The effect of phencyclidine on dopamine metabolism in the mouse brain

Life Sciences, Vol. 28, pp. 361-369 Printed in the U.S.A.

Pergamon Pres~

THE EFFECT OF PHENCYCLIDINE ON DOPAMINE METABOLISM IN THE MOUSE BRAIN Kenneth M. Johnson and Kevln C. Oeffinger Department of Pharmacology and Toxicology The University of Texas Medical Branch Galveston, TX 77550 (Received in final form November 7, 1980)

Summary The a d m i n i s t r a t i o n of p h e n c y c l i d i n e (PCP) to mice r e s u l t e d in no change in b r a i n l e v e l s of t y r o s i n e , dopamine (DA), norepinephrJne (NE), or homovanillic acid (HVA). Although PCP reduced plasma tyrosine levels, no effect of PCP on the utilization of DA or NE after blockade of synthesis with ~-methyl-p-tyrosine (AMPT) was observed. In addition, PCP did not affect the probenecid-induced accumulation of HVA. However, PCP was observed to potentiate the haloperidolinduced increase in HVA concentration, and the haloperidol-induced decline in DA levels after AMPT. The former effect was blocked by baclofen, suggesting that PCP mobilizes BA for impulse-dependent release. This effect could not be attributed to an antagonism of presynaptic DA receptors. These effects are similar to those of the "non-amphetamine" stimulant class of drugs. The r e s u l t s of both behavioral and biochemical studies on the rodent suggest t h a t the c e n t r a l e f f e c t s of p h e n c y c l i d i n e (PCP) may be mediated through dopaminergic ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 1 0 ) , noradrenergic ( 3 , 4 , 1 1 , 1 2 , 1 3 ) , s e r o t o n e r g i c ( 4 , 1 4 , 1 5 , 1 6 , 1 7 ) , c h o l i n e r g i c ( 2 , 1 8 , 1 9 , 2 0 , 2 1 , 2 2 ) , GABA-ergic (23,24), and/or enk e p h a l i n e r g i c neurons (20,25). At t h i s time, i t is unknown which, i f any, o f these systems is p r i m a r i l y responsible f o r the expression o f the behavioral e f f e c t s of PCP. However, many i n v e s t i g a t o r s feel that the "PCP psychosis" which o c c a s i o n a l l y r e s u l t s a f t e r acute i n t o x i c a t i o n resembles paranoid schizoprenia (26), and t h i s , in t u r n , implies the involvement of dopamine (DA) neurons. A l though the e f f e c t s of PCP on catecholamine (CA) f u n c t i o n have been the most w i d e l y s t u d i e d , i t s e f f e c t s are s t i l l poorly understood. The behavioral l i t e r a t u r e suggests that PCP acts as an i n d i r e c t dopaminergic a g o n i s t , s i m i l a r to amphetamine. For example, PCP produces i p s i l a t e r a l r o t a t i o n in rats w i t h e i t h e r e l e c t r o l y t i c (2) or 6-hydroxydopamine (7) lesions of the n i g r o - s t r i a t a l DA pathway. This e f f e c t is attenuated w i t h s - m e t h y l - p t y r o s i n e (AMPT,2,7) or h a l o p e r i d o l (HAL,2). S i m i l a r l y , PCP-induced s t e r o t y p i c behavior in the monkey (5) and rat (6) is blocked by n e u r o l e p t i c s . Interesti n g l y some PeP-induced behaviors have also been attenuated w i t h a low dose of apomorphine suggesting t h a t PCP may block p r e s y n a p t i c DA receptors (10). Studies using biochemical techniques also show some s i m i l a r i t i e s between PCP and amphetamine. Like amphetamine, PCP has been shown to i n h i b i t synaptosomal CA uptake (3,4,8,9) and to f a c i l i t a t e synaptosomal DA release ( 8 , 9 ) . In a d d i t i o n , i t has been demonstrated that the release of synaptosomal DA is associated w i t h an increase in t y r o s i n e hydroxylase (TH) a c t i v i t y (8), an e f f e c t 0024-3205/81/040361-09502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

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shared with amphetamine (27), presumably due to a decrease in end-product feedback inhibition. Since reserpine can block the stimulatory effects on TH by PCP (8), but not by amphetamine (27), PCP appears to be more similar to methylphenidate than to amphetamine (27). Although one group reported that PCP partially blocked the conversion of 14C-tyrosine into I~C-CA (I), another group failed to observe any effect of PCP on the decline of CA after AMPT (11). In the study reported here, we have attempted to clarify the effects of PCP on DA (and to some extent NE) turnover and utilization in vivo. We now report that PCP has little effect on CA steady-state concentrations or turnover, but, like the non-amphetamine stimulants methylphenidate and amfonelic acid, appears to mobilize DA for impulse-dependent release. Methods Male ICR mice (22-32 g) obtained from Texas Inbred Mouse Co., Houston, TX, were used in all experiments. The mice were housed in large group cages under constant lighting conditions (lights were on from 0800 h - 2000 h) for at least 10 days before the start of an experiment. At least 24 h before an experiment, the mice were transferred to the experimental room. All drugs were administered in a counter balanced design and the time of death remained constant (between 1000 and 12OO h). The mice were killed by decapitation, the brains removed, weighed, and homogenized in 0.4N HCI04 containing 0.05% disodium EDTA and sodium metabisulfite. The homogenate was kept cold until centrifugation at 12,000 x g for 15 minutes. The supernatant was frozen at -70°C until assayed. In all experiments in which homovanillic acid (HVA) was measured the forebrains (including the midbrains) of 2-3 mice were pooled for analysis. The extracts from the cerebella were used as tissue blanks for the assay of DA and HVA. In other experiments the assay of DA and/or norepinephrine (NE) was performed on the whole brain from individual mice. Catecholamines were adsorbed onto alumina at pH 8.4-8.6 and eluted with 0.2 N acetic acid (28). The CA were assayed fluorometrically according to Shellenberger and Gordon (29). HVA was extracted from the perchloric acid fraction after removal of CA according to Spano and Neff (30), except that ethyl acetate was substituted for butyl acetate. HVA was analyzed fluorometrically (31). In some experiments tyrosine was assayed in percholic acid fractions of plasma and brain tissue according toWaalkes and Udenfriend (32). Phencyclidine HCI (NIDA), d-amphetamine sulfate (NIDA), (±) baclofen HCI (Ciba-Geigy), y-butyrolactone (Sigma Chemical Co.) and DL-~-methyl-p-tyrosine (Sigma Chemical Co.) were dissolved in 0.9% NaCI (saline). Apomorphine HCI (Sigma Chemical Co.) was dissolved in saline containing O.1% ascorbic acTd. Probenecid (Sigma Chemical Co.) was dissolved in phosphate buffer and Na0H and the pH was adjusted to 8.0 with HCI; haloperidol (McNeil) was purchased in an injectable form. All drugs were administered in the form listed above in a volume of 10 ml/kg (s.c.). Comparison of group means was accomplished with the use of Dunnett's modification of the t-test which uses the overall error estimate from the analysis of variance. Differences were considered significant if the t value exceeded the value for P < 0.05 (two-tailed). All values reported are the means ± S.E.M.

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Results In this study we used 2,4,8 and 16 mg/kg PCP° Gross observation of these mice indicates that the lowest dose produces a very slight increased activity and no stereotypic behaviors. After 4 mg/kg the mice showed considerable hyperactivity and some stereotypic activity consisting chiefly of increased sniffing and side-to-side head movements. They also are slightly ataxic. At 8 mg/kg the mice demonstrated less locomotor activity, but were more ataxic and demonstrated a greater frequency of stereotypic behaviors than those given 4 mg/kg. After 8 mg/kg the mice were very ataxic after one hour, but most appeared to recover after 2.5 hours. At even higher doses (e.g. 16 mg/kg) the mice become grossly ataxic and were dyskinetic. In our experience, the same sequential constellation of behaviors can be observed in the rat after PCP, but the rat appears to be about 1.5-2.O times more sensitive to PCP than the mouse. TABLE I Effect of PCP on Plasma and Brain Tyros±he and Brain Catecholamines Time (hr)

PCP (mg/kg)

Tyr (ug/9) Plasma Brain

CA (ng/g) DA

NE

I 1 I

0 4 8

22.8±1.0 18.3±I.O~ 18.7±1.4""

17.9±1.5 15.0±1.2 14.7±0.5

576±34 583±21 652±32

284±15 276± 8 323± 8

1,4 a I 4

O 8 8

17.1±1.6 13.1±0.7 13.9±1.8

13.0±1.O 13.4±1.6 12.6±1.4

674±28 704±50 699±63

348±17 367±20 347±21

aMice were treated with saline (O) and half were killed at one hour and half at four hours. The data represent the results of two separate experiments. ,N=6 or 7 in each group Significantly different from saline (P
ng/9 N 13 3 8 3 3

HVA 129±10 124±11 115± 7 119±16 162±11

DA 886± 47 1025± 50 905± 36 883±107 963± 72

aThe experimental mice were injected with 8 mg/kg PCP and killed at the indiboated times. Saline (10 ml/kg) was administered to these mice. Eight were killed after 60 minutes and five after 180 minutes.

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The effect of PCP on the probenecid-induced accumulation of HVA was examined in two experiments (data not shown). In the first, mice were injected with either saline or PCP (8 mg/kg) at zero minutes, followed by probenecid (200 mg/ kg) at 90 minutes: the mice were killed at 180 minutes. In the second experiment, mice were administered probenecid (200 mg/kg) at 0 minutes, then either saline or PCP (8 mg/kg) at 50 minutes, and the mice were killed at 100 minutes. In neither experiment did PCP have any effect on either HVA levels or the HVA/DA ratio. For example, in the second experiment the HVA/DA values were 0.307±0.21 and 0.315±0.019 for the saline and PCP treated groups, respectively. In addition, the effect of PCP on the AMPT-induced decline of DA levels was tested in an experiment which utilized five groups of six mice. One hour after saline or AMPT (100 mg/kg) or AMPT plus either 0,4,8 or 16 mg/kg PCP, the mice were sacrificed. AMPT alone depleted DA levels from 930±40 ng/g to 730±50 ng/g. No dose of PCP plus AMPT was significantly different from AMPT alone (data not shown). TABLE 3 The Effect of PCP and Haloperidol

on the AMPT - induced Decline of Brain DA

Treatment I Hour Exp. Saline AMPT AMPT + PCP AMPT + HAL AMPT + HAL + PCP

DA (% Control) a b

100+4 (10) 74±2 (10) 76±2 ( 5 ) 56±3 (12)~i..~ 44±4 (12) ....

3 Hour Exp.

c

100±4 (11) 64±4 (12) 55±4 (8) 50±5 (12)* 43±4 (12)*

aThese data are the mean±S.E.M. (N) as compiled from five independent experibments. In these two experiments AMPT (250 mg/kg), PCP (8 mg/kg), and HAL (I mg/kg) were injected either alone or in combination in the same syringe. The mice were decapitated after one hour. Cln these experiments AMPT or saline was administered at time zero, HAL or saline at two minutes, and PCP or saline at four minutes (all in the same dose as ,the one hour experiment) and the animals were decapitated after 180 minutes. ,§ignificantly different from AMPT alone (P
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PCP, at a dose which has no effect on either HVA or DA concentration in the brain, significantly potentiates the increase in DA metabolism by HAL.

TABLE 4 Effect of PCP on the Increase in DA Metabolism by Haloperidol Treatment a Saline Haloperidol Haloperidol + PCP

N

HVA

8 12 8

137±14 252±11" 321±15"+

ng/g

DA 975±53 863±31" 689±17"+

aHaloperidol (I mg/kg) was aministered either alone or in the same syringe ,with PCP (8 mg/kg). The mice were killed one hour later. Significantly different from saline treated control (P
The dependence of this potentiation on impulse flow was examined by testing the effect of HAL and HAL plus PCP on DA metabolism (HVA/DA ratio) in the presence and absence of (±) baclofen. These data are shown in Figure I. As before, PCP stimulated DA metabolism when administered with HAL. This effect was blocked with 10 mg/kg baclofen, a dose known to block PCP-induced locomotor activity (24). However, this dose also blocked the induction of DA metabolism by HAL. A dose of baclofen (2.5 mg/kg) which does not block the HAL effect, had no effect on the potentiation of HAL by PCP. The reported ability of PCP to act as an antagonist of presynaptic DA receptors (10) was tested using the model described by Walters and Roth (33) in which the role of postsynaptic receptors is minimized by the reducation of impulse flow by 7-butyrolactone (GBL). The results of this experiment are shown in Table 5. As can be seen apomorphine (APO) partially reversed the GBL-induced elevation in DA levels. PCP was unable to antagonize this effect. Furthermore, PCP had no agonist-like activity of its own, as it had no effect on DA levels in the GBL treated mice.

TABLE 5 Effect of PCP and Apomorphine on GBL-Induced Elevation of Brain DA Treatment a

N

DA (ng/g)

GBL GBL + APO GBL + PCP GBL + APO + PCP

5 6 5 6

1560±55 1114±58 ~ 1603±99. 1077±58 ~

aGBL (680 mg/kg), apomorphine (I mg/kg), and PCP (8 mg/kg) were administered in ,the indicated combinations and the mice were killed after one hour. Significantly different from GBL alone (P
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4(;

30 ,e=.,. x

> '-r

20

10 HAL HAL PCP

HAL HAL 2.5B 2.5B PCP

HAL HAL 10B 10B PCP

FIG. I. The Potentiation of HAL-Induced DA Metabolism by PCP is Blocked by Baclofen HAL (I mg/kg), PCP (8 mg/kg), and baclofen (B) in the indicated dose (mg/kg), were administered one hour before the mice were killed. Results are express,ed as the mean ± S.E.M. with N inset in the bar. ~.Significantly different from HAL (P
Discussion The lack of effect of PCP on the mouse brain NE concentration is in agreement with a study which utilized guinea pigs (34), but is at variance with one that showed a reduction in rat brain NE levels (11). This study showed no effect of PCP on,AMPT-induced delcine of NE, which is in agreement with a similar study in rats (11) which also showed that PCP had no effect on normethanephrine levels. Utilizing the conversion of radiolabeled tyrosine to radiolabeled CA and metabolites as an index of synthesis, one group has reported a decrease in NE synthesis in the mouse brain (35). PCP has also been reported to decrease

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normetanephrine l e v e l s in the r a t (36) and to increase the formation o f Z4C-normetanephrine from 14C-tyroslne (1). Thus, no c l e a r p i c t u r e o f the e f f e c t s o f PCP on NE has emerged and i t is probably safe to assume that there is no major e f f e c t d i s c e r n i b l e at the whole b r a i n l e v e l o f a n a l y s i s . Similarly, the effects of PCP on DA synthesis and metabolism are unclear. Using both the probenecid-induced accumulation of HVA and the decline of DA after AMPT as indices of DA turnover, the results of this study suggest that PCP has no discernible effect on DA turnover in the mouse brain. This is in agreemerit with other workers who utilized the AMPT technique in the rat (11, and H.Y. Meltzer, personal communication) and the conversion of radiolabeled tyrosine technique in the mouse (35). However, in direct distinction to these studies, Meltzer (personal communication) has observed that PCP blocks the accumulation of DOPA after decarboxylase inhibition, and Hitzemann et al. (I) reported that PCP blocks the conversion of 14C-tyrosine to I~C-DA in the rat. Thus, the effects of PCP on DA turnover may be species dependent and may be subtle enough to require verification by more than one technique. In spite of the apparent lack of effect of PCP on DA turnover in the mouse, PCP was observed to potentiate both the HAL-induced decline in DA levels after AMPT and the HAL-induced elevation of HVA and HVA/DA. Certain non-amphetamine stimulants such as cocaine, methylphenidate, and amfonelic acid (AFA) have been reported te have similar effects (37). For example, AFA administration results in no change in striatal DA levels (37), a slight increase in DA metabolites, HVA (37) and dihydroxyphenylacetic acid (37,38), and no change in DA levels after AMPT (37). However, AFA greatly potentiated the decline in DA concentration after AMPT produced by HAL (37), and potentiated the increase in DA metabofires after HAL (37,39) or spiperone (38). These results are in marked contrast to those observed after administration of amphetamine (37,38). It has been postulated that, since HAL is believed to increase DA turnover by reflexly stimulating nigrostriatal impulse flow, AFA acts to increase the amount of DA available for impulse-induced DA release (37). This hypothesis was supported by the demonstration that drugs which block impulse flow (apornorphine and T-hydroxybutyric acid) were able to block the potentiating effect of AFA on HAL-induced DA turnover (37). Baclofen (B-p-chlorophenyl GABA) is an agent known to increase DA synthesis (40) and reduce DA release (40,41), apparently as a consequence of reduced impulse flow in the nigrostriatal pathway (42). Thus, the similarities between baclofen and ¥-hydroxybutyric acid (41), and the report that baclofen blocked PCP-induced motor activity (24), led to the experiment described in Fig. I. The blockade of the HAL-PCP effect on DA metabolism by baclofen suggests that, like AFA, the effect of PCP is dependent on impulse flow. That baclofen could only block the effect of PCP at a dose which also blocked the HAL effect suggests that PCP may not be acting as a GABA antagonist as others have suggested (24). However, baclofen probably does not act directly on GABA receptors (43), so. the potential GABA antagonist activity of PCP can not be ruled out by this experiment On the basis o f behavloral studies showing that a very low dose o f apomorphine (0.04 mg/kg) completely reversed PCP-induced locomotor a c t i v i t y , Garey et a l . r e c e n t l y p o s t u l a t e d t h a t PCP was a c t i n g as an i n d i r e c t DA agonls t by blocking presynaptic DA receptors which normally f u n c t i o n to i n h i b i t DA release (10). Our study (Table 5) showing that PCP did not antagonize the a c t i o n of apomorphine in GBL t r e a t e d mice, suggests that PCP does not act as an antagon i s t o f p r e s y n a p t l c receptors. However, since we measured DA in the whole b rai n (which consists p r i m a r i l y o f s t r i a t a l DA), i t is p o s s i b l e t h a t a l t e r a t i o n s in s ma l l e r DA areas (e.g. the nucleus accumbens) would be missed using

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this model. Alternatively, since these studies show the dopaminergic effects of PCP to be dependent on impulse flow, the inhibition of PCP-induced locomotor activity by apomorphine (10) may rely on the ability of apomorphine to inhibit nigrostriatal impulse flow (44). Although these results and those of a very recent study conducted in the rat (45) underscore the ability of PCP to release DA, in vitro tests show PCP to be a more potent inhibitor of DA uptake (8,45). Also, there appears to be a rather good correlation between the inhibition of DA uptake and the potentiation of neuroleptic-induced DA metabolism in the rat by the non-amphetamine stimulants (46). In this regard, amfonelic acid appears to be several times more potent PCP in facilitating HAL promotion of HVA accumulation (37) and, in fact, is about 25 times more potent than PCP as an inhibitor of striatal DA uptake (8,46). However, the mechanism underlying this correlation remains unclear since amfonelic acid inhibits DA uptake in both rat and rabbit synaptosomes, but potentiates HAL induction of DA release only in the rat (46). Since HAL increases the synaptic concentration of DA, the influence of a competitive inhibitor of DA uptake such as PCP would be reduced. Apparently, only under these conditions is the ability of PCP to promote DA release uncovered (this study and 45). In conclusion, the effects of PCP on DA metabolism are unmasked only in the presence of DA receptor blocking agent (HAL) which enhances nigrostriatal impulse flow. Under these conditions PCP appears to mobilize DA for release. The precise mechanism for this effect remains unclear. Although other durgs have been described which share this action (e.g. cocaine, methylphenidate, and AFA) there are reported effects of PCP which make it difficult to classify PCP along with the methylphenidate group. For example, the behavioral effects of this group of non-amphetamine stimulants can be blocked by reserpine, but not AMPT (47). Since AMPT has been reported to partially block the PCP-induced circling behavior of rats with nigrostriatal lesions (2,7), we suggest that PCP fits into neither the amphetamine nor methylphenidate class of stimulants. This uniqueness is likely the result of the combined actions of PCP on the several neurotransmitter systems which mediate the affected behaviors. Acknowled@ement This work was supported by a grant from the USPHS (DA-02073). References I. 2.

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