- Email: [email protected]
Biochemical and behavioral changes in rats during and after chronic d-amphetamine exposure
Drug and Alcohol Dependence, 15 (1985) 245-253 Elsevier Scientific Publishers Ireland Ltd.
245
BIOCHEMICAL AND BEHAVIORAL CHANGES IN RATS DURING AND AFTER CHRONIC d-AMPHETAMINE EXPOSURE
WOLFGANG H. VOGEL, JOANNE MILLER, HOWARD WAXMAN and EDWARD GOTTHEIL Department of Pharmacology Thomas Jefferson University,
and Department of Psychiatry Philadelphia, PA 19107 (U.S.A.)
and Human
Behavior,
(Received September 19th, 1984)
SUMMARY
Two groups of rats were implanted with ALZET@ minipumps to deliver vehicle or a theoretical amount of 1 mg/kg per h of d-amphetamine (A) for 12 days. After 3 days of A-exposure, motor movements and stereotypic behavior were markedly increased. Subsequent testing during A-exposure showed that motor movements and stereotypic behavior remained significantly increased but declined. After removal of the pumps, these effects disappeared and no differences at rest, during stress or A challenge, were apparent in either group. Animals sacrificed after 3 days of drug exposure, showed a drastic decrease in cardiac, but not adrenal, catecholamine levels. In the brain, norepinephrine (NE) levels were markedly decreased in the frontal cortex, hypothalamus, caudate, pons-medulla and cerebellum. Epinephrine (E) levels were unaffected and dopamine (DA) levels were decreased in most areas without reaching statistical significance. Plasma corticosterone levels were similar in both groups. Animals in both groups sacrificed about 25 days after pump removal were biochemically similar. Under our conditions, A-exposure produced marked behavioral and biochemical changes but there was no evidence of residual abnormalities after cessation of drug treatment. Key words: Biogenic amines - Behavior - Rats - d-Amphetamine INTRODUCTION
A-induced changes in behavior and biochemistry of animals have been well investigated because of their possible contributions to the understanding of amphetamine psychosis and schizophrenia in man. Until recently, there has been less interest in the chronic use of A and in the possibility of behavioral and neurochemical effects persisting after the discontinuation of the 0376-8716/85/$03.30 o 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
246
drug. However, recent clinical observations of progressive personality deterioration and the development of schizophrenic symptoms in some chronic amphetamine abusers following chronic drug abuse, have suggested that the chronic abuse of amphetamines may indeed pose risks for the development of persistent alterations in brain and behavior [l-3]. Most of the initial studies of chronic amphetamine administration employed repeated daily injections of the drug [ 4-141. Under these conditions, an augmentation (sensitization, reverse tolerance) of the drug effect was observed under drug treatment, followed in some cases by residual behavioral and/or neurochemical changes after cessation of drug treatment. Since repeated daily injections do not resemble the intake patterns of human addicts and result in sensitization which is unlike the human situation where tolerance develops, other studies were performed using implanted, slow release pellets, silicone reservoirs or Alzet minipumps [6,9,15--171. The neurochemical and/or behavioral changes in these studies demonstrated the development of tolerance during treatment and in some cases residual effects after A-administration had stopped. Most of the above studies concentrated either on the behavioral or the biochemical effects of amphetamine exposure, and cannot be easily compared since they used different doses, delivery systems and species or strains. This was emphasized in a recent review in which it was noted that the results of existing studies differ markedly and that it is difficult at present to draw any definite conclusions about the neurochemical effects of A [ 51. Furthermore, few of the studies explored the possibility of residual effects. The present study, therefore, was designed to simultaneously investigate the behavioral and neurochemical effects of A in the same animals (a) during a la-day period of A-administration by Alzet minipumps, and (b) at various points up to 26 days following removal of the drug delivering minipumps. The catecholamine system in different brain areas was studied during and after drug exposure and testing for current and residual behavioral effects was performed under conditions of rest, during stress and following an A-challenge. MATERIALS
AND METHODS
Male Sprague-Dawley rats (175-200 g) were purchased from Perfection Breeders, Douglasville, PA, U.S.A., housed individually before, during and after the experiment, and were provided with water and food ad libitum. They were randomly assigned to 2 groups. After establishing a behavioral baseline, all animals were lightly anesthetized and implanted with Alzet mini-osmotic pumps (Model 2002) from the Alza Corp., Palo Alto, CA, U.S.A., in the scapular region. One group (n = 18) received minipumps filled with polyethylene-glycol (PEG) and one group (n = 33) received pumps filled with A in PEG. The pumps were removed after 12 days using the same anesthetic.
241
d-Amphetamine sulfate was a gift from Smith, Kline and French Laboratories. It was dissolved in water and NaOH was added. The A-base was extracted 3 times with ether. The ether was removed in vacua. The pumps were filled to deliver a theoretical amount of 1 mg/kg per h of A. However, the actual amount released is less [ 171 and is probably 0.5 mg/kg per h. For the behavioral studies, animals were always tested between 0900 h and 1200 h. Fine and gross motor activities were measured on a StoeltingActivity Meter for 5 min after a 1-min acclimation period and measurements were recorded as artificial measures of the equipment. Stereotypic behavior consisting of grooming, scratching, ‘wet dog shakes’, rearing, limb flicks and biting were observed for 30 10-s periods and periods during which these behaviors occurred repetitively were recorded. These behavioral tests were performed on the animals of both groups without manipulations on days 3,4 and 12 during implantation. Behavioral measures were also performed on experimental days 17, following a 5-min restraint period on experimental day 33; 10 and 90 min after i.p. injections of 4 mg/kg of A on experimental day 34, and after 1 mg/kg of A on experimental day 40. For the biochemical data, randomly chosen animals from both groups were killed by decapitation after 3 days of A-exposure (experimental day 6), and 26-30 days after removal of the pumps (experimental day 40). Heparinized plasma, adrenals, and hearts were obtained, immediately frozen and stored at -70°C. Brains were dissected on ice and also frozen. Within 2 months, the tissues were homogenized in 0.4 N perchloric acid containing 5 mM reduced glutathione. Catecholamines were determined using the Cat-a-Kit from Upjohn Diagnostics, Kalamazoo, MI, U.S.A. Corticosterone levels were determined using the procedure and antiserum from Endocrine Sciences, Tarzana, CA, U.S.A. RESULTS The weights of the animals were followed during the entire experiment. Although the A-treated animals lost a small amount of weight during Aexposure, this was not statistically different. After pump removal, the weights of these animals quickly increased and became equal to those of the control animals. Table I shows the effects of A on fine and gross movements and stereotype before, during and after chronic A-exposure. A significant increase in gross and fine movements was observed during exposure to the drug. Maximal effects were observed on the first test on experimental day 6 or after 3 days of drug exposure. Thereafter, motor movements declined but remained significantly elevated during the A-exposure days. After removal of the pumps, behavioral differences between groups disappeared and did not differ even in response to challenge with a restraint stressor or A. Stereotypy was significantly increased at day 6 or on day 3 of A-exposure. No residual behavioral effects were noticed in the A-treated rats.
248 TABLE
II
EFFECTS OF CHRONIC A-EXPOSURE (rig/g))) HEART (ng/g) AND ADRENALS
ON CATECHOLAMINE (rg/g) IN RATS
Values represent the mean r S.D. Groups of 3-7 and about 25 days (day 40) after pump removal.
animals were sacrificed
Day 6
Adrenal
Frontal cortex Hypet haiamus
Caudate
Polls Medulla Cerebellum
*Comparison
NE E DA NE E DA
S.D.
between
M
SD.
A
M
S.D.
M
SD
196 146 23 165 186
201 53 132 438 ill?
+ f f f
52* 41* 33 121 42
1105 t 267 r 213 t 977 t 431 t
349 152 49 101 134
894 328 166 864 405
f 265 f 234 + 55 * 149 f 72
375 f 1246 +
56 285
166 + 954 r
23* 540
360 1228
114 736
408 1327
* 60 t 854
1788 106 396 895 283 16704
+ 501 f. 54 f 255 15 * 54 f 483
1345 114 372 138 360 21896
NE DA NE DA
during (day 6)
+ + + + f
872 241 97 575 246
NE DA
Control
A
M NE E NE E DA
OF BRAIN
Day 40
Control
Heart
LEVELS
+ 187 f 43 + 84 +_ 18 * 229 +_8573
643 f 197 + 319 r 23+ groups;
75 100 53 14
883 66 285 64 364 17436 433 163 131 30
* 221* f 24* * 102 t 20* + 289 t 4379 + + f +
P < 0.05 (Student’s
96* 98 21* 11
* r
1663 + 444 105 c 31 366 + 99 93+ 14 277 + 94 16947 t 5923 517 * 164 r 255 + 19+
93 16 32 6
604 + 153 159 + 39 267 * 26 25?; 11
t-test)
Table II shows the biochemical data obtained on experimental days 6 and 40. The values for the vehicle-treated animals are’ similar to those reported in the literature. After 3 days of A-exposure (experimental day 6), NE and E levels decreased significantly in the hearts of the drugged animals. No changes were found in the catecholamine levels in the adrenals. Significant decreases in NE levels were seen in the frontal cortex, hypothalamus, caudate, pons medulla and cerebellum. DA levels of the A-treated were lower than those of the control group in the frontal cortex, hypothalamus, caudate and pons-medulla, but none of these differences were statistically significant. At experimental day 40, no biochemical differences between the 2 groups of animals were apparent. There were no significant differences in the plasma corticosterone levels w between drugged and control animals on experimental day 6 (19.9 + 7.6 pg/ 100 ml vs. 17.9 + 6.5 pg/lOO ml) or on experimental day 40 (20.2 f 5.1 erg/ 100 ml vs. 17.3 f 5.4 c(g/lOO ml).
EFFECTS OF CHRONIC A-EXPOSURE IN RATS
B B B B B B B S A-l 0 A-90 B A-10 A-90
Test
27 11 18 9 16 8 31 43 112 92 25 118 81
r 31 c 13 * 24 ? 12 f 17 + 18 r 42 + 36 ? 35 +_54 t 35 * 66 r 64
(18) (18) (17) (11) (11) (11) (7) (6) (7) (7) (7) (7) (7)
34 f. 27 (33) 23 + 26 (33) 93 r 52 (31) 64 t 65* (25) 42 ? 44* (18) 14 f 22 (12) 53 t 55 (7) 43 + 35 (6) 94*50(7) 107 + 58 (7) 59 r 57 (7) 97 + 66 (7) 86 f 61 (7)
86 + 74 44 + 44 58 + 64 32 + 37 46 t 50 28 +_ 69 86 + 115 131 + 91 286 r 80 249 r 65 8* 85 262 t 93 181 + 34
V-Group
V-Group
A-Group
Fine movements
Gross movements
*Comparison between groups;P < 0.05 (Rank Sum Test).
39 40
1 2 6 9 12 17 32 33 34
Day
100 71 247 150 110 42 130 119 290 290 169 247 219
* 63 f 68 f 115* + 126* r 98* 2 62 + 132 ?- 81 t 97 * 105 t 168 + 110 f. 152
A-Group
-
0 0 0 0 0 0 0 0 4.6 t 11.2 5.3 r 10.6 0 0 0
V-Group
0 0 3.8 0.4 0.3 0 0 0 0 8.9 0 0 0
? 14.5
? 4.6* + 1.4 * 1.2
A-Group
Stereotypic behavior
Animais were tested on days 1 and 2 prior to minipump implantation. They were tested for 3 days (days 6,9 and 12) under the influence of A and on the indicated days after the pumps had been removed. Testing was performed without manipulation (B), after 5 min of restraint stress (S) or 10 (A-10) or 90 (A-90) min after i.p. injection of 4 mg/kg (day 34) or 1 mg/kg (day 40). Values represent the mean t S.D.; numbers in parenthesis indicate number of animals tested.
BEHAVIORAL
TABLE I
250
DISCUSSION
The implantation of minipumps delivering an actual amount of about 0.5 mg/kg per h of A into the rat during the first experimental week [ 171 produced some marked biochemical changes in our animals during cirrug exposure. The heart was one of the most affected organs and a drastic decrease in both NE and E was observed after 3 days of A exposure. The synthesis of NE apparently could not keep up with the increased demand suggesting that A can seriously compromise the homeostasis of this organ. This is in agreement with the physiological observations that A can markedly increase cardiac sympathetic activity causing tachycardia and, in rare cases, acute cardiac failure as a result of excessive myocardial stimulation followed by exhaustion [18,19]. No changes in the catecholamine levels of the adrenals were observed. No differences in plasma corticosterone levels were noted during and after A-exposure. The lack of increased corticosterone levels during A-exposure is surprising since the animals were quite excited and since plasma catecholamine levels are increased after A-administration (unpublished observation). In the brain, the NE system was most affected whereas the DA and E systems showed relatively little change. NE was significantly reduced in frontal cortex, hypothalamus, caudate, pons-medulla and cerebellum. DA was reduced in most areas but the reduction did not reach statistical significance. After the pumps were removed, and the animals were allowed to recover from the drug, all of these effects disappeared and no differences between vehicle-treated and A-treated animals were found. The findings suggest that the biochemical changes seen during A-exposure are reversible under our experimental conditions. A comparison of our results with those reported in the literature reveals some similarities and some differences. Rats pretreated with A releasing pellets for 4 l/2 days and sacrificed 12 h later showed a significant decrease in caudate DA levels [20] . Release of A (3.5 mg/day) by silicone pellets implanted in rats for ‘10 days resulted in decreases in caudate, but not in hypothalamic and cortical, DA levels as well as decreases in hypothalamic and cortical NE levels. After pellet removal these values returned to normal. A priming dose of 15 mg/kg, i.p., followed by 16 h of A-infusion from minipumps produced a marked decrease in striatal DA which lasted for at least 24 weeks [21] ; whole brain concentrations of A of about 25 rig/g after i.p. injection were very high. Continuous administration of A to mice by minipumps decreased whole brain DA levels during and 60 days after A-exposure [ 221. Twenty-eight daily injections of A into rats reduced NE in hippocampus and hypothalamus, and DA in the caudate; 1 week after cessation of A-treatment, no differences between control and A-treated animals were seen [6] . Semichronic (2X daily for 3 days) A-treatment of rats produced no changes in hypothalamic but increases in caudal DA levels while no effects on NE levels were noted [ 41. Chronic A-treatment (twice daily for 10 days) of rats produced decreased NE levels in the hypo-
251
thalamus, but no changes in the caudate and cortex; DA levels were significantly increased in the caudate with no changes in the other two areas [7]. Three daily injections of A did not change striatal DA levels in rats [ 231. In vivo measurements of DA turnover in mice showed that DA is released by A from a pool of DA which can be maintained rather well by increased DA synthesis [9]. This brief review of some existing results indicates that the response of the brain catecholamine system to A is complex. One possible explanation of some of the differences observed is that the response of the different neurochemical systems to A depends not only on the dosing schedule but is also multiphasic; for example, initial decreases in DA may be quickly compensated for by increased DA synthesis which could lead to increases followed by normal DA levels. This was also suggested by Ellison and his group [6] . Furthermore, our study shows clearly that systems other than the DA system must not be neglected, and that the NE system may play a more important role in A-intoxication than is usually recognized. Again, this suggestion is in agreement with those of others [4,15,24]. The behavioral effects seen in this study of initial increases in activity and stereotypy followed by decreases in these behaviors during continuous Aexposure are very similar to those reported in the literature [6,9,15,17,20]. The development of tolerance with continuous administration is in contrast to repeated, single injections of A which result in sensitization [ 6,8] , i.e. a gradual increase in these behaviors in response to the same dose. However, it was found that if repeated injections are continued, sensitization will eventually give way to the development of tolerance manifested by a recurrence of stereotypy [22] . Thus, it seems quite firmly established that A produces stereotyped behaviors which will show tolerance or reverse tolerance depending on the dose, schedule of injections, and duration of exposure [23-251. After removal of the pumps, the rats were tested for residual behavioral effects following rest, restraint stress or A-challenge. No differences in locomotor activity and stereotype were observed between groups indicating that the behavioral changes seen with our animals during A-treatment were reversible. This is in agreement with another study [lo], which also did not find residual effects of chronic A-exposure on locomotor activity. In other studies, however, chronic A-exposure was shown to affect stereotype behavior after drug cessation. Stereotypy was increased after chronic Atreatment had been stopped for about 5 days and was augmented following a subsequent challenge with the drug [5,6,8,10,25]. Again, this effect seems to depend on the administration schedule. Cessation of daily injections (28 days) of A caused augmentation of stereotypic behavior by A-challenge, whereas cessation of A-pellet implantation for the same time caused decreased stereotypic behavior [6]. We did not find any residual effects on stereotype. One reason could be that our A-exposure time lies between the usual short-term (4-6 days) and the long-term (28-day) treatment schedules.
252
Furthermore, known individual and strain differences to the effects of A as well as inherit problems with behavioral testing procedures [26] could also have contributed to the results [10,13,25]. It must be noted that we found no statistically significant differences between groups, we did find that a small number of individual animals showed marked residual behavioral abnormalities. In humans, chronic A-abuse has been associated with residual psychopathology [l--3]. However, it is difficult to distinguish in man whether the psychopathology existed before A-abuse and was merely aggravated by the drug, or whether it did not exist before and was the sole consequence of the abuse of A for long periods. Experiments that have been done with animals are not conclusive and the occurrence of after-effects seems to depend strongly on the administration schedule as well as on the strain of animal used [lo 1. Even within one strain, animals have been identified which respond differently to A [27] or metabolize the drug differently [28]. Furthermore, previous experience such as escapable vs. inescapable shock [ 291 or group vs. single housing [ 301 can produce a differential response. The complexity of genetic, environmental and experimental factors does not currently permit us to explain why some chronic A-users experience residual psychopathology and others do not. A worthwhile strategy might be to select individual animals which are particularly affected by A and study their behavioral and neurochemical responses in detail over time. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
J. Deveaugh-Geiss and A. Pandurangi, Am. J. Psychiatry, 139 (1982) 1190. A.T. McLellan, G.E. Woody, and C.P. O’Brien, New Engl. J. Med., 301 (1979) 1310. M. Sato et al., Biol. Psychiatry, 18 (1983) 429. L.W. Chuang, F. Karoum and R.J. Wyatt, Eur. J. Pharmacol., 81 (1982) 385. C. Demellweek and A.J. Goudie, Psychopharmacol., 80 (1983) 287. G. Ellison and W. Morris, Eur. J. Psychopharmacol., 74 (1981) 207. F. Karoum et al., J. Pharmacol. Exp. Ther., 223 (1982) 432. R. Kuczenskiet al., Pharmacol.Biochem. Behav., 17 (1982) 547. N.J. Leith and R. Kuczenski, Pharmacol. Biochem. Behav., 15 (1981) 399. N.J. Leith and R. Kuczenski, Psychopharmacol., 76 (1982) 310. T.E. Robinson and J.B. Becker, Eur. J. Pharmacol., 85 (1982) 253. D.S. Segal and A.J. Mandell, Pharmacol. Biochem. Behav., 2 (1974) 249. SM. Sorenson, S.W. Johnson and R. Friedman, Brain Res., 243 (1982) 365. M.E. Trulson and B.L. Jacobs, J. Pharmacol. Exp. Ther., 211 (1979) 375. M.S. Eison and G. Ellison, Gen. Pharmacol., 13 (1982) 407. H.S. Huberman et al., Eur. J. Pharmacol., 45 (1977) 237. E.B. Nielsen, Pharmacol. Biochem. Behav., 15 (1981) 161. T.D. Call et a., Ann. Int. Med., 97 (1982) 559. N. Weiner in: L.S. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, McMillan Publishing Co., Sixth edn, 1980, p. 160. 20 G. Ellison and R. Ratan, Life Sci., 31 (1982) 771. 21 L.R. Steranka, Eur. J. Pharmacol., 96 (1983) 159. 22 E. Nwanze and G. Jonsson, Neurosci. Lett., 26 (1981) 163.
253 23 R. Kucenzski and N.J. Leith, Pharmacol. Biochem. Behav., 15 (1981) 405. 24 L. Korinidas and H. Anisman, Neurosci. Biobehav. Rev., 5 (1981) 449. 25 S.M. Antelman and L.A. Chiado, in: I. Creese (Ed.), Stimulants: Neurochem. Behav. Clin. Prospect., Raven Press, NY, 1983, pp. 269-299. 26 G.V. Rebec and T.R. Bashore, Neurosci. Behav. Rev., 8 (1984) 153. 27 C.M. Quirce et al., Res. Commun. Substance Abuse, 3 (1982) 49. 28 S. Pashko and W.H. Vogel, Biochem. Pharm., 29 (1980) 221. 29 A.J. McLennan and S.F. Maier, Science, 219 (1983) 1091. 30 G.W. Kraemer et al., in: Ethnopharmacology: Primate Models of Neuropsychiatric Disorders, Alan R. Liss, Sot., 150 Fifth Avenue, New York, pp. 199-218.
245
BIOCHEMICAL AND BEHAVIORAL CHANGES IN RATS DURING AND AFTER CHRONIC d-AMPHETAMINE EXPOSURE
WOLFGANG H. VOGEL, JOANNE MILLER, HOWARD WAXMAN and EDWARD GOTTHEIL Department of Pharmacology Thomas Jefferson University,
and Department of Psychiatry Philadelphia, PA 19107 (U.S.A.)
and Human
Behavior,
(Received September 19th, 1984)
SUMMARY
Two groups of rats were implanted with ALZET@ minipumps to deliver vehicle or a theoretical amount of 1 mg/kg per h of d-amphetamine (A) for 12 days. After 3 days of A-exposure, motor movements and stereotypic behavior were markedly increased. Subsequent testing during A-exposure showed that motor movements and stereotypic behavior remained significantly increased but declined. After removal of the pumps, these effects disappeared and no differences at rest, during stress or A challenge, were apparent in either group. Animals sacrificed after 3 days of drug exposure, showed a drastic decrease in cardiac, but not adrenal, catecholamine levels. In the brain, norepinephrine (NE) levels were markedly decreased in the frontal cortex, hypothalamus, caudate, pons-medulla and cerebellum. Epinephrine (E) levels were unaffected and dopamine (DA) levels were decreased in most areas without reaching statistical significance. Plasma corticosterone levels were similar in both groups. Animals in both groups sacrificed about 25 days after pump removal were biochemically similar. Under our conditions, A-exposure produced marked behavioral and biochemical changes but there was no evidence of residual abnormalities after cessation of drug treatment. Key words: Biogenic amines - Behavior - Rats - d-Amphetamine INTRODUCTION
A-induced changes in behavior and biochemistry of animals have been well investigated because of their possible contributions to the understanding of amphetamine psychosis and schizophrenia in man. Until recently, there has been less interest in the chronic use of A and in the possibility of behavioral and neurochemical effects persisting after the discontinuation of the 0376-8716/85/$03.30 o 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
246
drug. However, recent clinical observations of progressive personality deterioration and the development of schizophrenic symptoms in some chronic amphetamine abusers following chronic drug abuse, have suggested that the chronic abuse of amphetamines may indeed pose risks for the development of persistent alterations in brain and behavior [l-3]. Most of the initial studies of chronic amphetamine administration employed repeated daily injections of the drug [ 4-141. Under these conditions, an augmentation (sensitization, reverse tolerance) of the drug effect was observed under drug treatment, followed in some cases by residual behavioral and/or neurochemical changes after cessation of drug treatment. Since repeated daily injections do not resemble the intake patterns of human addicts and result in sensitization which is unlike the human situation where tolerance develops, other studies were performed using implanted, slow release pellets, silicone reservoirs or Alzet minipumps [6,9,15--171. The neurochemical and/or behavioral changes in these studies demonstrated the development of tolerance during treatment and in some cases residual effects after A-administration had stopped. Most of the above studies concentrated either on the behavioral or the biochemical effects of amphetamine exposure, and cannot be easily compared since they used different doses, delivery systems and species or strains. This was emphasized in a recent review in which it was noted that the results of existing studies differ markedly and that it is difficult at present to draw any definite conclusions about the neurochemical effects of A [ 51. Furthermore, few of the studies explored the possibility of residual effects. The present study, therefore, was designed to simultaneously investigate the behavioral and neurochemical effects of A in the same animals (a) during a la-day period of A-administration by Alzet minipumps, and (b) at various points up to 26 days following removal of the drug delivering minipumps. The catecholamine system in different brain areas was studied during and after drug exposure and testing for current and residual behavioral effects was performed under conditions of rest, during stress and following an A-challenge. MATERIALS
AND METHODS
Male Sprague-Dawley rats (175-200 g) were purchased from Perfection Breeders, Douglasville, PA, U.S.A., housed individually before, during and after the experiment, and were provided with water and food ad libitum. They were randomly assigned to 2 groups. After establishing a behavioral baseline, all animals were lightly anesthetized and implanted with Alzet mini-osmotic pumps (Model 2002) from the Alza Corp., Palo Alto, CA, U.S.A., in the scapular region. One group (n = 18) received minipumps filled with polyethylene-glycol (PEG) and one group (n = 33) received pumps filled with A in PEG. The pumps were removed after 12 days using the same anesthetic.
241
d-Amphetamine sulfate was a gift from Smith, Kline and French Laboratories. It was dissolved in water and NaOH was added. The A-base was extracted 3 times with ether. The ether was removed in vacua. The pumps were filled to deliver a theoretical amount of 1 mg/kg per h of A. However, the actual amount released is less [ 171 and is probably 0.5 mg/kg per h. For the behavioral studies, animals were always tested between 0900 h and 1200 h. Fine and gross motor activities were measured on a StoeltingActivity Meter for 5 min after a 1-min acclimation period and measurements were recorded as artificial measures of the equipment. Stereotypic behavior consisting of grooming, scratching, ‘wet dog shakes’, rearing, limb flicks and biting were observed for 30 10-s periods and periods during which these behaviors occurred repetitively were recorded. These behavioral tests were performed on the animals of both groups without manipulations on days 3,4 and 12 during implantation. Behavioral measures were also performed on experimental days 17, following a 5-min restraint period on experimental day 33; 10 and 90 min after i.p. injections of 4 mg/kg of A on experimental day 34, and after 1 mg/kg of A on experimental day 40. For the biochemical data, randomly chosen animals from both groups were killed by decapitation after 3 days of A-exposure (experimental day 6), and 26-30 days after removal of the pumps (experimental day 40). Heparinized plasma, adrenals, and hearts were obtained, immediately frozen and stored at -70°C. Brains were dissected on ice and also frozen. Within 2 months, the tissues were homogenized in 0.4 N perchloric acid containing 5 mM reduced glutathione. Catecholamines were determined using the Cat-a-Kit from Upjohn Diagnostics, Kalamazoo, MI, U.S.A. Corticosterone levels were determined using the procedure and antiserum from Endocrine Sciences, Tarzana, CA, U.S.A. RESULTS The weights of the animals were followed during the entire experiment. Although the A-treated animals lost a small amount of weight during Aexposure, this was not statistically different. After pump removal, the weights of these animals quickly increased and became equal to those of the control animals. Table I shows the effects of A on fine and gross movements and stereotype before, during and after chronic A-exposure. A significant increase in gross and fine movements was observed during exposure to the drug. Maximal effects were observed on the first test on experimental day 6 or after 3 days of drug exposure. Thereafter, motor movements declined but remained significantly elevated during the A-exposure days. After removal of the pumps, behavioral differences between groups disappeared and did not differ even in response to challenge with a restraint stressor or A. Stereotypy was significantly increased at day 6 or on day 3 of A-exposure. No residual behavioral effects were noticed in the A-treated rats.
248 TABLE
II
EFFECTS OF CHRONIC A-EXPOSURE (rig/g))) HEART (ng/g) AND ADRENALS
ON CATECHOLAMINE (rg/g) IN RATS
Values represent the mean r S.D. Groups of 3-7 and about 25 days (day 40) after pump removal.
animals were sacrificed
Day 6
Adrenal
Frontal cortex Hypet haiamus
Caudate
Polls Medulla Cerebellum
*Comparison
NE E DA NE E DA
S.D.
between
M
SD.
A
M
S.D.
M
SD
196 146 23 165 186
201 53 132 438 ill?
+ f f f
52* 41* 33 121 42
1105 t 267 r 213 t 977 t 431 t
349 152 49 101 134
894 328 166 864 405
f 265 f 234 + 55 * 149 f 72
375 f 1246 +
56 285
166 + 954 r
23* 540
360 1228
114 736
408 1327
* 60 t 854
1788 106 396 895 283 16704
+ 501 f. 54 f 255 15 * 54 f 483
1345 114 372 138 360 21896
NE DA NE DA
during (day 6)
+ + + + f
872 241 97 575 246
NE DA
Control
A
M NE E NE E DA
OF BRAIN
Day 40
Control
Heart
LEVELS
+ 187 f 43 + 84 +_ 18 * 229 +_8573
643 f 197 + 319 r 23+ groups;
75 100 53 14
883 66 285 64 364 17436 433 163 131 30
* 221* f 24* * 102 t 20* + 289 t 4379 + + f +
P < 0.05 (Student’s
96* 98 21* 11
* r
1663 + 444 105 c 31 366 + 99 93+ 14 277 + 94 16947 t 5923 517 * 164 r 255 + 19+
93 16 32 6
604 + 153 159 + 39 267 * 26 25?; 11
t-test)
Table II shows the biochemical data obtained on experimental days 6 and 40. The values for the vehicle-treated animals are’ similar to those reported in the literature. After 3 days of A-exposure (experimental day 6), NE and E levels decreased significantly in the hearts of the drugged animals. No changes were found in the catecholamine levels in the adrenals. Significant decreases in NE levels were seen in the frontal cortex, hypothalamus, caudate, pons medulla and cerebellum. DA levels of the A-treated were lower than those of the control group in the frontal cortex, hypothalamus, caudate and pons-medulla, but none of these differences were statistically significant. At experimental day 40, no biochemical differences between the 2 groups of animals were apparent. There were no significant differences in the plasma corticosterone levels w between drugged and control animals on experimental day 6 (19.9 + 7.6 pg/ 100 ml vs. 17.9 + 6.5 pg/lOO ml) or on experimental day 40 (20.2 f 5.1 erg/ 100 ml vs. 17.3 f 5.4 c(g/lOO ml).
EFFECTS OF CHRONIC A-EXPOSURE IN RATS
B B B B B B B S A-l 0 A-90 B A-10 A-90
Test
27 11 18 9 16 8 31 43 112 92 25 118 81
r 31 c 13 * 24 ? 12 f 17 + 18 r 42 + 36 ? 35 +_54 t 35 * 66 r 64
(18) (18) (17) (11) (11) (11) (7) (6) (7) (7) (7) (7) (7)
34 f. 27 (33) 23 + 26 (33) 93 r 52 (31) 64 t 65* (25) 42 ? 44* (18) 14 f 22 (12) 53 t 55 (7) 43 + 35 (6) 94*50(7) 107 + 58 (7) 59 r 57 (7) 97 + 66 (7) 86 f 61 (7)
86 + 74 44 + 44 58 + 64 32 + 37 46 t 50 28 +_ 69 86 + 115 131 + 91 286 r 80 249 r 65 8* 85 262 t 93 181 + 34
V-Group
V-Group
A-Group
Fine movements
Gross movements
*Comparison between groups;P < 0.05 (Rank Sum Test).
39 40
1 2 6 9 12 17 32 33 34
Day
100 71 247 150 110 42 130 119 290 290 169 247 219
* 63 f 68 f 115* + 126* r 98* 2 62 + 132 ?- 81 t 97 * 105 t 168 + 110 f. 152
A-Group
-
0 0 0 0 0 0 0 0 4.6 t 11.2 5.3 r 10.6 0 0 0
V-Group
0 0 3.8 0.4 0.3 0 0 0 0 8.9 0 0 0
? 14.5
? 4.6* + 1.4 * 1.2
A-Group
Stereotypic behavior
Animais were tested on days 1 and 2 prior to minipump implantation. They were tested for 3 days (days 6,9 and 12) under the influence of A and on the indicated days after the pumps had been removed. Testing was performed without manipulation (B), after 5 min of restraint stress (S) or 10 (A-10) or 90 (A-90) min after i.p. injection of 4 mg/kg (day 34) or 1 mg/kg (day 40). Values represent the mean t S.D.; numbers in parenthesis indicate number of animals tested.
BEHAVIORAL
TABLE I
250
DISCUSSION
The implantation of minipumps delivering an actual amount of about 0.5 mg/kg per h of A into the rat during the first experimental week [ 171 produced some marked biochemical changes in our animals during cirrug exposure. The heart was one of the most affected organs and a drastic decrease in both NE and E was observed after 3 days of A exposure. The synthesis of NE apparently could not keep up with the increased demand suggesting that A can seriously compromise the homeostasis of this organ. This is in agreement with the physiological observations that A can markedly increase cardiac sympathetic activity causing tachycardia and, in rare cases, acute cardiac failure as a result of excessive myocardial stimulation followed by exhaustion [18,19]. No changes in the catecholamine levels of the adrenals were observed. No differences in plasma corticosterone levels were noted during and after A-exposure. The lack of increased corticosterone levels during A-exposure is surprising since the animals were quite excited and since plasma catecholamine levels are increased after A-administration (unpublished observation). In the brain, the NE system was most affected whereas the DA and E systems showed relatively little change. NE was significantly reduced in frontal cortex, hypothalamus, caudate, pons-medulla and cerebellum. DA was reduced in most areas but the reduction did not reach statistical significance. After the pumps were removed, and the animals were allowed to recover from the drug, all of these effects disappeared and no differences between vehicle-treated and A-treated animals were found. The findings suggest that the biochemical changes seen during A-exposure are reversible under our experimental conditions. A comparison of our results with those reported in the literature reveals some similarities and some differences. Rats pretreated with A releasing pellets for 4 l/2 days and sacrificed 12 h later showed a significant decrease in caudate DA levels [20] . Release of A (3.5 mg/day) by silicone pellets implanted in rats for ‘10 days resulted in decreases in caudate, but not in hypothalamic and cortical, DA levels as well as decreases in hypothalamic and cortical NE levels. After pellet removal these values returned to normal. A priming dose of 15 mg/kg, i.p., followed by 16 h of A-infusion from minipumps produced a marked decrease in striatal DA which lasted for at least 24 weeks [21] ; whole brain concentrations of A of about 25 rig/g after i.p. injection were very high. Continuous administration of A to mice by minipumps decreased whole brain DA levels during and 60 days after A-exposure [ 221. Twenty-eight daily injections of A into rats reduced NE in hippocampus and hypothalamus, and DA in the caudate; 1 week after cessation of A-treatment, no differences between control and A-treated animals were seen [6] . Semichronic (2X daily for 3 days) A-treatment of rats produced no changes in hypothalamic but increases in caudal DA levels while no effects on NE levels were noted [ 41. Chronic A-treatment (twice daily for 10 days) of rats produced decreased NE levels in the hypo-
251
thalamus, but no changes in the caudate and cortex; DA levels were significantly increased in the caudate with no changes in the other two areas [7]. Three daily injections of A did not change striatal DA levels in rats [ 231. In vivo measurements of DA turnover in mice showed that DA is released by A from a pool of DA which can be maintained rather well by increased DA synthesis [9]. This brief review of some existing results indicates that the response of the brain catecholamine system to A is complex. One possible explanation of some of the differences observed is that the response of the different neurochemical systems to A depends not only on the dosing schedule but is also multiphasic; for example, initial decreases in DA may be quickly compensated for by increased DA synthesis which could lead to increases followed by normal DA levels. This was also suggested by Ellison and his group [6] . Furthermore, our study shows clearly that systems other than the DA system must not be neglected, and that the NE system may play a more important role in A-intoxication than is usually recognized. Again, this suggestion is in agreement with those of others [4,15,24]. The behavioral effects seen in this study of initial increases in activity and stereotypy followed by decreases in these behaviors during continuous Aexposure are very similar to those reported in the literature [6,9,15,17,20]. The development of tolerance with continuous administration is in contrast to repeated, single injections of A which result in sensitization [ 6,8] , i.e. a gradual increase in these behaviors in response to the same dose. However, it was found that if repeated injections are continued, sensitization will eventually give way to the development of tolerance manifested by a recurrence of stereotypy [22] . Thus, it seems quite firmly established that A produces stereotyped behaviors which will show tolerance or reverse tolerance depending on the dose, schedule of injections, and duration of exposure [23-251. After removal of the pumps, the rats were tested for residual behavioral effects following rest, restraint stress or A-challenge. No differences in locomotor activity and stereotype were observed between groups indicating that the behavioral changes seen with our animals during A-treatment were reversible. This is in agreement with another study [lo], which also did not find residual effects of chronic A-exposure on locomotor activity. In other studies, however, chronic A-exposure was shown to affect stereotype behavior after drug cessation. Stereotypy was increased after chronic Atreatment had been stopped for about 5 days and was augmented following a subsequent challenge with the drug [5,6,8,10,25]. Again, this effect seems to depend on the administration schedule. Cessation of daily injections (28 days) of A caused augmentation of stereotypic behavior by A-challenge, whereas cessation of A-pellet implantation for the same time caused decreased stereotypic behavior [6]. We did not find any residual effects on stereotype. One reason could be that our A-exposure time lies between the usual short-term (4-6 days) and the long-term (28-day) treatment schedules.
252
Furthermore, known individual and strain differences to the effects of A as well as inherit problems with behavioral testing procedures [26] could also have contributed to the results [10,13,25]. It must be noted that we found no statistically significant differences between groups, we did find that a small number of individual animals showed marked residual behavioral abnormalities. In humans, chronic A-abuse has been associated with residual psychopathology [l--3]. However, it is difficult to distinguish in man whether the psychopathology existed before A-abuse and was merely aggravated by the drug, or whether it did not exist before and was the sole consequence of the abuse of A for long periods. Experiments that have been done with animals are not conclusive and the occurrence of after-effects seems to depend strongly on the administration schedule as well as on the strain of animal used [lo 1. Even within one strain, animals have been identified which respond differently to A [27] or metabolize the drug differently [28]. Furthermore, previous experience such as escapable vs. inescapable shock [ 291 or group vs. single housing [ 301 can produce a differential response. The complexity of genetic, environmental and experimental factors does not currently permit us to explain why some chronic A-users experience residual psychopathology and others do not. A worthwhile strategy might be to select individual animals which are particularly affected by A and study their behavioral and neurochemical responses in detail over time. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
J. Deveaugh-Geiss and A. Pandurangi, Am. J. Psychiatry, 139 (1982) 1190. A.T. McLellan, G.E. Woody, and C.P. O’Brien, New Engl. J. Med., 301 (1979) 1310. M. Sato et al., Biol. Psychiatry, 18 (1983) 429. L.W. Chuang, F. Karoum and R.J. Wyatt, Eur. J. Pharmacol., 81 (1982) 385. C. Demellweek and A.J. Goudie, Psychopharmacol., 80 (1983) 287. G. Ellison and W. Morris, Eur. J. Psychopharmacol., 74 (1981) 207. F. Karoum et al., J. Pharmacol. Exp. Ther., 223 (1982) 432. R. Kuczenskiet al., Pharmacol.Biochem. Behav., 17 (1982) 547. N.J. Leith and R. Kuczenski, Pharmacol. Biochem. Behav., 15 (1981) 399. N.J. Leith and R. Kuczenski, Psychopharmacol., 76 (1982) 310. T.E. Robinson and J.B. Becker, Eur. J. Pharmacol., 85 (1982) 253. D.S. Segal and A.J. Mandell, Pharmacol. Biochem. Behav., 2 (1974) 249. SM. Sorenson, S.W. Johnson and R. Friedman, Brain Res., 243 (1982) 365. M.E. Trulson and B.L. Jacobs, J. Pharmacol. Exp. Ther., 211 (1979) 375. M.S. Eison and G. Ellison, Gen. Pharmacol., 13 (1982) 407. H.S. Huberman et al., Eur. J. Pharmacol., 45 (1977) 237. E.B. Nielsen, Pharmacol. Biochem. Behav., 15 (1981) 161. T.D. Call et a., Ann. Int. Med., 97 (1982) 559. N. Weiner in: L.S. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, McMillan Publishing Co., Sixth edn, 1980, p. 160. 20 G. Ellison and R. Ratan, Life Sci., 31 (1982) 771. 21 L.R. Steranka, Eur. J. Pharmacol., 96 (1983) 159. 22 E. Nwanze and G. Jonsson, Neurosci. Lett., 26 (1981) 163.
253 23 R. Kucenzski and N.J. Leith, Pharmacol. Biochem. Behav., 15 (1981) 405. 24 L. Korinidas and H. Anisman, Neurosci. Biobehav. Rev., 5 (1981) 449. 25 S.M. Antelman and L.A. Chiado, in: I. Creese (Ed.), Stimulants: Neurochem. Behav. Clin. Prospect., Raven Press, NY, 1983, pp. 269-299. 26 G.V. Rebec and T.R. Bashore, Neurosci. Behav. Rev., 8 (1984) 153. 27 C.M. Quirce et al., Res. Commun. Substance Abuse, 3 (1982) 49. 28 S. Pashko and W.H. Vogel, Biochem. Pharm., 29 (1980) 221. 29 A.J. McLennan and S.F. Maier, Science, 219 (1983) 1091. 30 G.W. Kraemer et al., in: Ethnopharmacology: Primate Models of Neuropsychiatric Disorders, Alan R. Liss, Sot., 150 Fifth Avenue, New York, pp. 199-218.