Brain Research, 521 (1990) 175-184 Elsevier

175

BRES 15660

Stimulant-conditioned locomotion is not affected by blockade of and/or D 2 dopamine receptors during conditioning

D 1

Mathew Thomas Martin-Iverson and David John McManus Neurochemical Research Unit and PMHAC Research Unit, Department of Psychiatry, University of Alberta, Edmonton, Alta. (Canada) (Accepted 9 January 1990) Key words: Amphetamine; (+)-4-Propyl-9-hydroxynaphthoxazine; Haloperidol; SCH 23390; Conditioned locomotion; Rat; Dopamine; Dl-receptor; D2-receptor; Dopamine receptor

A series of experiments were conducted to investigate the role of dopamine (DA) D 1 and D 2 receptor subtypes in stimulant-conditioned locomotion in rats. Expt. 1 demonstrated that locomotion could be induced by a testing situation when that situation was previously paired with (+)-amphetamine (1.5 mg/kg, s.c.) or a D 2 receptor selective agonist (PHNO, 15 or 30/~g/kg, s.c.), but not when the drug treatments were given 3 h after exposure to the situation. The selective D 2 receptor antagonist, haloperidol (50/~g/kg, i.p.), and the D 1 receptor antagonist, SCH 23390 (20 ag/kg, s.c.), blocked amphetamine-induced locomotion during the pairing process, but failed to block amphetamine-conditioned locomotion as assessed during a drug-free test in Expt. 2. This was true when the antagonists were given separately or together. The results of Expts. 3 and 4 showed that doses of the D 1 (20/~g/kg, s.c.) and D 2 antagonist (250 ag/kg, i.p.) that blocked the unconditioned locomotor effects of PHNO failed to block its conditioned locomotion. It is concluded that neither D 1 nor D 2 DA receptors are essential for the development of stimulant-conditioned locomotion. INTRODUCTION Repeated ( + ) - a m p h e t a m i n e injections to rats in an initially novel environment leads to the conditioning of the locomotor stimulant effects of the drug to the environmental stimuli 12'15'18. Conditioned locomotion is revealed on a subsequent test day when the animals are placed in the environment without drug. If the animals exhibit higher rates of locomotion than controls, then it is concluded that conditioned locomotion occurred, providing that control groups rule out a non-environment specific increase in locomotion. This conditioning effect likely contributes to the 'sensitization' phenomenon (gradual augmentation of behavioral effects) often observed with chronic amphetamine treatments, and thought by some investigators to be related to stimulantinduced psychoses, as reviewed by Robinson and Becker 14. Beninger and H a h n 2 observed that pimozide, a dopamine ( D A ) D 2 receptor antagonist, given during conditioning to block the unconditioned stimulant effects of ( + ) - a m p h e t a m i n e , attenuated conditioned locomotor activity. However, we have observed that haloperidol, a neuroleptic that is also selective for the D 2 receptor at lower doses 1, failed to block locomotion conditioned with amphetamine or methylphenidate 9. This occurred despite

the fact that the doses of haloperidol used effectively blocked the direct m o t o r stimulant effects of the stimulants. These previous experiments were not designed to directly address the conditioned locomotion phenomenon, and therefore appropriate control groups to rule out increases in locomotion independent of conditioning were not included. In the present experiments, we directly investigate the conditioning of amphetamine-induced locomotion, and the effects of haloperidol and SCH 23390, a D 1 receptor antagonist 6, given separately or together during the conditioning procedure. In addition, we investigated whether or not the locomotor stimulant effects of (+)-4-propyl-9-hydroxynaphthoxazine ( P H N O ) , a direct D2 agonist 7, could be conditioned to environmental stimuli, and whether such conditioning could be blocked with a D 1 and/or a D 2 antagonist. Four experiments were conducted in total. Expt. 1 was designed to determine whether or not repeated injections with one dose of ( + ) - a m p h e t a m i n e or each of two doses of P H N O could produce conditioned locomotor activity. Additional groups were included to assess whether or not the pairing of drug effects with the injection ritual + test boxes stimulus complex was necessary to produce an increase in locomotor activity on the test day. Expt. 2 examined the possibility that haloperidol, a selective D 2

Correspondence: M.T. Martin-Iverson, Neurochemical Research Unit and PMHAC Research Unit, Department of Psychiatry, University of Alberta, Edmonton Alta. T6G 2B7, Canada. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

176 a n t a g o n i s t at t h e d o s e u s e d , o r S C H 23390, a s e l e c t i v e D~ a n t a g o n i s t , g i v e n a l o n e o r in c o m b i n a t i o n c o u l d b l o c k t h e u n c o n d i t i o n e d a n d c o n d i t i o n e d l o c o m o t o r s t i m u l a n t effects

of

(+)-amphetamine

or

PHNO,

using

30-min

c o n d i t i o n i n g sessions. E x p t . 3 t e s t e d t h e effects o f t h e a n t a g o n i s t s at b l o c k i n g t h e u n c o n d i t i o n e d t i o n e d l o c o m o t o r effects o f P H N O ,

and condi-

increasing the dura-

t i o n o f t h e e x p o s u r e s to t h e test b o x e s d u r i n g c o n d i t i o n ing to 60 m i n . T h i s e x p e r i m e n t was c o n d u c t e d b e c a u s e t h e u n c o n d i t i o n e d l o c o m o t o r s t i m u l a n t effects of P H N O a r e n o t a p p a r e n t d u r i n g t h e first 40 m i n p o s t i n j e c t i o n . E x p t . 4 was a f u r t h e r s t u d y of t h e effects of h a l o p e r i d o l o n t h e u n c o n d i t i o n e d a n d c o n d i t i o n e d effects of P H N O . In this last e x p e r i m e n t , r a t s w e r e t r e a t e d with a h i g h e r dose of haloperidol and a lower dose of PHNO than used in t h e p r e v i o u s e x p e r i m e n t .

MATERIALS AND METHODS

Animals, apparatus and drugs Male Sprague-Dawley rats (n = 11 or 12 for each group) weighing 250-350 g were used in all experiments. Locomotor activity counts were made in test boxes equipped with two infrared photocell assemblies in each, with a sensitivity set so that rapid interruptions did not register. This setting was chosen so that only gross locomotor activity would be counted; rapid movements of the head, paws or tail were not counted. The test boxes (48 in total) were made of stainless steel, with one wall being Plexiglas, and the floor made of a steel mesh. The dimensions of these boxes were 25 (W) x 25 (H) x 30 (L) cm, with the two photocells placed 3 cm from the floor on the side walls, spaced 14 cm apart, and equidistant from the end walls. Photobeam interruptions were recorded in 5 min blocks by a Commodore 64 computer. (+)-Amphetamine sulfate was provided courtesy of Dr. R. T. Coutts (Pharmaceutical Sciences, University of Alberta), (+)-4-propyl-9-hydroxynaphthoxazine HCI (PHNO) was courtesy of Merck Sharp and Dohme, haloperidol (Haldol) was purchased from McNeil as 1 ml ampoules containing 5 mg haloperidol dissolved in a solution of methylparaben (1.8 rag), propylparaben (0.2 rag) and lactic acid, SCH 23390 was purchased from Research Biochemicals Inc. All drugs were dissolved or diluted (in the case of haloperidol) with distilled water. Dosages of the drugs used were chosen on the basis of pilot studies, and previous experience of the investigators with these compounds. The doses of haloperidol and SCH 23390 were chosen in order to adequately block the unconditioned effects of the stimulants. Amphetamine and PHNO doses were chosen to produce optimal photobeam interruption counts, over the course of the 10 days of treatment.

Procedures Four conditioning experiments were conducted, all of which consisted of two phases: a drug conditioning phase, and a test day on which no drugs were given. The conditioning phase consisted of repeated daily drug or vehicle injections and 60 min (Expts. 1, 3, 4) or 30 min (Expt. 2) placements in the (initially) novel testing boxes for a total of 10 consecutive days. The test day, on which the rats were placed into the test boxes for 60 min (all experiments), occurred on the fourth day after the last conditioning injection (i.e. 3 intervening non-testing days), to allow for drug clearance. No drugs were administered on this day, although most groups received an injection of vehicle prior to placement into the test boxes (see below for the exceptions). Expt. 1 consisted of 8 treatment groups as follows: groups 1 and 5 (vehicle, i.e. distilled water), groups 2 and 6 (1.5 mg/kg

(+)-amphetamine), groups 3 and 7 (15 !~g/kg PttNO), groups 4 and 8 (30 ~g/kg PHNO), with all weights expressed as the salts, and all injections s.c. During the conditioning phase, groups 1-4 were given injections 10 rain prior to placement in the test boxes, while groups 5-8 received injections in their home cages 3 h after removal from the test boxes. On the test day, groups 1-4 received vehicle injections 10 min prior to placement in the test boxes, while groups 5-8 did not. This was to avoid the possibility of measuring locomotor activity conditioned to the injection ritual in the groups receiving home cage injections. A randomized block design was followed in Expt. 2 to examine the effects of DA antagonists on stimulant-conditioned locomotion. There were 12 treatment groups, with each animal receiving 3 injections, consisting of vehicle (i.p.) or haloperidol (i.p.) 70 rain before placement in the test boxes, vehicle (s.c.) or SCH 23390 (s.c.) 40 rain before placement in the test boxes, and vehicle (s.c.), (+)-amphetamine (s.c.) or PHNO (s.c.) 10 rain before placement in the test boxes. Thus, the groups were as follows, where V = vehicle, A = (+)-amphetamine 1.5 mg/kg, P = PHNO 30 ug/kg, S = SCH 23390 20,ug/kg, and H = haloperidol 50Bg/kg: group I, VVV; group 2, VVA; group 3, VVP; group 4, VSV; group 5, VSA; group 6, VSP; group 7, HVV; group 8, HVA; group 9, HVP; group 10, HSV; group 11, HSA; and group 12, HSE In this experiment, the 10 conditioning sessions were each of 30 rain duration. This time was chosen because previous pilot studies indicated that it was sufficient to produce conditioned locomotion. Expt. 3 further tested the ability of the selective antagonists to block the unconditioned and conditioned effects of PHNO (30 ug/kg). Conditioning sessions were of a 60 min duration, as the effects of the antagonists on the locomotor activity elicited by PHNO were not testable with the 30 min conditioning trials used in Expt. 2. In the last experiment (Expt. 4), the effect of a higher dose of haloperidol (200 ug/kg) on PHNO-conditioned activity was assessed. There were 4 groups in Expt. 4, VV, HV, VP and HP, with the dose of PHNO being 15 ug/kg. Ten daily 60-min conditioning trials were followed 3 days later by a test day consisting of a 60-rain test, 10 rain after an injection of vehicle. The photobeam interruptions recorded in 5 rain blocks for each rat in every experiment were combined to produce daily totals, and these daily totals were subjected to appropriate ANOVAs with post-hoe comparisons between groups by Tukey's test or Dunnen's test, as appropriate. Data from the conditioning trials (as opposed to the test days) were also subjected to various multivariate tests for any term that involved the days effect. This was done because repeated measures ANOVA is inappropriate when the assumption of homogeneity of covariances is violated. Violation of this assumption is the usual case during repeated drug studies or learning studies, when the response is dependent upon its place in the sequence of measures. The 3 multivariate tests employed were Pillais Trace, Hotellings T and Wilks Lambda. Probability values determined by ANOVA were confirmed by these methods, unless specified otherwise.

RESULTS

Expt. 1: conditioning of amphetamine and PHNO activity Expt. 1 included a 3-factor conditioning design. The first i n d e p e n d e n t

f a c t o r w a s t h e p l a c e a n d t i m i n g of

i n j e c t i o n , w i t h a n i m a l s i n j e c t e d in t h e t e s t r o o m 10 m i n p r i o r to p l a c e m e n t in t h e l o c o m o t o r b o x e s , o r in t h e i r h o m e c a g e s in t h e a n i m a l q u a r t e r s , 3 h a f t e r r e m o v a l f r o m t h e l o c o m o t o r cages. T h i s f a c t o r is c a l l e d ' p a i r i n g ' to i n d i c a t e w h e t h e r o r n o t t h e d r u g e f f e c t s w e r e p a i r e d with the testing environment. The second independent f a c t o r was t h e d r u g g i v e n t o t h e r a t s ( v e h i c l e , a m p h e t -

177 PHOTOBEAMINTERRUpTIONS 5OO •

450-

VEH

~-

AMPH

~-

PHNO (16 uo/kg)

~

PHNO (30 ug/kg)

400 350300250 200 ~ 1504 100 50

+-

02

0

4

---+

I

I

6 8 CONSECUTIVE DAYS

10

12

Fig. 1. The effects of 10 consecutive days of injections (s.c.) of vehicle (VEH), amphetamine (AMPH, 1.5 mg/kg), and two doses of a selective D2 agonist (PHNO, dose in/~g/kg) on locomotion (photobeam interruptions) of rats unpaired with the test boxes. Injections were made 3 h after 1 h in the test boxes. There was no significant effect of drug treatments, although there was a trend for all groups given stimulants to exhibit decreased locomotion on the last 4 days.

amine, P H N O (15 /~g/kg) or P H N O (30/~g/kg)). The third factor (dependent) was days of treatment. A N O V A revealed that the Pairing x Drug × Days interaction was significant (F27,792 ~-- 4.25, P < 0.001). Locomotor activity from the groups of rats given drug injections in the home cage (unpaired) is displayed in Fig. 1 and the locomotor activity of the groups receiving paired injections is shown in Fig. 2. All groups exhibited increased activity on Day 1 as compared to Day 2, an

PHOTOBEAMINTERRUPTIONS 500

400 350 ! 300 250 200 150

.

.

......

100 50

°

VEHICLE

2

- e - AMPH

4

~-

PHNO (15 ug/kg)

6

~-

8

PHNO (30 ug/kg)

10

12

CONSECUTIVE DAYS

Fig. 2. The effects of 10 consecutive days of injections (s.c.) of vehicle, amphetamine (AMPH, 1.5 mg/kg), and two doses of a selective D 2 agonist (PHNO, dose in /~g/kg) on locomotion (photobeam interruptions) of rats paired with the test boxes. Injections were made 10 rain prior to 1 h in the test boxes. AMPH significantly increased locomotion relative to controls on all days, whereas both doses of PHNO increased locomotion only on days 6--10.

expected habituation effect, with the exception of the two groups receiving the highest dose of PHNO. It is of interest that this habituation effect was not evident in either of the groups receiving the highest dose of PHNO, regardless of whether or not the injection was paired with the testing apparatus. Post-hoc comparisons indicate no sustained significant differences between the vehicle group and those receiving any of the other drug injections unpaired with the test boxes. However, from days 5-10 all 3 drug-treated groups exhibited less activity than controls, which may suggest a trend towards conditioning of compensatory mechanisms in anticipation of drug treatment. This trend was not statistically significant for the most part (only the unpaired amphetamine-treated group exhibited significantly less activity and only on days 7 and 9). The locomotor activity pattern in those groups receiving paired injections (Fig. 2) was quite different from those given unpaired injections. The amphetamineinjected group exhibited significantly greater activity than vehicle throughout drug treatments. There was an increase in activity after Day 2, which peaked by Day 4 and then decreased from Days 8-10. This decrease appeared to coincide with the emergence of informally observed focussed oral stereotypies. The two groups treated with paired injections of P H N O exhibited a pattern of activity different from the group receiving amphetamine. Both groups given paired P H N O injections maintained a relatively sustained level of activity not significantly different from that of the vehicle group until Day 6. On this day both groups exhibited a sharp increase in activity; this increase was greater in the high-dose group and was maintained at that level thereafter. The smaller increase in locomotion observed on Day 6 in the low-dose group was followed on subsequent days with further increases until the maximal level of locomotion was the same for the two PHNO-treated groups. A N O V A revealed a significant Pairing x Drug interaction for locomotor activity on the test day (F3,ss = 4.49, P < 0.01). All groups given unpaired injections of drugs during conditioning exhibited activity levels lower than the locomotion level of the unpaired vehicle-injected group on the test day. None of these differences were significant (Fig. 3). On the other hand, all 3 paired stimulant-injected groups exhibited levels of locomotion significantly greater than the locomotion exhibited by the paired vehicle group. The differences between these groups and the appropriate unpaired stimulant-injected groups were also significant. Therefore, rats given repeated injections of amphetamine or P H N O paired with the testing apparatus exhibited greater activity than controls when tested in the same apparatus after a vehicle injection.

178 Expt. 2: effects o l D t and D 2 antagonists on amphetamine and PHNO-induced unconditioned and conditioned locomotion The results of the conditioning phase of this experiment were subjected to A N O V A with 4 factors. The factors were: (1) haloperidol pretreatment ( H A L ) consisting of 2 levels (vehicle or 50 ~g/kg); (2) SCH 23390 pretreatment (SCH) with 2 levels (vehicle or 20 tzg/kg); (3) stimulant treatment (STIM) with 3 levels (vehicle, 1.5 mg/kg amphetamine or 30/~g/kg P H N O ) ; and (4) days of treatment (DAYS) with 10 levels. There was one significant 3-way interaction, SCH x STIM x D A Y S ([;'18,117o = 3.04, P < 0.001). Thus, the effect of SCH 23390 depended upon both the stimulant (or vehicle) administered and on the day of administration. The following groups receiving the D~ antagonist exhibited consistently lower locomotion than the VVV control group: VSV, HSV, H S A , VSP. The decrease in locomotion produced by SCH 23390 varied from day to day in the VSA and HSP groups. The effects of the stimulants depended upon the day of administration (STIM x DAYS: F l s , l l 7 0 = 4.26, P < 0.001). Amphetamine increased locomotion as compared to the V V V control group consistently across days, but this effect was not significant on days 3 and 4. The effects of haloperidol depended upon whether vehicle or one of the stimulants were administered ( H A L × STIM: FE,13o = 8.76, P < 0.001). Neither the H A L x SCH x STIM x D A Y S nor the H A L x SCH x STIM interactions were

PHOTOBEAM INTERRUPTIONS

250

200

150

100

50

O

VEHICLE

AMPHETAMINE P H N O (15) DRUG TREATMENT

PHNO (30)

Fig. 3. The effects of 10 days of previous treatments with vehicle, amphetamine (1.5 mg/kg, s.c,) or one of two doses of PHNO ~g/kg, s.c.) unpaired and paired with the test boxes on locomotion (photobeam interruptions) on a test day with no drug treatments. Previous treatment with amphetamine or either dose of PHNO, but not vehicle, produced increases in locomotion on the day with no drug treatments when drug treatments were previously paired with the test boxes, but not when unpaired. Thus, conditioned locomotion occurred with all 3 treatments. *Paired-treatment group significantly different from unpaired group, P < 0.05; **significantly different from paired vehicle group, P < 0.05.

PHOTOBEAM INTERRUPTIONS 250 ~ . . . . . . . . •

VVV

~)

VSV

~

HVV

:5

HSV

200 --

,4),

1so r 1

\ ~oo +

0

2

4

6 CONSECUTIVE DAYS

8

10

12

Fig. 4. Effects of 3 injections of vehicle (V), SCH 23390, a D 1 receptor antagonist (S, 20 /xg/kg, s.c.) and/or haloperidol, a D: receptor antagonist (H, 50/xg/kg, i.p.) on 10 consecutive days of locomotion (photobeam interruptions) during 30-rain periods. The injections were made 70 (V or H), 40 (V or S) and 10 (V) min prior to placement in the test boxes. Both groups receiving SCH 23390 (VSV and HSV) displayed locomotion significantly less than the VVV group.

significant. As can be seen in Fig. 4, the dose of haloperidol used did not affect locomotor activity in the rats treated with H V V (haloperidol + vehicle + vehicle), but both vehicle-injected groups also receiving SCH 23390 (VSV and HSV) displayed lower than vehicle levels of locomotion. On the other hand, haloperidol did block locomotion of the group receiving H V A (Fig. 5), with this group displaying locomotion levels similar to the VVV group. Both amphetamine-injected groups treated with SCH 23390 (VSA and H S A ) also exhibited a block of amphetamine-induced activity, with almost no activity in the H S A group. Fig. 6 shows that the group receiving P H N O (VVP) did not exhibit locomotion levels higher than the vehicle group (VVV). No locomotor stimulant effects of P H N O were therefore apparent during a 30-min conditioning period, and there was no measured effect for the antagonists to block during conditioning. Fig. 7 displays the conditioned activity (i.e. locomotion induced by exposure to the test boxes only, with a single vehicle injection) of the 12 groups. Analysis of variance, with the same factors as during conditioning except DAYS, ( H A L , SCH and STIM) indicated that there was a significant main effect of ST1M (F2,130 = 11.26, P < 0.001). No other main effect or interaction was significant. All rats treated with amphetamine (VVA, HVA, VSA and H S A ) displayed significant increases in locomotion, relative to the control group (VVV). Likewise, all groups treated with P H N O (VVP, HVP, VSP and HSP) exhibited levels of locomotion significantly above that of the control group. None of the vehicle-injected

179 PHOTOBEAM INTERRUPTIONS

LOCOMOTION % CONTROL

400 400 -

300 300

250 200 200 150

lOO

100

5o

0 0

2

4

6 8 C O N S E C U T I V E DAYS

10

12

rats receiving one or both of the antagonists (HVV, VSV, HSV) exhibited such increases. Thus, all stimulanttreated groups exhibited conditioned increases in locomotion, regardless of antagonist pretreatment. This was observed in spite of the fact that either antagonist alone or both in combination completely blocked the uncon-

LOCOMOTION % CONTROL 400 " -~-VSP

~VVP

-~-HVP

-~-HSP

300 250 200 150 100 50 0

0

VS

HV

HS

ANTAGONIST P R E T R E A T M E N T

Fig. 5. Effects of 3 injections of vehicle (V), and/or SCH 23390, a D 1 receptor antagonist (S, 20/~g/kg, s.c.) and/or haloperidol, a D 2 receptor antagonist (H, 50 ~g/kg, i.p.), and amphetamine (A, 1.5 mg/kg, s.c.) on 10 consecutive days of locomotion (photobeam interruptions) as a percent of the control group (VVV, see Fig. 4), during 30-min periods. The injections were made 70 (V or H), 40 (V or S) and 10 (A) min prior to placement in the test boxes. The group receiving injections of vehicle and amphetamine (VVA) exhibited significantly greater activity than all 3 other groups receiving antagonists as well as the control group. The antagonists reduced amphetamine-induced locomotion to control levels or below.

350 -

VV

I

t

I

I

I

2

4

6 CONSECUTIVE DAYS

8

10

12

Fig. 6. Effects of 3 injections of vehicle (V), and/or SCH 23390, a D 1 receptor antagonist (S, 20/~g/kg, s.c.) and/or haloperidol, a D 2 receptor antagonist (H, 50 pg/kg, i.p.), and PHNO, a selective D2 agonist (P, 30 pg/kg, s.c.) on 10 consecutive days of locomotion (photobeam interruptions) as a percent of the control group (VVV, see Fig. 4) during 30-min periods. The injections were made 70 (V or H), 40 (V or S) and 10 (P) min prior to placement in the test boxes. Both the D 2 agonist (P) and antagonist (H) were without significant effects, while the D1 antagonist (S) reduced locomotion, relative to the control group.

Fig. 7. The effects of 10 days of previous pretreatments with vehicle (V), SCH 23390, a D 1 antagonist (S, 20 pg/kg, s.c.), and/or haloperidol, a D 2 antagonist (H, 50 /xg/kg, i.p.) on vehicle, amphetamine (1.5 mg/kg, s.c.) or PHNO (30/xg/kg, s.c.) treatments paired with the test boxes on locomotion (photobeam interruptions) on a test day with no drug treatments. Previous treatment with amphetamine or PHNO produced increases in locomotion on the day with no drug treatments relative to the control group (VV vehicle) regardless of antagonist treatment. ANOVA revealed significant effects of both amphetamine and PHNO, but no effects of either antagonist. Thus, neither antagonist, even when given concomitantly during conditioning, had any effects on conditioned locomotion.

ditioned motor stimulation produced by amphetamine during the conditioning trials. The results were particularly surprising for the P H N O - t r e a t e d groups, since there was no test box paired locomotion induced by this drug during the conditioning trials.

Expt. 3: effects of D 1 and D 2 antagonists on P H N O induced unconditioned and conditioned locomotion with increased conditioning session durations The results of the conditioning phase of Expt. 3 were subjected to A N O V A with 4 factors. The factors were: (1) haloperidol pretreatment ( H A L ) consisting of 2 levels (vehicle or 50/~g/kg); (2) SCH 23390 pretreatment (SCH) with 2 levels (vehicle or 20/~g/kg); (3) stimulant treatment (STIM) with 2 levels (vehicle or 30/ag/kg P H N O ) ; and (4) days of treatment (DAYS) with 10 levels. As in Expt. 2, the SCH × STIM × D A Y S interaction was significant ( F 9 , 7 9 2 = 8.91, P < 0.001), as was the STIM x D A Y S interaction (F9,792 ~- 6.31, P < 0.001). However, no term involving H A L was significant in this case. Fig. 8 displays locomotion in rats treated with the antagonists but not with P H N O (VVV, VSV, H V V and HSV). The effects in these groups were found to be similar to those shown by equivalent groups in Expt. 2 (Fig. 4). It was again found that haloperidol had no effect when given in conjunction with vehicle, but SCH 23390, the D I antagonist, (groups VSV and HSV) reduced locomotion below that of the V V V group.

180 PHOTOBEAM iNTERRUPTIONS

500

"

VVV

'9

PHOTOBEAM INTERRUPTIONS r VSV

~

~J HSV

HVV

400 •

PHNOPRETREATMENT [-!VEHICLE ~ . , PNNOat

400 •

atSignificantly greater than vehicle

I

300 J 300 2oo

200 -~ !

~

100

•

lOO

*

I 0 ~

0 0

2

4

6 8 CONSECUTIVE DAYS

10

12

Fig. 8. Effects of 3 injections of vehicle (V), SCH 23390, a D 1 receptor antagonist (S, 20 #g/kg, s.c.) and/or haloperidol, a D 2 receptor antagonist (H, 50/~g/kg, i.p.) on 10 consecutive days of locomotion (photobeam interruptions) during 60-min periods. The injections were made 70 (V or H), 40 (V or S) and 10 (V) rain prior to placement in the test boxes. Both groups receiving SCH 23390 (VSV and HSV) displayed locomotion significantly less than the VVV group. The group receiving P H N O (VVP) over 60 min conditioning sessions displayed significant increases in locomotion from Days 3 - 1 0 (Fig. 9). The locomotion of the rats in this group exhibited a gradual increase from days 1 to 6, after which an asymptotic level of activity was a p p a r e n t l y reached. The group receiving haloperidol and P H N O ( H V P ) displayed activity patterns equivalent to

LOCOMOTION % OF CONTROL (VVV) 500

-

-~

400

I

300

--

VVP

~

~

VSP

~-

HVP

~

HSP

~

2OO

0

. . . . . . . . . . . . . . . . . . . .

0

2

4

~

....

6 8 CONSECUTIVE DAYS

4

10

12

Fig. 9. Effects of 3 injections of vehicle (V), and/or SCH 23390, a D 1 receptor antagonist (S, 20 pg/kg, s.c.) and/or haloperidol, a D e receptor antagonist (H, 50/~g/kg, i.p.), and PHNO, a selective D2 agonist (P, 30/~g/kg, s.c.) on 10 consecutive days of locomotion (photobeam interruptions) as a percent of the control group (VVV, see Fig. 8) during 60-min periods. The injections were made 70 (V or H), 40 (V or S) and 10 (P) min prior to placement in the test boxes. The D 2 agonist (P) significantly increased locomotion relative to the control group on days 3-10, but the antagonist (H) was without significant effect. The D 1 antagonist (S) significantly blocked PHNO-induced increases of locomotion over the control group.

VV

VS HV ANTAGONIST PRETREATMENT

HS

Fig. 10. The effects of 10 days of previous pretreatments with vehicle (V), SCH 23390, a D 1 antagonist (S, 20 #g/kg, s.c.), and/or haloperidol, a D 2 antagonist (H, 50/tg/kg, i.p.) on vehicle or PHNO (30/~g/kg, s.c.) treatments paired with the test boxes on locomotion (photobeam interruptions) on a test day with no drug treatments. Previous treatment with amphetamine or PHNO produced increases in locomotion on the day with no drug treatments relative to the control group (VV vehicle) regardless of antagonist treatment. ANOVA revealed a significant effect of PHNO, but no effects of either antagonist. Thus, neither antagonist, even when given concomitantly during conditioning, had any effects on PHNOconditioned locomotion. the V V P group; no attenuation of the locomotion p r o d u c e d by the D 2 agonist was o b s e r v e d with this dose of the D 2 antagonist. On the o t h e r hand, both groups receiving S C H 23390 and P H N O (VSP and HSP) displayed levels of l o c o m o t i o n less than or equivalent to those of the control group (VVV). Thus, the unconditioned effects of the D 2 agonist were blocked by the D 1 antagonist (at the dose of 20 /~g/kg), but not by a relatively low dose of the D 2 antagonist (50 ktg/kg). The conditioned locomotion test day results are displayed in Fig. 10. As found previously, P H N O t r e a t m e n t during conditioning increased l o c o m o t i o n when rats were placed in the testing e n v i r o n m e n t with only one vehicle injection (STIM: F1,88 = 41.6, P < 0.001). This effect was not blocked by the D1 antagonist, although in this case, the conditioning session injections of the D 1 antagonist itself p r o d u c e d an overall increase in locomotion on the test day (SCH: FI,SS = 4.94, P < 0.05). N o other term in the A N O V A was significant. The D 2 antagonist, haloperidol, had no effect on conditioned locomotion in either the vehicle or the P H N O - t r e a t e d groups.

Expt. 4: effects of a high dose haloperidol and low dose of P H N O on unconditioned and conditioned locomotion The results of the conditioning phase of this experiment were subjected to A N O V A with 2 i n d e p e n d e n t and 1 d e p e n d e n t factors. The first b e t w e e n - f a c t o r was haloperidol t r e a t m e n t ( H A L ) with two levels (vehicle or 0.25 mg/kg), the second i n d e p e n d e n t factor was P H N O

181 peridol attenuated this increase (HP group). On the other hand, as can be seen in Fig. 12, haloperidol treatment during the conditioning phase did not influence the PHNO-conditioned locomotion as revealed by a drug-free exposure to the testing apparatus. A N O V A revealed that there was a significant conditioned locomotion by P H N O (F~,44 = 27.4, P < 0.001). There was no effect of haloperidol and no interaction between haloperidol and P H N O . Therefore, haloperidol, at the appropriate doses, can block P H N O - i n d u c e d unconditioned locomotion, without influencing its conditioned locomotion.

P H O T O B E A M INTERRUPTIONS

500450

°

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- ~ - HV

- B - HP

400 350 300 • 250 200 150 100 50 0 0

I

I

I

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I

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5 CONSECUTIVE DAYS

8

10

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Fig. 11. Effects of injections of vehicle (V) or a high dose of haloperidol, a D 2 receptor antagonist (H, 250 gg/kg, i.p.), on locomotion (photobeam interruptions) induced with vehicle or a tow dose of PHNO, a selective D 2 agonist (P, 15/zg/kg, s.c.) during 60-min sessions over 10 consecutive days. The injections were made 70 (V or H) and 10 (V or P) min prior to placement in the test boxes. The D 2 agonist (P) significantly increased locomotion relative to the control group on days 3-10. The antagonist (H) significantly blocked PHNO-induced locomotion.

treatment, again with 2 levels (vehicle or 15/~g/kg), and the third (dependent) factor was D A Y S (10 levels). Analysis revealed only one significant interaction involving the D A Y S factor ( P H N O x DAYS: F 9 , 3 9 6 = 6.53, P < 0.001). The H A L x P H N O interaction was also significant (F1,44 = 5.25, P < 0.05). As can be seen in Fig. 11, P H N O (VP group) gradually increased locomotion over days with the 60 min conditioning sessions. Halo-

PHOTOBEAMINTERRUPTIONS 350 AGONIST PRETREATMENT 300

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HALOPERIDOL ANTAGONIST PRETREATMENT

Fig. 12. The locomotor (photobeam interruptions) effects of 10 days of previous pretreatments with vehicle or haloperidol, a D E antagonist (250/~g/kg, i.p.) combined with vehicle or PHNO (15 /~g/kg, s.c.) treatments paired with the test boxes on a test day with no drug treatments. Previous treatment with PHNO produced increases in locomotion on the day with no drug treatments regardless of haloperidol pretreatment. ANOVA revealed a significant effect of PHNO, but no effects of haloperidol. Thus, the DE antagonist did not block the development of PHNO-conditioned locomotion.

DISCUSSION It has been previously reported 2'5'1a'15'19 that repeated associations between the stimuli of a testing situation and amphetamine-induced locomotion can result in the elicitation of locomotion by exposure to the stimuli alone. Expt. 1 of the present report verified this finding: amphetamine injections to rats paired with exposure to a unique environment resulted in the environment alone eliciting an increase in locomotion. This effect was not observed in a group of rats given amphetamine treatments unpaired with the environment, Furthermore, Expt. 1 also indicated that a similar increase in locomotion can be induced by an environment previously paired with repeated injections of a selective D 2 receptor agonist, P H N O . Again, only exposure to an environment paired with P H N O could elicit this response. If the environment and P H N O treatments were unpaired, no increase in locomotion was observed upon exposure to the environment. The ability of an environment previously paired with stimulants to increase locomotion has been thought to be due to a Pavlovian conditioning process. The conditioned stimulus (CS) is the stimulus complex associated with the testing environment, and the conditioned response (CR) is usually thought to be the measured behavior (locomotion, in this case). It has never been completely clear what constitutes the unconditioned stimulus (UCS) and the unconditioned response ( U C R ) in the 'conditioning' of drug effects. The UCS may be the direct interaction of the drug at one or all of its sites of action. The U C R could be a measured behavior, or some subset of the effects on the central nervous system produced by the drug's action. The factors that determine the direction of the C R are not known. For example, the more-or-less direct effects of some drugs are conditioned (e.g. motor stimulant effects of psychomotor stimulants), while the indirect compensatory effects of other drugs are conditioned (e.g. tolerance to the cataleptic effects of neuroleptics and to the analgesic effects of morphine). Fur-

182 thermore, conditioning is not produced by all centrally active and behaviorally potent drugs (e.g. the locomotor depressant effects of SCH 23390). The reasons for this are unknown. Another problem with stimulant-conditioning, raised by investigators in Koob's laboratory 5, is that the stimulant-conditioned locomotor response seldom reaches the same level as drug-induced locomotion, and seldom displays a clear dose-response relationship. Rather, conditioned locomotion usually attains the same levels as occur in animals first introduced to a novel environment, as found in the present experiment. The lack of a clear dose-response relationship with stimulant conditioning may result from a changing pattern of behavioral responses to the drug (see for example the PHNO-induced locomotion in Fig. 2). If the unconditioned effects of a drug vary from day to day, then a dose-response relationship for a conditioned effect may not be expected. In this respect, it is noteworthy that doubling the dose of P H N O did not increase the maximal response observed after 10 days of treatment (Expt. 1). There is yet another difficulty with stimulant-conditioned locomotion. In Pavlovian conditioning, a stimulus previously neutral with regards to the UCR is associated with the UCS until it can produce a CR. However, in stimulant-conditioned locomotion, the stimulus is not initially neutral with regards to the behavioral response. A novel environment of the types most commonly used in locomotor conditioning experiments induces locomotion, as can be observed in the relatively high levels of locomotion in vehicle-treated rats on day 1, which decreases on subsequent days (Figs. 1, 2, 4, 8 and 11). Both this effect and the process of habituation to this effect are intrinsic in studies of conditioned locomotion. In spite of these problems, the conditioning hypothesis has been considered the most likely explanation for the process of acquired drug effects. The only viable alternative so far presented for the case of psychomotor stimulant-conditioned locomotion is that stimulants enhance the motivational significance of novel stimuli, and thereby retard the usual process of habituation that normally decreases locomotion in the testing apparatus 5. On the other hand, normal habituation appears to occur in rats treated with amphetamine; a decrease in locomotion from days 1 to day 2 occurs parallel to the habituation effect in vehicle-treated rats (Fig. 2). Beninger and Hahn 2 reported that pimozide, a selective D 2 antagonist, blocked the conditioned locomotion produced by associating amphetamine injections to rats with a novel environment. This block of conditioned locomotion occurred when pimozide was given during conditioning, but not when it was injected on the test day (a day on which the animals do not receive amphet-

amine). A similar pattern of results was observed with cocaine-conditioned locomotion :~. They concluded from these findings that the indirect action of amphetamine on DA receptors is necessary for the development of conditioned locomotion, but not the expression of this phenomenon, once established. The finding of Expt. 2 in the present report contradicts this conclusion. Doses of the D 2 antagonist (haloperidol) and the D~ antagonist (SCH 23390), given independently or concomitantly, which are effective at blocking the unconditioned locomotor response to amphetamine, failed to block conditioned locomotion. This finding is in agreement with our previous findings 9 with haloperidol, amphetamine and methylphenidate, and with an unpublished pilot study also using haloperidol and SCH 23390 as antagonists and amphetamine as a stimulant. It is possible that the difference between the two D 2 antagonists used in the two studies may be an important factor determining these conflicting results. There is evidence that pimozide has an aversive component to its actions 16"17, while haloperidol apparently does not 9. More importantly, another laboratory has also found that pimozide does not block conditioned locomotion on the test day, but that both haioperidol and sulpiride, another D 2 antagonist, do block this effect on the test day 13. Therefore at least two mutually opposed conclusions are possible, depending upon which drug is used: (1) actions on DA receptors are necessary for the establishment of amphetamine-conditioned locomotion, but not its expression, once established (Beninger and Hahn 2 with pimozide); or (2) actions on DA receptors are necessary for the expression of amphetamine-conditioned locomotion, but not its establishment (present data and Poncelet et al. 13 with haloperidol). The story becomes more complicated. Gold et al. 5 observed that lesions of the D A terminals in the nucleus accumbens induced with 6-hydroxydopamine (6-OHDA) blocked amphetamine-conditioned locomotion when the lesions were made prior to conditioning or after conditioning but before the test day. They concluded that the mesolimbic D A system is critical to both the development and expression of the conditioned locomotor response to amphetamine. However, it should be remembered that lesioning the animals before conditioning produces a lesioned animal on the test day as well. Since lesioning rats after conditioning and before the test day blocks conditioned locomotion, it is not clear whether or not a lesion made before conditioning has any effect. That is, one must be able to deplete DA during conditioning and then return it to normal on the test day to investigate the role of presynaptic DA in the establishment of conditioned locomotion with amphetamine. Therefore, it c a n n o t be concluded that the release of

183 presynaptic DA in the nucleus accumbens is essential to the development of the conditioned locomotor response. The present results as well as a previous paper 9 indicate that conditioning of amphetamine-induced locomotion can occur even when the unconditioned locomotion is blocked during conditioning. Therefore, one can conclude that locomotion is n o t the UCR, and is probably not the CR either, even if that is the effect measured. The UCR must be some event in the central nervous system prior to the expression of the behavior. Conditioning occurred even under conditions of concomitant blockade of both D~ and D z subtypes of DA receptors. It is clear that if DA is necessary for amphetamine-conditioned locomotion then both the UCR and the CR must be physiological effects of amphetamine that precede the actions of synaptic DA at its receptors. Since the direct action of amphetamine is on the presynaptic release of DA, then it appears plausible (at first) that the presynaptic release of DA is conditioned to the environmental stimuli. During conditioning in the presence of antagonists, this release of DA is not blocked 2°. On the test day, the behavioral effects of this conditioned DA release are unmasked in the absence of the antagonists. The experiments with PHNO, a direct D 2 agonist, were an attempt to indirectly test this interpretation. PHNO does not release DA; indeed, it inhibits the release of DA via actions on inhibitory presynaptic D 2 autoreceptors 7. Furthermore, PHNO is a highly selective D 2 agonist with no other major presently known effects at the doses employed in this study. If PHNO-conditioned locomotion occurs via actions on the DA system, then presumably what is conditioned must occur at the postsynaptic receptor level or further down the chain of causal events. It was expected that the receptor antagonists, especially haloperidol, would block the PHNOconditioned locomotion. Such an effect would support the view that the UCR in amphetamine-conditioning is a presynaptic event, since the presynaptic-acting amphetamine was not blocked and the postsynaptic agonist would (we presumed) be blocked. This was not observed. Indeed, combined D 1 and D 2 receptor antagonists failed to influence PHNO-conditioned locomotion (Expts. 2-4), just as they had failed to block amphetamine-conditioned locomotion. Since DA receptors are not apparently necessary for the development of environmentally-elicited locomotion conditioned by a direct DA agonist, it is also doubtful that presynaptic DA release is essential for the development of amphetamine-conditioned locomotion. Of course, it cannot be ruled out that the conditioned locomotion induced by amphetamine and PHNO occur by completely independent mechanisms. However, the similarity of the conditioned effects of the drugs and the rule of parsimony argue against this possibility.

The dose of haloperidol (0.05 mg/kg, i.p.) that effectively blocked amphetamine-induced unconditioned locomotion failed to block PHNO-induced unconditioned locomotion (Expt. 3). This was thought to be the result of the rather low dose of haloperidol, coupled with a high affinity of PHNO for the D 2 receptor. Expt. 4 validated this assumption, by showing that a higher dose of haloperidol could block locomotion induced by a lower dose of PHNO. In spite of the blockade of PHNOinduced unconditioned locomotion during the conditioning phase, conditioned locomotion was unaffected. The conclusion that DA is essential to the expression of the amphetamine-conditioned locomotor response is also questionable. As mentioned above, pimozide does not block the expression of such a C R 2"13, although haloperidol and sulpiride do. It has also been shown that the noradrenergic a 2 agonist, clonidine, can block the expression of amphetamine-conditioned behaviors at doses that had no effect on the unconditioned response to amphetamine 13. The a a receptor functions largely as a presynaptic inhibitor of noradrenaline release TM. This may be important, since the 6-hydroxydopamine (6OHDA) lesions of the accumbens by Gold et al. 5 also depleted noradrenaline by about 50%. It is not certain whether or not the blockade of conditioned locomotion by the 6-OHDA lesions is a function of the DA or the noradrenaline decrease. A number of other findings of the present series of experiments are of interest. One is that repeated injections of appropriate doses of antagonists selective for D 1 and D E receptor subtypes (SCH 23390 or haloperidol, respectively) given in combination or independently can block the unconditioned locomotion induced by either amphetamine or PHNO, a selective D E receptor agonist. This finding is consistent with the growing body of literature describing D1/D 2 receptor interactions which indicates that the motor stimulant effects of DA agonists, including PHNO 8 require activation of both types of receptor subtypes4. A second point of interest is that neither dose of PHNO induced locomotion on the first few days, but gradually increased locomotion on successive days, with both doses producing a similar asymptotic level of activity. That there was a dose-dependency in the rate at which the asymptote of locomotion was reached, but not in the asymptote itself, is puzzling. It is likely not a ceiling effect since the peak effect of amphetamine was higher than that produced by PHNO. Why the locomotor effects of a selective D 2 agonist require some days to develop is also not dear. Another point of interest is that the unconditioned induction of locomotion by PHNO did not have to be paired with the environment in order to produce conditioned locomotion, as long as the exposure to the environment preceded the locomotor response (Expt. 2),

184 and did not antecede it. This is not altogether surprising if the conditioned locomotion is indeed a Pavlovian conditioning process. The temporal association between CS and UCS does not require an overlapping period for conditioning to occur. It is notable that conditioned decreases in locomotion did not occur with the pairing of the D~ antagonist, SCH 23390. This drug reliably reduced locomotion below the level of vehicle controls in both Expts. 2 and 3, but this reduction in locomotion was not conditioned to the e n v i r o n m e n t . One possible explanation is that rats treated with SCH 23390 were not exposed to the e n v i r o n m e n t a l stimuli because the sedation prevented them from attending to the stimuli. If this were the case, then levels of locomotion should have been higher on the test day, due to a failure to habituate to the novelty of the e n v i r o n m e n t . That is, if they fail to attend to the stimuli to such a degree that conditioning cannot occur, then that same failure of attention should prevent habituation. This was not observed. Tolerance to catalepsy induced by relatively high doses of neuroleptics can be conditioned to e n v i r o n m e n t a l stimuli ~1, although sedation at these doses would be greater than that produced with SCH 23390 at the dose used in the present study. Furthermore, a decrease in locomotion produced by the autoreceptor

REFERENCES 1 Andersen, P.H., Nielsen, E.B., Gronvald, EC. and Braestrup, C., Some atypical neuroleptics inhibit [3H]SCH 23390 binding in vivo, Eur. J. Pharmacol., 120 (1986) 143-144. 2 Beninger, R.J. and Hahn, B., Pimozide blocks establishment but not expression of amphetamine-produced environment-specific conditioning, Science, 220 (1983) 1304-1306. 3 Beninger, R.J. and Herz, R.S., Pimozide blocks establishment but not expression of cocaine-produced environment-specific conditioning, Life Sci., 38 (1986) 1425-1431. 4 Clark, D. and White, F.J., Review: D1 dopamine receptor - the search for a function: a critical review of the D1/D2 dopamine receptor classification and its functional implications, Synapse, 1 (1987) 347-388. 5 Gold, L.H., Swerdlow, N.R. and Koob, G.E, The role of mesolimbic dopamine in conditioned locomotion produced by amphetamine, Behav. Neurosci., 102 (1988) 544-552. 6 Iorio, L.C., Barnett, A., Leitz, F.H., Houser, V.P. and Korduba, C.A., SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopaminergic systems. J. Pharmacol. Exp. Ther., 226 (1983) 462-468. 7 Martin, G.E., Williams, M., Pettibone, D.J., Yarbrough, G.G., Clineschmidt, B.V. and Jones, J.H., Pharmacologic profile of a novel potent dopamine agonist (+)-4-propyl-9-hydroxynaphthoxazine ([+]-PHNO), J. Pharmacol. Exp. Ther., 230 (1984) 569-576. 8 Martin-Iverson, M.T., Iversen, S.D. and Stahl, S.M., Long-term motor stimulant effects of (+)-4-propyl-9-hydroxynaphthoxazine (PHNO), a dopamine D-2 receptor agonist: interactions with a dopamine D-1 receptor antagonist and agonist, Eur. J. Pharmacol., 149 (1988) 25-31. 9 Mithani, S., Martin-Iverson, M.T., Phillips, A.G. and Fibiger, H.C., The effects of haloperidol on amphetamine- and methylphenidate-induced conditioned place preference and locomotor

agonist, B-HT 920, can also be conditioned to environmental stimuli 1°. We therefore do not have a plausible explanation for the failure of SCH 23390 to produce conditioned decreases in locomotion. This lack of generality of drug conditioning is an important problem with Pavlovian conditioning explanations of drug ~conditioning' p h e n o m e n a . In conclusion, the present data do not support the view that D A systems are essential for the conditioning of locomotion with a m p h e t a m i n e or a selective D 2 agonist. While our data do not provide evidence as to what other system(s) are essential for this p h e n o m e n o n , Poncelet et a1.13 have suggested that noradrenergic ¢x2 receptors may be critical. The observation that a D 2 antagonist had no effect on the development of PHNO-conditioned locomotion also raises questions as to the neurotransmitter specificity of PHNO. Finally, we must express doubts concerning the role of Pavlovian processes in drug-conditioned locomotion. At present, the 'conditioning' of drug-induced behavior is at best a poorly understood phenomenon, without a clear account of the processes involved. Acknowledgements. This research was supported by the Provincial Mental Health Advisory Council of Alberta and Alberta Heritage Foundation for Medical Research. We are grateful for the excellent technical support provided by Richard Strel.

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