, a phenomenon termed latent inhibition [19]. If latent inhibition interferes with the association between sucrose and the physiological effects induced by wheel running, then sucrose pre-exposure should weaken the conditioned, but not the unconditioned, sucrose suppression induced by wheel introduction. In this case one might see a zigzag pattern of sucrose consumption with alternate-day wheel access. Experiment 3 investigates how several days of pre-exposure to the sucrose solution changes the effect of the alternate-day wheel access on consumption of that food. 4.1. Procedure Twenty-four rats similar to those in the first two experiments were used for this study. Three days after arrival, 24% sucrose solution was offered to all the rats. Ad lib food and tap water were also available. After 4 days of sucrose exposure, 6 days of baseline were started. The rats were randomly assigned to one of three conditions, Home, Wheel, and Alternate-W. The 8 rats in the Home group remained in their wire cages. The 8 rats in the Wheel group were transferred into the wire cages, attached to wheels, with the wheels locked. The 8 rats in the Alternate-W group started the first day and consecutive odd days in the locked wheel cages; and on the second day and consecutive even days were in the hanging wire home cages. Four of the rats
Fig. 3. A. Mean (±SEM) daily food intake (Kcal left axis, grams right axis) of the three groups of rats in Experiment 3. The vertical line indicates the point of wheel introduction. B. Mean (±SEM) daily 24% sucrose solution intake (Kcal left axis, grams solution right axis) of the three groups of rats in Experiment 3. C. Mean (±SEM) daily percentage of calories consumed as sucrose of rats in all three groups.
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The food intake ANOVA for days 1 to 6 revealed a significant effect of block, F(2,42) = 3.70, p < 0.05, and condition, F(1, 21) = 16.96, p < 0.01. The block × group, F(4, 42) = 3.47, p < 0.05, condition × group, F(2, 21) = 13.47, p < 0.01, and block × condition interactions, F(2, 42) = 6.82, p < 0.05 were all significant. Thus, separate ANOVAs were performed for each group over the first 6 days of the experiment. The conditions by blocks ANOVA of the Home group rats found a significant main effect of block, F(2,14) = 7.31, p < 0.05, and it appears from Fig. 3A that rats in the Home group were gradually decreasing food intake over this period. The ANOVA of the Wheel group rats found significant effects of block, F(2, 14) = 7.02, p < 0.01, and condition, F(1, 7) = 7.40, p < 0.05. As evident in Fig. 3A, the rats in the Wheel group decreased and then increased their food consumption over the first 6 days of the experiment the typical feeding suppression seen at wheel introduction. The ANOVA of the Alternate-W group revealed a significant effect of condition F(1, 7) = 15.76, p < 0.05, and a significant block × condition interaction, F(2, 14) = 6.92, p < 0.05. When rats are given alternate-day wheel access, familiar food consumption is suppressed on wheel days and is increased on non-wheel, home cage days, a difference that becomes more pronounced over the first few cycles. Fig. 3A reveals this zigzag pattern of food consumption in the Alternate-W group. 4.2.2. Familiar sucrose calorie intake The last 4 days (baseline) of sucrose intake data immediately before the introduction of the wheel was analyzed by a 3 × 4 ANOVA and found significant effects of day, F(3, 63) = 4.64, p < 0.01, indicating that the rats in the three groups, as shown in Fig. 3B, all changed their sucrose intake over the baseline period, but that the groups did not differ. An ANOVA of the sucrose consumption for the first 6 days of the experiment showed a significant effect of group, F(2, 21) = 5.21, p < 0.05, block, F(2,42) = 4.34, p < 0.05, and condition, F(1, 21) = 8.76, p < 0.05. The block × group, F(4, 42) = 4.19, p < 0.05, condition × group, F(2, 21) = 10.16, p < 0.05, and block × condition interactions, F(2, 42) = 5.61, p < 0.05 were all significant. Thus, there was a significant difference in sucrose consumption of the three groups for the first 6 days of the experiment. The results of the three planned ANOVAs, for each group for the first 6 days of the experiment, were as follows: The planned ANOVA for the Home group rats revealed only a significant effect of block, F(2, 14) = 4.4, p < 0.05. As shown in Fig. 3B, rats in Home group were increasing their sucrose intake over these 6 days. The ANOVA of the Wheel group rats revealed a significant effect of block, F (2, 14) = 4.76, p < 0.05, and a significant block × condition interaction, F(2, 14) = 5.5, p < 0.05. As is evident from Fig. 3B, the initial suppression of sucrose intake recovers by day 6 and this change in consumption was responsible for both significant effects, thus in this experiment the sucrose and familiar food were affected equally by wheel introduction. The ANOVA of the Alternate-W group rats revealed a significant effect of condition, F(1, 7) = 13.54, p < 0.05, and a significant block × condition interaction, F(2, 14) = 4.37, p < 0.05, indicat-
ing that there was a decreased consumption of sucrose on odd (wheel) days relative to that on even (non-wheel, home cage) days over these 6 days. As evident from Fig. 3B, the AlternateW group was showing a zigzag pattern of sucrose consumption that was becoming more pronounced over the three blocks and maintained for the duration of this experiment. 4.2.3. Percentage of calories consumed as sucrose Fig. 3C shows the percentage of kcal consumed as sucrose in the three groups over the entire study. It is evident that, as previous experiments, about 50% of the Home group rats' calorie intake was from the sucrose consumption. The sucrose preference of the Wheel group rats in this study was somewhat suppressed initially, like their absolute sucrose consumption, but recovered fast. The Alternate-W rats' sucrose preference was similar to the Wheel group rats initial and only changed to a zigzag pattern of preference on the last few days of study. 4.2.4. Wheel running and body weight As seen in Experiment 2, the bottom section of Table 1 shows that in this experiment animals with alternate-day wheel access increase their running over days in a similar manner to that of animals with continuous wheel access, F < 1. The average body weights of the rats in the three groups for the 3 baseline days and for last 3 days of the experiment are presented in the bottom section of Table 1. The ANOVA for the baseline days revealed only a significant days effect, F (2, 42) = 121.32, p < 0.001, with rats gaining weight over these days. The ANOVA of days 16 to 18 revealed significant days, F(2, 42) = 32.63, p < 0.001, and wheel, F(2, 21) = 3.73, p < 0.05, effects. In this experiment only the animals in the Alternate-W group weighted less than the Home cage group rats (Tukey p < 0.05) and the body weights of the wheel group animals were in-between the other two groups. 5. Discussion The present study looked at the effect of wheel running on consumption of two sources of food (familiar rat chow and novel sucrose). In all three experiments, the control animals without wheel access given sucrose solution as the second source of food consumed considerable amounts of novel sucrose from day 1, and maintained this consumption as long as they had sucrose access (gradually increasing the amount over days) (Figs. 1B, 2B, 3B). The first day of sucrose consumption was sometimes a bit lower than subsequent days (Fig. 1B), sometimes not (Fig. 2B), but it was high for all animals almost from the first exposure. Even though the rats in these groups suppressed their intake of familiar rat chow in favour of the palatable novel sucrose (Figs. 1A, 2A, 3A), their total daily calorie intake was increased from day 1 and maintained at this level in all three studies, increasing from about 100 Kcal at baseline to about 120–130 Kcal after sucrose introduction. The sucrose consumption of these control rats indicates that this solution is highly palatable and that rats initiate consumption of this novel sucrose almost immediately.
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By way of contrast, novel sucrose consumption of the rats with ad lib or continuous wheel access in the first two experiments was almost completely suppressed, dropping further from day 1 to 2, and then increased only gradually toward the end of the experiments (Figs. 1B, 2B). As sucrose consumption increased for rats with wheel access their chow consumption gradually decreased, possibly due to consumption of sucrose. Given that the animals had two food sources in these experiments, it is not clear how strong a novel food feeding suppression would be induced by wheel access with only a single novel food. When in the first experiment, sucrose was introduced to rats with 26 days of wheel experience, there was no feeding suppression, suggesting that it was the novelty of the wheel running that resulted in the initial sucrose suppression at the start of the experiment (Fig. 1B). Wheel preexposure has been shown previously in traditional conditioned avoidance experiments to prevent the conditioned avoidance [15,16]. In experiment one (and the other two) the intake of the familiar rat chow of the rats with wheel access was also suppressed relative to baseline and to No Sucrose-No Wheel control animals. The consumption of this familiar rat chow was as suppressed, and recovered as rapidly, as it did in other wheel access studies that did not involve a second food source [4–6]. Even though the rats avoided the novel sucrose, they also initially avoided the familiar rat chow, but the recovery of chow feeding was more rapid than that of the novel sucrose (Fig. 1A and B). In the third experiment, when the animals were familiar with the sucrose solution, the wheel induced sucrose suppression was similar to that of the familiar rat chow. Thus despite the strong sucrose suppression wheel running still had a general unconditioned affect on feeding. Ad lib wheel introduction induced a much stronger and longer lasting suppressive effect on the intake of a novel compared to a familiar food. Whereas the suppression of the familiar rat chow recovered after only a few days, the intake suppression of the novel sucrose was very strong, almost complete, and long lasting. The stronger suppression of novel sucrose consumption suggests that while the wheel running may induce a “sickness” state that directly suppresses food intake [11], it may also support a learned suppression. This learned suppression in combination with the direct suppression, makes the suppression of novel foods much stronger than that seen with familiar foods. Due to latent inhibition [19] this learned effect is not evident with the familiar food, and the suppression is just a consequence of the direct unconditioned effects of the wheel. Unlike other studies, in which traditional procedure for conditioned taste avoidance have been applied [10–14], the rats in the present study were given ad lib access to water, food, sucrose solution and wheels and our results suggest that a learned suppression can be evident even when food and wheel access are not explicitly paired by the experimenter. With Experiment 2 and 3, we also examined the effect of alternate-day wheel access on consumption of novel and familiar food to further explore the role of learning. The group of rats with alternate-day wheel exposure showed a zigzag pattern of familiar food intake from day 1; suppressing
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their intake of the chow on wheel days and increasing it on the intervening no wheel days (Figs. 2A, 3A). However, in Experiment 2 with novel sucrose, rats suppressed their sucrose intake on both no wheel (home cage) and wheel days. In other words, the rats avoided the sucrose whether they were in the wheels or home cages. These results are in agreement with a role for learned conditioned taste avoidance in the feeding suppression. Evidently, if the food is novel it supports a more pronounced suppression than if the food is familiar, as would be predicted based on an involvement of learning. The animals associate the state change, induced by running, with the novel food, rather than the familiar food. The fact that the group of rats with alternate-day wheel exposure suppressed their intake of the novel sucrose, on both wheel and no wheel days, also supports the conditioned taste avoidance hypothesis. On wheel days, an association is formed between the novel sucrose and the unconditioned effect of running. Thus, animals avoid consuming sucrose on wheel days in response to the unconditioned state change induced by running (“sickness”). The days when rats are in their home cages, sucrose solution is still avoided but now in response to the learned association between the taste and the unconditioned state change induced by running. Such an association is not formed when the food is familiar due to latent inhibition [19]. Therefore, the rats consume normal amount of the familiar food when the wheels are not available. However, on wheel days, the unconditioned state change induced by running, occurs and directly influences all of their food consumption. Experiment 3 investigated the intake suppression induced by alternate-day wheel running after sucrose pre-exposure. Latent inhibition would suggest that CS pre-exposure should reduce the conditioned taste avoidance [19]. The rats with alternate-day wheel exposure showed a zigzag consumption pattern of the now-familiar sucrose throughout the entire study; suppressed on wheel days compared to their more normal consumption on non-wheel days (Fig. 3B). As the rats were familiarized with sucrose prior to the introduction of the alternate-day wheel access, they did not form an association between the unconditioned effect of wheel running and the sucrose solution. Therefore, on non-wheel days rats consumed normal amount of familiar sucrose solution, but on wheel days, the unconditioned effect of running still directly suppressed consumption. These animals showed a similar alternate-day pattern of the even more familiar rat chow intake at the beginning of the experiment; however, toward the end of the study rat chow consumption had stabilized, consumption was no longer affected by wheel access. Thus, toward the end of the study, rats were only suppressing their intake of the sucrose solution and not the rat chow on wheel days. On non-wheel, home cage days the rats were consuming the same amount of rat chow, but increasing their sucrose consumption considerably compared to the preceding wheel day. This may be either the result of a difference in preference for sucrose and rat chow or the result of differing degrees of familiarity with these foods. Note that the rats, with alternate-day wheel exposure in both Experiment 2 and 3, showed levels of running that were similar to running on the equivalent days in the rats with continuous wheel exposure.
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Thus, differences in food intake of these two groups are unlikely to be due to differences in running. The results of these experiments indicate that wheel running supports a conditioned taste avoidance, as has been previously suggested [11]. Even though, wheel running can act as an unconditioned stimulus to support the development of a CS-US association, it is not clear what internal changes are induced by wheel running. Opiate systems seem to be directly involved in wheel running. For example it has been shown that morphineinduced analgesia is reduced by wheel running [20]. In fact, a number of lines of evidence suggest that wheel running may induce a state similar to that induced by addictive drugs. Rats lever press to gain wheel access suggesting it is rewarding [21]. With ad lib access in cocaine and heroin self-administration, the rate of self-administration increases over days, leading to death from an overdose [22]. This escalation in consumption over days is a classic feature of addictive behaviour [23], and parallels the transition to addiction in humans. Similarly, with ad lib wheel access; rats come to increase their running over a number of days to an excessive level in a manner similar to that which happens with addicting drugs, it only occurs if the animals have at least a few hours of access a day [24]. In addition, rats develop a conditioned place preference for an environment associated with the after-effects of both wheel running [25] and addictive drugs such as morphine and cocaine [26]. This suggests that wheel running, like addictive drugs, produces a positive affect that outlasts wheel access and supports a place preference. Wheel running seems to have parallels with many aspects of drug self-administration. This has led to the suggestion that wheel running may serve as an animal model of addiction [24,27]. Thus, it may be possible that the novel food avoidance induced by wheel running, from our experiments, is similar to that induced by rewarding drugs. However it should be noted that in studies looking at the feeding suppression and weight loss evident with restricted feeding and ad lib wheel access (the activity anorexia procedure) many other hormonal and neural systems are disrupted by wheel access [28–30]. Finally it should be noted that the wheel running-induced feeding suppression had the expected effect on the weight of the rats. While sucrose access did not significantly increase body weight, wheel access in Experiment 1 caused a pronounced weight reduction. It is interesting that in the shorter second and third experiment alternate-day wheel access which had a longer lasting effect on food consumption also produced a more pronounced weight reduction. This is different from previous work in our laboratory [9] where alternate-day and continuous wheel access had similar effects on body weight. As many aspects of these experiments were different (the previous work looked at a 32 day wheel exposure for example) it is not clear what is responsible for the inconsistent results. In conclusion, three experiments in this paper suggest that in addition to the direct unconditioned effects of wheel running on feeding, learning factors may influence the feeding suppression observed. In situation where learning is more likely to occur this causes the animals to avoid the food almost completely at all
times for an extended period. This learned suppression is evident in situations where no explicit attempt is made to pair consumption with wheel running. This learning may occur either because in the feeding system learned associations can occur with long intervals between the CS and US or because voluntary running occurs in close temporal proximity to feeding. Future studies may illuminate some of these issues and help demonstrate some of the parameters influencing the wheel induced feeding suppression. Acknowledgments This research was funded, in part, by the Science and Technology Endowment Program at Wilfrid Laurier University to ES and by the Natural Sciences and Engineering Research Council funding to RE. Portions of this research were presented in a poster at the 2004 Society for Neuroscience meeting (#427.6). References [1] Sherwin CM. Voluntary wheel running: a review and novel interpretation. Anim Behav 1998;56:11–27. [2] Stewart CC. Variations in daily activity produced by alcohol and by changes in barometric pressure and diet with a description of recording methods. Am J Physiol 1898;1:40–56. [3] Richter CP. Animal behavior and internal drives. Q Rev Biol 1927;2: 307–43. [4] Afonso VM, Eikelboom R. Relationship between wheel running, feeding, drinking, and body weight in male rats. Physiol Behav 2003;80:19–26. [5] Levitsky DA. Feeding patterns of rats in response to fasts and changes in environmental conditions. Physiol Behav 1970;5:291–300. [6] Looy H, Eikelboom R. Wheel running, food intake, and body weight in male rats. Physiol Behav 1989;45:403–5. [7] Premack D, Premack AJ. Increased eating in rats deprived of running. J Exp Anal Behav 1963;6:209–12. [8] Bauman RA. The effects of wheel running, a light/dark cycle, and the instrumental cost of food on the intake of food in a closed economy. Physiol Behav 1992;52:1077–83. [9] Mueller DT, Loft A, Eikelboom R. Alternate-day wheel access: effects on feeding, body weight, and running. Physiol Behav 1997;62:905–8. [10] Hayashi H, Nakajima S, Urushihara K, Imada H. Taste avoidance caused by spontaneous wheel running: effects of duration and delay of wheel confinement. Learn Motiv 2002;33:390–409. [11] Lett BT, Grant VL. Wheel running induces conditioned taste aversion in rats trained while hungry and thirsty. Physiol Behav 1996;59:699–702. [12] Lett BT, Grant VL, Gaborko LL. Wheel running simultaneously induces CTA and facilitates feeding in non-deprived rats. Appetite 1998;31: 351–60. [13] Lett BT, Grant VL, Koh MT, Smith JF. Wheel running simultaneously produced conditioned taste aversion and conditioned place preference in rats. Learn Motiv 2001;32:129–36. [14] Nakajima S, Hayashi H, Kato T. Taste aversion induced by confinement in a running wheel. Behav Processes 2000;49:35–42. [15] Salvy SJ, Pierce WD, Heth DC, Russell JC. Pre-exposure to wheel running disrupts taste aversion conditioning. Physiol Behav 2002;76:51–6. [16] Baysari M, Boakes R. Flavour aversion produced by running and attenuated by prior exposure to wheels. Q J Exp Psychol B 2004;57: 273–86. [17] Nikoletseas MM. Exercise-induced sucrose suppression in the rat. Physiol Behav 1981;26:145–6. [18] Jennings WA, McCutcheon LE. Novel food and novel running wheels: conditions for inhibition of sucrose intake in rats. J Comp Physiol Psychol 1974;87:100–5.
E. Satvat, R. Eikelboom / Physiology & Behavior 89 (2006) 428–437 [19] Lubow RE, Moore AU. Latent inhibition: the effect of nonreinforced preexposure to the conditional stimulus. J Comp Physiol Psychol 1959; 52:451−19. [20] Kanarek RB, Gerstein AV, Wildman RP, Mathes WF, D'Anci KE. Chronic running-wheel activity decreases sensitivity to morphine-induced analgesia in male and female rats. Pharmacol Biochem Behav 1998;61:19–27. [21] Iversen IH. Techniques for establishing schedules with wheel running as reinforcement in rats. J Exp Anal Behav 1993;60:219–38. [22] Bozarth MA, Wise RA. Toxicity associated with long-term intravenous heroin and cocaine self-administration in the rat. JAMA 1985;254:81–3. [23] Ahmed SH, Koob GF. Transition from moderate to excessive drug intake: change in hedonic set point. Science 1998;282:298–300. [24] Eikelboom R, Lattanzio SB. Wheel access duration in rats: II. Day–night and within-session changes. Behav Neurosci 2003;117:825–32. [25] Lett BT, Grant VL, Byrne MJ, Koh MT. Pairings of a distinctive chamber with the aftereffect of wheel running produce conditioned place preference. Appetite 2000;34:87–94.
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[26] Mucha RF, van der Kooy D, O'Shaughnessy M, Bucenieks P. Drug reinforcement studied by the use of place conditioning in rat. Brain Res 1982;243:91–105. [27] Lattanzio SB, Eikelboom R. Wheel access duration in rats: I. Effects on feeding and running. Behav Neurosci 2003;117:496–504. [28] Broocks A, Schweiger U, Pirke KM. Hyperactivity aggravates semistarvation-induced changes in corticosterone and triiodothyronine concentrations in plasma but not luteinizing hormone and testosterone levels. Physiol Behav 1990;48:567–9. [29] Watanabe K, Hara C, Ogawa N. Feeding conditions and estrous cycle of female rats under the activity-stress procedure from aspects of anorexia nervosa. Physiol Behav 1992;51:827–32. [30] Aravich PF, Stanley EZ, Doerries LE. Exercise in food-restricted rats produces 2DG feeding and metabolic abnormalities similar to anorexia nervosa. Physiol Behav 1995;57:147–53.
Dissociation of conditioned and unconditioned factors in the running-induced feeding suppression Elham Satvat, Roelof Eikelboom ⁎ Department of Psychology, Wilfrid Laurier University, 75 University Ave West, Waterloo ON, Canada N2L 3C5 Received 12 October 2005; received in revised form 6 June 2006; accepted 5 July 2006
Abstract In adult rats, running wheel introduction induces a 7 to 10 day feeding suppression, either due to a learned conditioned taste avoidance or to the direct unconditioned effects of wheel running. The three experiments investigated the effects of wheel introduction on familiar (rat chow) and novel (24% sucrose solution) food consumption (Experiment 1), and then explored how alternate-day wheel access affected sucrose consumption when it was novel (Experiment 2) or familiar (Experiment 3). When paired with wheel introduction the consumption of a novel sucrose solution was completely suppressed for an extended period, whether the rats had continuous or alternate-day wheel access. In contrast, familiar food consumption was suppressed, for a limited period, only on wheel days. When rats were pre-exposed to the sucrose, consumption was suppressed only on wheel days. The results suggest that in addition to the direct unconditioned effects of wheel running on feeding, learning factors may influence the feeding suppression observed and thus wheel introduction supports a learned conditioned taste avoidance. © 2006 Elsevier Inc. All rights reserved. Keywords: Wheel running; Feeding; Novel sucrose; Associative learning; Taste avoidance; Male rats; Anorexia; Latent inhibition
1. Introduction Rats will voluntarily and spontaneously run soon after being given access to running wheels [1–3]. A substantial body of evidence demonstrates that wheel running influences energy balance in adult male and female rats [4–7]. The voluntary running would be expected to increase energy expenditure and an elevation in food intake might be expected. Paradoxically, when a wheel is first made available to rats, a noticeable suppression in food intake is evident [4–9]. With ad lib wheel and food access, feeding is suppressed by up to 30% when rats are first introduced to wheels. This feeding suppression disappears after about 10 days, and the rats gradually increase their food intake to a level similar to, or more than, those of nonrunning rats [4,6,9]. It is not yet clear why running induces this temporary feeding suppression. In a number of studies, the food suppression induced by wheel running has been suggested to be the result of a conditioned taste avoidance [10–16]. In these studies the ⁎ Corresponding author. Tel.: +1 519 884 1970x3465; fax: +1 519 746 7605. E-mail address: [email protected] (R. Eikelboom). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.07.005
traditional procedure for demonstrating taste avoidance conditioning has been employed, involving limited access to the novel CS (flavoured solution) followed by short term exposure to the US (wheel running). It was proposed that wheel running may produce a “sickness” state [11]. If wheel running induces a “sickness” state that supports a conditioned taste avoidance, then wheel running should have two effects on feeding, first the direct unconditioned suppression induced by the running, and second running should support a learned suppression. This learned suppression could add to the direct suppression, making the reduction in feeding with novel foods stronger than that seen with familiar foods (where learning would be reduced). The learned suppression should also be evident in feeding at times when animals do not have wheel access. Novelty is important in taste avoidance learning; it has been shown that prior exposure to the wheel running disrupts a conditioned taste avoidance [15,16] and food novelty is important for developing a strong taste avoidance. Given that familiar food consumption is suppressed by wheel access it becomes important to consider the effects of ad lib wheel access on novel food consumption. Two such studies exist [17,18] both using 32% sucrose solution as their novel food.
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However, these earlier experiments were complex and neither experiment was designed to compare the effects of initial wheel exposure on feeding of both familiar and novel foods and so lacked controls to address this issue. One study used only 4 rats and had no control animals [17]. The other study lacked animals with only familiar food access, given wheel or no wheel experience [18]. Moreover, while only intake of the novel sucrose was reported, the rats also had access to familiar rat chow and it is not clear what effect running had on consumption of this familiar food. In other words, from these studies the relative degree of wheel induced suppression of novel and familiar foods was not clear. This comparison would permit the exploration of the learned and unconditioned (direct) effects of wheel access. Our first experiment therefore, investigated the effects of wheel running on consumption of both familiar and novel food. This experiment was continued until the consumption of the novel and familiar food had stabilized. At this point it was possible to see what effect novel food introduction would have in animals with extensive wheel experience. 2. Experiment 1—the effect of continuous wheel access on intake of novel and familiar food 2.1. Animals Twenty-four male Sprague–Dawley rats (200–225 g, 47– 49 days old) from Charles River, Canada were used in this study. Upon arrival, rats were housed in pairs in standard plastic shoebox cages (48 × 27 × 20 cm) in a colony room, maintained at 21°–22 °C with a 12 h light–dark cycle (lights on at 0700 h). During this habituation period and throughout the study rats had ad lib access to food (Harlan Teklad, 8640, 22/5 Rodent Diet, 3.11 Kcal/g metabolizable energy) and tap water. They were weighed daily between 1000 h and 1200 h to become accustomed to handling. At the daily weighing of rats, food, sucrose, and water consumption were recorded by measuring the difference between the weight of the food, sucrose (24% w/w, 0.9 Kcal/g of solution), and water when introduced to the rats, and the weight remaining after 24 h. Small visible pieces of food spillage were included in the food intake measures but food powder was left on the tray as previous work had suggested such spillage was similar for all rats and weighed less than a gram [9]. All the procedures (in all these experiments) were approved by the Wilfrid Laurier University Animal Care Committee following the Canadian Council of Animal Care guidelines.
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(25 × 17 × 20 cm) were used as home cages for the animals without wheel access. 2.3. Procedure After 1 week of habituation, the rats (n = 6) were assigned to one of four conditions in a 2 × 2 design: Wheel–Sucrose, Wheel–No Sucrose, No Wheel–Sucrose, and No Wheel–No Sucrose, and were transferred to their assigned cages. For three baseline days (Days—2 to 0), the wheels were locked. On Day 0, sucrose solution, wheel or both became available ad lib to the animals, which were assigned to receive sucrose, wheel or both. The rats given sucrose access had one bottle of water and one bottle of sucrose solution, whereas the rats with no sucrose access had two bottles of water. To avoid having any sucrose spill on the rat chow, the sucrose bottles were always placed on the side away from the rat chow and the water bottles were always placed on top of the food hopper on the other side. However, such arrangements of the bottles did not allow controlling for bottle position preferences. On day 25 rats that had not had sucrose access (in Wheel–No Sucrose and No Wheel–No Sucrose Groups) were also introduced to the sucrose solution and consumption measured for another 3 days (till day 28). Therefore, during the last 3 days of the experiment, all the animals had ad lib access to sucrose and water. 2.4. Analysis Consumption was analyzed based on the caloric values of food (3.11 Kcal/g) and sucrose (0.9 Kcal/g of solution) for this and subsequent experiments, but in the figures we provide the actual consumption in grams on the right hand axis. Rat chow consumption was analyzed for the days around the environmental changes, using a 2 (wheel vs. no wheel) × 2 (sucrose vs. no sucrose) × 3 (days) mixed analysis of variance (ANOVA), for the baseline (days—2 to 0), days 1 to 3, days 23 to 25 and the last 3 days (days 26 to 28). The sucrose intake was analyzed using a 2 × 3 (days) mixed ANOVA for day 1 to 3 and day 23 to 25. For the last 3 days (day 26 to 28) when all the rats were given sucrose access, sucrose intake was analyzed, using a 2 × 2 × 3 mixed ANOVA. 2.5. Results and discussion One of the rats in the No Wheel–Sucrose condition broke its front teeth and its data were eliminated from analysis, resulting in five animals for this group.
2.2. Apparatus Twelve standard wire cages (25 × 17 × 20 cm), each with a custom-built wheel (diameter 30 cm, width 11 cm) attached, were used for the animals with wheel access in the first and subsequent studies. The number of wheel turns was recorded using a magnetic contact closure system, in 5-s bins by Dataquest III, a Mini-Mitter Co. data collection system. The cages were mounted on a three-tiered rack. Identical wire cages
2.5.1. Familiar food intake Fig. 1A shows the mean food calorie intake of the four groups over the entire study. The ANOVA of the 3 days of baseline found only a significant days effect, F(2, 38) = 4.81, p < 0.05. It is evident that the rats in the four groups all increased their feeding over these days. For the first 3 days of the experiment, the ANOVA of the food intake revealed a significant main effect for sucrose,
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p < 0.05, with wheel access animals eating more than animals without wheel access. After 23 days of wheel and sucrose availability, wheel access no longer had a major effect on food consumption, but sucrose access caused a pronounced suppression in food consumption. ANOVA of food intake for the last 3 days when all animals had sucrose access revealed a significant interaction between days and wheel, F(2, 38) = 5.23, p < 0.05, and a three way interaction approaching significance, F(2, 38) = 3.00, p = 0.06. Fig. 1A shows a sharp drop in the mean food consumption of the two groups that had no prior experience with sucrose. However, the interactions suggest that it took a few days for animals, introduced to sucrose, to completely readjust their feeding. 2.5.2. Unfamiliar sucrose intake The ANOVA of sucrose intake for days 1 to 3 showed a significant effect of wheel F(1, 10) = 170.50, p < 0.001, and a significant interaction, F(2, 20) = 12.02, p < 0.001. Fig. 1B demonstrates that whereas the rats with wheel access hardly consumed any sucrose at the start of the experiment, actually decreasing their consumption over these days, the rats with no wheel access consumed a large amount of sucrose with an increase from day 1 to 2. The ANOVA of sucrose intake for day 23 to 25 found no significant effects indicating, as is evident from Fig. 1B, that by this time sucrose consumption had increased and stabilized for animals with wheel access at levels similar to sucrose consumption in no wheel rats. For the last 3 days of the study, ANOVA for the sucrose calorie intake found only a significant main effect of wheel, F(1, 19) = 12.73, p < 0.01, with wheel access rats consuming more sucrose. Fig. 1. A. Mean (±SEM) daily food intake (Kcal left axis, grams right axis) of the four groups of rats in Experiment 1. The first vertical line indicates the point of wheel and sucrose solution introduction, the second line the point at which the no sucrose groups started to receive sucrose. B. Mean (± SEM) daily 24% sucrose solution intake (Kcal left axis, grams solution right axis) of the Wheel– Sucrose and No Wheel–Sucrose group in Experiment 1. On day 26 the no sucrose groups started to receive sucrose solution. C. Mean (±SEM) daily percentage of calories consumed as sucrose of rats that had access to this solution.
F(1, 19) = 14.53, p < 0.01, and a significant wheel × sucrose interaction, F(1, 19) = 24.71, p < 0.001. It is evident from Fig. 1A that the interaction is due to the fact that while both wheel groups showed a similar feeding suppression over these 3 days, for the rats in the no wheel groups, those with sucrose availability showed an even more marked suppression of the food intake relative to No Wheel-No Sucrose rats. The ANOVA of the food intake for days 23 to 25 found a significant effect for sucrose, F(1, 19) = 138.17, p < 0.001 and a days × wheel × sucrose interaction, F(2, 38) = 4.91, p < 0.05. As is evident in Fig. 1A, at this point in the experiment both groups given sucrose access consumed significantly less food than did the groups without sucrose access. Separate ANOVAs for each of these 3 days revealed significant sucrose effect for each day, and significant wheel effect only for day 23, F(1, 19) = 4.64,
2.5.3. Percentage of calories consumed as sucrose Fig. 1C represents the percentage of calories consumed as sucrose (100 × sucrose Kcal / total Kcal intake) in the four groups over the study. A ratio of 100 indicates consumption of only sucrose, a ratio of 0 indicates consumption of only rat chow. It is evident that about 50% of the no wheel group rats' calorie intake was from the sucrose consumption, but that the calorie intake of the rats with wheel access was initially almost completely from rat chow but they eventually increased their sucrose intake to the level of rats without wheel access. These results parallel almost exactly the results of the actual sucrose consumption (compare Fig. 1B and C). 2.5.4. Wheel running and body weight Table 1 presents the average wheel running for the first and last 3 days (days 23 to 25) of this experiment. The wheel running data were analyzed and revealed that while, as expected, the running increased over the experiment the running of the rats with and without sucrose access was similar, both F s < 1, suggesting that sucrose availability did not influence wheel running. The average body weights of the rats in the four groups for the 3 days of baseline and for days 23 to 25 are also presented in
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Table 1 Experimental groups
Average wheel running First 3 days
Body weight Last 3 days
3-day baseline
Last 3 days
1344.8 ± 106.01 1303.2 ± 161.63
4449.7 ± 950.04 4561.3 ± 1782.77
301.0 ± 7.14 295.4 ± 5.21 294.4 ± 6.34 299.9 ± 9.06
450.5 ± 9.34 473.7 ± 16.88 420.9 ± 14.16 434.8 ± 19.39
Experiment 2 Home Wheel Alternate-W
1582.8 ± 152.00 1473.9 ± 136.18
4570.7 ± 136.18 4000.5 ± 885.35
312.1 ± 3.06 315.0 ± 4.50 309.8 ± 2.58
424.5 ± 7.32 402.5 ± 9.92 369.1 ± 6.08
Experiment 3 Home Wheel Alternate-W
1426.3 ± 233.00 1843.9 ± 366.88
4751.7 ± 1241.16 5501.0 ± 1527.67
304.6 ± 9.95 302.6 ± 6.84 300.5 ± 4.71
405.2 ± 24.80 373.8 ± 13.93 340.5 ± 5.75
Experiment 1 No Wheel–No Sucrose No Wheel–Sucrose Wheel–No Sucrose Wheel–Sucrose
Table 1. The ANOVA for the baseline days revealed only a significant days effect, F(2, 38) = 5.64, p < 0.01, with rats gaining weight over these days. The ANOVA of days 23 to 25 revealed a significant days, F(2, 38) = 19.61, p < 0.001, and wheel, F(1, 19) = 4.61, p < 0.05, effects. While wheel access reduced body weight, the availability of sucrose did not cause a significant increase in body weight.
avoidance [11], then introducing rats to a novel food, in combination with alternate-day wheel access, should induce a suppression of the novel food, regardless of wheel availability. Thus, this experiment aims to examine the effect of the alternate-day wheel access on the consumption of novel and familiar food by comparing rats with alternate-day wheel access to rats with either no or continuous wheel access.
3. Experiment 2—the effect of alternate-day wheel access on intake of novel and familiar food
3.1. Procedure
The effects of alternate-day wheel access on consumption of a familiar food have been explored [9]. If learning has a role in the feeding suppression one might expect the suppression to be evident on both wheel and non-wheel days as learned effects should also be evident on the non-wheel days. In this study, however, for rats with alternate-day wheel exposure there was a significant difference in the food consumption on wheel and non-wheel days. These rats suppressed their food intake on the wheel days, but on the intervening non-wheel days they initially consumed as much as, and gradually more than, no wheel control rats. Thus, with a familiar food while wheel access had direct effects on feeding, it had no carry-over or learned effects on the non-wheel days. Rats with alternate-day wheel exposure had a total of 16 days of wheel running over the 32 days of the study. In contrast to animals with continuous wheel access where the feeding suppression only lasted 10 days, the feeding of the alternate day rats was suppressed each time these rats were given wheel access, with little change in the scale of the suppression. Note there was no difference in the amount of running between the two groups on the days when both groups had wheel access. The alternate-day wheel access study suggests that wheel running induces a direct anorexia lasting for about a day [9]. If wheel access only has direct effects on feeding then giving rats a novel food in combination with alternate-day wheel exposure, should induce a suppression of the novel food on wheel days and increased consumption of this food on non-wheel days. However, if the running can also support a conditioned taste
Thirty-six rats, similar to those in the first experiment, were tested in two replications for this study. Unspecified procedures and equipment were similar to those in Experiment 1. Following a week of habituation, rats were randomly assigned to one of three conditions, Home, Wheel, and Alternate-W. Rats were transferred from pair-housing to their assigned cages; Home group rats into the home cages; Wheel group rats into the wheel cages; Alternate-W group rats spent the first and consecutive odd days in the wheel cages and the second and consecutive even days of the experiment in the home cages. In the first replication there were 8 rats in Home group, 8 rats in Wheel group, and 4 rats in Alternate-W group. In the second replication there were 4 rats in the Home and Wheel groups and 8 rats in the Alternate-W group (sharing the wheel cages on alternate days, with the four lightest animals 1 day behind in the experiment), resulting in 12 rats in each group. After the four baseline days during which the wheels were locked, wheels were unlocked and all the animals were given ad lib access to a 24% sucrose solution, in addition to ad lib food and water. Wheel turns, body weight, and consumptions were measured daily for 18 days. 3.2. Analysis Because feeding suppression induced by wheel running is only evident for about a week [4,6,9], it was decided to focus our analysis on the effects of wheel running on consumptions over the first 6 days (3 cycles). Food calorie data for the baseline were analyzed using a 3 (groups) × 4 (days) mixed ANOVA. For
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the main part of the experiment, food calorie and sucrose calorie data were analyzed separately by a 3 (groups) × 2 (conditions, odd vs. even days) × 3 (blocks of 2 days) mixed ANOVAs for the first 6 days of the experiment. This was followed by three planned 2 (conditions) × 3 (blocks) ANOVAs looking at each group of rats individually. 3.3. Results and discussion 3.3.1. Familiar food intake Fig. 2A shows the mean food intake of the three groups over the complete experiment. The ANOVA of baseline food intake found a significant days effect, F(3, 99) = 26.61, p < 0.01 and a days × group interaction, F(6, 99) = 2.22, p < 0.05, suggesting that consumption differed from day to day. The food intake data for day 1 to 6 of the experiment (shown in Fig. 2A) showed, in a mixed ANOVA, a significant effects of group, F(2, 33) = 30.67, p < 0.01, and condition, F (1, 33) = 19.07, p < 0.001. The block × group, F(4, 66) = 4.66,
Fig. 2. A. Mean (±SEM) daily food intake (Kcal left axis, grams right axis) of the three groups of rats in Experiment 2. The vertical line indicates the point of wheel and sucrose solution introduction. B. Mean (±SEM) daily 24% sucrose solution intake (Kcal left axis, grams solution right axis) of the three groups of rats in Experiment 2. C. Mean (± SEM) daily percentage of calories consumed as sucrose of rats in all three groups.
p < 0.01, condition × group, F(2, 33) = 16.02, p < 0.01, and triple interactions, F(4, 66) = 3.91, p < 0.01 were all significant. The planned ANOVA of the Home group rats revealed no significant main effects or interactions, indicating that their food intake did not change over these 6 days of the experiment. It is also evident in Fig. 2A that the rats in the Wheel group decreased (relative to baseline), and then gradually increased their food consumption over the first part of the experiment. An ANOVA of these Wheel group rats found only a significant block effect, F(2, 22) = 7.18, p < 0.01. The ANOVA of the Alternate-W group rats revealed a significant effect of condition F(1,11) = 22.33, p < 0.01, and a significant block × condition interaction, F(2, 22) = 9.1, p < 0.01. Fig. 2A shows that when rats were given alternate-day wheel access, familiar food consumption was suppressed on wheel days and increased on non-wheel, home cage days. This difference became more pronounced over the initial few blocks. 3.3.2. Unfamiliar sucrose intake Fig. 2B shows the mean sucrose intake of the three groups of rats for the entire study. Sucrose consumption for the first 6 days of sucrose access was analysed as for the food, and showed a significant main effect of group, F(2,33) = 149.56, p < 0.001, and a significant block × condition interaction, F(2, 66) = 6.57, p < 0.01. It is clear that the rats without wheel access consumed significantly more sucrose in these 6 days of the experiment than did the rats in the other two groups, and that the drop in sucrose consumption over the first few days for the rats in the two wheel groups may have resulted in the significant interaction. The planned ANOVA for the Home group and the AlternateW group rats revealed no significant effects. As is evident in Fig. 2B, rats in both groups showed constant sucrose consumption over these 6 days. The Home group rats consumed about 60 Kcal/day while the Alternate-W group only 6 Kcal reflecting the almost complete suppression in this group on both wheel and no wheel days. Only in the latter part of this experiment did they start to show a zigzag pattern but with a low level of consumption. The ANOVA of the Wheel group rats revealed a significant effect of condition, F(1, 22) = 7.54, p < 0.05, and block × condition interaction, F(2, 22) = 11.15, p < 0.01. The rats in the Wheel group consumed an average of 18.4 ± 4.9 Kcal of sucrose on the first day of the experiment and their mean sucrose consumption decreased to 3.7 ± .8 Kcal by the second day and stayed low for day 3 to day 6. This decrease in consumption from day 1 to day 2 is most likely responsible for both significant effects. Only towards the end of this experiment did sucrose consumption in this group start to increase (at about the same rate as in Experiment 1). 3.3.3. Percentage of calories consumed as sucrose Fig. 2C shows the percentage of calories consumed as sucrose in the three groups over the entire study. It is evident that about 50% (slowly increasing) of the Home group rats' calorie intake was from the sucrose consumption, but that the calorie intake of the rats with continuous wheel access was mainly from the rat chow only gradually increasing their percentage sucrose consumption towards the end of this
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experiment. The Alternate-W rats' calorie intake is also initially almost completely from the rat chow but by the end of the study they are showing a zigzag pattern of sucrose preference. The rats come to show an increased preference for sucrose on home cage days and decreased preference on wheel days. As in the first experiment the percentage sucrose consumption parallels almost exactly the absolute sucrose consumption. 3.3.4. Wheel running and body weight As seen in previous work [9], the middle section of Table 1 shows that animals with alternate-day wheel access increase their running over days in a similar manner to that of animals with continuous wheel access, F < 1. The average body weights of the rats in the three groups for the 3 baseline days and for last 3 days of the experiment are presented in the middle of Table 1. The ANOVA for the baseline days revealed only a significant days effect, F(2, 66) = 221.12, p < 0.001, with rats gaining weight over these days. The ANOVA of days 16 to 18 revealed significant days, F(2, 66) = 35.09, p < 0.001, wheel, F(2, 33) = 12.37, p < 0.001, and interaction, F(4, 66) = 4.01, p < 0.01, effects. Interestingly while wheel access reduced body weight, the weight reduction was only significant in comparing the Alternate-W rats to animals in the other two groups. Alternate-day wheel access seemed to have a more profound suppressant effect on body weight than did continuous wheel access (Tukey p < 0.05).
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in this group that were lightest in weight, at the start of habituation, started the experiment 1 day later than the other animals permitting the 8 rats to share 4 wheel cages. Body weight, sucrose and food intakes were recorded daily for the 6 days of baseline. After 10 days of sucrose pre-exposure (4 days of habituation and 6 days of baseline), wheels were unlocked. Body weight, food, water and sucrose consumption of each rat was measured as before for 18 days. The results of this third experiment were analyzed in the same way as the results of the second experiment (using the last 4 days of the baseline period). 4.2. Results and discussion 4.2.1. Familiar food intake Fig. 3A shows the mean food intake for the three groups over the experiment. In this experiment the last 4 days of baseline food intake revealed only a significant day effect, F(3, 63) = 6.13, p < 0.01.
4. Experiment 3—the effect of alternate-day wheel access on intake of familiar sucrose and food Conditioned stimulus pre-exposure has been shown to reduce the strength of a conditioned taste avoidance, a phenomenon termed latent inhibition [19]. If latent inhibition interferes with the association between sucrose and the physiological effects induced by wheel running, then sucrose pre-exposure should weaken the conditioned, but not the unconditioned, sucrose suppression induced by wheel introduction. In this case one might see a zigzag pattern of sucrose consumption with alternate-day wheel access. Experiment 3 investigates how several days of pre-exposure to the sucrose solution changes the effect of the alternate-day wheel access on consumption of that food. 4.1. Procedure Twenty-four rats similar to those in the first two experiments were used for this study. Three days after arrival, 24% sucrose solution was offered to all the rats. Ad lib food and tap water were also available. After 4 days of sucrose exposure, 6 days of baseline were started. The rats were randomly assigned to one of three conditions, Home, Wheel, and Alternate-W. The 8 rats in the Home group remained in their wire cages. The 8 rats in the Wheel group were transferred into the wire cages, attached to wheels, with the wheels locked. The 8 rats in the Alternate-W group started the first day and consecutive odd days in the locked wheel cages; and on the second day and consecutive even days were in the hanging wire home cages. Four of the rats
Fig. 3. A. Mean (±SEM) daily food intake (Kcal left axis, grams right axis) of the three groups of rats in Experiment 3. The vertical line indicates the point of wheel introduction. B. Mean (±SEM) daily 24% sucrose solution intake (Kcal left axis, grams solution right axis) of the three groups of rats in Experiment 3. C. Mean (±SEM) daily percentage of calories consumed as sucrose of rats in all three groups.
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The food intake ANOVA for days 1 to 6 revealed a significant effect of block, F(2,42) = 3.70, p < 0.05, and condition, F(1, 21) = 16.96, p < 0.01. The block × group, F(4, 42) = 3.47, p < 0.05, condition × group, F(2, 21) = 13.47, p < 0.01, and block × condition interactions, F(2, 42) = 6.82, p < 0.05 were all significant. Thus, separate ANOVAs were performed for each group over the first 6 days of the experiment. The conditions by blocks ANOVA of the Home group rats found a significant main effect of block, F(2,14) = 7.31, p < 0.05, and it appears from Fig. 3A that rats in the Home group were gradually decreasing food intake over this period. The ANOVA of the Wheel group rats found significant effects of block, F(2, 14) = 7.02, p < 0.01, and condition, F(1, 7) = 7.40, p < 0.05. As evident in Fig. 3A, the rats in the Wheel group decreased and then increased their food consumption over the first 6 days of the experiment the typical feeding suppression seen at wheel introduction. The ANOVA of the Alternate-W group revealed a significant effect of condition F(1, 7) = 15.76, p < 0.05, and a significant block × condition interaction, F(2, 14) = 6.92, p < 0.05. When rats are given alternate-day wheel access, familiar food consumption is suppressed on wheel days and is increased on non-wheel, home cage days, a difference that becomes more pronounced over the first few cycles. Fig. 3A reveals this zigzag pattern of food consumption in the Alternate-W group. 4.2.2. Familiar sucrose calorie intake The last 4 days (baseline) of sucrose intake data immediately before the introduction of the wheel was analyzed by a 3 × 4 ANOVA and found significant effects of day, F(3, 63) = 4.64, p < 0.01, indicating that the rats in the three groups, as shown in Fig. 3B, all changed their sucrose intake over the baseline period, but that the groups did not differ. An ANOVA of the sucrose consumption for the first 6 days of the experiment showed a significant effect of group, F(2, 21) = 5.21, p < 0.05, block, F(2,42) = 4.34, p < 0.05, and condition, F(1, 21) = 8.76, p < 0.05. The block × group, F(4, 42) = 4.19, p < 0.05, condition × group, F(2, 21) = 10.16, p < 0.05, and block × condition interactions, F(2, 42) = 5.61, p < 0.05 were all significant. Thus, there was a significant difference in sucrose consumption of the three groups for the first 6 days of the experiment. The results of the three planned ANOVAs, for each group for the first 6 days of the experiment, were as follows: The planned ANOVA for the Home group rats revealed only a significant effect of block, F(2, 14) = 4.4, p < 0.05. As shown in Fig. 3B, rats in Home group were increasing their sucrose intake over these 6 days. The ANOVA of the Wheel group rats revealed a significant effect of block, F (2, 14) = 4.76, p < 0.05, and a significant block × condition interaction, F(2, 14) = 5.5, p < 0.05. As is evident from Fig. 3B, the initial suppression of sucrose intake recovers by day 6 and this change in consumption was responsible for both significant effects, thus in this experiment the sucrose and familiar food were affected equally by wheel introduction. The ANOVA of the Alternate-W group rats revealed a significant effect of condition, F(1, 7) = 13.54, p < 0.05, and a significant block × condition interaction, F(2, 14) = 4.37, p < 0.05, indicat-
ing that there was a decreased consumption of sucrose on odd (wheel) days relative to that on even (non-wheel, home cage) days over these 6 days. As evident from Fig. 3B, the AlternateW group was showing a zigzag pattern of sucrose consumption that was becoming more pronounced over the three blocks and maintained for the duration of this experiment. 4.2.3. Percentage of calories consumed as sucrose Fig. 3C shows the percentage of kcal consumed as sucrose in the three groups over the entire study. It is evident that, as previous experiments, about 50% of the Home group rats' calorie intake was from the sucrose consumption. The sucrose preference of the Wheel group rats in this study was somewhat suppressed initially, like their absolute sucrose consumption, but recovered fast. The Alternate-W rats' sucrose preference was similar to the Wheel group rats initial and only changed to a zigzag pattern of preference on the last few days of study. 4.2.4. Wheel running and body weight As seen in Experiment 2, the bottom section of Table 1 shows that in this experiment animals with alternate-day wheel access increase their running over days in a similar manner to that of animals with continuous wheel access, F < 1. The average body weights of the rats in the three groups for the 3 baseline days and for last 3 days of the experiment are presented in the bottom section of Table 1. The ANOVA for the baseline days revealed only a significant days effect, F (2, 42) = 121.32, p < 0.001, with rats gaining weight over these days. The ANOVA of days 16 to 18 revealed significant days, F(2, 42) = 32.63, p < 0.001, and wheel, F(2, 21) = 3.73, p < 0.05, effects. In this experiment only the animals in the Alternate-W group weighted less than the Home cage group rats (Tukey p < 0.05) and the body weights of the wheel group animals were in-between the other two groups. 5. Discussion The present study looked at the effect of wheel running on consumption of two sources of food (familiar rat chow and novel sucrose). In all three experiments, the control animals without wheel access given sucrose solution as the second source of food consumed considerable amounts of novel sucrose from day 1, and maintained this consumption as long as they had sucrose access (gradually increasing the amount over days) (Figs. 1B, 2B, 3B). The first day of sucrose consumption was sometimes a bit lower than subsequent days (Fig. 1B), sometimes not (Fig. 2B), but it was high for all animals almost from the first exposure. Even though the rats in these groups suppressed their intake of familiar rat chow in favour of the palatable novel sucrose (Figs. 1A, 2A, 3A), their total daily calorie intake was increased from day 1 and maintained at this level in all three studies, increasing from about 100 Kcal at baseline to about 120–130 Kcal after sucrose introduction. The sucrose consumption of these control rats indicates that this solution is highly palatable and that rats initiate consumption of this novel sucrose almost immediately.
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By way of contrast, novel sucrose consumption of the rats with ad lib or continuous wheel access in the first two experiments was almost completely suppressed, dropping further from day 1 to 2, and then increased only gradually toward the end of the experiments (Figs. 1B, 2B). As sucrose consumption increased for rats with wheel access their chow consumption gradually decreased, possibly due to consumption of sucrose. Given that the animals had two food sources in these experiments, it is not clear how strong a novel food feeding suppression would be induced by wheel access with only a single novel food. When in the first experiment, sucrose was introduced to rats with 26 days of wheel experience, there was no feeding suppression, suggesting that it was the novelty of the wheel running that resulted in the initial sucrose suppression at the start of the experiment (Fig. 1B). Wheel preexposure has been shown previously in traditional conditioned avoidance experiments to prevent the conditioned avoidance [15,16]. In experiment one (and the other two) the intake of the familiar rat chow of the rats with wheel access was also suppressed relative to baseline and to No Sucrose-No Wheel control animals. The consumption of this familiar rat chow was as suppressed, and recovered as rapidly, as it did in other wheel access studies that did not involve a second food source [4–6]. Even though the rats avoided the novel sucrose, they also initially avoided the familiar rat chow, but the recovery of chow feeding was more rapid than that of the novel sucrose (Fig. 1A and B). In the third experiment, when the animals were familiar with the sucrose solution, the wheel induced sucrose suppression was similar to that of the familiar rat chow. Thus despite the strong sucrose suppression wheel running still had a general unconditioned affect on feeding. Ad lib wheel introduction induced a much stronger and longer lasting suppressive effect on the intake of a novel compared to a familiar food. Whereas the suppression of the familiar rat chow recovered after only a few days, the intake suppression of the novel sucrose was very strong, almost complete, and long lasting. The stronger suppression of novel sucrose consumption suggests that while the wheel running may induce a “sickness” state that directly suppresses food intake [11], it may also support a learned suppression. This learned suppression in combination with the direct suppression, makes the suppression of novel foods much stronger than that seen with familiar foods. Due to latent inhibition [19] this learned effect is not evident with the familiar food, and the suppression is just a consequence of the direct unconditioned effects of the wheel. Unlike other studies, in which traditional procedure for conditioned taste avoidance have been applied [10–14], the rats in the present study were given ad lib access to water, food, sucrose solution and wheels and our results suggest that a learned suppression can be evident even when food and wheel access are not explicitly paired by the experimenter. With Experiment 2 and 3, we also examined the effect of alternate-day wheel access on consumption of novel and familiar food to further explore the role of learning. The group of rats with alternate-day wheel exposure showed a zigzag pattern of familiar food intake from day 1; suppressing
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their intake of the chow on wheel days and increasing it on the intervening no wheel days (Figs. 2A, 3A). However, in Experiment 2 with novel sucrose, rats suppressed their sucrose intake on both no wheel (home cage) and wheel days. In other words, the rats avoided the sucrose whether they were in the wheels or home cages. These results are in agreement with a role for learned conditioned taste avoidance in the feeding suppression. Evidently, if the food is novel it supports a more pronounced suppression than if the food is familiar, as would be predicted based on an involvement of learning. The animals associate the state change, induced by running, with the novel food, rather than the familiar food. The fact that the group of rats with alternate-day wheel exposure suppressed their intake of the novel sucrose, on both wheel and no wheel days, also supports the conditioned taste avoidance hypothesis. On wheel days, an association is formed between the novel sucrose and the unconditioned effect of running. Thus, animals avoid consuming sucrose on wheel days in response to the unconditioned state change induced by running (“sickness”). The days when rats are in their home cages, sucrose solution is still avoided but now in response to the learned association between the taste and the unconditioned state change induced by running. Such an association is not formed when the food is familiar due to latent inhibition [19]. Therefore, the rats consume normal amount of the familiar food when the wheels are not available. However, on wheel days, the unconditioned state change induced by running, occurs and directly influences all of their food consumption. Experiment 3 investigated the intake suppression induced by alternate-day wheel running after sucrose pre-exposure. Latent inhibition would suggest that CS pre-exposure should reduce the conditioned taste avoidance [19]. The rats with alternate-day wheel exposure showed a zigzag consumption pattern of the now-familiar sucrose throughout the entire study; suppressed on wheel days compared to their more normal consumption on non-wheel days (Fig. 3B). As the rats were familiarized with sucrose prior to the introduction of the alternate-day wheel access, they did not form an association between the unconditioned effect of wheel running and the sucrose solution. Therefore, on non-wheel days rats consumed normal amount of familiar sucrose solution, but on wheel days, the unconditioned effect of running still directly suppressed consumption. These animals showed a similar alternate-day pattern of the even more familiar rat chow intake at the beginning of the experiment; however, toward the end of the study rat chow consumption had stabilized, consumption was no longer affected by wheel access. Thus, toward the end of the study, rats were only suppressing their intake of the sucrose solution and not the rat chow on wheel days. On non-wheel, home cage days the rats were consuming the same amount of rat chow, but increasing their sucrose consumption considerably compared to the preceding wheel day. This may be either the result of a difference in preference for sucrose and rat chow or the result of differing degrees of familiarity with these foods. Note that the rats, with alternate-day wheel exposure in both Experiment 2 and 3, showed levels of running that were similar to running on the equivalent days in the rats with continuous wheel exposure.
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Thus, differences in food intake of these two groups are unlikely to be due to differences in running. The results of these experiments indicate that wheel running supports a conditioned taste avoidance, as has been previously suggested [11]. Even though, wheel running can act as an unconditioned stimulus to support the development of a CS-US association, it is not clear what internal changes are induced by wheel running. Opiate systems seem to be directly involved in wheel running. For example it has been shown that morphineinduced analgesia is reduced by wheel running [20]. In fact, a number of lines of evidence suggest that wheel running may induce a state similar to that induced by addictive drugs. Rats lever press to gain wheel access suggesting it is rewarding [21]. With ad lib access in cocaine and heroin self-administration, the rate of self-administration increases over days, leading to death from an overdose [22]. This escalation in consumption over days is a classic feature of addictive behaviour [23], and parallels the transition to addiction in humans. Similarly, with ad lib wheel access; rats come to increase their running over a number of days to an excessive level in a manner similar to that which happens with addicting drugs, it only occurs if the animals have at least a few hours of access a day [24]. In addition, rats develop a conditioned place preference for an environment associated with the after-effects of both wheel running [25] and addictive drugs such as morphine and cocaine [26]. This suggests that wheel running, like addictive drugs, produces a positive affect that outlasts wheel access and supports a place preference. Wheel running seems to have parallels with many aspects of drug self-administration. This has led to the suggestion that wheel running may serve as an animal model of addiction [24,27]. Thus, it may be possible that the novel food avoidance induced by wheel running, from our experiments, is similar to that induced by rewarding drugs. However it should be noted that in studies looking at the feeding suppression and weight loss evident with restricted feeding and ad lib wheel access (the activity anorexia procedure) many other hormonal and neural systems are disrupted by wheel access [28–30]. Finally it should be noted that the wheel running-induced feeding suppression had the expected effect on the weight of the rats. While sucrose access did not significantly increase body weight, wheel access in Experiment 1 caused a pronounced weight reduction. It is interesting that in the shorter second and third experiment alternate-day wheel access which had a longer lasting effect on food consumption also produced a more pronounced weight reduction. This is different from previous work in our laboratory [9] where alternate-day and continuous wheel access had similar effects on body weight. As many aspects of these experiments were different (the previous work looked at a 32 day wheel exposure for example) it is not clear what is responsible for the inconsistent results. In conclusion, three experiments in this paper suggest that in addition to the direct unconditioned effects of wheel running on feeding, learning factors may influence the feeding suppression observed. In situation where learning is more likely to occur this causes the animals to avoid the food almost completely at all
times for an extended period. This learned suppression is evident in situations where no explicit attempt is made to pair consumption with wheel running. This learning may occur either because in the feeding system learned associations can occur with long intervals between the CS and US or because voluntary running occurs in close temporal proximity to feeding. Future studies may illuminate some of these issues and help demonstrate some of the parameters influencing the wheel induced feeding suppression. Acknowledgments This research was funded, in part, by the Science and Technology Endowment Program at Wilfrid Laurier University to ES and by the Natural Sciences and Engineering Research Council funding to RE. Portions of this research were presented in a poster at the 2004 Society for Neuroscience meeting (#427.6). References [1] Sherwin CM. Voluntary wheel running: a review and novel interpretation. Anim Behav 1998;56:11–27. [2] Stewart CC. Variations in daily activity produced by alcohol and by changes in barometric pressure and diet with a description of recording methods. Am J Physiol 1898;1:40–56. [3] Richter CP. Animal behavior and internal drives. Q Rev Biol 1927;2: 307–43. [4] Afonso VM, Eikelboom R. Relationship between wheel running, feeding, drinking, and body weight in male rats. Physiol Behav 2003;80:19–26. [5] Levitsky DA. Feeding patterns of rats in response to fasts and changes in environmental conditions. Physiol Behav 1970;5:291–300. [6] Looy H, Eikelboom R. Wheel running, food intake, and body weight in male rats. Physiol Behav 1989;45:403–5. [7] Premack D, Premack AJ. Increased eating in rats deprived of running. J Exp Anal Behav 1963;6:209–12. [8] Bauman RA. The effects of wheel running, a light/dark cycle, and the instrumental cost of food on the intake of food in a closed economy. Physiol Behav 1992;52:1077–83. [9] Mueller DT, Loft A, Eikelboom R. Alternate-day wheel access: effects on feeding, body weight, and running. Physiol Behav 1997;62:905–8. [10] Hayashi H, Nakajima S, Urushihara K, Imada H. Taste avoidance caused by spontaneous wheel running: effects of duration and delay of wheel confinement. Learn Motiv 2002;33:390–409. [11] Lett BT, Grant VL. Wheel running induces conditioned taste aversion in rats trained while hungry and thirsty. Physiol Behav 1996;59:699–702. [12] Lett BT, Grant VL, Gaborko LL. Wheel running simultaneously induces CTA and facilitates feeding in non-deprived rats. Appetite 1998;31: 351–60. [13] Lett BT, Grant VL, Koh MT, Smith JF. Wheel running simultaneously produced conditioned taste aversion and conditioned place preference in rats. Learn Motiv 2001;32:129–36. [14] Nakajima S, Hayashi H, Kato T. Taste aversion induced by confinement in a running wheel. Behav Processes 2000;49:35–42. [15] Salvy SJ, Pierce WD, Heth DC, Russell JC. Pre-exposure to wheel running disrupts taste aversion conditioning. Physiol Behav 2002;76:51–6. [16] Baysari M, Boakes R. Flavour aversion produced by running and attenuated by prior exposure to wheels. Q J Exp Psychol B 2004;57: 273–86. [17] Nikoletseas MM. Exercise-induced sucrose suppression in the rat. Physiol Behav 1981;26:145–6. [18] Jennings WA, McCutcheon LE. Novel food and novel running wheels: conditions for inhibition of sucrose intake in rats. J Comp Physiol Psychol 1974;87:100–5.
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