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Reverse tolerance to stimulant-induced abnormal behavior
Life Sciences Vol . 20, pp . 1063-1076, 1977 . Printed in the U .S .A .
Pergamon Prese
REVERSE TOLERANCE TO STIMULANT-INDUCED ABNORMAL BEHAVIOR M . Marlyne Kilbey and Everett H . Ellinwood, Jr . Duke University Medical Center Box 3870 Durham, North Carolina 27710
(Received in final form February 14, 1977)
Changes in the behavior of female rats induced by repeated, daily administrations of d-amphetamine or cocaine were determined in two experiments . Potentia tion of stereotyped behavior in the initial post-injection period, which suggests a decrease in the latency to reach maximum stereotyped behavior, was found during and following daily treatment with both drugs . In addition, the maximum level of stereotyped behavior was increased during and following treatment with cocaine . In two experiments, behavior induced by apomorphine was potentiated during the initial post-injection period following a course of repeated, daily administrations of d-amphetamine or cocaine, suggesting the development of supersensitive dopaminergic post-synaptic mechanisms . Repeated administrations of stimulant drugs have been reported to result in tolerance, reverse tolerance, or no change occurring in the pattern of stereotyped behavior associated with the drug (1,2 ;3,4,5,6,7) . The discrepancies may be due, in part, to the use of a variety of behaviors to represent the drug state and a variety of methods to quantify the behaviors . Stimulant-induced abnormal behavior (SIAB) refers to repetitive patterns of locomotion, rearing, sniffing, gnawing, or biting . Generally, mechanical measures of activity or observational rating measures have been used to evaluate SIAB . Few of the rating scales have been shown to yield significantly different scores as a function of dose, and photocell activity measures are sensitive only to the hyperactivity forms of SIAB while missing the in-place, repetitious behavior forms characteristic of intense stereotyped responses (2) . However, a unidimensional rating scale of nine behavioral items ranked in order of progression from normal sleep, through hyperactivity (rated 5) and stereotyped behaviors, to dyskinetic-reactive behaviors (rated 9), has been described (8) . Using this scale, the intensity of abnormal behavior has been found to vary significantly as a function of d-amphetamine or cocaine dose (8,9) . Recently, Kilbey and Ellinwood (submitted for publication) have shown that while age-related factors contribute significantly to the duration of cocaine-induced stereotypies as measured by the rating method of Ellinwood and Balster (8), onset and intensity of 1063
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maximum stereotypy are not influenced by age-related factors . Thus, these two parameters may be evaluated in a within-S design to determine the influence of chronic treatment on SIAB in rats and subsequent retests or challenges with dopaminergic agonists . These measures, therefore, were used to determine the effects of d-amphetamine or cocaine administered daily over a two-week period, as well as the effects of readministration following a drugfree period . d-Amphetamine was chosen in order to attempt replication of response augmentation (2,3) using parameters not sensitive to age effects and to evaluate the phenomena in a different species . Cocaine was selected on the basis of work indicating that its behavioral and pharmacological actions may differ from those of d-amphetamine (11) . In order to test the possibility of dopamine receptor alteration following chronic administration of d-amphetamine and cocaine, apomorphine, which has been shown to have direct dopaminergic receptor agonist properties (12), was administered after a drug-free period . Methods The subjects were female Sprague-Dawley rats purchased from Zivic Miller, Pittsburgh, PA, weighing approximately 120-130 grams at the inception of the study . The rats were isolated in a pri vate temperature-controlled room and housed in plastic cages, 40x2Ox2Ocm with food and water ad lib . A twelve hour light-dark cycle was maintained . Rats wereweighed every two or three days throughout an experiment . Cocaine hydrochloride (40 mg/kg), damphetamine sulfate (7 mg/kg), and apomorphine hydrochloride (1 or 3 mg/kg) were administered i .p . as aqueous solutions in saline at a volume of 1 ml/kg . Dosages to induce SIAB were chosen on the basis of prior work of this laboratory (8,9) . The dosage of apomorphine was chosen on the basis of work showing a dose-dependent increase in stereotyped behavior using i .p . doses of drug from 0 .5 to 4 .0 mg/kg (13) . Four experiments were completed . The numbers of subjects used and the treatment protocol are presented in Table I . In each experiment, saline was administered daily during the 14-day interval preceding the post-treatment test . In all experiments, behavior ratings were made in the early afternoon, midway through the light cycle, according to a method described by Ellinwood and Balster (8) by a single observer who was not informed of the experimental treatments . Data were analyzed for significance of main effects using a 2-way ANOVA for repeated measures (14) . When these were significant, specific time periods were compared for a difference over days using total stereotypy scores ; i .e ., the sum of the behavior ratings for a specific period, using a 1-way ANOVA for repeated measures (14) . When the day effect in this analysis reached significance, a Dunnett's t procedure (14) was used to compare the ratings on the initial treatment day with those on later days . In Experiment 2, comparisons were made between independent groups using similar procedures . Experiment 1 . The data are presented in Figure 1 . The standard deviation or the points graphed ranged between 0 .0 and 1 .79 . Amphetamine-induced stereotyped behavior as a function of time consists of three periods : an onset period, during which the intensity of stereotypy is increasing ; a period of maximum level SIAB ; and an offset period, during which the intensity of the response is decreasing . Analysis of the data shows that the maximum
Vol . 20, No . 6, 1977
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level of stereotypy after 7 mg/kg d-amphetamine is not increased by lengthening the duration of treatment . However the rapidity with which this level is reached does change and there appears to be a faster onset of maximum level stereotyped behavior with repeated drug administrations . When Ss were readministered damphetamine following four days of saline administration, the more rapid onset of maximum stereotypy was still evident . The latency at which maximum stereotypy was reached was 31 .2 t 7 .9 minutes post-injection on day one . This was 8 .6 t 1 .9 and 6 .3 t 2 .5 on day 14 and . 5 days post-treatment, respectively . These changes in the temporal pattern of stereotyped behavior with repeated drug administrations resulted in statistically significant day, time, and day x time factors in an ANOVA for repeated measures (p _~ 0 .01, respectively) . Individual comparisons of the total stereotypy score affirmed that a faster onset of stereotyped behavior (Table II) is the major change in temporal characteristics of SIAB over the ex perimental period . During the first hour post-injection, total stereotypy scores were greater on day 14 of treatment and five days following cessation of daily treatment than they were follow-
d-AMPHETAMINE
Day 1 --- Day 14 Day 5 Post-Treatment
10 [
PRE-INJ.
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40 60
75 90 1 120 150 180 210 240 280 300
TIME (Minutes)
FIG . 1 Mean behavior rating on days 1 and 14 of daily treatment with 7 mg/kg d-amphetamine (i .p .) and the fifth post-treatment day . n-9 .
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ing initial treatment . For the period between 120-300 minutes post-injection, i .e ., the offset period, only the behavior recorded on the fifth post-treatment day was significantly more stereotyped than thât initially observed . Thus, these data indicate that daily treatment with a constant dose of d-amphetamine results in significant augmentation of the stereotypy score in the initial onset period both during and following the treatment period . TABLE II F Value and Probability Levels Obtained by Analysis of Rated Amphetamine-Induced Abnormal Behavior
ANOVA (1-way, 1 repeated measure) Total Stereotypy Score : 3 to 60 min . post-injection Day Dunnett's t :
F Value, Degrees of Freedom
Probability Level of F
1`1 .31 (2,8)
0 .01
Day 1 vs 14, t = 4 .1 . p s 0 .005 ; Day 1 vs SPT, t- 4 .0, p < 0 .05
Total Stereotypy Score : 120 to 300 min . post-injection Day Dunnett's t :
7 .11
(2,8)
0 .01
Day 1 vs 14, t - 0 .7 n .s . ; Day 1 vs 5PT, t 2 .8, p : 0 .001
Experiment 2 . The data are presented in Figure 2 . The s .d . of the data points graphed ranged between 0 .0 and 2 .2 . The results Stereoof the statistical analyses are presented in Table III . typed behavior induced by 1 or 3 mg/kg apomorphine did not vary as a function of 14 days' prior treatment with saline or d-amphetamine, nor did this treatment significantly affect the temporal pattern of stereotyped behavior induced by 1 mg/kg apomorphine, although the mean latency to maximum stereotypy in the saline pretreated group was 9 .8 t 9 .7 minutes in contrast with a mean latency of 6 .6 t 2 .9 minutes in the d-amphetamine pretreated group . The significant treatment x time interaction factor for the 3 mg/kg apomorphine group indicates that five days after cessation of daily treatment the time course of apomorphine-induced stereotyped behavior for Ss that previously had been administered amphetamine was significantly different from that of Ss pretreated with saline . As indicated in Figure 2, Ss pretreated with d-amphetamine showed maximum stereotyped behavior at an average latency of 3 .6 minutes post-injection t 2 .0 minutes when tested with 3 mg/kg apomorphine, while latency in the saline pretreated Ss was 6 .0 t 2 .1 minutes post-injection .
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Reverse Tolerance/Stimulant-Induced Bebavior
106 9
TABLE III F Value and Probability Levels Obtained by Analyses of Ratings of Apomorphine-Induced Stereotyped Behavior, as a Function of Dose, in Rats Treated for 14 Days with Saline or d-Amphetamine
ANOVA (2 way, 1 repeated measure) F Value, Degrees of Freedom
Factor Pretreatment Condition (P)
Probability Level of F
1 mg/kg Apomorphine Test 0 .8 (1, 16)
Time (T) P x T
n .s .
36 .7 (14,
224)
0 .01
0 .4 (14,
224)
n .s .
3 mg/kg Apomorphine Test Pretreatment Condition (P) Time
0 .8 (1, 16)
CrI
P x T
n .s .
80 .3 (14, 224)
0 .01
3 .6 (14, 224)
0 .01
TABLE IV F Value and Probability Levels Obtained by Analyses of Ratings of Apomorphine-Induced Stereotyped Behavior Summed for Specific Time Intervals in Rats Treated for 14 Days with Saline or d-Amphetamine ANOVA (1 way, 0 repeated measures) F Value, Degrees of Freedom
Factor Total Stereotypy Score
Probability Level of F
3 mg/kg Apomorphine Test
3-15 min . Post-injection 20-45 min . Post-injection 60-90 min . Post-injection
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0 .01
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Vol . 20, No . 6, 1977
cant day, time, and day x time effects (p s 0 .01) . Analysis of the total stereotypy score (Table V) for the three major periods showed that stereotyped activity was intensified in the onset and maximum level periods as a function of day of treatment or posttreatment while stereotyped behavior during the offset period did not change . Individual comparisons of the total stereotypy score for the initial period and the period of maximum stereotypy demonstrated that treatment day 13 and post-treatment day 5 scores were significantly higher than those obtained on the initial treatment day over these periods (Table V) . These data indicate that an index of reverse tolerance seen during chronic treatment with d-amphetamine (i .e ., augmentation of stereotypy scores during the initial onset period) is seen with chronic cocaine treatment . However, with the dose level of cocaine used in this study, augmentation of maximum level of stereotypy occurs, also . On the fifth day following cessation of daily treatment, these indices of reverse tolerance are still present .
COCAINE
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60
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FIG . 3 Mean behavior ràting on days 1 and 13 during 14 daily treatments with 40 mg/kg cocaine (i .p .) and on the fifth post-treatment day . n - 11 .
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TABLE V F Value and Probability Level for Behavior Ratings Summed over Specific Durations Post-Injection as a Function of Treatment- or Post-Treatment Day
ANOVA (1-way, 1 repeated measure) Total Stereotypy Score : 3-15 min . post-injection
34 .0 (2,20)
Day Dunnett's t :
Probability Level of F 0 .01
Day 1 vs Day 13, t - 4 .3, p S 0 .005 Day 1 vs Day 5PT, t - 4 .8, p s 0 .005
Total Stereotypy Score : 20-45 min . post-injection Dunnett's t :
F Value Degrees of Freedom
10 .9 (2,20)
0 .01
Day 1 vs Day 13, t - 3 .5, p s 0 .005 Day 1 vs Day 5PT,, t - 3 .5, p S 0 .005
Total Stereotypy Score : 60-150 min . post-injection
0 .34 (2,20)
n.s .
Experiment 4 . The data for the cocaine treated Ss are presented in Figure The s .d . of the points graphed ranged between 0 .0 and 2 .7 . Analysis of the data shows that treatment with cocaine for 14 days results in a significantly augmented response to apomorphine (p _< 0 .01) . The time course of the response is not changed significantly, however, as the treatment by time effect is not significant (p > 0 .01) . In contrast, treatment with saline for 14 days resulted in no significant changes in the response to apomorphine . Analysis of the three periods within the cocaine treated group indicated that augmentation of the response during the onset period was the principle change resulting from chronic treatment (Table VI) . The latency of onset of maximum apomorphine -induced stereotypy pre- and post-cocaine treatment was 7 .6 ± 3 .5 and 5 .0 t 2 .5 minutes, respectively . In summary, the data of this experiment show that one aspect of the reverse tolerance seen with chronic stimulant administration, augmentation of the initial phase of the stereotyped response, is obtained by administration of apomorphine, a dopamine receptor agonist .
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Vol . 20, No . 6, 1977
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TABLE VI F Values and Probability Levels for Summed Ratings of Apomorphine-Induced Behavior Pre- or Post-Treatment With Cocaine as a Function of Specific Intervals Post-Injection
ANOVA (1 way, 1 repeated measure) F Value De grees of Freedom
Probability Level of F
3-15 min . Post-injection
6 .9 (1,8)
0 .01
20-40 min . Post-injection
1 .8 (1,8)
n .s .
45-90 Min . Post-injection
0 .8 (1,8)
n .s .
Cocaine Treated Pre- vs Post-
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Reverse Tolerance/Stianlant-Induced Behavior
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Discussion The major findings of the four studies reported are that daily stimulant administration results in augmentation of the stereotypy elicited by unit doses of d-amphetamine and cocaine and that characteristics of these augmented responses can be induced by a dopamineraic receptor agonist . Previous work (8,9) has shown that one characteristic of increases in dose of d-amnhetaAlthough mine or cocaine is increased intensity rating of SIAB . neither study compared the time of onset of maximum stereotypy, inspection of the figures included suggests that earlier onset The present work also may be a characteristic of increased dose . indicates that during and following daily treatment with 7 mg/kg d-amphetamine or 40 mg/kg cocaine, maximum stereotyped behavior occurs significantly faster . Possibly due to the use of a relatively high dose of d-amphetamine, the maximum stereotypy level for this drug did not vary during the experimental period . A smaller unit dose might, in fact, show augmentation of maximum level similar to that seen in experiment three with 40 mg/kg cocaine . Experiments two and four indicate that dopamine receptor supersensitivity may underlie at least one aspect (i .e ., augmentation of the response during the initial period) of the al tered stereotyped response seen during and following chronic treatment with .stimulants . Interestingly, Klawans and Margolin (3) present data in theft Table 1 which show that in guinea -pigs readministratión of d-am hetamine following cessation'of'daily : treatment resulted in a aster onset and an increase in intensity of stereotypy, while administration of apomorphine resulted in only a faster onset . Our findings using cocaine in rats are similar . Their results in conjunction with the data presented in this report may suggest that supersensitivity of dopamine receptors underlies, at east in part, the decrease in latency but probably does not account for all indices of reverse tolerance as the increase in maximum stereoty Y seen during chronic administration and readministration of simulants is not reproduced by apomorphine . Twice daily treatment with d-amphetamine for 28 days (6) results in significantly lower brain levels of dopamine when measured 24 hours post-treatment . Treatment daily for seven days with cocaine results in decreased levels of homovanillic acid (15) . Under similar conditions, tyrosine hydroxylase activity in the striatum and hypothalamus-thalamus increases significantly (15) . These data raise the possibility that the treatment parameters used in the studies of this report may have resulted in decreased dopamine activity at the post-synaptic receptor following the main drug effect, which could result, in turn, in supranormal response to subsequent drug-induced dopamine effects (16) . Suggestive as these data are, however, it remains to be shown whether or not biosynthesis of catecholamines, especially dopamine, is altered during and after treatment with psychomotor stimulants using the parameters employed in these studies . Furthermore, while studies implicate supersensitivity of the dopaminergic receptor in the intensification of the initial response to apomorphine following chronic treatment with stimulants, this does not, of course, preclude in any way the possibility that
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changes in relative amounts of metabolites or in drug absorption characteristics"following chronic treatment contribute to the altered stimulant-induced-behavior . Although our data show no alterations in the offset of drug effects which might be expected if metabolic processes are altered by chronic treatment, changes following chronic treatment in metabolites of d-amphetamine in the rat brain have been shown (17) and in metabolites of cocaine in the brain of dogs (18) . Once again, whether or not our treatment produces altered metabolic processes which persist for periods as long as the two months for which augmented behavioral responses have been shown (9) remains to be tested . Acknowledgements This work was supported by Grant number DA00057 from the National Institute on Drug Abuse . Parts of these data were presented at the Contemporary Issues in Stimulant Research Conference at Duke University, November 10-12, 1975 . The authors wish to thank Nancy Wagoner, who rated the behavior of the animals reported in this paper . The animals were cared for in accordance with Federal regulations and guidelines . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
12 . 13 . 14 . 15 .
16 .
L . MAGOS, Eur . J . Pharmacol . 6 200-201 (1969) . D . S . SEGA an ELL, Pharm. Biochem . Behav . 2 249-255 (1974) . H . L . KLAWANS and D . I . MARGOLIN, Arch . Gen . Psychiatry 32 725-732 (1975) . L . M . GUNNE and T . LEWANDER, Res . Pubs . Ass . Res . Nerv . Ment . Dis . 4 6 106-116 (1966) . T_._UEWANDER, Ps cho harmacolo is 13 394-407 (1968) . T . LEWANDER, aunyn- c mie e ergs Arch . Pharmak . 271 211-233 (1971) . T . C . LU, B . T . HO, and W . M. McISSAC, Experientia 28 1461 (1972) . E . H . ELLINWOOD, JR . and R . L . BALSTER, Eur . J . Pharmacol . 28 35-41 (1974) . M . M . KILBEY and E . H . ELLINWOOD, JR ., Cocaine and Other Stimulants (E . H . Ellinwood, Jr . and M . il ey, Editors), pp . 4097-W, Plenum Press, New York (1977) . E . SCHIORRING, Behaviour 39 1-17 (1971) . J . SCHEEL-KRUGET,_7C_ .'1`RUP, M . NIELSEN, K . GOLEMBIOWSKA, and E . MOGILNICKA, Cocaine and Other Stimúlants (E . H . Ellinwood, JY . and M. M. Kilbey, Editors pp . 373-408, Plenum Press, New York (1977) . N . E . ANDEN, A . RUBENSSON, F . FUXE, AND T . HOKFELT, J . Pharm . Pharmacol . 19 627-629 (1967) . T._KU9=SIZY, Life Sciences 17 43-48 (1975) . B . J . WINER, Statistical rince les in Experimental Desi n McGraw Hill, New York B . T . HO, D . L . TAYLOR, V . S . ESTEVEZ, L . F . ENGLERT, AND M . L . McKENNA, Cocaine and Other Stimulants (E . H . Ellinwood, Jr . and M. M . Ki ey, Editors) pp . Plenum Press, New York (1977) . S . Z . LANGER, Handbook of Ps cho harmacolo (L . L . Iversen, S . D . Iversen, andS . ny er, Editors) pp 245-280, Plenum Press, New York (1975) .
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Reverse Tolerance/Stimulant-Induced Behavior
C . M . KUHN and S . M . SCHANBERG, Cocaine (E . H . Ellinwood, Jr . and M . M . i ey, Plenum Press, New York (1977) . 18 . S . J . MULE and A . L . MISRA, Cocaine and H . Ellinwood and M . M . Kilbey, Editors) Press, New York (1977) . 17 .
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and Other Stimulants Editors) Pp .161-178, Other Stimulants (E . pp . 5-2 , enum
Pergamon Prese
REVERSE TOLERANCE TO STIMULANT-INDUCED ABNORMAL BEHAVIOR M . Marlyne Kilbey and Everett H . Ellinwood, Jr . Duke University Medical Center Box 3870 Durham, North Carolina 27710
(Received in final form February 14, 1977)
Changes in the behavior of female rats induced by repeated, daily administrations of d-amphetamine or cocaine were determined in two experiments . Potentia tion of stereotyped behavior in the initial post-injection period, which suggests a decrease in the latency to reach maximum stereotyped behavior, was found during and following daily treatment with both drugs . In addition, the maximum level of stereotyped behavior was increased during and following treatment with cocaine . In two experiments, behavior induced by apomorphine was potentiated during the initial post-injection period following a course of repeated, daily administrations of d-amphetamine or cocaine, suggesting the development of supersensitive dopaminergic post-synaptic mechanisms . Repeated administrations of stimulant drugs have been reported to result in tolerance, reverse tolerance, or no change occurring in the pattern of stereotyped behavior associated with the drug (1,2 ;3,4,5,6,7) . The discrepancies may be due, in part, to the use of a variety of behaviors to represent the drug state and a variety of methods to quantify the behaviors . Stimulant-induced abnormal behavior (SIAB) refers to repetitive patterns of locomotion, rearing, sniffing, gnawing, or biting . Generally, mechanical measures of activity or observational rating measures have been used to evaluate SIAB . Few of the rating scales have been shown to yield significantly different scores as a function of dose, and photocell activity measures are sensitive only to the hyperactivity forms of SIAB while missing the in-place, repetitious behavior forms characteristic of intense stereotyped responses (2) . However, a unidimensional rating scale of nine behavioral items ranked in order of progression from normal sleep, through hyperactivity (rated 5) and stereotyped behaviors, to dyskinetic-reactive behaviors (rated 9), has been described (8) . Using this scale, the intensity of abnormal behavior has been found to vary significantly as a function of d-amphetamine or cocaine dose (8,9) . Recently, Kilbey and Ellinwood (submitted for publication) have shown that while age-related factors contribute significantly to the duration of cocaine-induced stereotypies as measured by the rating method of Ellinwood and Balster (8), onset and intensity of 1063
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maximum stereotypy are not influenced by age-related factors . Thus, these two parameters may be evaluated in a within-S design to determine the influence of chronic treatment on SIAB in rats and subsequent retests or challenges with dopaminergic agonists . These measures, therefore, were used to determine the effects of d-amphetamine or cocaine administered daily over a two-week period, as well as the effects of readministration following a drugfree period . d-Amphetamine was chosen in order to attempt replication of response augmentation (2,3) using parameters not sensitive to age effects and to evaluate the phenomena in a different species . Cocaine was selected on the basis of work indicating that its behavioral and pharmacological actions may differ from those of d-amphetamine (11) . In order to test the possibility of dopamine receptor alteration following chronic administration of d-amphetamine and cocaine, apomorphine, which has been shown to have direct dopaminergic receptor agonist properties (12), was administered after a drug-free period . Methods The subjects were female Sprague-Dawley rats purchased from Zivic Miller, Pittsburgh, PA, weighing approximately 120-130 grams at the inception of the study . The rats were isolated in a pri vate temperature-controlled room and housed in plastic cages, 40x2Ox2Ocm with food and water ad lib . A twelve hour light-dark cycle was maintained . Rats wereweighed every two or three days throughout an experiment . Cocaine hydrochloride (40 mg/kg), damphetamine sulfate (7 mg/kg), and apomorphine hydrochloride (1 or 3 mg/kg) were administered i .p . as aqueous solutions in saline at a volume of 1 ml/kg . Dosages to induce SIAB were chosen on the basis of prior work of this laboratory (8,9) . The dosage of apomorphine was chosen on the basis of work showing a dose-dependent increase in stereotyped behavior using i .p . doses of drug from 0 .5 to 4 .0 mg/kg (13) . Four experiments were completed . The numbers of subjects used and the treatment protocol are presented in Table I . In each experiment, saline was administered daily during the 14-day interval preceding the post-treatment test . In all experiments, behavior ratings were made in the early afternoon, midway through the light cycle, according to a method described by Ellinwood and Balster (8) by a single observer who was not informed of the experimental treatments . Data were analyzed for significance of main effects using a 2-way ANOVA for repeated measures (14) . When these were significant, specific time periods were compared for a difference over days using total stereotypy scores ; i .e ., the sum of the behavior ratings for a specific period, using a 1-way ANOVA for repeated measures (14) . When the day effect in this analysis reached significance, a Dunnett's t procedure (14) was used to compare the ratings on the initial treatment day with those on later days . In Experiment 2, comparisons were made between independent groups using similar procedures . Experiment 1 . The data are presented in Figure 1 . The standard deviation or the points graphed ranged between 0 .0 and 1 .79 . Amphetamine-induced stereotyped behavior as a function of time consists of three periods : an onset period, during which the intensity of stereotypy is increasing ; a period of maximum level SIAB ; and an offset period, during which the intensity of the response is decreasing . Analysis of the data shows that the maximum
Vol . 20, No . 6, 1977
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Reverse Tolerance/Stimulant-Induced Behavior
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Vol . 20, No . 6, 1977
level of stereotypy after 7 mg/kg d-amphetamine is not increased by lengthening the duration of treatment . However the rapidity with which this level is reached does change and there appears to be a faster onset of maximum level stereotyped behavior with repeated drug administrations . When Ss were readministered damphetamine following four days of saline administration, the more rapid onset of maximum stereotypy was still evident . The latency at which maximum stereotypy was reached was 31 .2 t 7 .9 minutes post-injection on day one . This was 8 .6 t 1 .9 and 6 .3 t 2 .5 on day 14 and . 5 days post-treatment, respectively . These changes in the temporal pattern of stereotyped behavior with repeated drug administrations resulted in statistically significant day, time, and day x time factors in an ANOVA for repeated measures (p _~ 0 .01, respectively) . Individual comparisons of the total stereotypy score affirmed that a faster onset of stereotyped behavior (Table II) is the major change in temporal characteristics of SIAB over the ex perimental period . During the first hour post-injection, total stereotypy scores were greater on day 14 of treatment and five days following cessation of daily treatment than they were follow-
d-AMPHETAMINE
Day 1 --- Day 14 Day 5 Post-Treatment
10 [
PRE-INJ.
0
0 8 z 7 oc 6 0
â
W
5 4
m z 3 Q W
2 1 u
n
13
n
n
n
6
9
12
1
r
n
15 20
n
30
n
1
m
40 60
75 90 1 120 150 180 210 240 280 300
TIME (Minutes)
FIG . 1 Mean behavior rating on days 1 and 14 of daily treatment with 7 mg/kg d-amphetamine (i .p .) and the fifth post-treatment day . n-9 .
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Reverse Tolerance/Stimulant-Induced Behavior
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ing initial treatment . For the period between 120-300 minutes post-injection, i .e ., the offset period, only the behavior recorded on the fifth post-treatment day was significantly more stereotyped than thât initially observed . Thus, these data indicate that daily treatment with a constant dose of d-amphetamine results in significant augmentation of the stereotypy score in the initial onset period both during and following the treatment period . TABLE II F Value and Probability Levels Obtained by Analysis of Rated Amphetamine-Induced Abnormal Behavior
ANOVA (1-way, 1 repeated measure) Total Stereotypy Score : 3 to 60 min . post-injection Day Dunnett's t :
F Value, Degrees of Freedom
Probability Level of F
1`1 .31 (2,8)
0 .01
Day 1 vs 14, t = 4 .1 . p s 0 .005 ; Day 1 vs SPT, t- 4 .0, p < 0 .05
Total Stereotypy Score : 120 to 300 min . post-injection Day Dunnett's t :
7 .11
(2,8)
0 .01
Day 1 vs 14, t - 0 .7 n .s . ; Day 1 vs 5PT, t 2 .8, p : 0 .001
Experiment 2 . The data are presented in Figure 2 . The s .d . of the data points graphed ranged between 0 .0 and 2 .2 . The results Stereoof the statistical analyses are presented in Table III . typed behavior induced by 1 or 3 mg/kg apomorphine did not vary as a function of 14 days' prior treatment with saline or d-amphetamine, nor did this treatment significantly affect the temporal pattern of stereotyped behavior induced by 1 mg/kg apomorphine, although the mean latency to maximum stereotypy in the saline pretreated group was 9 .8 t 9 .7 minutes in contrast with a mean latency of 6 .6 t 2 .9 minutes in the d-amphetamine pretreated group . The significant treatment x time interaction factor for the 3 mg/kg apomorphine group indicates that five days after cessation of daily treatment the time course of apomorphine-induced stereotyped behavior for Ss that previously had been administered amphetamine was significantly different from that of Ss pretreated with saline . As indicated in Figure 2, Ss pretreated with d-amphetamine showed maximum stereotyped behavior at an average latency of 3 .6 minutes post-injection t 2 .0 minutes when tested with 3 mg/kg apomorphine, while latency in the saline pretreated Ss was 6 .0 t 2 .1 minutes post-injection .
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1 mg/kg APOMORPHM ~" SO/km PhNreoled
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Reverse Tolerance/Stimulant-Induced Bebavior
106 9
TABLE III F Value and Probability Levels Obtained by Analyses of Ratings of Apomorphine-Induced Stereotyped Behavior, as a Function of Dose, in Rats Treated for 14 Days with Saline or d-Amphetamine
ANOVA (2 way, 1 repeated measure) F Value, Degrees of Freedom
Factor Pretreatment Condition (P)
Probability Level of F
1 mg/kg Apomorphine Test 0 .8 (1, 16)
Time (T) P x T
n .s .
36 .7 (14,
224)
0 .01
0 .4 (14,
224)
n .s .
3 mg/kg Apomorphine Test Pretreatment Condition (P) Time
0 .8 (1, 16)
CrI
P x T
n .s .
80 .3 (14, 224)
0 .01
3 .6 (14, 224)
0 .01
TABLE IV F Value and Probability Levels Obtained by Analyses of Ratings of Apomorphine-Induced Stereotyped Behavior Summed for Specific Time Intervals in Rats Treated for 14 Days with Saline or d-Amphetamine ANOVA (1 way, 0 repeated measures) F Value, Degrees of Freedom
Factor Total Stereotypy Score
Probability Level of F
3 mg/kg Apomorphine Test
3-15 min . Post-injection 20-45 min . Post-injection 60-90 min . Post-injection
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0 .01
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n .s
Fl r 16 ' 0 .2
n .s .
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Vol . 20, No . 6, 1977
cant day, time, and day x time effects (p s 0 .01) . Analysis of the total stereotypy score (Table V) for the three major periods showed that stereotyped activity was intensified in the onset and maximum level periods as a function of day of treatment or posttreatment while stereotyped behavior during the offset period did not change . Individual comparisons of the total stereotypy score for the initial period and the period of maximum stereotypy demonstrated that treatment day 13 and post-treatment day 5 scores were significantly higher than those obtained on the initial treatment day over these periods (Table V) . These data indicate that an index of reverse tolerance seen during chronic treatment with d-amphetamine (i .e ., augmentation of stereotypy scores during the initial onset period) is seen with chronic cocaine treatment . However, with the dose level of cocaine used in this study, augmentation of maximum level of stereotypy occurs, also . On the fifth day following cessation of daily treatment, these indices of reverse tolerance are still present .
COCAINE
Day 1 Day 13 --- Day S Pbst-Treatment ------
PRE- INJ 0 0 13
0c
Ó
âx
6 5 4
Z 3 Q 2
a
1
e
0
PRE-101 3
6
9
12
1520
25 30
35 40 45 (Minutes) TIME
60
75
90 105 120 135 150
FIG . 3 Mean behavior ràting on days 1 and 13 during 14 daily treatments with 40 mg/kg cocaine (i .p .) and on the fifth post-treatment day . n - 11 .
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Reverse Tolerance/Stimulant-Induced Behavior
107 1
TABLE V F Value and Probability Level for Behavior Ratings Summed over Specific Durations Post-Injection as a Function of Treatment- or Post-Treatment Day
ANOVA (1-way, 1 repeated measure) Total Stereotypy Score : 3-15 min . post-injection
34 .0 (2,20)
Day Dunnett's t :
Probability Level of F 0 .01
Day 1 vs Day 13, t - 4 .3, p S 0 .005 Day 1 vs Day 5PT, t - 4 .8, p s 0 .005
Total Stereotypy Score : 20-45 min . post-injection Dunnett's t :
F Value Degrees of Freedom
10 .9 (2,20)
0 .01
Day 1 vs Day 13, t - 3 .5, p s 0 .005 Day 1 vs Day 5PT,, t - 3 .5, p S 0 .005
Total Stereotypy Score : 60-150 min . post-injection
0 .34 (2,20)
n.s .
Experiment 4 . The data for the cocaine treated Ss are presented in Figure The s .d . of the points graphed ranged between 0 .0 and 2 .7 . Analysis of the data shows that treatment with cocaine for 14 days results in a significantly augmented response to apomorphine (p _< 0 .01) . The time course of the response is not changed significantly, however, as the treatment by time effect is not significant (p > 0 .01) . In contrast, treatment with saline for 14 days resulted in no significant changes in the response to apomorphine . Analysis of the three periods within the cocaine treated group indicated that augmentation of the response during the onset period was the principle change resulting from chronic treatment (Table VI) . The latency of onset of maximum apomorphine -induced stereotypy pre- and post-cocaine treatment was 7 .6 ± 3 .5 and 5 .0 t 2 .5 minutes, respectively . In summary, the data of this experiment show that one aspect of the reverse tolerance seen with chronic stimulant administration, augmentation of the initial phase of the stereotyped response, is obtained by administration of apomorphine, a dopamine receptor agonist .
Reverse Tolerance/Stimulant-Induced Behavior
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Vol . 20, No . 6, 1977
10 9 f- 8 Q oc 7 Ó 6
m
s
4
z 3 a W
2
1
O 1-L74 .
30 3s âo âs
6
7s
.J
90
TIME (Minuta)
FIG . 4 9) Mean behavior rating to 3 mg/kg apomorphine (i .p .) pre- (9 and post-treatment (o----o) with 40 mg/kg cocaine for 14 days and n - 9. saline for an additional four days .
TABLE VI F Values and Probability Levels for Summed Ratings of Apomorphine-Induced Behavior Pre- or Post-Treatment With Cocaine as a Function of Specific Intervals Post-Injection
ANOVA (1 way, 1 repeated measure) F Value De grees of Freedom
Probability Level of F
3-15 min . Post-injection
6 .9 (1,8)
0 .01
20-40 min . Post-injection
1 .8 (1,8)
n .s .
45-90 Min . Post-injection
0 .8 (1,8)
n .s .
Cocaine Treated Pre- vs Post-
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Reverse Tolerance/Stianlant-Induced Behavior
107 3
Discussion The major findings of the four studies reported are that daily stimulant administration results in augmentation of the stereotypy elicited by unit doses of d-amphetamine and cocaine and that characteristics of these augmented responses can be induced by a dopamineraic receptor agonist . Previous work (8,9) has shown that one characteristic of increases in dose of d-amnhetaAlthough mine or cocaine is increased intensity rating of SIAB . neither study compared the time of onset of maximum stereotypy, inspection of the figures included suggests that earlier onset The present work also may be a characteristic of increased dose . indicates that during and following daily treatment with 7 mg/kg d-amphetamine or 40 mg/kg cocaine, maximum stereotyped behavior occurs significantly faster . Possibly due to the use of a relatively high dose of d-amphetamine, the maximum stereotypy level for this drug did not vary during the experimental period . A smaller unit dose might, in fact, show augmentation of maximum level similar to that seen in experiment three with 40 mg/kg cocaine . Experiments two and four indicate that dopamine receptor supersensitivity may underlie at least one aspect (i .e ., augmentation of the response during the initial period) of the al tered stereotyped response seen during and following chronic treatment with .stimulants . Interestingly, Klawans and Margolin (3) present data in theft Table 1 which show that in guinea -pigs readministratión of d-am hetamine following cessation'of'daily : treatment resulted in a aster onset and an increase in intensity of stereotypy, while administration of apomorphine resulted in only a faster onset . Our findings using cocaine in rats are similar . Their results in conjunction with the data presented in this report may suggest that supersensitivity of dopamine receptors underlies, at east in part, the decrease in latency but probably does not account for all indices of reverse tolerance as the increase in maximum stereoty Y seen during chronic administration and readministration of simulants is not reproduced by apomorphine . Twice daily treatment with d-amphetamine for 28 days (6) results in significantly lower brain levels of dopamine when measured 24 hours post-treatment . Treatment daily for seven days with cocaine results in decreased levels of homovanillic acid (15) . Under similar conditions, tyrosine hydroxylase activity in the striatum and hypothalamus-thalamus increases significantly (15) . These data raise the possibility that the treatment parameters used in the studies of this report may have resulted in decreased dopamine activity at the post-synaptic receptor following the main drug effect, which could result, in turn, in supranormal response to subsequent drug-induced dopamine effects (16) . Suggestive as these data are, however, it remains to be shown whether or not biosynthesis of catecholamines, especially dopamine, is altered during and after treatment with psychomotor stimulants using the parameters employed in these studies . Furthermore, while studies implicate supersensitivity of the dopaminergic receptor in the intensification of the initial response to apomorphine following chronic treatment with stimulants, this does not, of course, preclude in any way the possibility that
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changes in relative amounts of metabolites or in drug absorption characteristics"following chronic treatment contribute to the altered stimulant-induced-behavior . Although our data show no alterations in the offset of drug effects which might be expected if metabolic processes are altered by chronic treatment, changes following chronic treatment in metabolites of d-amphetamine in the rat brain have been shown (17) and in metabolites of cocaine in the brain of dogs (18) . Once again, whether or not our treatment produces altered metabolic processes which persist for periods as long as the two months for which augmented behavioral responses have been shown (9) remains to be tested . Acknowledgements This work was supported by Grant number DA00057 from the National Institute on Drug Abuse . Parts of these data were presented at the Contemporary Issues in Stimulant Research Conference at Duke University, November 10-12, 1975 . The authors wish to thank Nancy Wagoner, who rated the behavior of the animals reported in this paper . The animals were cared for in accordance with Federal regulations and guidelines . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
12 . 13 . 14 . 15 .
16 .
L . MAGOS, Eur . J . Pharmacol . 6 200-201 (1969) . D . S . SEGA an ELL, Pharm. Biochem . Behav . 2 249-255 (1974) . H . L . KLAWANS and D . I . MARGOLIN, Arch . Gen . Psychiatry 32 725-732 (1975) . L . M . GUNNE and T . LEWANDER, Res . Pubs . Ass . Res . Nerv . Ment . Dis . 4 6 106-116 (1966) . T_._UEWANDER, Ps cho harmacolo is 13 394-407 (1968) . T . LEWANDER, aunyn- c mie e ergs Arch . Pharmak . 271 211-233 (1971) . T . C . LU, B . T . HO, and W . M. McISSAC, Experientia 28 1461 (1972) . E . H . ELLINWOOD, JR . and R . L . BALSTER, Eur . J . Pharmacol . 28 35-41 (1974) . M . M . KILBEY and E . H . ELLINWOOD, JR ., Cocaine and Other Stimulants (E . H . Ellinwood, Jr . and M . il ey, Editors), pp . 4097-W, Plenum Press, New York (1977) . E . SCHIORRING, Behaviour 39 1-17 (1971) . J . SCHEEL-KRUGET,_7C_ .'1`RUP, M . NIELSEN, K . GOLEMBIOWSKA, and E . MOGILNICKA, Cocaine and Other Stimúlants (E . H . Ellinwood, JY . and M. M. Kilbey, Editors pp . 373-408, Plenum Press, New York (1977) . N . E . ANDEN, A . RUBENSSON, F . FUXE, AND T . HOKFELT, J . Pharm . Pharmacol . 19 627-629 (1967) . T._KU9=SIZY, Life Sciences 17 43-48 (1975) . B . J . WINER, Statistical rince les in Experimental Desi n McGraw Hill, New York B . T . HO, D . L . TAYLOR, V . S . ESTEVEZ, L . F . ENGLERT, AND M . L . McKENNA, Cocaine and Other Stimulants (E . H . Ellinwood, Jr . and M. M . Ki ey, Editors) pp . Plenum Press, New York (1977) . S . Z . LANGER, Handbook of Ps cho harmacolo (L . L . Iversen, S . D . Iversen, andS . ny er, Editors) pp 245-280, Plenum Press, New York (1975) .
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Reverse Tolerance/Stimulant-Induced Behavior
C . M . KUHN and S . M . SCHANBERG, Cocaine (E . H . Ellinwood, Jr . and M . M . i ey, Plenum Press, New York (1977) . 18 . S . J . MULE and A . L . MISRA, Cocaine and H . Ellinwood and M . M . Kilbey, Editors) Press, New York (1977) . 17 .
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and Other Stimulants Editors) Pp .161-178, Other Stimulants (E . pp . 5-2 , enum