Psychopharmacotogy (1994) 116:152-160 Psychopharmacology © Springer-Verlag 1994

Ontogenetic effects of EEDQ on amphetamine-induced behaviors of rats: role of presynaptic processes Cynthia A. Crawford 1'*, Sanders A. McDougall 2, Michael T. Bardo 1

1 Department of Psychology, University of Kentucky, Lexington, KY 40506, USA, 2 Department of Psychology, California State University, San Bernardino, CA 92407, USA

Received: 30 July 1993 / Final Version: t5 December 1993

Abstract. Previous research has shown that the alkylating agent N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ) affects dopamine (DA) synthesis and metabolism in both preweanling and adult rats. In the present study, we attempted to determine the behavioral relevance of EEDQ's presynaptic actions. To that end, t7- and 90- day-old rats were injected with either EEDQ (7.5 mg/kg, IP) or its vehicle 30 rain after half the rats were pretreated with the selective DA antagonists SCH 23390 and sulpir- ide. (SCH 23390/sulpiride pretreatment was used to protect D1 and D 2 receptors from EEDQ-induced inac- tivation.) The behavioral effects of amphetamine (0, 0.1, 0.3, or 1.0 mg/kg, IP) were then assessed 1, 2, 4, and 8 days after EEDQ treatment. Amphetamine-induced behaviors were used to assess EEDQ's presynaptic actions, because amphetamine does not directly bind to the DA receptor, but rather releases DA from the presynaptic terminal. Further, since half of the EEDQ-treated rats had a full complement of DA receptors (i.e., those rats pretreated with SCH 23390/sulpiride), EEDQ's actions in the presynaptic terminal could be dissociated from actions at pre- and postsynaptic receptors. In general, the results showed that EEDQ blocked most of the amphetamine- induced behaviors of both 17- and 90-day-old rats. Sur- prisingly, pretreatment with SCH 23390 and sulpiride only protected the amphetamine-induced behaviors of adult rats, but not the behaviors of 17-day-old rat pups. When considered together, these results suggest that EEDQ's presynaptic effects are not behaviorally relevant to the adult rat, but may be responsible for eliminating amphetamine-induced behaviors in the 17-day-old rat pup.

Key words: EEDQ - Dopamine - Amphetamine - Ontogeny - Rat pups

*Present address: Mental Retardation Research Center, Neuro- psychiatric Institute, University of California, Los Angeles, CA 90024, USA Correspondence to. S.A. McDougall

The alkylating agent N-ethoxycarbonyt-2-ethoxy-l,2- dihydroquinoline (EEDQ) has been used in the adult rat to study the behavioral rote of dopamine (DA) D1 and D a receptors and the time-course for behavioral recovery after DA receptor inactivation (Hambtin and Creese 1983; Arnt et at. 1988; Bordi et al. 1989; Cameron and Crocker 1989; Meller et al. 1989; Giorgi and Biggio 1990a,b). Recently, EEDQ has also been used to study the DA- mediated behaviors of preweanling rat pups (McDougall et al. 1992, 1993; Mestlin and McDougall 1993). Surpri- singly, this irreversible antagonist has very different be- havioral effects in young, as opposed to adult animals. For example, in adult rats, EEDQ eliminates behaviors in- duced by nonselective DA receptor agonists [e.g., apomorphine or R-propylnorapomorphine (NPA)] (Hamblin and Creese 1983; Meller et al. 1989; McDougall et al. 1992). Presumably, the DA agonists are unable to activate behavior because an insufficient number of DA receptors are left functioning after EEDQ treatment. In contrast, EEDQ does not block the agonist-induced behav- iors of 11- and 17-day-old rat pups. That is, in the preweanling rat, behaviors induced by nonselective and selective (e.g., SKF 38393 or quinpirole) DA agonists are not diminished by prior treatment with EEDQ (McDougall et al. 1992, 1993; Mestlin and McDougall 1993).

At present, it is uncertain why EEDQ induces these unusual ontogenetic differences; however, it is apparently not due to age-dependent changes in drug efficacy, as EEDQ inactivates a substantial percentage of D1 and D2 receptors in the 11- and 17-day-old rat pup (Crawford et at. 1992a, 1994). Instead, actions at the presynaptic terminal may be involved in EEDQ's age-dependent be- havioral effects. For example, EEDQ decreases striatal DA levels, and increases striatal DA synthesis, of both adult and preweanling rats (Crawford et al. 1992a, b). The decreased DA levels occur even when D1 and D2 recep- tors are protected from EEDQ-induced inactivation. In addition, many of EEDQ's nonspecific presynaptic effects vary according to age (Crawford et al. 1992a, b), sugges- ting that the behavioral differences shown by preweanting and adult rats may be due to the presynaptic actions of EEDQ.

153

To de te rmine the funct ional significance of E E D Q ' s p resynap t i c neurochemica l effects, the present s tudy assessed the behav io ra l ac t ions of a m p h e t a m i n e in E E D Q - t r e a t e d 17- and 90-day-o ld rats. A m p h e t a m i n e was employed because it does no t d i rect ly b ind to D A receptors , bu t ra ther acts as an indirect agonis t by releas- ing D A from the p resynap t i c t e rmina l (Ze t te rs t rom et al. 1983; P a r k e r and C u b e d d u 1986). P r i o r to E E D Q treat- ment , hal f of the ra ts were p re t r ea ted with reversible D1 and D2 receptor an tagon is t s in o rde r to p ro tec t D A recep- tors f rom E E D Q ' s recep tor deple t ing effects (see Mel ler et al. 1985; Satler et al. 1989; Crawford et al. 1994). Thus, E E D Q ' s ac t ions on a m p h e t a m i n e - i n d u c e d behaviors were assessed in rats with n o r m a l levels of D A receptors (i.e., in rats p re t r ea t ed with reversible D1 and D2 antagonis ts ) and in ra ts with depressed levels of D A receptors (i.e., in rats p re t r ea ted with saline pr io r to E E D Q treatment) . I t was p red ic ted tha t E E D Q would b lock the amphe t a mine - induced behaviors of rats possessing a n o r m a l comp- lement of D A receptors . This pa t t e rn of results would indicate tha t at least some of E E D Q ' s behav io ra l effects were med ia t ed by ac t ions in the p resynap t i c terminal , ra ther than by act ions at pre- and pos t synap t i c D A receptors .

a 25-min testing session by an observer blind to treatment condi- tions. Photobeam interruptions were measured for the entire 25 rain; whereas, the occurrence of stereotyped sniffing, locomotor activity, and rearing were measured every 2 min using time-sampling. Behav- ioral intensity was also assessed every 2 min using the following rating system: 0 = asleep or inactive, 1 = normal exploratory activity (no repetitive behaviors), 2 = discontinuous activity with prominent (repetitive) sniffing or rearing, 3 = continuous activity with repetitive sniffing and rearing in a fixed path, 4 = con- tinuous activity with repetitive sniffing and rearing in the same location, 5 = continuous activity with repetitive chewing or gnaw- ing in a fixed path, 6 = continuous activity with repetitive chewing or gnawing in the same location (Creese and Iversen 1973).

Because each animal was exposed to the drug treatment and the testing apparatus multiple times, four additional groups of 17- and 90-day-old rats were assessed for the possible contributions of con- ditioned activity and behavioral sensitization. All of these rats were nonprotected and given an injection of EEDQ (7.5 mg/kg). Behav- ioral testing occurred 1, 2, 4, and 8 days after EEDQ pretreatment, with rats receiving one of four drug sequences: group DW-DW received distilled water on all test days; group AMP-DW received amphetamine (1.0 mg/kg) on days 1, 2, and 4, and distilled water on day 8; group DW-AMP received distilled water on days 1, 2, and 4, and amphetamine on day 8; group AMP-AMP received ampheta- mine on all test days. The DW-DW and AMP-DW groups were used to assess conditioning effects of the drug-environment pairings; whereas, the DW-AMP and AMP-AMP groups were used to assess behavioral sensitization.

Materials and methods

Subjects. Subjects were 224 mate and female rats of Sprague-Dawley descent (Harlan) bred at the University of Kentucky. Litters were culled to eight to ten pups at 3 days of age. Rats were initially injected with drugs at 16 or 89 days of age and began behavioral testing when either 17 or 90 days old. The rat pups were kept with the dam throughout behavioral testing; whereas the older rats were group caged with same-sexed litter mates. Assignment of subjects was random, with each litter providing subjects for both the 17- and 90-day-old age groups. Importantly, no more than one rat from each litter was placed into a particular treatment condition, thus minimiz- ing the confounding effects of litter. The colony room was main- tained at 23-25 °C and kept under a 14-h light: 10-h dark cycle.

Apparatus. Behavioral testing was done in an activity chamber made of plywood (30 x 30 x 42 cm), with a wood floor and an open top. The floor and walls were painted white. Located 2 cm above the floor were two photocells which divided the activity chamber into four equal parts. Interruption of a photobeam resulted in one count being automatically recorded on a counter.

Procedure. At 16 or 89 days of age, rats were randomly placed into a protected or nonprotected condition. In the protected (PROT) condition, rat pups were given an initial injection of the D2 antago- nist sulpiride (100 mg/kg) followed, 30 rain later, by an injection of the D~ antagonist SCH 23390 (1.0 mg/kg). Rats in the nonprotected (NONPROT) condition received two injections of saline. Thirty minutes after the second injection, rats were injected with EEDQ (7.5 mg/kg) or its vehicle. Only nonprotected rats were given vehicle, since we have previously shown that protection treatment does not significantly affect the behavior of nonEEDQ-treated animals (McDougall et al. 1992).

For each rat, behavioral testing occurred 1, 2, 4, and 8 days after initial drug treatments. On each of these test days, rats were habitu- ated to the testing apparatus for 30 min. After habituation, rats were injected with distilled water or amphetamine (0.1, 0.3, or t.0 mg/kg) and immediately placed back in the testing apparatus. Stereotyped (head-down) sniffing, locomotor activity (defined as forward or backward ambulation), photobeam interruptions, behavioral inten- sity (a measure of stereotypy), and rearing were then assessed during

Drugs. All drugs were injected intraperitoneally (IP) and, for the rat pups, were given at a volume of 5.0 ml/kg. For adult rats, (_+)- sulpiride and R( + )-SCH 23390 HCI were given at a volume of 2.0ml/kg, EEDQ at 4.0mt/kg, and amphetamine sulfate at 1.0 ml/kg. SCH 23390 was dissolved in saline; whereas, sulpiride was dissolved in a minimal amount of glacial acetic acid and diluted with saline. Amphetamine was dissolved in distilled water and EEDQ was dissolved in 95% ethanol:distilled water (1:4). Sulpiride and SCH 23390 were acquired from Research Biochemicals (Natick, Mass., USA). EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2-dihydro- quinoline) and d-amphetamine were acquired from Sigma (St Louis, MO., USA).

Statistics. Analyses of variance (ANOVAs) were used for statistical analysis of stereotyped sniffing, locomotor activity, photobeam in- terruptions, behavioral intensity scores, and rearing data. The photobeam interruption and locomotor activity data provided sim- ilar results and conclusions, so only the locomotor activity data are presented. Amphetamine did not affect the rearing of the 17-day-old rats on test day 1, so this data was also not presented. For clarity, two sets of ANOVAs were performed: the first assessed day 1 effects only and the second assessed changes across days. Significant two- and three-way interactions were further analyzed by lower-order ANOVAs. When appropriate, Tukey tests were used for making post hoc comparisons (P < 0.05).

Results

Behavioral intensity

Ninety-day-oIds. On day 1, the behav io ra l intensi ty scores of a m p h e t a m i n e - t r e a t e d 90-day-o ld rats in the N O N - P R O T / V E H and P R O T / E E D Q condi t ions d id no t differ (upper graph, Fig. 1). In bo th of these condi t ions , the groups of rats given 1.0 mg /kg a m p h e t a m i n e had scores s imilar to each other, bu t had scores which were signifi- cant ly greater than the dist i l led water con t ro l s [ D o s e x Cond i t i on interact ion, F(6,84) = 6.82, P < 0.001,

and T u k e y tests, P < 0.05]. Fu r the rmore , in the same

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Fig. 1. Mean behavioral intensity scores of 90- and 17-day-old rats given acute IP injections of amphetamine (0.t, 0.3, or 1.0 mg/kg) or distilled water immediately prior to testing. The testing sessions lasted 25 min and occurred 1, 2, 4, and 8 days after pretreatment with 7.5 mg/kg EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquino- line). Thirty minutes prior to EEDQ pretreatment, rats were given IP injections of vehicle (nonprotected condition; NONPROT) or 1.0 mg/kg SCH 23390 and 100 mg/kg sulpiride (protected condition; PROT). (n-m) 1.0 mg/kg amphetamine; (A A) 0.3 mg/kg am- phetamine; (O O) 0.1 mg/kg amphetamine; (O-O) distilled water

NONPROT/VEH and PROT/EEDQ conditions, groups of rats given 0.1 or 0.3 mg/kg amphetamine did not differ among themselves nor did they differ significantly from the groups given distilled water. In contrast, none of the doses of amphetamine (0.1, 0.3, or 1.0 mg/kg) increased the behavioral intensity scores of rats in the NON- PROT/EEDQ condition. In the NONPROT/EEDQ con- dition, the behavioral intensity scores of rats given 0.3 or 1.0mg/kg amphetamine were significantly lower than similarly treated rats from the NONPROT/VEH and PROT/EEDQ conditions (Dose x Condition interaction, and Tukey tests).

When assessed across test days the effects of condition was found to vary [Condition x Day interaction, F(6,252) = 6.94, P < 0.001]. More specifically, on test days 1 and 2, rats in the NONPROT/VEH and PROT/EEDQ conditions had significantly higher behav- ioral intensity scores than rats in the NONPROT/EEDQ condition (Tukey tests, P < 0.05). On test days 4 and 8, the differences due to treatment condition were no longer apparent. The effects of amphetamine also varied across days, as rats given 1.0 mg/kg amphetamine showed a pro- gressive increase in behavioral intensity scores across the first 4 days of testing [ D o s e x D a y interaction, F(9,252) = 5.59, P < 0.001, and Tukey tests, P < 0.05]. In

contrast, the lower doses of amphetamine (0.1 or 0.3 mg/kg) did not induce a significant day-dependent increase in behavioral intensity scores.

Seventeen-day-olds. On day 1, 17-day-old rats in the NONPROT/VEH condition showed significant increases in behavioral intensity scores after treatment with 1.0mg/kg amphetamine (lower graph, Fig. 1) [Dose x Condition interaction, F(6,84)= 2.22, P < 0.05, and

Tukey tests, P < 0.05]. The t.0 mg/kg dose of ampheta- mine did not have similar effects on 17-day-olds in the PROT/EEDQ and NONPROT/EEDQ conditions, as the behavioral intensity scores of rat pups given amphetamine (0.1, 0.3, or 1.0 mg/kg) did not differ from their distilled water controls. Importantly, among the groups receiving 1.0 mg/kg amphetamine, the rat pups in the NONPROT/ VEH condition had significantly higher behavioral inten- sity scores than pups in either the PROT/EEDQ or NONPROT/EEDQ conditions (Dose x Condition inter- action, and Tukey tests). The lower doses of amphetamine (0.1 or 0.3mg/kg) did not affect behavioral intensity scores.

When comparisons were made across days, the behav- ioral intensity scores of 17-day-olds in the NONPROT/ EEDQ conditions were found to increase progressively from test days 1 to 2 [Condition x D a y interaction, F(6,252) = 2.83, P < 0.01, and Tukey tests, P < 0.05]. This increase was only apparent in the NONPROT/EEDQ condition and asymptoted after test day 2. The effects of amphetamine also varied significantly across test days, as rat pups given 1.0 mg/kg amphetamine had behavioral intensity scores which were relatively smaller on test day 1 than on the subsequent 3 test days [Dose x Day interaction, F(9,252) = 6.99, P < 0.001, Tukey tests, P < 0.05]. On all test days, rat pups given 1.0 mg/kg amphetamine had higher behavioral intensity scores than pups given distilled water. The lower doses of amphetamine (0.1 or 0.3 mg/kg) did not significantly affect behavioral intensity scores on any of the 4 test days.

Stereotyped sniffing

Ninety-day-olds. On day t, the stereotyped sniffing scores of 90-day-old rats in the NONPROT/VEH condition were affected by amphetamine (upper graph, Fig. 2). More specifically, in the NONPROT/VEH condition, all three doses of amphetamine (0.1, 0.3, and 1.0 mg/kg) increased stereotyped sniffing relative to the distilled water controls [Condition x Dose interaction,/7(6,84) = 3.99, P < 0.00t, and Tukey tests, P < 0.05]. In contrast, amphetamine did not increase the stereotyped sniffing of rats in the NON- PROT/EEDQ condition. Comparisons among conditions showed that all of the groups in the NONPROT/VEH condition sniffed significantly more than their counter- parts in the NONPROT/EEDQ condition (Condi- tion x Dose interaction, and Tukey tests). Rats in the PROT/EEDQ condition had sniffing scores more like rats from the NONPROT/VEH condition, especially when the groups receiving 0.3 and 1.0 mg/kg amphetamine were compared. For example, rats in the PROT/EEDQ condi- tion given 0.3 or t.0 mg/kg amphetamine sniffed more

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Fig. 2. Mean stereotyped sniffing of 90- and 17-day-old rats. Drugs and testing conditions were the same as described for Fig. 1. ( • - • ) 1.0 mg/kg amphetamine; (A-•) 0.3 mg/kg amphetamine; (0 0) 0.1 mg/kg amphetamine; (© ©) distilled water

than their distilled water controls and, at the same time, had sniffing counts which were no different than similarly treated rats from the NONPROT/VEH condition (Condi- tion x Dose interaction, and Tukey tests). This was not true for rats given 0.1 mg/kg amphetamine, as rats from the PROT/EEDQ condition sniffed less than rats from the NONPROT/VEH condition.

When assessed across test days, 90-day-old rats from the NONPROT/VEH and PROT/EEDQ showed similar effects after being treated with 0.3 or 1.0 mg/kg ampheta- mine. In these conditions, 0.3 and 1.0 mg/kg amphetamine induced a stable, high-rate of stereotyped sniffing on all 4 test days [Dose x Condition x D a y interaction, F(18,252) = 2.82, P < 0.001, and Tukey tests, P < 0.05]. In contrast, when rats in the PROT/EEDQ condition were injected with 0.1 mg/kg amphetamine they had rela- tively few sniffing counts on the first test day and an increasing number of sniffing counts as the test days progressed. Rats in the NONPROT/EEDQ condition exhibited a different pattern of responding than rats from the other two conditions. On test day 1, all of the groups in the NONPROT/EEDQ conditions had a low number of sniffing counts; however, on subsequent test days, the amphetamine and distilled water groups showed a pro- gressive increase in stereotyped sniffing (Dose x Condi- tion x Day interaction, and Tukey tests). Importantly, by test day 2, 1.0mg/kg amphetamine increased the stereotyped sniffing of rats from the NONPROT/EEDQ condition to levels in excess of their distilled water con- trols. On test days 4 and 8, the rats receiving 1.0 mg/kg

amphetamine sniffed significantly more than their distilled water controls and had sniffing counts that were no differ- ent than similarly treated rats from the NONPROT/VEH and PROT/EEDQ conditions. In fact, by test days 4 and 8, treatment condition did not affect the responding of any of the groups.

Seventeen-day-olds. On day 1, the stereotyped sniffing scores of the 17-day-old rat pups were affected by both amphetamine and treatment condition (lower graph, Fig. 2). More specifically, in the NONPROT/VEH condi- tion, rat pups given 1.0mg/kg amphetamine sniffed significantly more than pups given distilled water [Condition x Dose interaction, F(6,84) = 3.05, P < 0.01, and Tukey tests, P < 0.05]. This was not true for rat pups from the PROT/EEDQ and NONPROT/EEDQ condi- tions, as none of the groups from these conditions differed significantly on test day 1. Importantly, comparisons across conditions showed that there were no differences among rat pups given 1.0 mg/kg amphetamine.

When assessed across test days, rat pups given 1.0 mg/kg amphetamine maintained a stable, high-rate of stereotyped sniffing, which was greater than the distilled water controls [Dose x Day interaction, F(9,252) = 3.62, P < 0.001, and Tukey tests, P < 0.05]. In contrast, rat pups treated with distilled water or 0.1 mg/~g ampheta- mine showed an initial increase in sniffing from test days 1 to 2, and then a subsequent decline over the last 2 test days. The 0.3 mg/kg dose of amphetamine did not induce any day-dependent changes in stereotyped sniffing. On none of the test days were the differences between the distilled water group and the 0.1 and 0.3 mg/kg ampheta- mine groups statistically significant.

Locomotor activity

Ninety-day-oIds. On day 1, the locomotor activity scores of 90-day-old rats in the NONPROT/VEH and PROT/EEDQ conditions were generally alike (upper graph, Fig. 3). For example, the two groups given 1.0mg/kg amphetamine had locomotor activity counts that were similar to each other, yet were significantly higher than their distilled water controls [Dose x Condi- tion interaction, F(6,84) = 3.78, P < 0.01, and Tukey tests, P < 0.05]. The groups of rats in the NONPROT/VEH and PROT/EEDQ conditions given 0.3 mg/kg ampheta- mine also responded similarly, but, in this case, were not different from the distilled water controls. The locomotor activity counts of rats in the PROT/EEDQ condition given 0.1 mg/kg amphetamine were depressed relative to their counterparts in the NONPROT/VEH condition, and neither of these groups was different from the distilled water groups. The performance of amphetamine-treated rats in the NONPROT/EEDQ condition were markedly dissimilar to rats from the other two conditions. More specifically, amphetamine (0.1, 0.3, and 1.0 mg/kg) did not increase the locomotor activity of rats in the NON- PROT/EEDQ condition. Importantly, comparisons across conditions indicated that rats from the NON- PROT/VEH and PROT/EEDQ conditions given 0.3 or 1.0 mg/kg amphetamine were more active than similarly

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treated rats from the N O N P R O T / E E D Q condition (Dose x Condition interaction, and Tukey tests).

When assessed across test days, there were no differ- ences in the locomotor activity counts of rats from the N O N P R O T / V E H and PROT/EEDQ conditions. In both of these conditions, rats given t.0 mg/kg amphetamine were significantly more active than their distilled water controls on each test day [Condition x Dose x Day inter- action, F(18,252)--2.65, P < 0.001, and Tukey tests, P < 0.05]. Further, neither of these 1.0 mg/kg ampheta- mine groups showed a significant day-dependent increase in locomotor activity. In contrast, rats in the NON- P R O T / E E D Q condition given 1.0 mg/kg amphetamine did not differ from their distilled water controls on test days 1 or 2; however, the locomotor activity of these rats increased substantially by test days 4 and 8 (Condi- tion x Dose x Day interaction, and Tukey tests). Indeed, by test days 4 and 8, the locomotor activity of rats given t.0 mg/kg amphetamine were similar regardless of treat- ment condition. Interestingly, when assessed across days, the locomotor activity of those rats given 0.1 or 0.3 mg/kg amphetamine did not differ according to either treatment condition or test day.

Seventeen-day-olds. On day 1, amphetamine did not inter- act with treatment condition to significantly affect the locomotor activity scores of the 17-day-old rat pups (lower graph, Fig. 3). Although the lack of a significant Dose x Condition interaction precluded further statistical analysis, rat pups from the N O N P R O T / V E H condition

Table 1. Mean behavior scores of 90- and 17-day-old rats given distilled water (DW) or amphetamine (AMP) on test days t, 2, and 4, followed by a single injection of distilled water or amphetamine on test day 8. Scores represent performance on day 8

DW-DW AMP-DW DW-AMP AMP-AMP

90-Day-olds BehavioraI intensity 5.88 4.88 26.25 24.25 Stereotyped sniffing 7.25 6.62 12.00 12.00 Locomotor activity 3.12 1.50 5.88 10.25 b Rears 1,12 0.75 3.38 6.25

17-Day-olds Behavioral intensity 150 8.50 ~ 24.38 21.88 Stereotyped sniffing 3,75 6.75 a 11.62 11.25 Locomotor activity 1.25 2.50 6.50 7.38 Rears 0,12 0.38 1.88 1.62

~Significantly different from the DW-DW group (P < 0.05) bSignificantly different from the DW-AMP group (P < 0.05)

showed a trend towards increased locomotor activity after treatment with t.0 mg/kg amphetamine. Amphetamine's effects varied according to test day, as rat pups given 1.0 mg/kg amphetamine showed a significant increase in activity from test day to test day 2 [Dose x Day interac- tion, F(9,252)=5.23, P < 0 . 0 0 1 , and Tukey tests, P < 0.05]. By the second test day, rat pups given 1.0 mg/kg amphetamine had higher activity scores than pups given distilled water. In contrast, rat pups receiving distilled water or 0.t mg/kg amphetamine showed a pro- gressive day-dependent decrease in locomotor activity (Dose x Day interaction, and Tukey tests). Rat pups given 0.3 mg/kg amphetamine maintained a stable level of re- sponding on the 4 test days. On none of the test days were the differences between the distilled water group and the 0.1 and 0.3 mg/kg amphetamine groups statistically significant.

Conditioned activity and behavioral sensitization

Conditioned activity was assessed by comparing the day 8 performance of rats in the DW-DW and AMP-DW groups. There was no evidence for any conditioned activ- ity when the behaviors of the 90-day-old rats were as- sessed (Table 1). In contrast, both the behavioral intensity scores and the stereotyped sniffing of the 17-day-old rats showed some conditioned activity. That is, rat pups re- ceiving prior injections of amphetamine (group AMP- DW) exhibited enhanced behavioral intensity scores and more stereotyped sniffing than pups receiving consecutive injections of distilled water (group DW-DW) [behavioral intensity: F(3,28) = 27.50, P < 0.001; stereotyped sniffing: F(3,28) = 36.19, P < 0.001].

Behavioral sensitization was assessed by comparing the day 8 performance of rats in the AMP-AMP and DW-AMP groups (Table 1). For the 90-day-old rats, behavioral sensitization was only apparent when locomo- tor activity was assessed, as rats receiving repeated am- phetamine injections were more active than rats getting amphetamine for the first time [F(3,28) = 18.31, P < 0.001]. The 17-day-old rat pups showed no evidence of behavioral sensitization.

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Fig. 4. Mean rearing scores of 90- and 17-day-old rats. Drugs and testing conditions were the same as described for Fig. 1. ( I - ! ) 1.0 mg/kg amphetamine; ( i - i ) 0.3 mg/kg amphetamine; ( O - t ) 0.1 mg/Ng amphetamine; ((2)-(2)) distilled water

Discussion

Previous studies have shown that the alkylating agent EEDQ inactivates DA receptors and affects the presynap- tic functioning (e.g., neurotransmitter synthesis and meta- bolism) of DA neurons (Hamblin and Creese 1983; Arnt et al. 1988; Saller et al. 1989; Giorgi and Biggio 1990a; Crawford et al. 1992a, b, 1994; McDougall et al. 1994. It is unclear, however, whether EEDQ's presynaptic actions have any behavioral relevance. Therefore in the present experiments, amphetamine, an indirect DA agonist, was used to dissociate the pre- and postsynaptic actions of EEDQ in 17- and 90-day-old rats. In general, the results showed that the amphetamine-induced behaviors (e.g., behavioral intensity scores, locomotor activity scores, photobeam interruptions, stereotyped sniffing, and rear- ing) of 90-day-old rats were blocked by EEDQ. The be- havior-disrupting effects of EEDQ were only temporary, as the amphetamine-induced behaviors of EEDQ-pre- treated adults returned to control values by either test day 2 or 4 (depending on the behavior). In adults, protecting D1 and Dz receptors with SCH 23390 and sulpiride was typically sufficient to eliminate EEDQ's behavior-disrupt- ing effects. Therefore, the pattern of these results was similar to previous studies using direct DA receptor agonists (e.g., apomorphine and NPA), as the ampheta- mine-induced behaviors of adult rats were temporarily eliminated by EEDQ (Hamblin and Creese 1983; Arnt et al. 1988; Meller et al. 1989; McDougall et al. 1992).

Importantly, for the first time, EEDQ was found to block certain DA agonist-induced behaviors of prewean-

ling rat pups. For example, EEDQ eliminated the am- phetamine-induced increases in behavioral intensity scores, locomotor activity scores, and photobeam inter- ruptions of 17-day-old rats. EEDQ also depressed the amphetamine-induced stereotyped sniffing of rat pups (relative to their distilled water controls), but these results provided interpretive difficulties because amphetamine (1.0 mg/kg) treated pups from the NONPROT/EEDQ and NONPROT/VEH conditions did not differ statist- ically (see Fig. 2). However, when considered together, it is apparent that a number of the behaviors induced by amphetamine were blocked by EEDQ. This contrasts with previous experiments using the preweanling rat pup, which have consistently shown that EEDQ does not block behaviors induced by direct DA agonists (e.g., NPA, quin- pirole, and SKF 38393) (see McDougall et al. 1992, 1993; Mestlin and McDougall 1993). Surprisingly, pretreating the 17-day-olds with SCH 23390 and sulpiride did not protect all of these amphetamine-induced behaviors from EEDQ's disruptive effects (e.g., compare the PROT/EEDQ and NONPROT/EEDQ groups in Fig. 1).

Therefore, when combined, these developmental psychopharmacological studies provide a number of important findings: first, EEDQ blocked the NPA-in- duced behaviors of adult, but not preweanting, rats; second, amphetamine-induced behaviors of both preweanling and adult rats were diminished by EEDQ; and third, only the behaviors of adult rats were consis- tently protected by SCH 23390 and sulpiride pretreat- ment. Thus, when considered together, it appears that EEDQ's ability to block the amphetamine-induced be- haviors of 17-day-old rat pups may not be entirely due to actions at the postsynaptic DA receptor, because EEDQ does not block behaviors induced by direct DA agonists (e.g., NPA and apomorphine) (McDougall et al. 1992, 1993; Mestlin and McDougall 1993). Instead, these results suggest an interesting possibility: that EEDQ's presynap- tic actions may be responsible for diminishing the am- phetamine-induced behaviors of preweanling rat pups. Although the precise mechanism is uncertain, one possible explanation is that an EEDQ-induced reduction in presynaptic DA (see Crawford et al. 1992a, b) may have made amphetamine behaviorally irrelevant to the preweanling rat. More specifically, amphetamine may have been unable to activate behavior, because there was an insufficient amount of DA in the presynaptic terminal available for release. In addition, EEDQ's presynaptic effects do not appear to be due to actions at the DA autoreceptor, because D1 and D2 receptor protection did not diminish EEDQ's ability to block amphetamine-in- duced behaviors (see Fig. 1). This is consistent with the inability of antagonist pretreatment to block the EEDQ- induced reduction in DA levels (Crawford et al. 1992a, b).

In adult rats, EEDQ's behavioral effects appear to be receptor mediated and not due to actions at the presynap- tic terminal. This conclusion is partially based on the fact that EEDQ blocks the actions of both the direct receptor agonist NPA and the indirect agonist amphetamine (Mel- ler et al. 1989; McDougall et al. 1992; present results). Furthermore, protecting DA receptors from EEDQ allows full NPA- and amphetamine-induced behaviors: again indicating that EEDQ's actions at DA receptors are

158

critical (Meller et al. 1989; McDougall et al. 1992; present results). Importantly, the results also show that the EEDQ-induced reduction in DA levels has no functional significance for the adult rat. If the reduction of intracellu- lar DA was important, amphetamine should not have affected the behaviors of rats with intact DA receptors. Instead, amphetamine activated the behaviors of adult rats in the protected/EEDQ condition.

Although EEDQ is typically used to study DA system functioning, this drug has also been shown to inactivate various other receptor types, including: ~-adrenergic, serotonin, and GABA receptors (Meller et al. 1985; Miller et al. 1991). Therefore, to control for EEDQ's lack of specificity, SCH 23390 (1.0 mg/kg) and sulpiride (100 mg/kg) were used to selectively protect DA U 1 and D2 receptors from EEDQ-induced inactivation. Receptor binding studies have shown that these doses of SCH 23390 and sulpiride seem to protect the D1 and D2 receptors of adult rats (Crawford et al. 1994). The protection afforded by sulpiride may be less than complete, however, because EEDQ is able to induce a nonsignificant decrease (of about 20%) in the number olD2 receptors (Crawford et al. 1994). Although not statistically significant, the latter re- sult leaves open the possibility that 100 mg/kg sulpiride may not be sufficient to completely protect the D2 recep- tors of adult rats. Inspection of the data from the present study supports this conclusion, as 90-day-old rats from the NONPROT/VEH condition, when compared to the PROT/EEDQ condition, had higher locomotor activity and stereotyped sniffing scores after treatment with 0.1 mg/kg amphetamine (see Figs. 2 and 3). However, when the more critical 1.0 mg/kg dose of amphetamine was used, the behaviors of 90-day-old rats from the NON- PROT/VEH and PROT/EEDQ conditions did not differ. Importantly, receptor binding experiments have clearly shown that SCH 23390/sulpiride pretreatment is sufficient to completely protect the D1 and D2 receptors of preweanling rat pups (Crawford et al. 1994). Therefore, EEDQ's ability to diminish the locomotor activity and behavioral intensity scores of amphetamine-treated rat pups was due to nonreceptor-mediated processes and not because of insufficient receptor protection. Ampheta- mine's neurotransmitter releasing properties are also not limited to the DA neuron, as amphetamine causes in- creased release of other monoamines including norepine- phrine and serotonin (Homan and Ziance 1981; Curet et al. 1992). This lack of specificity somewhat limits inter- pretation; however, in adult rats most of amphetamine's psychomotor stimulating effects appear to be mediated by the DA system (Gold et al. 1989, pp. 102-106).

Since rats were tested repeatedly, various control groups were used to assess the effects of environmental conditioning and behavioral sensitization. The 90-day-old rats showed no evidence of environmental conditioning; however, both the behavioral intensity scores and the stereotyped sniffing of the 17-day-old rats showed condi- tioned increases in activity. Thus, some of the day-depen- dent behavior changes exhibited by the 17-day-olds may have been due to the repeated pairing of amphetamine with the testing chamber. Even so, the scores of pups in the AMP-DW group were substantially less than those from the DW-AMP group, indicating that conditioned

effects were less pronounced than the acute effects of amphetamine alone. In general, there was little evidence of behavioral sensitization; however, adult rats given repeat- ed amphetamine treatments had higher locomotor activity scores on the final test day than rats receiving amphetamine for the first time. This indicates that behav- ioral sensitization may have been responsible for some of the day-dependent increases in activity exhibited by the 90-day-old rats. In contrast to adults, the 17-day-old rat pups showed no evidence of behavioral sensitization. This was interesting since we previously found that NPA indu- ces at least short-term behavioral sensitization in 17- day-old rat pups (McDougall et al. 1992). Thus, it appears that direct receptor agonists, but not indirect agonists, will induce behavioral sensitization in preweanling rats. This conclusion is supported by other research indicating that amphetamine fails to produce sensitization in rats under 30 days of age (Fujiwara et al. 1987; Kolta et al. 1990). Apparently, the mechanisms responsible for ampheta- mine-induced behavioral sensitization are not function- ally mature in the preweanling rat pup.

Developmental changes in neurotransmitter function are not unique to the DA system, as psychopharmacologi- cal responses to opiate (Caza and Spear 1980; Fanselow and Cramer 1988), serotonergic (Ristine and Spear 1985; Enters and Spear 1988), and cholinergic (Linville and Spear 1988; Fitzgerald and Hannigan 1989) drugs do not mature until after weaning. Similarly, most studies inves- tigating the ontogeny of DA functioning suggest that full, adult-like responding does not occur until at least 21 days of age. Prior to weaning, the DA mediated behaviors of rat pups are characterized by the emergence of new behav- iors (e.g., grooming) and the occurrence of age-specific behaviors (e.g., wall climbing) which disappear as the animal matures (Shalaby and Spear 1980; Moody and Spear 1992). This is not to say that all behaviors show qualitative age-dependent differences in response to DA- acting drugs. For example, direct DA receptor agonists (e.g., apomorphine and quinpirole) increase the locomotor activity of rat pups as young as 4 days of age (Shalaby and Spear 1980; Camp and Rudy 1987; Moody and Spear 1992); whereas, competitive DA receptor antagonists (e.g., haloperidol, SCH 23390, and sulpiride) reduce behavioral activation in both preweanling and adult rats (Burt et al. 1982; Fitzgerald and Hannigan 1989; McDougall et al. 1990). Even so, these psychopharmacological responses still show quantitative changes across ontogeny, as the potency of these DA-acting drugs are usually greater in older animals (see Shalaby and Spear 1980; Fitzgerald and Hannigan 1989).

The results of the present study are consistent with the developmental framework of DA functioning outlined above. For example, 1.0 mg/kg amphetamine increased the behavioral responsiveness of both 17-day-old and adult rats. Although direct statistical comparisons were not made between the age groups, the lower doses (0.1 and 0.3 mg/kg) of amphetamine were only able to increase the stereotyped sniffing of adult rats, suggesting that am- phetamine was more potent in the older animals. This interpretation is tentative, however, because Lanier and Isaacson (1977) reported that 2.0 mg/kg amphetamine in- duced greater locomotor activity in 18-day-old rats than

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adul t s (see also Spear 1979, pp. 139-141). M o r e impor - tantly, E E D Q ' s behav io ra l effects appea r to vary qua l i ta t - ively accord ing to age. The mos t d r ama t i c evidence of this is E E D Q ' s inabi l i ty to b lock the N P A - and quinpi ro le- induced behav iors of p reweanl ing rats (McDouga l t et al. 1992, 1993; Mest l in and M c D o u g a l l 1993). In the present s tudy, E E D Q did b lock a m p h e t a m i n e - i n d u c e d behav iors of bo th 17- and 90-day-o ld rats, bu t only the behaviors of the adu l t ra ts were p ro tec ted by S C H 23390/sulpir ide pre t rea tment . W e in te rpre ted these p sychopha rmaco log i - cal differences as being the resul t of age-dependen t changes in p resynap t ic funct ioning, but at the least, these results indicate tha t D A system funct ioning does no t reach full ma tu r i t y unti l after the p reweanl ing period.

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