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Tolerance to the discriminative stimulus properties of d-amphetamine
TOLERANCE TO THE DISCRIMINATIVE STIMULUS PROPERTIES OF d-AMPHETAMINE* R. J. BARRETTand NANCY J. LEITH Veterans Administration Medical Center and Department of Psychology and Pharmacology, Vanderbilt University, Nashville, TN 37203, U.S.A. (Accepted 13 August 1980) Summary-Male rats (F-344) responding for milk on a VI 20 set schedule of reinforcement were trained to discriminate which of two levers to press on the basis of whether they had been injected with d-amphetamine (0.50, 1.00 or 1.50 mg/kg) or saline 15 min prior to daily training sessions. Dose-response functions determined for each of the three (n = 6) training-dose groups indicated that ED,, values were directly correlated with training dose. Two days following chronic amphetamine injections (a total of 78 mg/kg over 4 days) rats were tested for tolerance at a dose which normally produced about 80% drug-lever responding. Rats in all three groups showed tolerance to the cue properties of amphetamine. In the 0.50 and I.00 mg/kg groups, complete tolerance was shown as evidenced by the fact that the drug lever responding did not differ from that which was appropriate following saline injections.
Previous studies with chronic administration of amphetamine have described two types of tolerance: (1) tolerance to the effects of amphetamine on physiological responses, such as hyperthermia (Harris, Ambrus and Ambrus, 1952), cardiovascular effects (Burn and Rand, 1958), changes in urinary excretion of catecholamines (Lewander, 1968) and its anorexigenic properties (Glick, 1973; Lu, Clayhorn and Schoolar, 1973); and (2) behavioral or “learned” tolerance which refers to a diminished drug effect on behavior reflecting the organism learning to accommodate or adapt to the drug condition (Schuster, Dockens and Wood, 1966). The latter develops only in situations where the drug action serves to disrupt the ability of the organism to meet the requirements of the task for reinforcement. For example, amphetamine has been shown to disrupt initially behavior in rats reinforced for correct temporal spacing of responding. However, daily administration of the drug given prior to each testing session results in a gradual return of responding to pre-drug baseline (Schuster and Zimmerman, 1961). Conversely, where the drug enhances the ability of the rat to meet the requirements for reinforcement, such as in active-avoidance tasks, responding does not return to the pre-drug baseline (Schuster et al., 1966). Recently, two experiments (Anderson, Leith and Barrett, 1978; Leith and Barrett, 1976) successfully demonstrated physiologically-mediated tolerance to the facilitation by amphetamine of responding for electrical self-stimulation of the brain and, in another experiment, (McCown and Barrett, 1980) tolerance to the reinforcing properties of d-amphetamine was demonstrated in an amphetamine self-administration * Supported by the Veterans Administration. Key words: discriminative stimulus, amphetamine, tolerance. 251
study with rats.. Both of these paradigms involve changes in response rate to assess tolerance develop ment. Since chronic administration of amphetamine can produce disruption of behavior due to general physical malaise, response-rate changes, especially decrements, can be difficult to interpret. The present experiment was designed to determine whether tolerance could be demonstrated in an operant two-lever drug discrimination paradigm. The use of d-amphetamine as a discriminative stimulus in a two-choice paradigm has been well documented in previous experiments (Harris and Balster, 1968; Huang and Ho, 1974; Jones, Hill and Harris, 1974; Kuhn, Appel and Greenberg, 1974; Schechter, 1978; Schechter and Cook, 1975; Schechter and Rosecrans, 1973). Also, other researchers have successfully used this rate-independent measure to show tolerance to the cue properties of cocaine (McKenna and Ho, 1977) and morphine (Miksic and Lal, 1977) following chronic administration. METHODS Subjects
Eighteen male F-344 rats, approx. 110 days old and weighing 230-25Og at the beginning of the experiment, were housed in individual cages and food deprived to 75 + 5% of their expected ad libitum weight. They were maintained on a 12-hr light-dark cycle (7:OOa.m.-7:OOp.m. light) and given enough food (Purina Lab Chow) immediately following tests sessions to maintain control weight throughout the experiment. The rats had free access to water at all times. Testing occurred at the same time each day, 5 days a week. On the days not tested the rats were given good sufficient to maintain 75% of their body weight. Injections of either d-amphetamine sulfate or
252
R. J.
BARRETT and NANCY
saline (0.9% sodium chloride) were administered intraperitoneally in volumes of 1 ml/kg body weight. Solutions of d-amphetamine sulfate were made by dissolving the drug in saline. All doses refer to amphetamine salt. Apparatus Three commercially available operant chambers (BRS/LVE Model No. RTC-022) each housed in a sound attenuating chamber were used for training rats on the discriminative stimulus task. On the front panel of each chamber two response levers were mounted 4.92cm above the floor and required a 24g force to activate. Reinforcements (Borden’s condensed milk diluted 1:I with water) were delivered by a liquid feeder (0.06ml) centered between the two levers. Electromechanical programming and recording equipment was located in an adjacent room. White noise was used in the experimental room to mask extraneous auditory stimuli. Procedure Preliminary training. Following gradual deprivation to 75% of ad Ii&turn body weight, the 18 rats were trained to lever press during daily 30 min sessions on a continuous schedule of reinforcement (CRF). They were given training on alternate days to each of the two levers. Acquisition of the d-amphetamine-saline discrimination. Once the lever press response was acquired, the reinforcement contingency was changed to a variable-interval 20 set (VI-20”) and discrimination training was initiated. Rats from each of three (n = 6) training-dose groups (0.50, 1.00, 1.50 mg/kg d-amphetamine sulfate) were injected intraperitoneally 1.5min prior to the start of daily 30min test sessions with either the drug or saline. For half the subjects in each group the right lever was designated the amphetamine-correct lever and the left lever the saline-correct one. This was reversed for the remaining half of the subjects. The first 2.5 min of each session consisted of an extinction period during which no lever presses were reinforced. This allowed for daily monitoring of discrimination acquisition unconfounded by reinforcement. During the remaining 27.5 min of the session, responses were reinforced on the correct lever with the exception that responses on the incorrect lever were punished by imposing a 20 set-delay before reinforcements were again available. Training was continued in this manner until the average percentage correct responses for each of the three groups was 85% or higher on both amphetamine and saline test days. Drug and saline injections were given on alternate days with the exception that, after every tenth test session, the sequence was reversed. This results in consecutive injections of either drug or saline once every 10 sessions and ensures that the rats are not responding to the temporal sequence of injection rather than to the drug-related cues. All discrimi-
J.
LEITH
nation training sessions were 30min long and were conducted 5 days per week (Monday to Friday). Generalization of the d-amphetamine cue to different doses of amphetamine. Once stable choice behavior was established, dose-response generalization functions were determined for the three groups during 5 min extinction sessions, i.e. no reinforcement was available to prevent the animal from receiving discrimination training to a new drug dose which might disrupt the original baseline discrimination. Immediately following the 5 min test session the rats were returned to their home cages and given their daily allotment of food. In addition to the training dose the four doses tested were: 0.10, 0.15,0.25, and 0.35 mg/kg for the 0.50mg/kg group; 0.25, 0.35, 0.50 and 0.75 mg/kg for the 1.0 mg/kg group; and 0.35, 0.50, 0.75 and 1.0 mg/kg for the 1.50 mg/kg training group. Order of testing was counterbalanced within each group and only one test dose was administered each week. Test days generally occurred on Wednesdays so that a drug and saline training day both preceded and followed each test session. Test for tolerance to the discriminative stimulus properties of amphetamine. Following completion of the dose-response function, testing was temporarily suspended and all subjects were injected chronically with d-amphetamine sulfate according to a schedule previously shown to produce tolerance to facilitation of responding by amphetamine for electrical brain stimulation (Leith and Barrett, 1976). The schedule consisted of three daily injections (8:00 a.m., 2:00 p.m., 8:OO p.m.) for 4 consecutive days. Following the first injection of 1 mg/kg, each subsequent injection was incremented by 1 mg/kg, so that the final injection on the fourth day was 12 mg/kg, totalling 78 mg/kg for the entire regimen. On days 2, 4 and 6 following the chronic injections all subjects were tested for tolerance during 5 min extinction sessions. The doses chosen to test for tolerance represented points on the dose-response curve where choice behavior had been shown to be sensitive to small changes in drug concentration. On days 3 and 5, regular saline training sessions were given. RESULTS
Acquisition of the d-amphetamine-saline discrimination Rats in all three groups received a total of 44 discrimination training sessions. A repeated measures analysis of variance on percentage of correct-lever responding during the 2)min extinction period at the beginning of each session indicated that although there were no significant differences between the three groups in percentage correct responses by the end of training, a significant training dose x days interaction [F(66,495) = 9.16, P < O.OOl] confirmed the expectation that the rate of acquiring the discrimination would be dose-related. Inspection of the acquistion data for the three groups clearly showed that the 1.0 and 1.5 mg/kg training groups, while not different
253
Tolerance to amphetamine cues
0.5mg/kg
d-AMPH
I.Omp
1
kg
1.5 mg/k@
d-AMPH
d-AMPH
I loo-•PM OPOS!
FL s
Chronic
d- AMPH oDAY
0
DAY6
6
4
60
k G P b
40-
/’
ODAY4
l
.
/ 2oi-
l
CDAYZ
o DAY2 18 0.100.15
I 0.25
, 0.36
! 0.50
II 0.25035
I 0.50
1 075
Dose of d-amphetamine
1 1.0
I,, 0.35(150
0.75
I 1.0
I I3
fmg/kg)
Fig. 1. Mean percentage drug-lever responding as a function of five test doses for each of three training dose groups. Data points labelfed Day 2, Day 4, and Day 6 represent the results of the three tests for tolerance following chronic administration of d-amphetamine.
from each other, were both superior to the 0.50 mg/kg group early in training. By the end of training all three groups consistently averaged between 8595% correct-lever responding on both saline and drug sessions during the 24 min test period.
Generalization of the d-amphetamine doses of amphetamine
cue to different
The mean percentage d-amphetamine-lever responding is presented in Figure 1 for the 5 min test sessions as a function of training dose and four test doses. As can be seen, subjects trained on 0.50 mg/kg d-amphetamine were extremely sensitive to small reductions from the training dose compared to subjects in either the 1.00 or 1.50 mg/kg training groups. The effect of the training dose on the dose-response curve is best illustrated by comparing estimates for each group of the dose resulting in 50% drug-lever responding (ED,,). The EDso values were estimated using a procedure {Barry, 1974) which involves interpolating between the log transformations of the two doses which bracket the 50% point. The resulting EDSo doses were 0.23, 0.30 and 0.37mg/kg for the 0.50, 1.00 and 1.50 mg,kg training groups.
Test for tolerance stimulus properties
to d-~phet~mine
A dose of 0.35 mg/kg of d-amphetamine was chosen to test for tolerance in the 0.50 and 1.00 mg/kg training groups while 0.50 mg,kg was used for testing the 1.50 mg/kg group. These doses produced comparable decrements in drug-lever responding of 8, 11 and 13% from training dose levels for the 0.5, 1.0 and 1.50mg/kg groups respectively (see Fig. 1) prior to chronic amphetamine treatment. In Figure 1 it can be seen that when tested on Day 2 following chronic amphetamine administration, subjects in all three groups showed tolerance to the cue value of the drug. The changes in percentage choice of the drug-lever from pre-chronic to the first post-chronic test session on Day 2 were 79 to 10% for the 50 mg/kg subjects, 77 to 12% for the 1.00 mg/kg group, and from 84 to 42% for the 1.50 mg,kg training group. For the 0.50 and l.OOmg/kg groups, these values were indistinguishable from the 5-10% amphetamine-lever responding typically observed following saline injections indicating complete tolerance to the cue value of 0.35 mg/kg of d-amphetamine. The 1.5 mg/kg training group tested on 0.50 mg/kg demonstrate responding equivalent to that normally
Table 1. Comparison between mean number of responses + SE made on the d-amphetamine (dA) and saline (Sal) levers during 5 min extinction periods before and on Day 2 after chronic amphetamine Training group 0.5 mgjkg 1.Omg/kg 1.5 mg/kg
n 6 ::
Test dose (mg/kg) 0.35 0.35 0.50
Pre-chronic dA Sal lever dA lever 59 f 10 56 Jo 23 62 k 16
discriminative
25 f 18 44 f 37* 10 f 4
Post-chronic dA dA lever Sal lever 5*2 9*1 14 ?L:4
59 f 23 86 & 19 41 f 16
* One deviant rat which made 229 responses on the Sal lever and only 3 on the drug lever accounts for this high mean and SE.
254
R. J. BARRETT and NANCYJ. LEITH
found for 0.35 mg,kg. A repeated measures analysis of variance across the means for the one pre- and three post-chronic drug test sessions was significant for all three groups EF(3.15) = 2.89, 11.6 and 6.3, P < 0.01, for 0.51, 1.00 and iSOmg/kg groups in that order). Post analysis comparison of the individual means to assess the rate of recovery used the Neuman-Keuls test. These tests indicated that by Day 4 the 1.00 and 1.50 mg/kg subjects had recovered to pre-treatment discrimination levels while it was not until Day 6 that the 0.50 mg/kg group no longer showed tolerance. Another result of interest was that while the pattern of responding on the amphetamine and saline levers was essentially reversed on Day 2 following the chronic regimen, compared to pre-chronic values, the total number of responses made during the 5 min test sessions did not change significantly (t-test for correlated means, P > 0.10). These data are presented in
in describing and quan~f~ng tolerance to the cue properties of d-amphetamine. Furthermore, the attenuation of the cue properties of amphetamine as measured in the present paradigm would seem to reflect pharmac~~amic tolerance rather than “learned” tolerance. Although both processes deserve further study, examples of the former have been difficult to demonstrate in animal experiments studying chroni~lly administered amphetamine. Finally, if the discriminative stimulus properties of the drug are based on its euphoria or rewarding effects, then this paradigm might prove useful in understanding the physiolo~~l processes responsible for the rapid development of tolerance to the mood elevating or euphoric properties of amphetamine in man (Schick, Smith and Wesson, 1973).
Table 1.
Acknowledgements-The author acknowledges the expert technical assistance of MS Vicky Feinstein who was responsible for the data collection. This work was supported by the Veterans Administration and the Tennessee Department of Mental Health and Mental Retardation.
DISCUSSION
In the present experiment it was found that rats learned to discriminate 1.00 and 1.50 mg/kg damphetamine from saline more rapidly than when injected with 0,5Omg/kg of d-amphetamine when given training in a two-lever drug discrimination task. However, by the end of training the three groups were all discriminating equally well. The effect of different training doses was most pronoun~d on the dose-response functions generated for each of the three groups. These data, in agreement with a previous report (Miksic and Lal, 1977) showed that the drug dose required to maintain 50% responding on the drug lever during extinction testing was directly correlated with training dose. The drug-dose generalization functions also illustrate the importance of selecting a drug dose when testing for tolerance, from which small changes prior to chronic drug treatment can be shown to reliably produce corresponding changes in lever-choice behavior. For example, in the present experiment, it is unlikely that tolerance would have been demonstrated in either the 1.00 or 1SOml mg/kg groups had they been tested for tolerance on their respective training dose since the generalization data show that no significant decrement in percentage drug-lever responding occurred until training doses were reduced by 65-70x. The finding that percentage drug-lever responding to the test dose gradually returned to pre-chronic injections levels, in all three groups without further training, is consistent with a tolerance interpretation of the decrement in percentage drug-lever responding observed on Day 2. These data indicate that tolerance to the cue value of d-amphetamine dissipated within 5 or 6 days following termination of the chronic injection regimen. In conclusion, the present experiment demonstrates the usefufness of the discriminative stimulus paradigm
REFERENCES
Anderson, J. L., L&h, N. J. and Barrett, R. J. (1978). Tolerance to amphetamine’s facilitation of self-stimulation responding: Anatomical specificity. Brain Rex 145: 3748. Barry, H. (1974). Classification of drugs according to their discriminable effects in rats. Fe& Proc. Frdn Am. Sots ex.wp.&of. 33: 1814-1824. Burn, J. H. and Rand, M. J. (1958). The action of sympathomimetic amines in animals treated with reserpine. .!. Physiol. Lond. 144: 314-336. Glick, S. D, (1973). Impaired tolerance to the effects of oral amphetamine intake in rats with frontal cortex ablations. Psychopharmacology
28: 363-371.
Haris, J. W. E., Ambrus, C. M. and Ambrus, J. L. (1952). Tolerance of rats toward amphetamine and methamphetamine. .I. Am. Pharm. Ass. 41: 539-541. Harris, R. T. and Balster, R. L. (1968). Discriminative control by d, f-amphetamine and saline of lever choice and response patterning. Psychonom. Sri. 10: 105-106. Huang, J. and Ho, B. T. (1974). Discriminative stimulus properties of d-amphetamine and related compounds in rats. Pharmac. Biochem. Behav. 2: 669-672. Jones, C. N., Hill, H. F. and Harris, R. T. (1974). Discriminative response control by d-amphetamine and related compounds in the rat. Psychopharmacologia 36: 347-356.
Kuhn, D. M., Appel, J. B. and Greenberg, f. (1974). An analysis of some discriminative properties of d-amphetamine. Psychopharmacologia 39: 57-66. Leith, N. J. and Barrett, R. J. (1976). Amphetamine tolerance and post-drug depression. Psychopharmacotogia 46: 19-25.
Lewander, T. (1968). Urinary excretion and tissue levels of catecholamines during chronic amphetamine intoxication. Psychopharmacologia 13: 394-407. Lu, T. C., Clayhorn, J. L. and Schootar, J. C. (1973). Chronic administration of d-amphetamine and chlorpromazine in rats. Eur. J. Pharmac. 21: 61-65. McCown, T. and Barrett, R. J. (1980). The development of tolerance to the rewarding effects of self-administered $+)-amphetamine. Pharma’c. Biochem. Behav. 12: 137-141.
McKenna, M. and Ho. B. T. (1977). Induced tolerance to
Tolerance to amphetamine cues the discriminative stimulus properties of cocaine. Pharmat. Biochem. Behav. 7: 273-216. Miksic, S. and Lal, H. (1977). Tolerance to morphineinduced discriminative stimuli and analgesia. Psychopharmacology
54,2
17-22
I.
Schechter, M. D. (1978). Stimulus properties of d-amphetamine as compared to I-amphetamine. Eur. J. Pharmac. 41: 461464. Schechter, M. D. and Cook, P. G. (1975). Dopaminergic mediation of the interoceptive cue produced by d-amphetamine in rats. Psychopharmacologia 42: 185-193. Schechter, M. D. and Rosecrans, J. A. (1973). d-Amphet-
255
amine as a discriminative cue: Drugs with similar properties. Eur. .I. Pharmuc. 21: 212-216. Shick, J. F. E., Smith, D. E. and Wesson, D. R. (1973). An analysis of amphetamine toxicity and patterns of use. In : Uppers and Downers (Smith, D. E. and Wesson, D. R., Eds). Prentice-Hall, Englewood Cliffs, NJ. Schuster, C. R. and Zimmerman, J. (1961). Timing behavior during prolonged treatment with d/-amphetamine. J. Exp. Behav. 4: 327-330.
Schuster, C. R., Dockens, W. S. and Woods, J. H. (1966). Behavioral variables affecting the developments of amphetamine tolerance. Psychopharmacologia 9: 17tX182.
Previous studies with chronic administration of amphetamine have described two types of tolerance: (1) tolerance to the effects of amphetamine on physiological responses, such as hyperthermia (Harris, Ambrus and Ambrus, 1952), cardiovascular effects (Burn and Rand, 1958), changes in urinary excretion of catecholamines (Lewander, 1968) and its anorexigenic properties (Glick, 1973; Lu, Clayhorn and Schoolar, 1973); and (2) behavioral or “learned” tolerance which refers to a diminished drug effect on behavior reflecting the organism learning to accommodate or adapt to the drug condition (Schuster, Dockens and Wood, 1966). The latter develops only in situations where the drug action serves to disrupt the ability of the organism to meet the requirements of the task for reinforcement. For example, amphetamine has been shown to disrupt initially behavior in rats reinforced for correct temporal spacing of responding. However, daily administration of the drug given prior to each testing session results in a gradual return of responding to pre-drug baseline (Schuster and Zimmerman, 1961). Conversely, where the drug enhances the ability of the rat to meet the requirements for reinforcement, such as in active-avoidance tasks, responding does not return to the pre-drug baseline (Schuster et al., 1966). Recently, two experiments (Anderson, Leith and Barrett, 1978; Leith and Barrett, 1976) successfully demonstrated physiologically-mediated tolerance to the facilitation by amphetamine of responding for electrical self-stimulation of the brain and, in another experiment, (McCown and Barrett, 1980) tolerance to the reinforcing properties of d-amphetamine was demonstrated in an amphetamine self-administration * Supported by the Veterans Administration. Key words: discriminative stimulus, amphetamine, tolerance. 251
study with rats.. Both of these paradigms involve changes in response rate to assess tolerance develop ment. Since chronic administration of amphetamine can produce disruption of behavior due to general physical malaise, response-rate changes, especially decrements, can be difficult to interpret. The present experiment was designed to determine whether tolerance could be demonstrated in an operant two-lever drug discrimination paradigm. The use of d-amphetamine as a discriminative stimulus in a two-choice paradigm has been well documented in previous experiments (Harris and Balster, 1968; Huang and Ho, 1974; Jones, Hill and Harris, 1974; Kuhn, Appel and Greenberg, 1974; Schechter, 1978; Schechter and Cook, 1975; Schechter and Rosecrans, 1973). Also, other researchers have successfully used this rate-independent measure to show tolerance to the cue properties of cocaine (McKenna and Ho, 1977) and morphine (Miksic and Lal, 1977) following chronic administration. METHODS Subjects
Eighteen male F-344 rats, approx. 110 days old and weighing 230-25Og at the beginning of the experiment, were housed in individual cages and food deprived to 75 + 5% of their expected ad libitum weight. They were maintained on a 12-hr light-dark cycle (7:OOa.m.-7:OOp.m. light) and given enough food (Purina Lab Chow) immediately following tests sessions to maintain control weight throughout the experiment. The rats had free access to water at all times. Testing occurred at the same time each day, 5 days a week. On the days not tested the rats were given good sufficient to maintain 75% of their body weight. Injections of either d-amphetamine sulfate or
252
R. J.
BARRETT and NANCY
saline (0.9% sodium chloride) were administered intraperitoneally in volumes of 1 ml/kg body weight. Solutions of d-amphetamine sulfate were made by dissolving the drug in saline. All doses refer to amphetamine salt. Apparatus Three commercially available operant chambers (BRS/LVE Model No. RTC-022) each housed in a sound attenuating chamber were used for training rats on the discriminative stimulus task. On the front panel of each chamber two response levers were mounted 4.92cm above the floor and required a 24g force to activate. Reinforcements (Borden’s condensed milk diluted 1:I with water) were delivered by a liquid feeder (0.06ml) centered between the two levers. Electromechanical programming and recording equipment was located in an adjacent room. White noise was used in the experimental room to mask extraneous auditory stimuli. Procedure Preliminary training. Following gradual deprivation to 75% of ad Ii&turn body weight, the 18 rats were trained to lever press during daily 30 min sessions on a continuous schedule of reinforcement (CRF). They were given training on alternate days to each of the two levers. Acquisition of the d-amphetamine-saline discrimination. Once the lever press response was acquired, the reinforcement contingency was changed to a variable-interval 20 set (VI-20”) and discrimination training was initiated. Rats from each of three (n = 6) training-dose groups (0.50, 1.00, 1.50 mg/kg d-amphetamine sulfate) were injected intraperitoneally 1.5min prior to the start of daily 30min test sessions with either the drug or saline. For half the subjects in each group the right lever was designated the amphetamine-correct lever and the left lever the saline-correct one. This was reversed for the remaining half of the subjects. The first 2.5 min of each session consisted of an extinction period during which no lever presses were reinforced. This allowed for daily monitoring of discrimination acquisition unconfounded by reinforcement. During the remaining 27.5 min of the session, responses were reinforced on the correct lever with the exception that responses on the incorrect lever were punished by imposing a 20 set-delay before reinforcements were again available. Training was continued in this manner until the average percentage correct responses for each of the three groups was 85% or higher on both amphetamine and saline test days. Drug and saline injections were given on alternate days with the exception that, after every tenth test session, the sequence was reversed. This results in consecutive injections of either drug or saline once every 10 sessions and ensures that the rats are not responding to the temporal sequence of injection rather than to the drug-related cues. All discrimi-
J.
LEITH
nation training sessions were 30min long and were conducted 5 days per week (Monday to Friday). Generalization of the d-amphetamine cue to different doses of amphetamine. Once stable choice behavior was established, dose-response generalization functions were determined for the three groups during 5 min extinction sessions, i.e. no reinforcement was available to prevent the animal from receiving discrimination training to a new drug dose which might disrupt the original baseline discrimination. Immediately following the 5 min test session the rats were returned to their home cages and given their daily allotment of food. In addition to the training dose the four doses tested were: 0.10, 0.15,0.25, and 0.35 mg/kg for the 0.50mg/kg group; 0.25, 0.35, 0.50 and 0.75 mg/kg for the 1.0 mg/kg group; and 0.35, 0.50, 0.75 and 1.0 mg/kg for the 1.50 mg/kg training group. Order of testing was counterbalanced within each group and only one test dose was administered each week. Test days generally occurred on Wednesdays so that a drug and saline training day both preceded and followed each test session. Test for tolerance to the discriminative stimulus properties of amphetamine. Following completion of the dose-response function, testing was temporarily suspended and all subjects were injected chronically with d-amphetamine sulfate according to a schedule previously shown to produce tolerance to facilitation of responding by amphetamine for electrical brain stimulation (Leith and Barrett, 1976). The schedule consisted of three daily injections (8:00 a.m., 2:00 p.m., 8:OO p.m.) for 4 consecutive days. Following the first injection of 1 mg/kg, each subsequent injection was incremented by 1 mg/kg, so that the final injection on the fourth day was 12 mg/kg, totalling 78 mg/kg for the entire regimen. On days 2, 4 and 6 following the chronic injections all subjects were tested for tolerance during 5 min extinction sessions. The doses chosen to test for tolerance represented points on the dose-response curve where choice behavior had been shown to be sensitive to small changes in drug concentration. On days 3 and 5, regular saline training sessions were given. RESULTS
Acquisition of the d-amphetamine-saline discrimination Rats in all three groups received a total of 44 discrimination training sessions. A repeated measures analysis of variance on percentage of correct-lever responding during the 2)min extinction period at the beginning of each session indicated that although there were no significant differences between the three groups in percentage correct responses by the end of training, a significant training dose x days interaction [F(66,495) = 9.16, P < O.OOl] confirmed the expectation that the rate of acquiring the discrimination would be dose-related. Inspection of the acquistion data for the three groups clearly showed that the 1.0 and 1.5 mg/kg training groups, while not different
253
Tolerance to amphetamine cues
0.5mg/kg
d-AMPH
I.Omp
1
kg
1.5 mg/k@
d-AMPH
d-AMPH
I loo-•PM OPOS!
FL s
Chronic
d- AMPH oDAY
0
DAY6
6
4
60
k G P b
40-
/’
ODAY4
l
.
/ 2oi-
l
CDAYZ
o DAY2 18 0.100.15
I 0.25
, 0.36
! 0.50
II 0.25035
I 0.50
1 075
Dose of d-amphetamine
1 1.0
I,, 0.35(150
0.75
I 1.0
I I3
fmg/kg)
Fig. 1. Mean percentage drug-lever responding as a function of five test doses for each of three training dose groups. Data points labelfed Day 2, Day 4, and Day 6 represent the results of the three tests for tolerance following chronic administration of d-amphetamine.
from each other, were both superior to the 0.50 mg/kg group early in training. By the end of training all three groups consistently averaged between 8595% correct-lever responding on both saline and drug sessions during the 24 min test period.
Generalization of the d-amphetamine doses of amphetamine
cue to different
The mean percentage d-amphetamine-lever responding is presented in Figure 1 for the 5 min test sessions as a function of training dose and four test doses. As can be seen, subjects trained on 0.50 mg/kg d-amphetamine were extremely sensitive to small reductions from the training dose compared to subjects in either the 1.00 or 1.50 mg/kg training groups. The effect of the training dose on the dose-response curve is best illustrated by comparing estimates for each group of the dose resulting in 50% drug-lever responding (ED,,). The EDso values were estimated using a procedure {Barry, 1974) which involves interpolating between the log transformations of the two doses which bracket the 50% point. The resulting EDSo doses were 0.23, 0.30 and 0.37mg/kg for the 0.50, 1.00 and 1.50 mg,kg training groups.
Test for tolerance stimulus properties
to d-~phet~mine
A dose of 0.35 mg/kg of d-amphetamine was chosen to test for tolerance in the 0.50 and 1.00 mg/kg training groups while 0.50 mg,kg was used for testing the 1.50 mg/kg group. These doses produced comparable decrements in drug-lever responding of 8, 11 and 13% from training dose levels for the 0.5, 1.0 and 1.50mg/kg groups respectively (see Fig. 1) prior to chronic amphetamine treatment. In Figure 1 it can be seen that when tested on Day 2 following chronic amphetamine administration, subjects in all three groups showed tolerance to the cue value of the drug. The changes in percentage choice of the drug-lever from pre-chronic to the first post-chronic test session on Day 2 were 79 to 10% for the 50 mg/kg subjects, 77 to 12% for the 1.00 mg/kg group, and from 84 to 42% for the 1.50 mg,kg training group. For the 0.50 and l.OOmg/kg groups, these values were indistinguishable from the 5-10% amphetamine-lever responding typically observed following saline injections indicating complete tolerance to the cue value of 0.35 mg/kg of d-amphetamine. The 1.5 mg/kg training group tested on 0.50 mg/kg demonstrate responding equivalent to that normally
Table 1. Comparison between mean number of responses + SE made on the d-amphetamine (dA) and saline (Sal) levers during 5 min extinction periods before and on Day 2 after chronic amphetamine Training group 0.5 mgjkg 1.Omg/kg 1.5 mg/kg
n 6 ::
Test dose (mg/kg) 0.35 0.35 0.50
Pre-chronic dA Sal lever dA lever 59 f 10 56 Jo 23 62 k 16
discriminative
25 f 18 44 f 37* 10 f 4
Post-chronic dA dA lever Sal lever 5*2 9*1 14 ?L:4
59 f 23 86 & 19 41 f 16
* One deviant rat which made 229 responses on the Sal lever and only 3 on the drug lever accounts for this high mean and SE.
254
R. J. BARRETT and NANCYJ. LEITH
found for 0.35 mg,kg. A repeated measures analysis of variance across the means for the one pre- and three post-chronic drug test sessions was significant for all three groups EF(3.15) = 2.89, 11.6 and 6.3, P < 0.01, for 0.51, 1.00 and iSOmg/kg groups in that order). Post analysis comparison of the individual means to assess the rate of recovery used the Neuman-Keuls test. These tests indicated that by Day 4 the 1.00 and 1.50 mg/kg subjects had recovered to pre-treatment discrimination levels while it was not until Day 6 that the 0.50 mg/kg group no longer showed tolerance. Another result of interest was that while the pattern of responding on the amphetamine and saline levers was essentially reversed on Day 2 following the chronic regimen, compared to pre-chronic values, the total number of responses made during the 5 min test sessions did not change significantly (t-test for correlated means, P > 0.10). These data are presented in
in describing and quan~f~ng tolerance to the cue properties of d-amphetamine. Furthermore, the attenuation of the cue properties of amphetamine as measured in the present paradigm would seem to reflect pharmac~~amic tolerance rather than “learned” tolerance. Although both processes deserve further study, examples of the former have been difficult to demonstrate in animal experiments studying chroni~lly administered amphetamine. Finally, if the discriminative stimulus properties of the drug are based on its euphoria or rewarding effects, then this paradigm might prove useful in understanding the physiolo~~l processes responsible for the rapid development of tolerance to the mood elevating or euphoric properties of amphetamine in man (Schick, Smith and Wesson, 1973).
Table 1.
Acknowledgements-The author acknowledges the expert technical assistance of MS Vicky Feinstein who was responsible for the data collection. This work was supported by the Veterans Administration and the Tennessee Department of Mental Health and Mental Retardation.
DISCUSSION
In the present experiment it was found that rats learned to discriminate 1.00 and 1.50 mg/kg damphetamine from saline more rapidly than when injected with 0,5Omg/kg of d-amphetamine when given training in a two-lever drug discrimination task. However, by the end of training the three groups were all discriminating equally well. The effect of different training doses was most pronoun~d on the dose-response functions generated for each of the three groups. These data, in agreement with a previous report (Miksic and Lal, 1977) showed that the drug dose required to maintain 50% responding on the drug lever during extinction testing was directly correlated with training dose. The drug-dose generalization functions also illustrate the importance of selecting a drug dose when testing for tolerance, from which small changes prior to chronic drug treatment can be shown to reliably produce corresponding changes in lever-choice behavior. For example, in the present experiment, it is unlikely that tolerance would have been demonstrated in either the 1.00 or 1SOml mg/kg groups had they been tested for tolerance on their respective training dose since the generalization data show that no significant decrement in percentage drug-lever responding occurred until training doses were reduced by 65-70x. The finding that percentage drug-lever responding to the test dose gradually returned to pre-chronic injections levels, in all three groups without further training, is consistent with a tolerance interpretation of the decrement in percentage drug-lever responding observed on Day 2. These data indicate that tolerance to the cue value of d-amphetamine dissipated within 5 or 6 days following termination of the chronic injection regimen. In conclusion, the present experiment demonstrates the usefufness of the discriminative stimulus paradigm
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