Tolerance to p-chloroamphetamine's effects on sidman avoidance performance and catecholamine metabolism

Neuropharmncology.

1977. 16. 761-769. Pergamon

Press. PrInted

in Great Brltam

TOLERANCE TO p-CHLOROAMPHETAMINE’S ON SIDMAN AVOIDANCE PERFORMANCE CATECHOLAMINE METABOLISM

EFFECTS AND

L. R. STERANKA, ELAINE SANDERS-BUSH and R. J. BARRETT Tennessee Neuropsychiatric Vanderbilt University School

Institute, Department of Pharmacology of Medicine, Veterans Administration Tennessee, U.S.A. (Accepted

17 March

and Psychology, Hospital, Nashville.

1977)

Summary-Rats trained to bar-press to avoid shock in a non-discriminated, Sidman avoidance paradigm were injected intraperitoneally with either saline or 5 mg/kg of PCA twice daily for 7.5 days. When tested 48 hr later, the administration of PCA increased response rates in saline-pretreated but not PCA-pretreated rats, indicating the development of physiological tolerance. Complete recovery from tolerance was observed within 2 weeks after the termination of chronic drug treatment. The rates of disappearance of PCA from the brains of tolerant and non-tolerant rats were similar, suggesting that tolerance was not due to an altered disposition of the drug. The effects of acute and chronic PCA administration on the conversion of [3H]-tyrosine to C3H]-norepinephrine and [3H]-dopamine and in uivo tyrosine hydroxylase activity were measured in whole brains 15 min after PCA administration. The acute administration of PCA increased the formation of both catecholamines and increased in uiuo tyrosine hydroxylase activity. However, neither of these effects was observed in tolerant rats challenged with PCA. These data suggest that tolerance to the facilitatory effect of PCA is associated with tolerance to the ability of the drug to increase the rate of synthesis of catecholamines.

The

halogenated

derivative

of amphetamine,

p-chlor-

(PCA), causes marked changes in the metabolism of brain catecholamines (CAs) and serotonin (5HT). Like amphetamine, PCA causes increases in the concentrations of normetanephrine (Strada, Sanders-Bush and Sulser, 1970) and 3-methoxytyramine (Scheel-Kruger, 1972) and a reduction in the deaminated metabolites of tritiated norepinephrine (Strada et al., 1970). Similar mechanisms may be responsible for these effects: release of catecholamines, blockade of their uptake, and perhaps inhibition of monoamine oxidase (MAO). In contrast to amphetamine, PCA causes a rapid and simultaneous decrease in brain levels of 5-HT and its principal metabolite, 5-hydroxyindole acetic acid (5-HIAA), which is due initially to a complex combination of at least four actions of PCA on brain serotonergic mechanisms: release of 5-HT (Pletscher, Da Prada, Burkard, Bartholini, Steiner, Bruderer and Bigler, 1966; Pletscher, Da Prada and Burkard, 1970; Wong, Horng and Fuller, 1973; Sanders-Bush, Gallager, and Sulser, 1974), blockade of its uptake (Carlsson, 1970; Wong et al., 1973; Meek and Fuxe, 1971; Meek, Fuxe and Carlsson, 1971), inhibition of MAO (Fuller, 1966; Fuller and Hines, 1970), and inhibition of 5-HT synthesis (Sanders-Bush and Sulser, 1970; Sanders-Bush, Bushing and Sulser, 1972a). In addition, PCA has been shown to produce long-term decreases in 5-HT, 5-HIAA and tryptophan hydroxylase activity oamphetamine

Key words: p-chloroamphetamine, tolerance, catecholamine turnover, in ho tyrosine hydroxylase activity, Sidman avoidance. 761

(Sanders-Bush et al., 1972b), high affinity uptake of 5-HT (Sanders-Bush, Bushing and Sulser, 1975), and a decrease in the turnover of 5-HT (Fuller and Snoddy, 1974). These changes persist for longer than 2 weeks after a single dose and are mediated by a mechanism different from the initial effects of the drug (Sanders-Bush et al., 1975; Sekerke, Smith, Bushing and Sanders-Bush, 1975; Gal, Christiansen and Yunger, 1974; Fuller, Perry and Molloy, 1975). Complex behavioural changes accompany these complex biochemical effects of PCA. Thus, the administration of PCA induces the usual amphetamine-like effects on behaviour such as stereotyped movements (Scheel-Kruger, 1972) and facilitation of continuous bar-press (Sidman) avoidance performance (Steranka, Barrett and Sanders-Bush, 1977) which are apparently related to drug-induced changes in catecholamine metabolism. In addition, recent behavioural studies indicate that PCA can produce a unique complex of abnormal behaviours which is related to the ability of the drug to enhance initially serotonergic transmission (Trulson and Jacobs, 1976; El-Yousef. Steranka, and Sanders-Bush, unpublished). The relative contribution of these qualitatively different types of responding in determining the effect of PCA on a particular behavioural measure depends upon the dose of the drug, the time since drug administration and the nature of the measure (Steranka et al., 1977). The present experiments were stimulated by the observation (Steranka, Sanders-Bush and Barrett, 1976) that the facilitatory effect of PCA on continuous, bar-press (Sidman) avoidance performance diminishes during the course of repeated, daily ad-

7hl

L. R.

STERAKRA. ELAINE SANDERS-BUSH and

ministrations of the drug. Interestingly, tolerance to the facilitatory effect of amphetamine on bar-press avoidance performance has not been demonstrated (Leith and Barrett, 1976; Schuster. Dockens and Woods, 1966). Since considerable evidence suggests that the behavioural effect of PCA, like that of amphetamine. is related to drug-induced changes in CA metabolism and, independent of its effects on 5-HT mechanisms (Steranka et al.. 1977). the observation of tolerance to PCA suggests another important difference in the behavioural effects of these two drugs. The present experiments were designed to characterize further the tolerance which develops to the facilitatory effect of PCA on Sidman avoidance performance and to investigate the possibility that this tolerance might be related to changes in the effects of the drug on the metabolism of CAs in the brain.

R. J.

BARRETT

were housed in sound-attenuated cubicles. and white noise was used in the experimental room to mask extraneous auditory stimuli. The behavioural contingencies were programmed using electromechanical equipment housed in an adjacent room Animals were trained. during either daily 2 hr scssions for approximately 20 days or daily 4-hr sessions for approximately 10 days, to bar press to avoid a 0.35-set duration footshock (60 HY a.~.) delivered to the grid foor through a Foringer. Model 1925 scrambler and rcgulatcd bq an autotransformer. A fixed resistor (270 kQ) in series with the animal provided a rclativcly constant current of 0.75 mA. Each bar press postponed shock onset for 30 set (rcsponse-shock interval). while failure to respond resulted in shock occurring every 3 set (shock shock interval) until a rcsponsc occurred.

METHODS Subject

The subjects were F344 male rats (Microbiological Associates, Walkersville, Maryland; Simenson Laboratories, Gilroy, California) which ranged in age from 75-160 days old at the start of the experiments. Rats were housed one per cage with constant access to food and water. A I?-hr light dark cycle was maintained in the animal quarters at all times and all cxperimental manipulations were done during the light phase of the cycle.

Drug dosages were calculated as the free base, and all solutions. except for the [3H]-tyrosine solution. were made in isotonic saline in a concentration which resulted in an injection volume of 1 ml/kg. (+)-/IChloroamphetaminc hydrochloride (PCA) was obtained from Regis Chemical Company and NSD-1015 (/?I-hydroxybenzylhydrazine hydrochloride) from Aldrich Chemical Company. l-(3.5-3H)-Tyrosinc (50 Ciimmol). purchased from Amersham/Searle, was purified prior to use by lyophilizing to dryness. dissolving in an isotonic solution of saline containing lo”,, 0.3 hl tris (Tris-saline) adjusted to pH X.4. and mixing with 200 mg of alumina for 5 min on a precipitate washer. After ccntrifugation (XOOg, 5 min). the supernatant was carefully removed, diluted with the Tris-saline solution to 0.4 mCi/ml. and adjusted to pH 7.4 with concentrated hydrochloric acid. This solution was injected intravenously via the tail vein in a volume of 2 ml/kg to provide a dose of 0.X mCi!kg.

Sidman (also called free-operant or non-discriminated) avoidance testing was performed in gridfloored operant chambers (26.7 cm high x 30.5 cm wide x 24.1 cm deep). each equipped with a single lever (2.3 x 2.9 x I.Ocm) located 4.9 cm above the floor and requiring a 249 force through an excursion of 0. I6 cm to activate the microswitch. The chambers

The

conversion

index

(CI), dcscribcd by Costa of the turnover rates ol‘ endogenous norepinephrine (NE) and dopamine (DA). The CL which is a relative measure of the ill biro rate of conversion of a pulse dose of [“HI-tyrosine into [“HI-NE and [“HI-DA was culculatcd as the ratio of radioactive catecholamine in disintegrations per minute per gram of tissue to the specific radioactivity 01‘ [3H]-tyrosine in brain. In the present experiments. rats received 0.X mCi.: kg-‘H-tyrosinc in ;I volume of 2 ml./kg just prior to drug treatment. Exactly 15 min after the tyrosine injection, rats were sacrificed by decapitation. and the brains were rapidly removed, frozen on dry ice, and stored frozen (- 70 C) until the time of assay. Endogenous and radioactive tyrosinc. NE and DA were determined by a modification of the procedure described by Neff. Spano. Groppetti. Wang and Costa (1971) as described previously (Steranka c’/ al.. 1077).

( 1971), was used l’or the estimation

The accumulation of dihydroxyphenylalanine (DOPA) in whole brain after an intraperitoneal injection of the aromatic amino acid decarboxylase inhibitor, NSD-1015 (/,I-hydroxybenLylhydrazine) was used as a measure of the irr t?ir~~rate of hydroxylation of tyrosine (Carlsson. Davis. Kchr. Lindqvist and Atack. 1977). Rats were injected with IOOmgikg 01 NSD-1015 15 min prior to drug treatment and sacrificed exactly 30 min after NSD- 1015 administration. The levels of DOPA in whole brain were determined as described previously (Steranka 01 trl.. 1977).

Brain levels ol’ 5-HT were dotermined by the specBogdanski. method ol’ trophotofluorometric Pletscher, Brodie and Udenfricnd (1956).

The levels ol” PCA in whole brain were dctermincd by the methyl orange procedure of Avelrod (1954).

Tolerance Table

I. Effect of acute

and chronic

injection

of PCA on norepinephrine

SAL-SAL Endogenous NE (nmol/g) NE (dpm/g) NE Conversion Index (nmol/g/hr) Endogenous DA (nmol:g) DA (dpmig) DA Conversion Index (nmol,‘g/hr) Brain Tyrosine Specific Activity (dpm/nmol)

3.10 3242 3.59 2.84 2137 2.35 3617

763

to p-chloroamphetamine

f k * * * f &

(6) 0.07 126 0.15 0.09 199 0.14 87

SALPPCA 3.44 4842 4.77 3.09 4240 3.88 4004

and dopamine PCAPSAL

(5)

3.08 5853 X.16 3.52 2991 4.05 3904

* 0.12; + 423’ * 0.34’ If: 0.1 I + 335h * 0.29h k 61

+ * i i_ k * f

conversion

index

PCAPPCA

(5)

3.28 5867 7.92 4.41 3807 4.17 3063

0.12 817’ 1.61” 0.24’ 184” 0.38” 157

) + + I * + f

(7)

0.06 1015’ 1.40” 0.20“ 197b 0.46h 283

Rats were injected intraperitoneally with either saline or PCA (5 mg;kg) at 12-14 hr intervals for 7.5 days (15 injections). Forty-eight hours after the last injection. animals from each drug condition were injected intraperitoneally with either saline or PCA (5 mgikg) immediately after the intravenous administration of 3H-tyrosine. Rats were killed 15 min after receiving ‘H-tyrosine. Values are expressed as the mean + S.E.M.: n is indicated in parentheses. “P < 0.05 compared to SAL-SAL group. "P < 0.01compared to SAL-SAL group. 'P < 0.07 compared to SAL- SAL group.

analyzed by means of appropriate analyses of variance using computational procedures outlined by Kirk (1968). Results from experiments involving repeated measures on a single subject and unequal cell frequencies were analyzed by the unweighted means analysis (Kirk, 1968, pp. 27k-277). Relevant pairwise comparisons following a significant F ratio were tested using Tukey’s HSD test (Kirk, 1968, pp. 9&9 I, 268-269. 277). An analysis of variance was performed on the reciprocals of the CI and dpm/g data for NE (Table 1) after tests of the assumption of homogeneity of variance (F,,, statistic, see Kirk, p. 62) revealed significant violations of this assumption (F,,,,,= 105.3, df = 417, P < 0.01 and F,,,;,,= 78.34, df = 417, P < 0.01, respectively). Results

were

RESULTS

PCA-induced ,functim

jiicilitutiorl

qf repeated

of

Sidrnm

moidmce

us

a

Lldmirlistrcrtion

stead, rats from each drug condition were tested only after the challenge injection of either saline or PCA (5 mg/kg) which was given 48 hr after the last injection of the chronic series and immediately prior to the session. Analysis of variance indicated that response rate (Fig. 2) varied as a function of drug condition (F = 15.50, df = 3/20, P < 0.001). That the decreased effect of PCA after chronic administration is due to the development of physiological tolerance to the drug is evidenced by the^finding that PCA produced a significant increase (Tukey’s HSD test on main effect means, P < 0.01) in responding in rats treated chronically with saline (SAL-PCA) but not in rats treated chronically with PCA (PCA-PCA). Analysis of variance of the shock data (Fig. 3) indicated that the termination of chronic PCA administration (PCA-SAL) resulted in an increase (F = 8.97, df = 3/20, P < 0.001) in the number of shocks received, and that this disruptive effect was offset by the administration of a challenge dose of PCA (PCAPCA).

As shown in Figure 1, PCA (5.0 mg/kg) given immediately prior to each of 9 daily 4-hr test sessions produced a marked increase in responding (F = 11.44, df = l/6, P < 0.02) which diminished gradually over days. As evidenced by the significant drug condition x days interaction (F = 4.33, df = 8/48, P < O.OOl), the drug no longer produced a facilitation of avoidance by the ninth day of testing. .4lthough the PCA-treated animals tended to receive fewer shocks than saline-treated rats, these differences were not statistically significant. Tolerance

to the ,facilitatory

ejjkt

I$ PCA

In order to test the extent to which the decreased effect of PCA after chronic administration could be attributed to the development of physiological tolerance, rats were not tested on Sidman avoidance during the period of chronic injections, which consisted of either saline or 5 mg/kg of PCA twice daily (l&l4 hr intervals) for 7.5 days (I 5 injections). ln-

4ooj 1

I I

2

3

4 DAILY

5 SESSIONS

/ 6

7

I 8

I 9

Fig. 1. Sidman avoidance responding after daily injections of PCA. Rats were injected intraperitoneally with either saline (n = 4) or 5,0mg/kg of PCA (n = 4) immediately prior to each of 9 daily 4 hr test sessions. All rats were injected with saline on day IO. Values presented are means of the 4-hourly values.

764

L. R. STERANKA,

ELAINE

SANDERS-BUSH and R. J. BARRETT

z

s

350-

In _ k 300@. g

250-

: 2 zoo2 CK 2 150z

50-

:: / , I I I I , , , 1 I , I , I, Cl25 0.500.75 1.001.251.50 1.752.00 2.25 2.50275 3.00 3.253.503.754.00 HOURS

Fig. 2. Effects of acute and chronic PCA on Sidman avoidance responding. Rats were injected intraperitonealiy with either saline (n = 12) or 5mg/kg of PCA (n = i2) twice daily (lo-14 hr intervals) for 7.5 days (15 injections). Forty-eight hours after the last injection, animals from each drug condition were injected intraperitoneally with either saline (n = 6) or PCA (5 mg/kg) (n = 6) immediately prior to a 4 hr test session. Values reported represent mean responding during each of the 15 min intervals. Recovery

from

tolerance

to the ,fhcilitatory

effect

of

PCA

In order to test for recovery from tolerance, the rats used in the previous experiment were not injected or run in Sidman avoidance for a period of 2 weeks and then were tested again under the drug conditions reported in the previous experiment (Fig. 4). Complete recovery from tolerance had occurred by this time as evidenced by the finding that PCA increased (F = 6.96, df = 3/20, P < 0.02) Sidman avoidance responding in chronically treated rats (PCA-PCAPCA) to the same extent as in rats not chronically 17 16 15

treated (SAL-PCA-PCA) with the drug. In addition, the increased number of shocks received by animals withdrawn from the drug 2 days before testing was no longer observed after 2 weeks (Fig. 5). Effect

of chronic

of disappearance

administration

qf PCA

on the rate

of the drug .from bruin

Rats administered either saline or 5 mg,&g of PCA Gee daily for 7.5 days were injected intraveneously

(48 hr later) with either saline or fOmg/kg of PCA and killed at various times after injection. In agreement with previous results (Miller, Sanders-Bush and 0 SAL-SAL A SAL-PC/L .PCA-SAL .PCA-PCA

I4

2

I 0.250.500.75 1.001.25 I.50 1.752.00 2.252.502.75 3.003.253.503.754.00 HOURS Fig.3. Effects of acute and chronic PCA on shocks received during Sidman avoidance testing. Values

reported represent mean shocks received during the test session represented in Figure 2.

Tolerance to p-chloroamphetamine 5oor 450 -

O SAL-SAL-SAL A SAL-PCA-PCA l PCA-SAL-SAL n PCA-PCAPCA

400 -

350 -

300

250 I

fI 0.25

11 0.50

075

fi 1.00

III) 1.25

“““‘I 1.50

1.75

2.00

2.25

2.50

2.75

3.00

X.25

3.50

3.75

4.00

HOURS

Fig. 4. Effect of PCA on Sidman avoidance responding after recovery from tolerance. After testing for the development of tolerance to the effect of PCA on Sidman avoidance as reported in Figures 2 and 3, these same rats were not injected or run in Sidman avoidance for a period of 2 weeks and then received the challenge dose again immediately prior to a 4 hr test session. Values reported are mean responses made during each of the I5 min periods,

Dingell, 1971) the levels of PCA in the brains of control rats declined monoexponentially with a half-life of 7.8 hr (Fig. 6). In the brains of rats treated chronically with PCA and injected intravenously with saline at various times before sacrifice, PCA could not be detected (i.e. the levels were less than 1 pg/g). More importantly, the absolute levels of PCA in the brains of tolerant and non-tolerant rats and the rates of disappearance of the drug were not different (Fig. 6).

Efict of acute and chronic PCA administration conversion index for NE and DA

Rats were injected intraperitoneally with either saline or 5 mg/kg of PCA twice daily (l&14 hr intervals) for 7.5 days (15 injections). Forty-eight hours after the last injection, animals from each drug condition were injected intraperitoneally with either saline or 5 mg/kg of PCA immediately after the intravenous administration of 3H-tyrosine.

0 SAL-SAL-SAL

16-

A SAL-PCA-PCA

15-

l PCA-SAL-SAL

14 z 5

n PCA-PCA-PCA

I3 12

lx IO 10 -L II9 .\ u

P7 * 6

0.25

0.50

0.75

1.00

1.25

1.50

I.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

HOURS

Fig. 5. Effect of PCA on shocks received Values reported are mean shocks

on the

during Sidman avoidance testing after recovery from tolerance. received during the test session represented in Figure 4.

L. R. STERANKA, ELAINE SANDERS-BUSH and R. J. BARRETT

766

NE and DA, which may reflect increased synthesis rates; and (3) PCA administered to animals chronically treated with the drug does not exert any additional effect on the synthesis rates of NE and DA relative to PCA-SAL animals. Effect qf‘ucute and chronic PCA on in vivo tprosinr hydroxylase activit?

I 2

1 4

I I6

8

I 24

HOURS

Fig. 6. Brain levels of PCA at various times after the administration of PCA to tolerant and non-tolerant rats. Rats were injected intraperitoneally with either saline or 5 mg/kg of PCA twice daily (IO-14 hr intervals) for 7.5 days (15 injections). Forty-eight hours after the last injection, animals from each drug condition were injected intravenously with IOmg/kg of PCA and killed either 2, 4, 8, 16 or 24 hr later. Values plotted are means k S.E.M. of

Rats treated chronically (twice daily for 7.5 days) with either saline or PCA (5 mg/kg) were injected intraperitoneally with either saline or PCA (5 mg/kg) 48 hr after the last injection of the chronic series. All rats were injected intraperitoneally with 100 mg/kg of NSD-1015 15 min prior to the challenge injection of either saline or PCA and were killed 30 min after the NSD-1015 injection. Results from this experiment are presented in Figure 7. Consistent with results from the conversion index experiment. acute PCA produced a significant increase (F = 10.31. df = .1./17. P < 0.001) in the rate of accumulation of DOPA. However, in sharp contrast to the previous results suggesting an increase in CA synthesis after chronic PCA administration (i.e. an increased CI, Table I). tyrosine hydroxylase activity was not altered in either of the chronic PCA groups.

4 or 5 determinations. Analysis of variance indicated that the CI (Table 1) for both NE (F = 5.14, df = J/19, P < 0.01) and DA (F = 10.60, df = 3/19, P < 0.001) varied significantly as a function of the drug-treatment condition. Consistent with previous results (Steranka et al., 1977), acute PCA produced a significant increase in the CI for DA (Tukey’s HSD test. P < 0.01) and a marginally significant (P < 0.07) increase in the CI for NE. More important, however, was the finding that the CI for both NE and DA was markedly increased (P < 0.05) in animals that were chronically treated with PCA, independent of whether the challenge injection was PCA or saline. Observed changes in the CI were consistently associated with changes in the dpm/g for the amine, while no significant changes in the specific activity of tyrosine were observed. With regard to the endogenous levels of the amines, analysis of variance indicated that endogenous NE (F = 3.96, df = 3/22, P < 0.03) and DA (F = 14.48, df = 3/19, P < 0.001) varied significantly as a function of drug condition. As shown previously (Steranka et ul., 1977), PCA produced a significant increase in the endogenous levels of NE (P < 0.05). In addition, DA was increased in chronically treated animals challenged with either saline (P < 0.07) or PCA (P < 0.05). In summary, analysis of the results from these experiments suggest that (1) acute PCA produces an increase in the rate of synthesis of DA and perhaps NE; (2) withdrawal of the drug after chronic administration results in an increase in the CI values for both

DISCUSSION

The administration of PCA prior to repeated daily test sessions resulted in the gradual decrease in the facilitatory effect of the drug on Sidman avoidance performance over a period of days until, by the ninth 400-

? 2

i

350-

% B f ;

300-

25cIr i

Fig. 7. Whole

S _-SAL

brain

SAL-PCA

levels

PCA-SAL

of DOPA

F

after

cn acute

and

Rats were injected intraperitoneally with either saline or 5 mg/kg PCA twice daily (lG14 hour inter-

chronic

PCA.

vals) for 7.5 days (15 injections). Forty-eight hours after the last injection, rats from each drug condition were injected intraperitoneally with either saline or PCA (5 mg/kg). All rats were injected intraperitoneally with 100 mg/kg of NSD-1015 15 min prior to the challenge injection of either saline or PCA and killed 30 min after the injection of NSD-1015. Values shown are means k S.E.M. of 4 or 5 determinations.

Tolerance to p-chloroamphetamine day of testing, no increase in response rate was observed. Although the repeated testing was necessary to determine what period of treatment would be required to produce behavioural tolerance to PCA, these results do not necessarily reflect the development of a physiological tolerance. Since the increased responding required additional effort with no apparent reward (the number of shocks received was not reduced), it is possible that the animal gradually learned, during repeated test sessions, to perform the task more efficiently, i.e. receive the same number of shocks for fewer responses. Thus, the argument could be made that the physiological action of the drug persists unchanged after chronic administration even though there is a decrease in the resultant behavioural effect. In order to test the extent to which true physiological tolerance develops to the facilitatory effect of PCA on Sidman avoidance performance, a second experiment was conducted in which no behavioural testing was performed during the period of chronic administration, so that any decreased drug effect could not be attributed to a learning process. p-chloramphetamine was given twice daily at lGl4 hr intervals in order to maintain a reasonably high level of the drug in brain throughout the chronic injection period (Ti = 7-X hr). Animals were given a challenge dose of either PCA or saline 48 hr after the last of the I5 injections of the chronic series, at which time practically all of the drug had disappeared from the brains of comparably treated animals. Results of this experiment indicate that physiological tolerance develops to the PCA-induced facilitation of Sidman avoidance performance. Thus. the facilitatory effect was significantly reduced by chronic administration; indeed, animals chronically treated with PCA and then given a challenge dose of the drug did not show a significant increase in response rate. It is important to note that the decreased effect of the drug in chronically treated animals cannot be attributed to general physical debilitation since the response rates of animals chronically treated with PCA and tested after a challenge injection of saline were not different from those of saline controls. Complete recovery from tolerance was observed when animals were tested after a 2-week drug-free period. Since the effects of PCA on serotonergic mechanisms persist for longer than 2 weeks (SandersBush et al., 1972b), this observation adds support to the hypothesis (Steranka et al., 1977) that the facilitatory effect of PCA on Sidman avoidance performance is not dependent upon the initial effects of the drug on serotonergic mechanisms. In addition, these results suggest that the development of tolerance to the facilitatory effect is unrelated to the effects of PCA on central 5-HT mechanisms. Interestingly, tolerance also develops to the increase in motor activity which occurs after PCA administration; however, this occurs after one or two doses of PCA and is apparently mediated by 5-HT mechanisms (El-Yousef rf a/.,

161

unpublished). A counterpart of this tolerance phenomenon has been demonstrated with the Sidman avoidance paradigm, i.e. a rapid disappearance in the initial disruptive effect of PCA occurs with repeated injections of the drug (Steranka et al., 1977). Such rapid tolerance phenomena are easily explained by a depletion of 5-HT by the previous injection of PCA; however, the more gradual reduction in the facilitatory effect of PCA must involve some other neurotransmitter systems, perhaps the CAs. In order to test the hypothesis that the effect of PCA on the metabolism of catecholamines might be altered by chronic drug administration, the conversion of [3H]-tyrosine to C3H]-NE and c3H]-DA and the in uiuo rate of hydroxylation of tyrosine were measured in whole brains after PCA administration. Consistent with previous results (Costa, Naimzada and Revuelta, 1971; Steranka et al., 1977) the acute administration of PCA increased the formation of C3H]-CAs and increased the in uiuo rate of hydroxylation of tyrosine. However, neither of these effects were observed in tolerant rats challenged with PCA. These data suggest that tolerance to the facilitatory effect of PCA on Sidman avoidance performance is associated with tolerance to the ability of the drug to increase the rate of synthesis of the CAs. However, neither the molecular mechanisms responsible for the increased formation of CAs and the increased in uiuo tyrosine hydroxylase activity after acute PCA administration nor those responsible for the tolerance to these effects are clearly understood. In contrast to amphetamine which releases CAs from a newly synthesized pool, PCA is thought to release from the large, intraneuronal storage pool (Strada and Sulser, 1971). However, the levels of DA and NE are not reduced by administration of PCA, presumably because of an increased rate of synthesis as demonstrated in the present study as well as previously (Costa et al., 1971, Steranka et cd., 1977). This increased synthesis after PCA administration does not occur in rats chronically treated with PCA. suggesting that release of CAs no longer occurs in tolerant rats. Other adaptive mechanisms such as changes in rates of CA metabolism or altered receptor sensitivity may also play a role in the behavioural tolerance. Indeed, the present results suggest that tolerant rats have a reduced catecholamine metabolic capacity. Thus, the chronic administration of PCA resulted in an increase in the amount of C3H]-NE and C3H]-DA formed from [3H]-tyrosine, independent of whether animals were given a challenge injection of saline or PCA. However, tyrosine hydroxylase activity was unchanged and the endogenous level of DA was increased after chronic drug administration, suggesting that the increased CI values reflect a decreased utilization or metabolism of the CAs rather than an increased rate of synthesis. Since PCA had practically disappeared from the brains of rats receiving comparable drug treatment, the increased CI values must reflect some adaptive response of the neurones resulting

76X

L. R. STEKANKA. ELAINE SANDERS-BUSH and R. .I. BARRETT

from previous, continuous exposure to the drug. Such a compensatory change may have functional significance with regard to the development of tolerance. The mechanisms of drug tolerance arc generally classified in two categories: altered physiological disposition of the drug and target tissue adaptation. In the former instance, drugs such as barbiturates (Remmer. 1962) cause an increase in the activity of liver microsomal drug-metabolizing enzymes and. by stimulating their own metabolism. produce a state of tolerance. This indirect mechanism of tolerance development is typically referred to as metabolic tolerance. Since chronic administration of PCA did not alter the half-life of the drug in brain, such a mechanism is apparently not responsible for the tolerance which occurs to the facilitatory effect of PCA on bar-press avoidance performance. Instead, this behavioural tolerance must reflect a change in the responsiveness of brain neurones to the presence of PCA. Indeed, the present studies show that a reduced response of CA neurones to PCA is associated with the behavioural tolerance which occurs after its chronic administration. Interestingly, amphetamine also increases bar-press avoidance behaviour. but true physiological tolerance to this effect has not been demonstrated (Leith and Barrett. 1976; Schuster rt rd.. 1966). If changes in the response of CA neurones do in fact mediate the tolerance which occurs after PCA administration, similar adaptive changes do not occur after the chronic administration of amphetamine. Ackrlowlrrl~/e,i2e,rts_These investigations were supported in part by U.S. Public Health Service Grants MH-08107. MH-I 1468. and MH-26463 and the Tennessee Department

of Mcntnl Health and Mental Retardation. Dr. E. SandersBush is recipient of NIMH Research Scientist Development Award MH-70X98. The authors wish to thank Mr. Michael Roh for skillful technical assistance. REFERENCES Axelrod, J. (1954). Studies on sympathomimetic amines. II. The biotransformation and physiological disposition of D-amphetamine. D-p-hydroxyamphetamine. and o-methamphetamine. J. Pharmrrc. esp. Thu. 110: 315-336. Bogdanski, D., Pletscher. A., Brodie, B. and Udenfriend, S. (1966). Identification and assay of serotonin in brain. J. Pharmw. r.~up.Thu. 117: X2-88. Carlsson, A. (1970). Structural specificity for inhibition of “C-S-hydroxytryptamine uptake by cerebral slices. J. Phcrrm. Phurmw. 22: 729-732. Carlsson, A., Davis. J. N.. Kehr. W.. Lindqvist. M. and Atack. C. V. (1972). Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in rlir)o using an inhibitor of the aromatic amimo acid decarboxylase. Naun~n-S[,kmirdrhergs Arch. P.Y~. Path. Phormnk. 275: I541 68. Costa, E. (1971). Methods for measuring indolealkylamine and catecholamine turnover rate in uiro. Adtx e\-p. Med. Biol. 13: 157-174. Costa. E.. Naimzadd, K. and Revuelta, A. (1971). Effect of phenmetrazine, aminorex, and (k ) p-chloroamphetamint on the motor activity and turnover rate of brain catecholamines. Br. J. Phnrmtrc. 43: 57@-579. Fuller. R. (1966). Serotonin oxidation by rat brain mono-

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