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Conditioned flavor avoidance as a measure of withdrawal in rats chronically exposed to a caffeine solution
Physiology & Behavior 95 (2008) 245–251
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Conditioned flavor avoidance as a measure of withdrawal in rats chronically exposed to a caffeine solution Sarah E. Dreumont-Boudreau ⁎, Rachel N. Dingle, Gillian M. Alcolado, Vincent M. LoLordo Dalhousie University, Halifax, Nova Scotia Canada B3H 4J1
A R T I C L E
I N F O
Article history: Received 22 October 2007 Received in revised form 12 May 2008 Accepted 10 June 2008 Keywords: Caffeine Dependence Withdrawal Conditioned flavor avoidance Rats
A B S T R A C T Rats were given 21 days of chronic oral caffeine. A novel flavor (Maintenance CS) was then paired with the continuation of caffeine, and a second flavor (Withdrawal CS) was paired with caffeine removal. Rats avoided the Withdrawal CS, and drank more of the Maintenance CS in a two-bottle test, suggesting that removing caffeine had induced withdrawal. The value of the Maintenance CS was investigated by comparing it to a novel flavor paired with water (Neutral CS). In a series of two-bottle tests, the Maintenance and Neutral CSs were equivalent when pitted against each other, and both were preferred to the Withdrawal CS. These results demonstrate that conditioned flavor avoidance is a useful procedure in assessing caffeine withdrawal, and by inference dependence, produced by chronic oral consumption. © 2008 Elsevier Inc. All rights reserved.
Caffeine has often been described as an atypical drug of dependence, and does not produce the same behavioral and physiological changes observed with more traditional drugs of dependence [1–3]. Given that caffeine is widely consumed by humans, it is important to gain a better understanding of tolerance, dependence, and withdrawal that occur to caffeine's pharmacological effects. Research with animals can contribute to this understanding. One focus of such research has been on understanding caffeine tolerance and dependence in the rat. Extended exposure to caffeine will produce tolerance to its stimulative effects on locomotion, and can potentially produce dependence and subsequent withdrawal symptoms when caffeine is removed. Holtzman [4] gave rats controlled access to caffeinated water for 10 min every 6 h over a period of 11 weeks. The caffeine concentration was gradually increased from 0.25 mg/ml to 3.0 mg/ml by the sixth week. A control group was given unadulterated water on the same schedule. Both groups were then given a challenge dose of caffeine by gavage, and locomotor activity was measured. The control group showed the expected increase in locomotor activity after the challenge dose, but the rats that had been given chronic caffeine exposure showed no increase in activity. Holtzman concluded that the rats from the chronic caffeine group had become tolerant to caffeine's stimulative effects as a result of their extended exposure to the drug. Spontaneous locomotor activity was also measured during the chronic ⁎ Corresponding author. Dalhousie University; Psychology Department, 1355 Oxford Street, Halifax, Nova Scotia Canada B3H 4J1. Tel.: +1 902 494 3441; fax: +1 902 494 6585. E-mail address: [email protected] (S.E. Dreumont-Boudreau). 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.06.008
caffeine period and immediately after the caffeinated water was removed. Rats in the chronic caffeine group showed depressed locomotor activity during the first 4 days following caffeine removal, suggesting that they were experiencing some sort of withdrawal symptoms, and thus that they had become caffeine dependent. Using a similar procedure, Finn and Holtzman [5] assessed the ability of three caffeine doses to produce tolerance and withdrawal. Rats received either 0.25, 0.5, or 1.0 mg/ml of chronic caffeine over a six week period. Various challenge doses were then delivered by gavage. Rats in the 0.25 mg/ml group continued to show an increase in locomotor activity to some of the lower challenge doses. However, the 0.5 and 1.0 mg/ml groups showed no change in locomotor activity to any of the challenge doses, indicating tolerance to caffeine's stimulant effects. Other studies have found similar results of tolerance to caffeine's stimulant effects on locomotion in the hole-board test [6], locomotion after unrestricted chronic access to caffeine [7], and wheelrunning activity [8]. In addition to tolerance, Finn and Holtzman [5] found that the 1.0 mg/ml group showed a decrease in spontaneous locomotor activity during the first 2 days after caffeine was removed compared to the 3 days prior to removal. As in Holtzman [4], the authors concluded that this result was evidence of withdrawal symptoms and thus of caffeine dependence. Repeated exposure to the pharmacological effects of caffeine also changes a rat's response to flavors associated with caffeine, and to flavors associated with its absence. Vitiello and Woods [9] demonstrated an avoidance of a taste that was paired with the absence of caffeine after repeated exposure to caffeine. For 14 days, groups of rats were given daily injections of saline or of varying caffeine concentrations following
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brief access to water. Then half the rats that had previously been injected with caffeine received access to a saccharin solution followed by a caffeine injection (C–C), while the other half received saccharin followed by a saline injection (C–S). The same treatment was given to the rats that had previously received only saline (S–C, S–S). All groups were then given a two-bottle test with saccharin and water. Saccharin was strongly preferred by group S–S. The S–C group avoided the saccharin [for recent related results see [10,11]]. The C–C group preferred saccharin as much as the S–S controls, showing no conditioned effects. Most important, the C–S group showed a strong avoidance of the saccharin. The authors concluded that the conditioned avoidance of saccharin by the C–S group came about through its association with negative caffeine withdrawal symptoms. While a conditioned preference for saccharin did not form in the C–C group, it was no longer aversive as it was in the S–C group that had no prior experience with caffeine. Naïve rats initially found caffeine's pharmacological effects to be aversive, but after repeated exposure this was no longer the case, and the absence of caffeine's pharmacological effects following a signal for caffeine became aversive instead. We sought to further explore caffeine dependence and withdrawal in conditioned flavor avoidance using an oral consumption procedure to establish dependence. The purpose of Experiment 1 was to determine whether a conditioned flavor avoidance paradigm would reveal evidence for caffeine dependence in rats that had 21 days of chronic exposure to caffeinated water. If the rats become dependent on caffeine, then they should experience withdrawal symptoms when the caffeine is taken away. In order to assess this, we used caffeine withdrawal to establish a conditioned avoidance of a novel flavor. Immediately after the chronic exposure period, the rats were given two 30 min exposures to a novel Kool-Aid flavor. The caffeinated drinking water was returned after each exposure, hence the novel flavor was termed the Maintenance CS because it was paired with the maintenance of access to caffeine. Rats were then given three exposures to a second novel Kool-Aid flavor. After the first exposure to the second Kool-Aid, the rats were given unadulterated tap water and the caffeinated water was never returned. We hypothesized that removal of caffeine would induce caffeine withdrawal, hence the second Kool-Aid flavor was termed the Withdrawal CS. If the rats become dependent on caffeine over the 21-day chronic exposure period, and subsequently experience withdrawal symptoms when the caffeinated water is permanently removed, then they should avoid the Withdrawal CS and consume more of the Maintenance CS when given a choice between the two CSs. 1. Experiment 1
Saccharin solutions contained 1.0 g/L saccharin sodium salt hydrate (Sigma, Canada) dissolved in tap water. The caffeine solution was made up of caffeine anhydrous powder (Sigma, Canada) dissolved in tap water at a concentration of 1.0 g/L. The Kool-Aid/saccharin solutions were made up of either Grape or Cherry Kool-Aid (Kraft Canada) at a concentration of 2.5 g/L, and 1.0 g/L saccharin sodium salt hydrate dissolved in tap water. 1.1.3. Procedure 1.1.3.1. Saccharin pre-exposure. Rats were pre-exposed to a saccharin– water solution to familiarize them with saccharin and eliminate any initial neophobia that might interfere with saccharin–Kool-Aid consumption in training and testing. First, a 24 h water consumption measurement was taken to determine normal drinking amounts. For the next 2 days, all rats were given a saccharin–water solution as their only source of fluid. Consumption was measured every 24 h. 1.1.3.2. Chronic caffeine exposure. All of the rats were given a caffeine– water solution as their only source of fluid for 21 days. The caffeine– water bottles were weighed every 24 h between 11:00 and 11:30 to determine daily consumption. The rats were weighed every 48 h at the same time of day, and this continued throughout the entire experiment. 1.1.3.3. Training. Immediately following the twenty-first day of chronic caffeine exposure, the rats were divided into two groups equated on the previous 24 h caffeine consumption. Group 1 received Cherry Kool-Aid and saccharin (CherrySacc) as the Maintenance CS, and Grape Kool-Aid and saccharin (GrapeSacc) as the Withdrawal CS. Group 2 received the opposite flavor assignments. Training began immediately after Day 21 of chronic caffeine, and took place over 5 days. Following the removal of the caffeine–water bottle at the end of Day 21, each rat received the appropriate Maintenance CS solution for 30 min. The caffeine–water solution was then placed back on the cage for the rest of the day. The same procedure was repeated on the second day of training, such that each group received two exposures to the Maintenance CS followed by the return of caffeine. On the next day, the caffeine–water bottle was removed and the appropriate Withdrawal CS was placed on each cage for 30 min. A bottle filled with tap water was then placed on each cage for the rest of the day. The same procedure was repeated on the next 2 days, except the tap water bottle was removed prior to the CS bottle going on the cage, such that each rat received three exposures to the Withdrawal CS. A third day of Withdrawal CS exposure was added in an effort to equate consumption of the Maintenance and Withdrawal CSs because the rats greatly reduced their Withdrawal CS consumption on the second exposure.
1.1.1. Subjects Twenty-five adult male Sprague–Dawley rats from Charles River Canada, Quebec were used. Rats were housed singly in standard shoebox cages under a 12:12 light/dark cycle with lights on at 0800 h. Food and fluid were available ad libitum throughout the entire experiment. Rats were weighed every other day for the duration of the experiment, and their mean weight at the beginning of the experiment was 359 g.
1.1.3.4. Testing. Immediately following the last training day, two tap water bottles were placed on each cage for 48 h. This was done to reduce any neophobia to having two bottles present on the cage simultaneously, and to ensure that if the rats had become dependent on the caffeine enough time (5 days) had elapsed for recovery from withdrawal symptoms prior to testing [4,5]. Water consumption was measured every 24 h. The water bottles were then removed and replaced with one CherrySacc bottle and one GrapeSacc bottle. The Kool-Aid solutions were left on the cages for 24 h, and bottle position was counterbalanced within each group.
1.1.2. Apparatus All training and testing were conducted in the homecage. The homecage was made of clear plastic with a wire top, and measured 46.5 cm × 24.5 cm × 21 cm. Each cage contained approximately 2 cm of wood shavings on the bottom and a 12 cm × 7.5 cm black PVC hiding tube. All solutions were delivered in 250 ml glass bottles with 3.5 cm black rubber stoppers. Each stopper contained a 6 cm long stainless steel sipper tube with a 0.5 mm opening at the end.
1.1.4. Statistical analysis Maintenance CS training data were analyzed using a 2(Day) × 2 (Flavor; CherrySacc or GrapeSacc as Maintenance CS) mixed ANOVA with Day as the within-subjects variable and Flavor as the betweensubjects variable. Withdrawal CS training data were analyzed with a 3 (Day) × 2(Flavor) mixed ANOVA with Day as the within-subjects variable, and Flavor as the between-subjects variable. Post hoc analysis was conducted using the Scheffe's test.
1.1. Methods
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Two-bottle Maintenance CS vs. Withdrawal CS test consumption data were analyzed using a 2(CS) ×2(Flavor) ×2(Maintenance Bottle Location) mixed ANOVA with CS as the within-subjects variable, and Flavor and Maintenance Bottle Location as the between-subjects variables.
70.0 mg/kg of caffeine consumed. On Day 21, the rats drank an average of 40.4 ml, resulting in an average of 93.9 mg/kg caffeine consumed. See Fig. 1A for the mean daily caffeine consumption in milliliters and mg/kg across the 21 days of chronic exposure.
1.2. Results
1.2.3. Training During training, consumption varied across days for both the Maintenance and the Withdrawal CSs. Consumption increased across days for the Maintenance CS, but decreased across days for the Withdrawal CS. See Fig. 1B. There was a significant main effect of Day for the Maintenance CS, with more consumption taking place on Day 2 than on Day 1, F(1, 46)= 7.09, p b 0.05. No other main effects or interactions reached significance. Withdrawal CS consumption data also revealed a significant main effect of Day, F(2, 69)=23.59, pb 0.01, with consumption dropping off dramatically on Days 4 and 5 compared to Day 3. Post hoc comparisons revealed
1.2.1. Saccharin pre-exposure During the baseline 24 h water consumption period, the rats drank an average of 45.1 ml of water. On the first day of 24 h saccharin exposure, the rats drank an average of 75.5 ml. On the second day, the average consumption had dropped to 64.8 ml. 1.2.2. Chronic caffeine exposure On Day 1 of chronic caffeine exposure, the rats drank an average of 25.1 ml of the caffeine–water solution. This resulted in a mean of
Fig. 1. (A) Mean daily consumption of caffeine across the 21-day chronic caffeine exposure, and during Maintenance CS training in Experiment 1. (B) Mean daily consumption of the Maintenance and Withdrawal CSs during training. (C) Mean consumption of the Maintenance and Withdrawal CSs in test. Error bars represent standard error of the mean.
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Day 3 consumption to be significantly higher than consumption on Days 4 and 5, which did not differ from each other. No other main effects or interactions reached significance. In order to compare total exposure to the Maintenance and Withdrawal CSs, consumption was summed across days for each CS. A 2(CS) × 2(Flavor) mixed ANOVA was run with CS as the within-subjects variable, and Flavor as the between-subjects variable. The only significant result was a main effect of CS, with rats drinking more of the Maintenance CS than the Withdrawal CS, F(1, 23) = 5.35, p b 0.05. 1.2.4. Testing Rats drank much more of the Maintenance CS compared to the Withdrawal CS during the Maintenance CS vs. Withdrawal CS twobottle test. See Fig. 1C. There was a significant main effect of CS, with more overall consumption of the Maintenance CS than the Withdrawal CS, F(1, 40)= 8.65, pb 0.05. No other main effects or interactions reached significance. 1.3. Discussion When given a choice between the Maintenance CS and the Withdrawal CS, rats avoided the Withdrawal CS and consumed more of the Maintenance CS in test. Therefore, the data strongly suggest that the rats experienced negative withdrawal symptoms when caffeine was no longer available. These symptoms were then associated with the Withdrawal CS. From these results we can infer that the oral 21-day chronic caffeine exposure period was enough to produce dependence in rats. One obvious alternative to this conclusion is that the rats simply drank more in test of the CS that was more familiar. In training, significantly more Maintenance CS was consumed than Withdrawal CS. This exposure effect could also produce the results that were observed in the two-bottle test, if the rats were simply choosing to drink the CS that they had the most experience with [12]. We examined whether relative exposure to the two CSs in training predicted preference in the two-bottle test by calculating the ratio of the amount of Withdrawal CS consumed in training divided by total consumption of the two CSs in training. The analogous ratio was calculated for the amount of Withdrawal CS consumed during the two-bottle test, and the correlation of the two was computed. The correlation was non-significant, r = 0.037, p N 0.05, suggesting that the observed preference is not an exposure effect. The increase in consumption of the Maintenance CS from the first training day to the second most likely reflects habituation of neophobia. Consumption of the Withdrawal CS on Day 3, before any withdrawal symptoms could have occurred, was comparable to consumption of the Maintenance CS. The reduction in consumption of the Withdrawal CS on Days 4 and 5 is consistent with the acquisition of a conditioned flavor avoidance based on aversive withdrawal symptoms. In this experiment the Withdrawal CS was pitted against the Maintenance CS in the two-bottle test. We chose the Maintenance CS as our neutral control because it did not signal a marked change in the availability of the caffeine solution. However, we had no evidence that the Maintenance CS was neutral. In Experiment 2 we sought to discover whether the Maintenance CS was neutral, or became positive after being paired with the continuation of caffeine. Rats were given chronic caffeine exposure, Maintenance CS, and Withdrawal CS training as in Experiment 1. But, several days after Withdrawal CS training a third CS, called the Neutral CS, was introduced, and was paired with plain tap water. Each rat was then given three two-bottle tests; Maintenance CS vs. Withdrawal CS, Withdrawal CS vs. Neutral CS, and Maintenance CS vs. Neutral CS. If the Maintenance CS became positive by signaling the continuation of caffeine, then the rats should prefer it to the Neutral CS in test. However, if the Maintenance CS was essentially neutral then there should be no preference between it and the Neutral CS when they are pitted against each other in test.
2. Experiment 2 2.1. Methods 2.1.1. Subjects Forty-nine adult male Sprague–Dawley rats from Charles River Canada, Quebec were used. Rats were housed and maintained as in Experiment 1. Rats were weighed every other day for the duration of the experiment, and their mean weight at the beginning of the experiment was 292 g. 2.1.2. Apparatus All training and testing were conducted in the homecage. Apparatus and solutions were as in Experiment 1, except that LemonLime Kool-Aid was also used. 2.1.3. Procedure 2.1.3.1. Saccharin pre-exposure. All rats were pre-exposed to saccharin for 6 days prior to the start of chronic caffeine exposure. First, a 24 h. water consumption measurement was taken to determine normal drinking amounts. For the next 3 days, all rats were given a saccharin– water solution as their only source of fluid. Consumptions were measured every 24 h. Following 24 h saccharin–water exposure, all rats were given one daily 30 min saccharin–water exposure for 3 days. The 30 min exposure was given during the same time that the experimental CS bottles would eventually be put on the cages during training. 2.1.3.2. Chronic caffeine. out as in Experiment 1.
The chronic caffeine procedure was carried
2.1.3.3. Training. Immediately following the twenty-first day of chronic caffeine exposure, the rats were divided into six groups equated on the previous 24 h caffeine consumption. Cherry, Grape, and LemonLime Kool-Aid flavors were assigned as Maintenance, Withdrawal, and Neutral CSs such that all possible combinations were used. See Table 1 for Kool-Aid flavor assignments in each group. Training began immediately after the twenty-first day of chronic caffeine, and took place over 11 days. Following the removal of the caffeine–water bottle at the end of Day 21, each rat received the appropriate Maintenance CS solution for 30 min. The caffeine–water solution was then placed back on the cage for the remainder of the 24 h period. The same procedure was repeated on Day 2 of training, such that each group received two exposures to the Maintenance CS. On Day 3, the caffeine–water bottle was removed and the appropriate Withdrawal CS was placed on each cage for 30 min. A bottle filled with tap water was then placed on each cage for the remainder of the 24 h period. The same procedure was repeated on Days 4 and 5, except that the tap water bottle was removed prior to the CS bottle going on the cage. Each rat received three exposures to the Withdrawal CS. A third day of Withdrawal CS exposure was added in an effort to equate Maintenance and Withdrawal CS consumption because the rats greatly reduced their Withdrawal CS consumption on the second
Table 1 Flavor assignment to Maintenance, Withdrawal, and Neutral CSs Group
1 2 3 4 5 6
CS Maintenance
Withdrawal
Neutral
Cherry Cherry LemonLime LemonLime Grape Grape
Grape LemonLime Grape Cherry Cherry LemonLime
LemonLime Grape Cherry Grape LemonLime Cherry
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exposure. On Days 6, 7, and 8 the tap water bottles were left on the cages and consumption was measured every 24 h. This was done to allow enough time (6 days) for any remaining symptoms of caffeine withdrawal to dissipate prior to Neutral CS training. On Day 9, the tap water bottles were removed from the cages and the appropriate Neutral CS was placed on each cage for 30 min. The tap water bottles were then placed back on the cages for the remainder of the 24 h period. The same procedure was repeated on Days 10 and 11, except that two bottles of tap water were placed on each cage after the Neutral CS was removed on Day 11. 2.1.3.4. Testing. The two tap water bottles were left on each cage for 24 h. Upon removal of the two water bottles, two-bottle CS testing began. Each rat received three 24 h two-bottle tests: Maintenance CS (M) vs. Withdrawal CS (W), Maintenance CS (M) vs. Neutral CS (N), and Withdrawal CS (W) vs. Neutral CS (N). There were 10 days between the last caffeine exposure and the first two-bottle test. To control for possible test order effects, for each of the six possible assignments of Kool-Aid flavors to the three CSs a rat was allocated to each of the six possible test orders. For example, one rat might receive the M vs. W test first, then the M vs. N test, and finally the W vs. N test. A second rat might receive the M vs. N test first, followed by the W vs. N test, and finally the M vs. W test. Extra rats were distributed among the various groups. In addition, bottle position was counterbalanced within each group such that the Maintenance CS in any given test was not always on the left side, for example. Test bottles were left on for 24 h, and were immediately replaced by new test bottles upon removal. 2.1.4. Statistical analysis Maintenance CS training data were analyzed using a 2(Day) × 3 (Flavor; Cherry, Grape, or LemonLime) mixed ANOVA with Day as the within-subjects variable, and Flavor as the between-subjects variable. Withdrawal and Neutral CS training data were analyzed using separate 3(Day) × 3(Flavor) mixed ANOVAs with Day as the withinsubjects variable, and Flavor as the between-subjects variable. Post hoc analysis was conducted using the Scheffe's test. Each two-bottle test was analyzed separately with a 2(CS) × 3(CS1 Flavor) × 3(CS2 Flavor) × 2(CS1 Bottle Location) mixed ANOVA, with CS as the within-subjects variable, and CS1 Flavor, CS2 Flavor, and CS1 Bottle Position as between-subjects variables. Effects of two-bottle CS test order were also analyzed separately for each two-bottle test using 2(CS) × 3(Order) mixed ANOVAs with CS as the within-subjects variable and Order as the between-subjects variables. The Order factor refers to where a particular test fell amongst the three twobottle tests for each rat. For example, when analyzing the Maintenance vs. Withdrawal test, one rat may have had that test first, while a second rat may have had that test third, and so on. 2.2. Results 2.2.1. Training Fig. 2A shows the mean consumptions of Maintenance, Withdrawal, and Neutral CSs during training. During presentation of the Maintenance CS, rats drank more on Day 2 than on Day 1. There was a significant main effect of Day, F(1, 92) = 13.76, p b 0.05. No other main effects or interactions reached significance. Consumption of the Withdrawal CS during training also varied across training days. Rats consumed more of the Withdrawal CS on Day 3 than on Days 4 and 5. There was a significant main effect of Day, F(2, 138) = 39.59, p b 0.05. A post hoc Scheffe's test revealed that the rats drank significantly more Withdrawal CS on Day 3 compared to Days 4 and 5 (p b 0.05), while Days 4 and 5 did not differ from each other (p N 0.05). No other main effects or interactions reached significance. During Neutral CS training, consumption of the CS did not differ across training days. However, rats that received Grape Kool-Aid– saccharin as their Neutral CS drank more than rats that received
Fig. 2. (A) Mean consumption of the Maintenance, Withdrawal, and Neutral CSs during training in Experiment 2. (B) Mean consumption of each CS in the Maintenance vs. Withdrawal, Neutral vs. Withdrawal, and Maintenance vs. Neutral tests. Error bars represent standard error of the mean.
Cherry Kool-Aid–saccharin or LemonLime Kool-Aid–saccharin. There was a significant main effect of Flavor, F(2, 138) = 9.18, p b 0.05. Post hoc Scheffe's tests showed that rats drank significantly more Grape Kool-Aid–saccharin compared to Cherry or LemonLime Kool-Aid– saccharin (p b 0.05), while Cherry and LemonLime Kool-Aid–saccharin consumptions did not differ from each other (p N 0.05). No other main effects or interactions reached significance. In order to compare total exposure to the Maintenance, Neutral, and Withdrawal CSs, consumption was summed across days for each rat within each CS. A one-way ANOVA was used to analyze the data. There was a significant difference in consumption between the three CSs, F(2, 138)= 7.57, pb 0.05. Post hoc Scheffe's tests revealed that there was no difference in total consumption between the Maintenance CS and the Withdrawal CS (p=.711). However, the rats consumed significantly less of the Neutral CS compared to the Maintenance CS (p=.018), and to the Withdrawal CS (p=.001). 2.2.2. Testing Fig. 2B shows the mean consumption of each CS during the Maintenance vs. Withdrawal, Withdrawal vs. Neutral, and Maintenance vs. Neutral two-bottle tests. Rats showed a preference for the Maintenance CS and the Neutral CS when they were pitted against the Withdrawal CS. However, rats showed no preference for either the Maintenance CS or the Neutral CS when given a choice between the two. The order in which the two-bottle tests were given did not affect CS consumption. Regardless of which test was given first, second, or third, rats still showed a preference for Maintenance and Neutral CSs over the Withdrawal CS, and no preference for the Maintenance CS over the Neutral CS or vice versa. For the Maintenance vs. Withdrawal two-bottle test, there was a significant main effect of CS, F(1, 37) = 11.43, p b 0.05, with rats consuming
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significantly more Maintenance CS than Withdrawal CS. There were no other significant main effects or interactions. For the Withdrawal vs. Neutral two-bottle test, there was a significant main effect of CS, F(1, 37) =7.75, p b 0.05, with rats consuming significantly more Neutral CS than Withdrawal CS. There were also significant main effects of Neutral Flavor, F(2, 37) =3.82, p b 0.05, and Withdrawal Flavor, F (2, 37)= 4.23, p b 0.05. Inspection of the data showed a small flavor preference for Grape Kool-Aid. Rats consumed more of the Neutral and Withdrawal CSs when they were Grape Kool-Aid than when they were Cherry or LemonLime Kool-Aids. No other main effects or interactions were significant. For the Maintenance vs. Neutral two-bottle test, there was no main effect of CS, F(1, 37) = 0.131, p N 0.05. No other main effects or interactions were significant. The test data were subjected to a second analysis to assess any effects of test order on CS consumption. Test order had no effect on the two-bottle consumption data. Maintenance and Neutral CSs were preferred to the Withdrawal CS, and neither the Maintenance CS or the Neutral CS was preferred over the other. For the Maintenance vs. Withdrawal two-bottle test there was a main effect of CS, F(1, 46) = 12.86, p b 0.05, but no main effect of Order, F(2, 46) = 1.22, p N 0.05, and no significant interactions. In the Neutral vs. Withdrawal two-bottle test, there was a significant main effect of CS, F(1, 46) = 7.28, p b 0.05, but no main effect of Order, F(2, 46) = 1.10, p N 0.05, and no significant interactions. For the Maintenance vs. Neutral two-bottle test, there was no main effect of CS, F(1, 46) = 0.261, p N 0.05, or Order, F(2, 46) = 1.80, p N 0.05, and no significant interactions. 2.3. Discussion The Maintenance CS was equally preferred to the Neutral CS in test. Hence, it is not inherently positive, but is essentially functioning like a Neutral CS when pitted against the Withdrawal CS. This is not surprising since very little changes for the rat when the Maintenance CS is presented in training. In separate two-bottle tests, both the Maintenance CS and the Neutral CS were preferred to the Withdrawal CS. During training, the patterns of consumption over days of the Maintenance CS and the Withdrawal CS were as seen in the previous experiment. However, there was no significant difference in total consumption of the two CSs in training. Nonetheless, there was still a robust avoidance of the Withdrawal CS in the Maintenance CS vs. Withdrawal CS test. In the present experiment this avoidance cannot be attributed to greater familiarity of the Maintenance CS. Consumption of the Neutral CS during training was surprisingly depressed, and rats consumed significantly less of it than the Maintenance CS and Withdrawal CS. Even though Neutral CS training was given after the physical symptoms of caffeine withdrawal should have worn off, the rats' previous experience with the Withdrawal CS could have generalized to the Neutral CS, resulting in lower consumption. Despite the depressed consumption of the Neutral CS in training, rats still showed a preference for the Neutral CS over the Withdrawal CS in test. Since the Neutral CS was the last CS presented in training; this preference implies that the avoidance of the Withdrawal CS observed in the first experiment could not have been simply avoidance of the most recent CS. Moreover, the Withdrawal and Neutral CSs had been presented three times in training, suggesting that the avoidance of the Withdrawal CS in the first experiment did not depend on the difference in the number of exposures of the two CSs in that experiment. There was also no preference for the Maintenance CS over the Neutral CS in test, even though rats consumed more of the Maintenance CS in training. 2.4. General discussion A conditioned flavor avoidance paradigm was successfully used to demonstrate caffeine withdrawal in rats. After 21 days of chronic free access to caffeine, rats avoided the Withdrawal CS flavor that had been
paired with the removal of caffeine, and drank more of the Maintenance CS. Presumably, this was due to the Withdrawal CS being associated with negative withdrawal symptoms stemming from the removal of caffeine, further suggesting that the rats had become dependent over the 21 day chronic caffeine period. The results support the Holtzman [4] and Finn and Holtzman [5] experiments, which examined changes in locomotor behavior, in demonstrating that chronic oral consumption of caffeine produces dependence and subsequent withdrawal when caffeine is removed. Finn and Holtzman [5] demonstrated that tolerance to the stimulatory effects of caffeine can happen after only 4 days of chronic caffeine. Dingle et al. [13] addressed the question whether comparably brief exposure to chronic caffeine with the current oral chronic caffeine procedure would produce dependence and withdrawal. Groups of rats were given 5, 10, 15, or 20 days exposure to 1.0 g/L caffeine solutions. A novel flavor (Withdrawal CS) was then associated with caffeine removal, and was later pitted against a second novel flavor (Neutral CS) in a two-bottle test. The rats that were given 20 days of chronic caffeine exposure were the only group to show a significant avoidance of the Withdrawal CS. The groups given 5 and 10 days of chronic caffeine showed no evidence of avoidance of the Withdrawal CS, while the groups given 15 days showed a weak avoidance. Although brief exposure to chronic caffeine is sufficient to produce tolerance to the stimulative effects of caffeine on locomotion [5], longer periods of chronic caffeine exposure are necessary to produce dependence. Finally, the Maintenance CS was determined to be equivalent to a Neutral CS that was simply paired with water, and did not carry any positive value after being paired with caffeine. This is not surprising, given that there was no change in the drug state during Maintenance CS training. Our Maintenance CS treatment parallels the treatment of the caffeine–caffeine (C–C) group of rats in Vitiello and Woods [9]. Rats in that group continued to receive caffeine injections after the daily water presentation was switched to a saccharin presentation, and responded like caffeine naive rats in a subsequent two-bottle test of saccharin against water. As in Vitiello and Woods [9], the present experiments produced a conditioned avoidance of a novel flavor that had previously been paired with caffeine withdrawal symptoms. However, instead of a single daily injection, we used a chronic oral procedure of caffeine consumption in which the rats determined when and how much caffeine they consumed at any given time. Even though our rats had no other fluid to drink but the caffeine solution, they maintained a level of control over caffeine dose via the amount of liquid consumed during individual drinking bouts. This is more analogous to the pattern of human caffeine consumption than is a single daily injection. In Vitiello and Woods [9], the onset of the drug effect was signaled by a discrete event, the daily drinking period. In the current experiments, rats had the opportunity to have many drinking bouts throughout the chronic caffeine exposure period. Prior to caffeine's drug effects taking place, the rats encountered the bitter taste of the caffeine itself. Hence, the bitter taste of caffeine likely became a signal for caffeine's drug effects, similar to the daily drinking bout signaling the drug effect in Vitiello and Woods' experiment. When the taste offered during the daily drinking bout changed from water to saccharin in Vitiello and Woods' experiment, and was then followed by no drug effect and subsequent withdrawal, the rats learned to avoid it. Our procedure is similar in that the rats received a novel Withdrawal CS followed by no drug effect, i.e., tap water. The rats learned to associate the negative state this put them in (caffeine withdrawal) with the Withdrawal CS, and avoided that CS in the two-bottle test. One could agree that the avoidance of the Withdrawal CS, relative to the Maintenance and Neutral CSs, observed in these experiments is a conditioned taste avoidance without agreeing that the basis of the avoidance is a caffeine withdrawal state. Instead one could argue that removal of any positive stimulus generates a negative after effect [14],
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and that a signal for that aftereffect becomes aversive. So, is access to a 1 g/L caffeine solution positive? Dreumont-Boudreau and LoLordo [15] gave rats access to a novel Kool-Aid–saccharin solution for 1 h, then gave them 24 h of access to the caffeine solution. Three days later the rats received 1 h access to a second Kool-Aid–saccharin solution followed by water. In a subsequent two-bottle test, the rats strongly avoided the flavor that signaled access to the novel caffeine solution [also see [10,11]]. Therefore, access to our caffeine solution is initially negative, not positive. But, is caffeine positive after 21 days? If it is not, then the alternative account does not apply to the present data. If it is positive after initially being negative, then that result implies caffeine dependence, which implies that the effect of removing caffeine is to induce withdrawal. Conditioned flavor/taste avoidance is a particularly useful procedure for detecting caffeine withdrawal, and has been successfully used with other drugs of dependence. Parker et al. [16] showed that a novel flavor paired with negative morphine withdrawal symptoms was subsequently avoided. Most research looking at caffeine dependence and withdrawal using chronic oral procedures to produce dependence has relied on changes in locomotor behavior to detect dependence and withdrawal [4,5,7,8]. The conditioned flavor/taste avoidance paradigm is a sensitive alternative to locomotor behavior for assessing withdrawal, particularly with chronic oral procedures of dependence. The next step in developing a rat model of caffeine dependence is to utilize the conditioned flavor/taste paradigm to assess a novel flavor that has been associated with recovery from negative caffeine withdrawal symptoms. Parker et al. [16] were successful in producing a preference for a novel flavor that had previously been associated with recovery from morphine withdrawal. Developing a model for caffeine withdrawal recovery is particularly important because there is compelling evidence in the human literature that relief from withdrawal symptoms drives habitual human consumption of caffeine [17,18]. While in withdrawal, humans report an increase in pleasantness of flavors that have been paired with ingestion of caffeine [17– 22]. It would be interesting to use the taste reactivity test [23–25] to investigate palatability shifts in rats for flavors/tastes that have been associated with caffeine withdrawal and recovery from withdrawal. Acknowledgements This research was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to Vincent M. LoLordo, and by a Student Research Award from the Nova Scotia Health Research Foundation to Sarah E. Dreumont-Boudreau. References [1] Daly JW, Fredholm BB. Caffeine—an atypical drug of dependence. Drug Alcohol Depend 1998;51:199–206. [2] Fredholm BB, Battig K, Holmen J, Nehlig A, Zvartau EE. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol Rev 1999;51:83–133.
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[3] Nehlig A, Boyet S. Dose–response study of caffeine effects on cerebral functional activity with a specific focus on dependence. Brain Res 2000;858:71–7. [4] Holtzman SG. Complete, reversible, drug-specific tolerance to stimulation of locomotor activity by caffeine. Life Sci 1983;33:779–87. [5] Finn IB, Holtzman SG. Tolerance to caffeine-induced stimulation of locomotor activity in rats. J Pharmacol Exp Ther 1985;238:542–6. [6] File SE, Baldwin HA, Johnston AL, Wilks LJ. Behavioral effects of acute and chronic administration of caffeine in the rat. Pharmacol Biochem Behav 1988;30:809–15. [7] Gasior MJ, Jaszyna M, Peters J, Goldberg SR. Changes in the ambulatory activity and discriminative stimulus effects of psychostimulant drugs in rats chronically exposed to caffeine: effect of caffeine dose. J Pharmacol Exp Ther 2000;295:1101–11. [8] Meliska CJ, Landrum RE, Landrum TA. Tolerance and sensitization to chronic and subchronic oral caffeine: effects on wheelrunning in rats. Pharmacol Biochem Behav 1990;35:477–9. [9] Vitiello MV, Woods SC. Evidence for withdrawal from caffeine by rats. Pharmacol Biochem Behav 1977;6:553–5. [10] Fedorchak PM, Mesita J, Plater SA, Brougham K. Caffeine-reinforced conditioned flavor preferences in rats. Behav Neurosci 2002;116:334–46. [11] Myers KP, Izbicki EV. Reinforcing and aversive effects of caffeine measured by flavor preference conditioning in caffeine-naïve and caffeine-acclimated rats. Physiol Behav 2006;88:585–96. [12] Domjan M. Determinants of the enhancement of flavored-water intake by prior exposure. J Exp Psychol Anim Behav Processes 1976;2:17–27. [13] Dingle RN, Dreumont-Boudreau SE, LoLordo VM. Caffeine dependence in rats: effects of exposure duration and concentration. Physiol Behav 2008;95:252–7, doi:10.1016/j.physbeh.2008.06.007. [14] Solomon RL, Corbit JD. The opponent-process theory of motivation. I. Temporal dynamics of affect. Psychol Rev 1974;81:119–45. [15] Dreumont-Boudreau SE, LoLordo VM. The role of caffeine exposure and dependence in conditioned flavor avoidance. Talk presented at the Annual Associative Learning Symposium, Gregynog, Wales; April 2007. [16] Parker L, Failor A, Weidman K. Conditioned preferences in the rat with an unnatural need state: Morphine withdrawal. J Comp Physiol Psychol 1973;82:294–300. [17] Rogers PJ, Richardson NJ, Elliman NA. Overnight caffeine abstinence and negative reinforcement of preference for caffeine-containing drinks. Psychopharmacology 1995;120:457–62. [18] Yeomans MR, Spetch H, Rogers PJ. Conditioned flavour preference negatively reinforced by caffeine in human volunteers. Psychopharmacology 1998;137:401–9. [19] Tinley EM, Durlach PJ, Yeomans MR. How habitual caffeine consumption and dose influence flavour preference conditioning with caffeine. Physiol Behav 2004;82:317–24. [20] Yeomans MR, Durlach PJ, Tinley EM. Flavour liking and preference conditioned by caffeine in humans. Q J Exp Psychol 2005;58B:47–58. [21] Mobini S, Elliman TD, Yeomans MR. Changes in the pleasantness of caffeineassociated flavours consumed at home. J Food Qual Pref 2005;16:659–66. [22] Chambers L, Mobini S, Yeomans MR. Caffeine deprivation state modulates expression of acquired liking for caffeine-paired flavours. Q J Exp Psychol 2007;60:1356–66. [23] Grill HJ, Norgren R. The taste reactivity test: I. Mimetic responses to gustatory stimuli in neurologically normal rats. Brain Res 1978;143:263–79. [24] Schwartz GJ, Grill HJ. Relationships between taste reactivity and intake in the neurologically intact rat. Chem Senses 1984;9:249–72. [25] Berridge KC. Modulation of taste affect by hunger, caloric satiety, and sensory specific satiety in the rat. Appetite 1991;16:103–20.
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Conditioned flavor avoidance as a measure of withdrawal in rats chronically exposed to a caffeine solution Sarah E. Dreumont-Boudreau ⁎, Rachel N. Dingle, Gillian M. Alcolado, Vincent M. LoLordo Dalhousie University, Halifax, Nova Scotia Canada B3H 4J1
A R T I C L E
I N F O
Article history: Received 22 October 2007 Received in revised form 12 May 2008 Accepted 10 June 2008 Keywords: Caffeine Dependence Withdrawal Conditioned flavor avoidance Rats
A B S T R A C T Rats were given 21 days of chronic oral caffeine. A novel flavor (Maintenance CS) was then paired with the continuation of caffeine, and a second flavor (Withdrawal CS) was paired with caffeine removal. Rats avoided the Withdrawal CS, and drank more of the Maintenance CS in a two-bottle test, suggesting that removing caffeine had induced withdrawal. The value of the Maintenance CS was investigated by comparing it to a novel flavor paired with water (Neutral CS). In a series of two-bottle tests, the Maintenance and Neutral CSs were equivalent when pitted against each other, and both were preferred to the Withdrawal CS. These results demonstrate that conditioned flavor avoidance is a useful procedure in assessing caffeine withdrawal, and by inference dependence, produced by chronic oral consumption. © 2008 Elsevier Inc. All rights reserved.
Caffeine has often been described as an atypical drug of dependence, and does not produce the same behavioral and physiological changes observed with more traditional drugs of dependence [1–3]. Given that caffeine is widely consumed by humans, it is important to gain a better understanding of tolerance, dependence, and withdrawal that occur to caffeine's pharmacological effects. Research with animals can contribute to this understanding. One focus of such research has been on understanding caffeine tolerance and dependence in the rat. Extended exposure to caffeine will produce tolerance to its stimulative effects on locomotion, and can potentially produce dependence and subsequent withdrawal symptoms when caffeine is removed. Holtzman [4] gave rats controlled access to caffeinated water for 10 min every 6 h over a period of 11 weeks. The caffeine concentration was gradually increased from 0.25 mg/ml to 3.0 mg/ml by the sixth week. A control group was given unadulterated water on the same schedule. Both groups were then given a challenge dose of caffeine by gavage, and locomotor activity was measured. The control group showed the expected increase in locomotor activity after the challenge dose, but the rats that had been given chronic caffeine exposure showed no increase in activity. Holtzman concluded that the rats from the chronic caffeine group had become tolerant to caffeine's stimulative effects as a result of their extended exposure to the drug. Spontaneous locomotor activity was also measured during the chronic ⁎ Corresponding author. Dalhousie University; Psychology Department, 1355 Oxford Street, Halifax, Nova Scotia Canada B3H 4J1. Tel.: +1 902 494 3441; fax: +1 902 494 6585. E-mail address: [email protected] (S.E. Dreumont-Boudreau). 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.06.008
caffeine period and immediately after the caffeinated water was removed. Rats in the chronic caffeine group showed depressed locomotor activity during the first 4 days following caffeine removal, suggesting that they were experiencing some sort of withdrawal symptoms, and thus that they had become caffeine dependent. Using a similar procedure, Finn and Holtzman [5] assessed the ability of three caffeine doses to produce tolerance and withdrawal. Rats received either 0.25, 0.5, or 1.0 mg/ml of chronic caffeine over a six week period. Various challenge doses were then delivered by gavage. Rats in the 0.25 mg/ml group continued to show an increase in locomotor activity to some of the lower challenge doses. However, the 0.5 and 1.0 mg/ml groups showed no change in locomotor activity to any of the challenge doses, indicating tolerance to caffeine's stimulant effects. Other studies have found similar results of tolerance to caffeine's stimulant effects on locomotion in the hole-board test [6], locomotion after unrestricted chronic access to caffeine [7], and wheelrunning activity [8]. In addition to tolerance, Finn and Holtzman [5] found that the 1.0 mg/ml group showed a decrease in spontaneous locomotor activity during the first 2 days after caffeine was removed compared to the 3 days prior to removal. As in Holtzman [4], the authors concluded that this result was evidence of withdrawal symptoms and thus of caffeine dependence. Repeated exposure to the pharmacological effects of caffeine also changes a rat's response to flavors associated with caffeine, and to flavors associated with its absence. Vitiello and Woods [9] demonstrated an avoidance of a taste that was paired with the absence of caffeine after repeated exposure to caffeine. For 14 days, groups of rats were given daily injections of saline or of varying caffeine concentrations following
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brief access to water. Then half the rats that had previously been injected with caffeine received access to a saccharin solution followed by a caffeine injection (C–C), while the other half received saccharin followed by a saline injection (C–S). The same treatment was given to the rats that had previously received only saline (S–C, S–S). All groups were then given a two-bottle test with saccharin and water. Saccharin was strongly preferred by group S–S. The S–C group avoided the saccharin [for recent related results see [10,11]]. The C–C group preferred saccharin as much as the S–S controls, showing no conditioned effects. Most important, the C–S group showed a strong avoidance of the saccharin. The authors concluded that the conditioned avoidance of saccharin by the C–S group came about through its association with negative caffeine withdrawal symptoms. While a conditioned preference for saccharin did not form in the C–C group, it was no longer aversive as it was in the S–C group that had no prior experience with caffeine. Naïve rats initially found caffeine's pharmacological effects to be aversive, but after repeated exposure this was no longer the case, and the absence of caffeine's pharmacological effects following a signal for caffeine became aversive instead. We sought to further explore caffeine dependence and withdrawal in conditioned flavor avoidance using an oral consumption procedure to establish dependence. The purpose of Experiment 1 was to determine whether a conditioned flavor avoidance paradigm would reveal evidence for caffeine dependence in rats that had 21 days of chronic exposure to caffeinated water. If the rats become dependent on caffeine, then they should experience withdrawal symptoms when the caffeine is taken away. In order to assess this, we used caffeine withdrawal to establish a conditioned avoidance of a novel flavor. Immediately after the chronic exposure period, the rats were given two 30 min exposures to a novel Kool-Aid flavor. The caffeinated drinking water was returned after each exposure, hence the novel flavor was termed the Maintenance CS because it was paired with the maintenance of access to caffeine. Rats were then given three exposures to a second novel Kool-Aid flavor. After the first exposure to the second Kool-Aid, the rats were given unadulterated tap water and the caffeinated water was never returned. We hypothesized that removal of caffeine would induce caffeine withdrawal, hence the second Kool-Aid flavor was termed the Withdrawal CS. If the rats become dependent on caffeine over the 21-day chronic exposure period, and subsequently experience withdrawal symptoms when the caffeinated water is permanently removed, then they should avoid the Withdrawal CS and consume more of the Maintenance CS when given a choice between the two CSs. 1. Experiment 1
Saccharin solutions contained 1.0 g/L saccharin sodium salt hydrate (Sigma, Canada) dissolved in tap water. The caffeine solution was made up of caffeine anhydrous powder (Sigma, Canada) dissolved in tap water at a concentration of 1.0 g/L. The Kool-Aid/saccharin solutions were made up of either Grape or Cherry Kool-Aid (Kraft Canada) at a concentration of 2.5 g/L, and 1.0 g/L saccharin sodium salt hydrate dissolved in tap water. 1.1.3. Procedure 1.1.3.1. Saccharin pre-exposure. Rats were pre-exposed to a saccharin– water solution to familiarize them with saccharin and eliminate any initial neophobia that might interfere with saccharin–Kool-Aid consumption in training and testing. First, a 24 h water consumption measurement was taken to determine normal drinking amounts. For the next 2 days, all rats were given a saccharin–water solution as their only source of fluid. Consumption was measured every 24 h. 1.1.3.2. Chronic caffeine exposure. All of the rats were given a caffeine– water solution as their only source of fluid for 21 days. The caffeine– water bottles were weighed every 24 h between 11:00 and 11:30 to determine daily consumption. The rats were weighed every 48 h at the same time of day, and this continued throughout the entire experiment. 1.1.3.3. Training. Immediately following the twenty-first day of chronic caffeine exposure, the rats were divided into two groups equated on the previous 24 h caffeine consumption. Group 1 received Cherry Kool-Aid and saccharin (CherrySacc) as the Maintenance CS, and Grape Kool-Aid and saccharin (GrapeSacc) as the Withdrawal CS. Group 2 received the opposite flavor assignments. Training began immediately after Day 21 of chronic caffeine, and took place over 5 days. Following the removal of the caffeine–water bottle at the end of Day 21, each rat received the appropriate Maintenance CS solution for 30 min. The caffeine–water solution was then placed back on the cage for the rest of the day. The same procedure was repeated on the second day of training, such that each group received two exposures to the Maintenance CS followed by the return of caffeine. On the next day, the caffeine–water bottle was removed and the appropriate Withdrawal CS was placed on each cage for 30 min. A bottle filled with tap water was then placed on each cage for the rest of the day. The same procedure was repeated on the next 2 days, except the tap water bottle was removed prior to the CS bottle going on the cage, such that each rat received three exposures to the Withdrawal CS. A third day of Withdrawal CS exposure was added in an effort to equate consumption of the Maintenance and Withdrawal CSs because the rats greatly reduced their Withdrawal CS consumption on the second exposure.
1.1.1. Subjects Twenty-five adult male Sprague–Dawley rats from Charles River Canada, Quebec were used. Rats were housed singly in standard shoebox cages under a 12:12 light/dark cycle with lights on at 0800 h. Food and fluid were available ad libitum throughout the entire experiment. Rats were weighed every other day for the duration of the experiment, and their mean weight at the beginning of the experiment was 359 g.
1.1.3.4. Testing. Immediately following the last training day, two tap water bottles were placed on each cage for 48 h. This was done to reduce any neophobia to having two bottles present on the cage simultaneously, and to ensure that if the rats had become dependent on the caffeine enough time (5 days) had elapsed for recovery from withdrawal symptoms prior to testing [4,5]. Water consumption was measured every 24 h. The water bottles were then removed and replaced with one CherrySacc bottle and one GrapeSacc bottle. The Kool-Aid solutions were left on the cages for 24 h, and bottle position was counterbalanced within each group.
1.1.2. Apparatus All training and testing were conducted in the homecage. The homecage was made of clear plastic with a wire top, and measured 46.5 cm × 24.5 cm × 21 cm. Each cage contained approximately 2 cm of wood shavings on the bottom and a 12 cm × 7.5 cm black PVC hiding tube. All solutions were delivered in 250 ml glass bottles with 3.5 cm black rubber stoppers. Each stopper contained a 6 cm long stainless steel sipper tube with a 0.5 mm opening at the end.
1.1.4. Statistical analysis Maintenance CS training data were analyzed using a 2(Day) × 2 (Flavor; CherrySacc or GrapeSacc as Maintenance CS) mixed ANOVA with Day as the within-subjects variable and Flavor as the betweensubjects variable. Withdrawal CS training data were analyzed with a 3 (Day) × 2(Flavor) mixed ANOVA with Day as the within-subjects variable, and Flavor as the between-subjects variable. Post hoc analysis was conducted using the Scheffe's test.
1.1. Methods
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Two-bottle Maintenance CS vs. Withdrawal CS test consumption data were analyzed using a 2(CS) ×2(Flavor) ×2(Maintenance Bottle Location) mixed ANOVA with CS as the within-subjects variable, and Flavor and Maintenance Bottle Location as the between-subjects variables.
70.0 mg/kg of caffeine consumed. On Day 21, the rats drank an average of 40.4 ml, resulting in an average of 93.9 mg/kg caffeine consumed. See Fig. 1A for the mean daily caffeine consumption in milliliters and mg/kg across the 21 days of chronic exposure.
1.2. Results
1.2.3. Training During training, consumption varied across days for both the Maintenance and the Withdrawal CSs. Consumption increased across days for the Maintenance CS, but decreased across days for the Withdrawal CS. See Fig. 1B. There was a significant main effect of Day for the Maintenance CS, with more consumption taking place on Day 2 than on Day 1, F(1, 46)= 7.09, p b 0.05. No other main effects or interactions reached significance. Withdrawal CS consumption data also revealed a significant main effect of Day, F(2, 69)=23.59, pb 0.01, with consumption dropping off dramatically on Days 4 and 5 compared to Day 3. Post hoc comparisons revealed
1.2.1. Saccharin pre-exposure During the baseline 24 h water consumption period, the rats drank an average of 45.1 ml of water. On the first day of 24 h saccharin exposure, the rats drank an average of 75.5 ml. On the second day, the average consumption had dropped to 64.8 ml. 1.2.2. Chronic caffeine exposure On Day 1 of chronic caffeine exposure, the rats drank an average of 25.1 ml of the caffeine–water solution. This resulted in a mean of
Fig. 1. (A) Mean daily consumption of caffeine across the 21-day chronic caffeine exposure, and during Maintenance CS training in Experiment 1. (B) Mean daily consumption of the Maintenance and Withdrawal CSs during training. (C) Mean consumption of the Maintenance and Withdrawal CSs in test. Error bars represent standard error of the mean.
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Day 3 consumption to be significantly higher than consumption on Days 4 and 5, which did not differ from each other. No other main effects or interactions reached significance. In order to compare total exposure to the Maintenance and Withdrawal CSs, consumption was summed across days for each CS. A 2(CS) × 2(Flavor) mixed ANOVA was run with CS as the within-subjects variable, and Flavor as the between-subjects variable. The only significant result was a main effect of CS, with rats drinking more of the Maintenance CS than the Withdrawal CS, F(1, 23) = 5.35, p b 0.05. 1.2.4. Testing Rats drank much more of the Maintenance CS compared to the Withdrawal CS during the Maintenance CS vs. Withdrawal CS twobottle test. See Fig. 1C. There was a significant main effect of CS, with more overall consumption of the Maintenance CS than the Withdrawal CS, F(1, 40)= 8.65, pb 0.05. No other main effects or interactions reached significance. 1.3. Discussion When given a choice between the Maintenance CS and the Withdrawal CS, rats avoided the Withdrawal CS and consumed more of the Maintenance CS in test. Therefore, the data strongly suggest that the rats experienced negative withdrawal symptoms when caffeine was no longer available. These symptoms were then associated with the Withdrawal CS. From these results we can infer that the oral 21-day chronic caffeine exposure period was enough to produce dependence in rats. One obvious alternative to this conclusion is that the rats simply drank more in test of the CS that was more familiar. In training, significantly more Maintenance CS was consumed than Withdrawal CS. This exposure effect could also produce the results that were observed in the two-bottle test, if the rats were simply choosing to drink the CS that they had the most experience with [12]. We examined whether relative exposure to the two CSs in training predicted preference in the two-bottle test by calculating the ratio of the amount of Withdrawal CS consumed in training divided by total consumption of the two CSs in training. The analogous ratio was calculated for the amount of Withdrawal CS consumed during the two-bottle test, and the correlation of the two was computed. The correlation was non-significant, r = 0.037, p N 0.05, suggesting that the observed preference is not an exposure effect. The increase in consumption of the Maintenance CS from the first training day to the second most likely reflects habituation of neophobia. Consumption of the Withdrawal CS on Day 3, before any withdrawal symptoms could have occurred, was comparable to consumption of the Maintenance CS. The reduction in consumption of the Withdrawal CS on Days 4 and 5 is consistent with the acquisition of a conditioned flavor avoidance based on aversive withdrawal symptoms. In this experiment the Withdrawal CS was pitted against the Maintenance CS in the two-bottle test. We chose the Maintenance CS as our neutral control because it did not signal a marked change in the availability of the caffeine solution. However, we had no evidence that the Maintenance CS was neutral. In Experiment 2 we sought to discover whether the Maintenance CS was neutral, or became positive after being paired with the continuation of caffeine. Rats were given chronic caffeine exposure, Maintenance CS, and Withdrawal CS training as in Experiment 1. But, several days after Withdrawal CS training a third CS, called the Neutral CS, was introduced, and was paired with plain tap water. Each rat was then given three two-bottle tests; Maintenance CS vs. Withdrawal CS, Withdrawal CS vs. Neutral CS, and Maintenance CS vs. Neutral CS. If the Maintenance CS became positive by signaling the continuation of caffeine, then the rats should prefer it to the Neutral CS in test. However, if the Maintenance CS was essentially neutral then there should be no preference between it and the Neutral CS when they are pitted against each other in test.
2. Experiment 2 2.1. Methods 2.1.1. Subjects Forty-nine adult male Sprague–Dawley rats from Charles River Canada, Quebec were used. Rats were housed and maintained as in Experiment 1. Rats were weighed every other day for the duration of the experiment, and their mean weight at the beginning of the experiment was 292 g. 2.1.2. Apparatus All training and testing were conducted in the homecage. Apparatus and solutions were as in Experiment 1, except that LemonLime Kool-Aid was also used. 2.1.3. Procedure 2.1.3.1. Saccharin pre-exposure. All rats were pre-exposed to saccharin for 6 days prior to the start of chronic caffeine exposure. First, a 24 h. water consumption measurement was taken to determine normal drinking amounts. For the next 3 days, all rats were given a saccharin– water solution as their only source of fluid. Consumptions were measured every 24 h. Following 24 h saccharin–water exposure, all rats were given one daily 30 min saccharin–water exposure for 3 days. The 30 min exposure was given during the same time that the experimental CS bottles would eventually be put on the cages during training. 2.1.3.2. Chronic caffeine. out as in Experiment 1.
The chronic caffeine procedure was carried
2.1.3.3. Training. Immediately following the twenty-first day of chronic caffeine exposure, the rats were divided into six groups equated on the previous 24 h caffeine consumption. Cherry, Grape, and LemonLime Kool-Aid flavors were assigned as Maintenance, Withdrawal, and Neutral CSs such that all possible combinations were used. See Table 1 for Kool-Aid flavor assignments in each group. Training began immediately after the twenty-first day of chronic caffeine, and took place over 11 days. Following the removal of the caffeine–water bottle at the end of Day 21, each rat received the appropriate Maintenance CS solution for 30 min. The caffeine–water solution was then placed back on the cage for the remainder of the 24 h period. The same procedure was repeated on Day 2 of training, such that each group received two exposures to the Maintenance CS. On Day 3, the caffeine–water bottle was removed and the appropriate Withdrawal CS was placed on each cage for 30 min. A bottle filled with tap water was then placed on each cage for the remainder of the 24 h period. The same procedure was repeated on Days 4 and 5, except that the tap water bottle was removed prior to the CS bottle going on the cage. Each rat received three exposures to the Withdrawal CS. A third day of Withdrawal CS exposure was added in an effort to equate Maintenance and Withdrawal CS consumption because the rats greatly reduced their Withdrawal CS consumption on the second
Table 1 Flavor assignment to Maintenance, Withdrawal, and Neutral CSs Group
1 2 3 4 5 6
CS Maintenance
Withdrawal
Neutral
Cherry Cherry LemonLime LemonLime Grape Grape
Grape LemonLime Grape Cherry Cherry LemonLime
LemonLime Grape Cherry Grape LemonLime Cherry
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exposure. On Days 6, 7, and 8 the tap water bottles were left on the cages and consumption was measured every 24 h. This was done to allow enough time (6 days) for any remaining symptoms of caffeine withdrawal to dissipate prior to Neutral CS training. On Day 9, the tap water bottles were removed from the cages and the appropriate Neutral CS was placed on each cage for 30 min. The tap water bottles were then placed back on the cages for the remainder of the 24 h period. The same procedure was repeated on Days 10 and 11, except that two bottles of tap water were placed on each cage after the Neutral CS was removed on Day 11. 2.1.3.4. Testing. The two tap water bottles were left on each cage for 24 h. Upon removal of the two water bottles, two-bottle CS testing began. Each rat received three 24 h two-bottle tests: Maintenance CS (M) vs. Withdrawal CS (W), Maintenance CS (M) vs. Neutral CS (N), and Withdrawal CS (W) vs. Neutral CS (N). There were 10 days between the last caffeine exposure and the first two-bottle test. To control for possible test order effects, for each of the six possible assignments of Kool-Aid flavors to the three CSs a rat was allocated to each of the six possible test orders. For example, one rat might receive the M vs. W test first, then the M vs. N test, and finally the W vs. N test. A second rat might receive the M vs. N test first, followed by the W vs. N test, and finally the M vs. W test. Extra rats were distributed among the various groups. In addition, bottle position was counterbalanced within each group such that the Maintenance CS in any given test was not always on the left side, for example. Test bottles were left on for 24 h, and were immediately replaced by new test bottles upon removal. 2.1.4. Statistical analysis Maintenance CS training data were analyzed using a 2(Day) × 3 (Flavor; Cherry, Grape, or LemonLime) mixed ANOVA with Day as the within-subjects variable, and Flavor as the between-subjects variable. Withdrawal and Neutral CS training data were analyzed using separate 3(Day) × 3(Flavor) mixed ANOVAs with Day as the withinsubjects variable, and Flavor as the between-subjects variable. Post hoc analysis was conducted using the Scheffe's test. Each two-bottle test was analyzed separately with a 2(CS) × 3(CS1 Flavor) × 3(CS2 Flavor) × 2(CS1 Bottle Location) mixed ANOVA, with CS as the within-subjects variable, and CS1 Flavor, CS2 Flavor, and CS1 Bottle Position as between-subjects variables. Effects of two-bottle CS test order were also analyzed separately for each two-bottle test using 2(CS) × 3(Order) mixed ANOVAs with CS as the within-subjects variable and Order as the between-subjects variables. The Order factor refers to where a particular test fell amongst the three twobottle tests for each rat. For example, when analyzing the Maintenance vs. Withdrawal test, one rat may have had that test first, while a second rat may have had that test third, and so on. 2.2. Results 2.2.1. Training Fig. 2A shows the mean consumptions of Maintenance, Withdrawal, and Neutral CSs during training. During presentation of the Maintenance CS, rats drank more on Day 2 than on Day 1. There was a significant main effect of Day, F(1, 92) = 13.76, p b 0.05. No other main effects or interactions reached significance. Consumption of the Withdrawal CS during training also varied across training days. Rats consumed more of the Withdrawal CS on Day 3 than on Days 4 and 5. There was a significant main effect of Day, F(2, 138) = 39.59, p b 0.05. A post hoc Scheffe's test revealed that the rats drank significantly more Withdrawal CS on Day 3 compared to Days 4 and 5 (p b 0.05), while Days 4 and 5 did not differ from each other (p N 0.05). No other main effects or interactions reached significance. During Neutral CS training, consumption of the CS did not differ across training days. However, rats that received Grape Kool-Aid– saccharin as their Neutral CS drank more than rats that received
Fig. 2. (A) Mean consumption of the Maintenance, Withdrawal, and Neutral CSs during training in Experiment 2. (B) Mean consumption of each CS in the Maintenance vs. Withdrawal, Neutral vs. Withdrawal, and Maintenance vs. Neutral tests. Error bars represent standard error of the mean.
Cherry Kool-Aid–saccharin or LemonLime Kool-Aid–saccharin. There was a significant main effect of Flavor, F(2, 138) = 9.18, p b 0.05. Post hoc Scheffe's tests showed that rats drank significantly more Grape Kool-Aid–saccharin compared to Cherry or LemonLime Kool-Aid– saccharin (p b 0.05), while Cherry and LemonLime Kool-Aid–saccharin consumptions did not differ from each other (p N 0.05). No other main effects or interactions reached significance. In order to compare total exposure to the Maintenance, Neutral, and Withdrawal CSs, consumption was summed across days for each rat within each CS. A one-way ANOVA was used to analyze the data. There was a significant difference in consumption between the three CSs, F(2, 138)= 7.57, pb 0.05. Post hoc Scheffe's tests revealed that there was no difference in total consumption between the Maintenance CS and the Withdrawal CS (p=.711). However, the rats consumed significantly less of the Neutral CS compared to the Maintenance CS (p=.018), and to the Withdrawal CS (p=.001). 2.2.2. Testing Fig. 2B shows the mean consumption of each CS during the Maintenance vs. Withdrawal, Withdrawal vs. Neutral, and Maintenance vs. Neutral two-bottle tests. Rats showed a preference for the Maintenance CS and the Neutral CS when they were pitted against the Withdrawal CS. However, rats showed no preference for either the Maintenance CS or the Neutral CS when given a choice between the two. The order in which the two-bottle tests were given did not affect CS consumption. Regardless of which test was given first, second, or third, rats still showed a preference for Maintenance and Neutral CSs over the Withdrawal CS, and no preference for the Maintenance CS over the Neutral CS or vice versa. For the Maintenance vs. Withdrawal two-bottle test, there was a significant main effect of CS, F(1, 37) = 11.43, p b 0.05, with rats consuming
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significantly more Maintenance CS than Withdrawal CS. There were no other significant main effects or interactions. For the Withdrawal vs. Neutral two-bottle test, there was a significant main effect of CS, F(1, 37) =7.75, p b 0.05, with rats consuming significantly more Neutral CS than Withdrawal CS. There were also significant main effects of Neutral Flavor, F(2, 37) =3.82, p b 0.05, and Withdrawal Flavor, F (2, 37)= 4.23, p b 0.05. Inspection of the data showed a small flavor preference for Grape Kool-Aid. Rats consumed more of the Neutral and Withdrawal CSs when they were Grape Kool-Aid than when they were Cherry or LemonLime Kool-Aids. No other main effects or interactions were significant. For the Maintenance vs. Neutral two-bottle test, there was no main effect of CS, F(1, 37) = 0.131, p N 0.05. No other main effects or interactions were significant. The test data were subjected to a second analysis to assess any effects of test order on CS consumption. Test order had no effect on the two-bottle consumption data. Maintenance and Neutral CSs were preferred to the Withdrawal CS, and neither the Maintenance CS or the Neutral CS was preferred over the other. For the Maintenance vs. Withdrawal two-bottle test there was a main effect of CS, F(1, 46) = 12.86, p b 0.05, but no main effect of Order, F(2, 46) = 1.22, p N 0.05, and no significant interactions. In the Neutral vs. Withdrawal two-bottle test, there was a significant main effect of CS, F(1, 46) = 7.28, p b 0.05, but no main effect of Order, F(2, 46) = 1.10, p N 0.05, and no significant interactions. For the Maintenance vs. Neutral two-bottle test, there was no main effect of CS, F(1, 46) = 0.261, p N 0.05, or Order, F(2, 46) = 1.80, p N 0.05, and no significant interactions. 2.3. Discussion The Maintenance CS was equally preferred to the Neutral CS in test. Hence, it is not inherently positive, but is essentially functioning like a Neutral CS when pitted against the Withdrawal CS. This is not surprising since very little changes for the rat when the Maintenance CS is presented in training. In separate two-bottle tests, both the Maintenance CS and the Neutral CS were preferred to the Withdrawal CS. During training, the patterns of consumption over days of the Maintenance CS and the Withdrawal CS were as seen in the previous experiment. However, there was no significant difference in total consumption of the two CSs in training. Nonetheless, there was still a robust avoidance of the Withdrawal CS in the Maintenance CS vs. Withdrawal CS test. In the present experiment this avoidance cannot be attributed to greater familiarity of the Maintenance CS. Consumption of the Neutral CS during training was surprisingly depressed, and rats consumed significantly less of it than the Maintenance CS and Withdrawal CS. Even though Neutral CS training was given after the physical symptoms of caffeine withdrawal should have worn off, the rats' previous experience with the Withdrawal CS could have generalized to the Neutral CS, resulting in lower consumption. Despite the depressed consumption of the Neutral CS in training, rats still showed a preference for the Neutral CS over the Withdrawal CS in test. Since the Neutral CS was the last CS presented in training; this preference implies that the avoidance of the Withdrawal CS observed in the first experiment could not have been simply avoidance of the most recent CS. Moreover, the Withdrawal and Neutral CSs had been presented three times in training, suggesting that the avoidance of the Withdrawal CS in the first experiment did not depend on the difference in the number of exposures of the two CSs in that experiment. There was also no preference for the Maintenance CS over the Neutral CS in test, even though rats consumed more of the Maintenance CS in training. 2.4. General discussion A conditioned flavor avoidance paradigm was successfully used to demonstrate caffeine withdrawal in rats. After 21 days of chronic free access to caffeine, rats avoided the Withdrawal CS flavor that had been
paired with the removal of caffeine, and drank more of the Maintenance CS. Presumably, this was due to the Withdrawal CS being associated with negative withdrawal symptoms stemming from the removal of caffeine, further suggesting that the rats had become dependent over the 21 day chronic caffeine period. The results support the Holtzman [4] and Finn and Holtzman [5] experiments, which examined changes in locomotor behavior, in demonstrating that chronic oral consumption of caffeine produces dependence and subsequent withdrawal when caffeine is removed. Finn and Holtzman [5] demonstrated that tolerance to the stimulatory effects of caffeine can happen after only 4 days of chronic caffeine. Dingle et al. [13] addressed the question whether comparably brief exposure to chronic caffeine with the current oral chronic caffeine procedure would produce dependence and withdrawal. Groups of rats were given 5, 10, 15, or 20 days exposure to 1.0 g/L caffeine solutions. A novel flavor (Withdrawal CS) was then associated with caffeine removal, and was later pitted against a second novel flavor (Neutral CS) in a two-bottle test. The rats that were given 20 days of chronic caffeine exposure were the only group to show a significant avoidance of the Withdrawal CS. The groups given 5 and 10 days of chronic caffeine showed no evidence of avoidance of the Withdrawal CS, while the groups given 15 days showed a weak avoidance. Although brief exposure to chronic caffeine is sufficient to produce tolerance to the stimulative effects of caffeine on locomotion [5], longer periods of chronic caffeine exposure are necessary to produce dependence. Finally, the Maintenance CS was determined to be equivalent to a Neutral CS that was simply paired with water, and did not carry any positive value after being paired with caffeine. This is not surprising, given that there was no change in the drug state during Maintenance CS training. Our Maintenance CS treatment parallels the treatment of the caffeine–caffeine (C–C) group of rats in Vitiello and Woods [9]. Rats in that group continued to receive caffeine injections after the daily water presentation was switched to a saccharin presentation, and responded like caffeine naive rats in a subsequent two-bottle test of saccharin against water. As in Vitiello and Woods [9], the present experiments produced a conditioned avoidance of a novel flavor that had previously been paired with caffeine withdrawal symptoms. However, instead of a single daily injection, we used a chronic oral procedure of caffeine consumption in which the rats determined when and how much caffeine they consumed at any given time. Even though our rats had no other fluid to drink but the caffeine solution, they maintained a level of control over caffeine dose via the amount of liquid consumed during individual drinking bouts. This is more analogous to the pattern of human caffeine consumption than is a single daily injection. In Vitiello and Woods [9], the onset of the drug effect was signaled by a discrete event, the daily drinking period. In the current experiments, rats had the opportunity to have many drinking bouts throughout the chronic caffeine exposure period. Prior to caffeine's drug effects taking place, the rats encountered the bitter taste of the caffeine itself. Hence, the bitter taste of caffeine likely became a signal for caffeine's drug effects, similar to the daily drinking bout signaling the drug effect in Vitiello and Woods' experiment. When the taste offered during the daily drinking bout changed from water to saccharin in Vitiello and Woods' experiment, and was then followed by no drug effect and subsequent withdrawal, the rats learned to avoid it. Our procedure is similar in that the rats received a novel Withdrawal CS followed by no drug effect, i.e., tap water. The rats learned to associate the negative state this put them in (caffeine withdrawal) with the Withdrawal CS, and avoided that CS in the two-bottle test. One could agree that the avoidance of the Withdrawal CS, relative to the Maintenance and Neutral CSs, observed in these experiments is a conditioned taste avoidance without agreeing that the basis of the avoidance is a caffeine withdrawal state. Instead one could argue that removal of any positive stimulus generates a negative after effect [14],
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and that a signal for that aftereffect becomes aversive. So, is access to a 1 g/L caffeine solution positive? Dreumont-Boudreau and LoLordo [15] gave rats access to a novel Kool-Aid–saccharin solution for 1 h, then gave them 24 h of access to the caffeine solution. Three days later the rats received 1 h access to a second Kool-Aid–saccharin solution followed by water. In a subsequent two-bottle test, the rats strongly avoided the flavor that signaled access to the novel caffeine solution [also see [10,11]]. Therefore, access to our caffeine solution is initially negative, not positive. But, is caffeine positive after 21 days? If it is not, then the alternative account does not apply to the present data. If it is positive after initially being negative, then that result implies caffeine dependence, which implies that the effect of removing caffeine is to induce withdrawal. Conditioned flavor/taste avoidance is a particularly useful procedure for detecting caffeine withdrawal, and has been successfully used with other drugs of dependence. Parker et al. [16] showed that a novel flavor paired with negative morphine withdrawal symptoms was subsequently avoided. Most research looking at caffeine dependence and withdrawal using chronic oral procedures to produce dependence has relied on changes in locomotor behavior to detect dependence and withdrawal [4,5,7,8]. The conditioned flavor/taste avoidance paradigm is a sensitive alternative to locomotor behavior for assessing withdrawal, particularly with chronic oral procedures of dependence. The next step in developing a rat model of caffeine dependence is to utilize the conditioned flavor/taste paradigm to assess a novel flavor that has been associated with recovery from negative caffeine withdrawal symptoms. Parker et al. [16] were successful in producing a preference for a novel flavor that had previously been associated with recovery from morphine withdrawal. Developing a model for caffeine withdrawal recovery is particularly important because there is compelling evidence in the human literature that relief from withdrawal symptoms drives habitual human consumption of caffeine [17,18]. While in withdrawal, humans report an increase in pleasantness of flavors that have been paired with ingestion of caffeine [17– 22]. It would be interesting to use the taste reactivity test [23–25] to investigate palatability shifts in rats for flavors/tastes that have been associated with caffeine withdrawal and recovery from withdrawal. Acknowledgements This research was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to Vincent M. LoLordo, and by a Student Research Award from the Nova Scotia Health Research Foundation to Sarah E. Dreumont-Boudreau. References [1] Daly JW, Fredholm BB. Caffeine—an atypical drug of dependence. Drug Alcohol Depend 1998;51:199–206. [2] Fredholm BB, Battig K, Holmen J, Nehlig A, Zvartau EE. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol Rev 1999;51:83–133.
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