Psychopharmacology (1994) 116: 483-490 Psychopharnmcology © Springer-Verlag 1994

Ontogeny of behavioral sensitization in the rat: effects of direct and indirect dopamine agonists Sanders A. McDougal l 1, Marcus A. Duke ~, Carlos A. Bolanos 1, Cynthia A. Crawford /

~Department of Psychology, California State University, San Bernardino, CA 92407, USA 2Mental Retardation Research Center, Neuropsychiatric Institute, University of California, Los Angeles, CA 90024, USA

Received: 12 November 1993 / Final version: 21 March 1994

Abstract. In the present study, the abilities of NPA (a direct DA receptor agonist) and amphetamine (an indirect DA receptor agonist) to induce short- and long-term be- havioral sensitization were assessed in 11- and t7-day-old rats (age at initial injection). Rats were injected on 4 con- secutive days with amphetamine (1.0, 2.5, or 5.0 mg/kg), NPA (1.0 mg/kg), or saline. A final test day occurred either 2 days (experiment 1) or 8 days (experiment 2) later. On the test day, rats given successive agonist injections received a single injection of the same agonist again; whereas rats given successive saline injections received either amphetamine or NPA for the first time. Five min- utes after injection, locomotor activity (line-crosses), stereotyped sniffing, and vertical activity were measured during a 30-min testing session. The results showed that 11- and 17-day-old rats exhibited behavioral sensitization when tested with NPA or amphetamine after a 2-day interval. In contrast, neither NPA nor amphetamine was able to sensitize the behaviors of preweanling rats when measured 8 days after initial drug treatments. Therefore, these results show that both direct and indirect DA ago- nists are able to induce short-term behavioral sensitiza- tion in preweanling rats, but that the mechanisms respon- sible for mediating long-term behavioral sensitization have not yet matured.

Key words" Behavioral sensitization - amphetamine N P A - Ontogeny Rat

Intermittent exposure to psychostimulants produces a phenomenon called behavioral sensitization in adult rats (Mattingly et al. 1988; Robinson and Becker 1988; Kalivas and Duffy 1989). This phenomenon is character- ized by a progressive and enduring enhancement of the locomotor-activating effects of a drug. This enhanced be- havioral response occurs after repeated treatment with direct [e.g., apomorphine and R-propylnorapomorphine (NPA)] or indirect (e.g., amphetamine and cocaine)

Correspondence to: S.A. McDougall

dopamine (DA) agonists and can be observed when be- haviors are tested from 1 day to several months later (Wilcox et al. 1980; Leith and Kuczenski 1982; Castro et al. 1985; Robinson and Becker 1986; Kalivas and Stewart 1991; Mattingly et al. 1991). Interestingly, the behavioral characteristics of apomorphine- and ampheta- mine-induced sensitization differ, perhaps indicating that these DA agonists induce sensitization via different neur- obiological processes (Mattingly et al. 1991; Rowlett et al. 1991). This would not be surprising, since NPA and amphetamine have different mechanisms of action. More specifically, NPA is a full agonist at both DA D1 and D2 receptors (Meller et al. 1987; Seeman and Grigoriadis 1987), whereas amphetamine releases DA and other monoamines from the presynaptic terminal (Homan and Ziance 1981; Zetterstrom et al. 1983; Parker and Cubeddu 1986; Curet et al. 1992).

Unexpectedly, indirect DA agonists do not induce adult-like behavioral sensitization in preweanling rats, as repeated injections of amphetamine are unable to increase the locomotor activity of these younger animals. For example, rats given five daily amphetamine treatments starting at 17 days of age (or younger) did not show behavioral sensitization when tested at 35 days of age (Fujiwara et al. 1987). Likewise, Kolta et al. (1990) found that 21-day-old rats did not exhibit amphetamine-induced sensitization when behavior was measured 15 days after initial agonist treatments. In contrast, direct DA agonists seem to induce behavioral sensitization in the preweanling rat, as NPA-treated rat pups exhibit enhanced responding when behaviors are measured 2 days later (McDougall et al. 1992). It is unclear, however, whether NPA and amphetamine are equally able (or unable) to induce be- havioral sensitization in the preweanting rat, because rats were tested with NPA after a 2-day interval (McDougall et al. 1992) and amphetamine after approximately a 15-day interval (Fujiwara et al. 1987; Kolta et al. 1990).

In order to assess the ontogenetic effects of direct and indirect agonists on behavioral sensitization, 11- and 17-day-old rats (age at initial injection) were given four daily injections of NPA or amphetamine and tested for behavioral sensitization after either a 2-day (experiment 1) or an 8-day (experiment 2) interval. This study should

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determine: first, whether direct and indirect DA agonists are able to produce short- and long-term behavioral sen- sitization in preweanting rats; and second, whether the behavioral sensitization induced by NPA and ampheta- mine appear at different ages.

General methods

Subjects. Subjects were i t - and 17-day-old (age at initial testing) male and t~male rats of Sprague-Dawley descent (Harlan). Litters were culled to eight to ten rat pups at 3 days of age. Rats were kept with the dana throughout behavioral testing. Assignment of male and female rats was random, with no more than one rat from each litter being placed into a particular group. The colony room was maintained at 23-25°C and kept under a 14 L:10 D cycle. Behav- ioral testing was conducted during the light phase of the cycle.

Apparatus. For the t7-day-olds, behavioral testing was done in grey plywood chambers (30 × 30 x 42 cm), with the floors divided by lines into four equal quadrants. For the 1 t-day-olds, behavioral testing was done in slightly smaller chambers (25x25x18 cm), with the floors also divided by lines into four equal quadrants. The smaller chambers were used to somewhat equate for differences in body size. The testing chambers for the younger animals were housed in a large glass-topped incubator. The incubator was maintained at 31°C, which is approximate thermoneutrality for 10- to 16-day-old rats (Conklin and Heggeness 1971).

Drugs. The drugs used in this study were d-amphetamine sulfate (Sigma Chemicals, St Louis, Mo.) and R-propylnorapomorphine (NPA; Research Biochemicals, Natick, Mass). All drugs were rejec- ted intraperitoneally (IP) and were given at a volume of 5.0 ml/kg. Both amphetamine and NPA were dissolved in saline.

Statistics. Analyses of variance (ANOVAs) and Student's ~-tests were used for statistical analysis of the behavioral data. Two sets of ANOVAs were performed: the first assessed effects on the final test day and the second assessed changes across days. The ANOVAs were done separately because, in some instances, drug-induced changes across days could be contbunded with maturational cha- nges in motor ability. When appropriate, Tukey tests were used for making post hoc comparisons (P < 0.05).

Exper iment 1. Short - term sensit izat ion: assessment after a 2-day interval

Methods

In the first experiment (n = 8), 1t- and 17-day-old rats (age at initial testing) were injected on 4 consecutive days with either saline or amphetamine (1.0, 2.5, or 5.0 mg/kg). Behavioral sensitization was then assessed on a final test day which occurred 2 days later. On the final test day, rats given successive saline injections were then given a single injection of saline, 1.0, 2.5, or 5.0 mg/kg amphetamine; whereas, rats given successive injections of the various doses of amphetamine received a single injection of the same agonist again. Two additional groups of 1 t- and 17-day-olds were given four consecutive injections of NPA (1.0 mg/kg) or saline and then tested with NPA 2 days later.

Behavioral testing occurred 5 rain after saline or drug injections. On each day, line-crosses, stereotyped (head- down) sniffing, and vertical activity were assessed during

a 30-rain testing session by an observer blind to treatment conditions. A single vertical activity count was defined as removing both front paws off the floor, thus this measure encompasses both rearing and wall climbing. Line-crosses and vertical activity were measured continuously across the testing session; whereas the occurrence of stereotyped sniffing was assessed every 20 s using a time sampling procedure.

Results and discussion

Eleven-day-olds

Line-crosses. On the final test day, 11-day-old rats given repeated injections of 1_0 mg/kg amphetamine had signifi- cantly more line-crosses than rats given an acute injection of 1.0 mg/kg amphetamine (upper graph, Fig. 1) [condi- tion effect, F (6 ,49)= 9.04, P < 0.001; and Tukey tests, P < 0.05]. Successive treatments with the higher doses of amphetamine (2.5 and 5.0 mg/kg) did not enhance activ- ity relative to control rats. All of the groups given am- phetamine were significantly more active than the groups given only saline.

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Fig. 1. Behavioral scores of rats given four daily injections of am- phetamine (1.0, 2.5, 5.0 mg/kg) or saline starting at 11 days of age. At 16 days of age (i.e., after a 2-day interval), rats given successive saline injections were then given a singte injection of saline, 1.0, 2.5, or 5.0 mg/kg amphetamine; whereas, rats given successive injections of the various doses of amphetamine received a single injection of the same agonist again. On each day, injections were given 5 min prior to a 30-min behavioral testing session. (O O) AMPH-AMPH (1.0mg/kg); (© ©) SAL-AMPH (1.0mg/kg); (A at) AMPH-AMPH (2.5 mg/kg); ( A - - - - A ) SAL-AMPH (2.5 mg/kg); ( l - - l ) AMPH-AMPH (5,0mg/kg); (D ....... []) SAL-AMPH (5.0 mg/kg); (V- - -V) SAL-SAL

On the first test day, all of the amphetamine-treated l l-day-olds showed increased line-crosses relative to the saline groups [Condition x Day interaction, F(24, 196) = 5.25, P < 0.001; and Tukey tests, P < 0.05]. When assessed across days, only those t l-day-olds given successive injections of 1.0 mg/kg amphetamine showed a progressive increase in line-crosses; whereas, rats given 2.5 or 5.0 mg/kg amphetamine maintained a stable level of responding across test days. Of course, rats given acute injections of amphetamine did not show increased line- crosses until the final test day.

Two additional groups of 11-day-olds received either successive NPA or saline injections on the initial 4 days and were tested with NPA when 16 days old (see Table 1). The 11-day-olds given successive NPA injections had sig- nificantly more line-crosses on the final test day than rats receiving an acute injection of NPA I t (14)= 4.74, P < 0.001]. Overall, NPA-treated rats had more total line-crosses than saline-treated rats on the initial four testing days [condition effect, F(1, 14) = 31.16, P < 0.05], but NPA did not induce a significant day-dependent in- crease in line-crosses [Condition x D a y interaction, F(4, 56) = 0.61, P > 0.05].

Stereotyped sniffing. On the final test day, 11-day-old rats given successive injections of 5.0 mg/kg amphetamine had significantly more stereotyped sniffing counts than rats given a single injection of 5.0 mg/kg amphetamine for the first time (middle graph, Fig. l) [condition effect, F(6, 49) = 6.39, P < 0.001; and Tukey tests, P < 0.05]. In contrast, rats given repeated injections of 1.0 or 2.5 mg/kg amphetamine did not have significantly more stereotyped sniffing counts than their controls.

When assessed across test days, 11-day-olds given 2.5 and 5 .0mg/kg amphetamine showed a day-dependent increase in stereotyped sniffing (when the first test day was compared to the last test day); whereas, the sniffing of rats

485

given 1.0 mg/kg amphetamine did not vary across test days [Condition x Day interaction, F(24, 196) = 5.36, P < 0.001; and Tukey tests, P < 0.05]. The stereotyped sniffing of rats given saline was almost non-existent on all test days.

In terms of responsiveness to NPA, behavioral sensi- tization was apparent when stereotyped sniffing was as- sessed after a 2 day testing interval (see Table 1). For example, l l-day-olds given four successive injections of NPA and tested when 16 days old had significantly more stereotyped sniffing counts than rats given an acute injec- tion of NPA on the final test day It (14) = 2.91, P < 0.05]. Further, rats given repeated NPA injections showed a progressive increase in sniffing across the initial test days, while rats given saline did not [Condition x Day interaction, F(4, 56) = 7.37, P < 0.001; and Tukey tests, P < 0.05].

Vertical activity. On the final test day, l 1-day-old rats given successive injections of amphetamine (1.0, 2.5, 5.0 mg/kg) had significantly more vertical activity counts than rats given acute injections of amphetamine for the first time (lower graph, Fig. l) [condition effect, F(6, 49) = 3.47, P < 0.01; and Tukey tests, P < 0.05]. When assessed across days, all of the groups receiving repeated amphetamine treatments showed a progressive increase in vertical activity (when performance on the first test day was compared to the last test day) [Condi- tion x Day interaction, F(24, 196) = 1.80, P < 0.05; and Tukey tests, P < 0.05].

When tested after a 2-day interval (i.e., at 16 days of age), the vertical activity counts of rats given successive NPA injections did not differ significantly from rats given NPA on only the final test day (see Table 1) It(14) = 1.98, P > 0.05]. In addition, rats given repeated injections of NPA did not show a day-dependent increase in vertical activity [Condition x Day interaction, F(4, 56) = 1.16, P > 0.05].

Table 1. Behavioral scores of preweanling rats given either successive injections of 1.0 mg/kg NPA (NPA- NPA) or an acute injection of 1.0 mg/kg NPA (SAL-NPA) on the final test day

Days of age 11 12 13 14 16

Line-crosses SAL-NPA 3.38 7.50 3.75 5.62 48.38 a NPA-NPA 36.50 39.75 44.25 54.25 85.50

Stereotyped sniffing SAL-NPA 0.38 0.12 0.25 0.38 30.00 ~ NPA-NPA 24.88 30.75 37.88 49.00 49.88

Vertical activity SAL-NPA 0.62 0.62 0.25 0.12 3.87 NPA-NPA 3.38 2.88 8.00 4.88 9.88

Days of age 17 18 19 20 22

Line-crosses SAL-NPA 88.12 2 4 .3 8 4 2 .2 5 43 .38 306.62 ~ NPA-NPA 119.25 250.27 318.56 411.50 589.25

Stereotyped sniffing SAL-NPA 17.00 5.88 8.38 9.37 59.87 a NPA-NPA 68.38 78.12 71.00 74.75 85.12

Vertical activity SAL-NPA 7.25 5.38 10.62 18.00 77.75 ~ NPA-NPA 18.50 56.75 116.88 182.00 200.50

a Significantly different from the final test day scores of rats in the NPA-NPA group, P < 0.05

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Seventeen-day-olds

Line-crosses. On the final test day, 17-day-old rats given successive treatments of 2.5 mg/kg or 5.0 mg/kg am- phetamine had significantly more line-crosses than same aged rats given a single acute injection of 2.5 mg/kg or 5.0mg/kg amphetamine, respectively (upper graph, Fig. 2) [condition effect, F (5 ,42)= 3.59, P < 0.01; and Tukey tests, P < 0.05]. In contrast, rats given successive injections of 1.0 mg/kg amphetamine did not show en- hanced activity relative to rats given an acute injection of the same dose of amphetamine.

Overall, the lower doses (1.0 and 2.5 mg/kg) of am- phetamine increased the line-crosses of 17-day-old rats on the first test day; however, only rats given 2.5 and 5.0 mg/kg amphetamine showed a significant increase in activity from the first to the last test day [Condition x Day interaction, F(20, 168) = 2.31, P < 0.01; and Tukey tests, P < 0.05].

Behavioral sensitization was also apparent with direct DA agonists, as 17-day-olds given successive NPA injec- tions had significantly more line-crosses on the final test day than rats receiving an acute injection of NPA (see Table 1) It(14) = 4.74, P < 0.001]. The line-crosses of the NPA- and saline-treated rats were similar on the first test day, but on all subsequent test days the NPA-treated rats showed a progressive increase in line-crosses ECondi-

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Fig, 2. Behavioral scores of rats initially injected at 17 days of age and tested for sensitization at 22 days of age (i.e., after a 2-day interval). Injection schedules were the same as described for Fig. 1. (O, O) AMPI t -AMPH (l,0mg/kg); ( O - - - O ) SAL-AMPH (l.0mg/kg); (A-----&) AMPH-AMPH (2.5mg/kg); (A A) SAL-AMPH (2.5 mg/kg); (11 - - - I I ) AMPH-AMPH (5.0mg/kg); (D--- -[Z) SAL-AMPH (5.0 mg/kg)

tion x Day interaction, F(4, 56)= 19.02, P < 0.001; and Tukey tests, P < 0.05~.

Stereotyped sniffing. On the final test day, 17-day-old rats injected with 5.0 mg/kg amphetamine had significantly more stereotyped sniffing counts than rats given 1.0 or 2.5 mg/kg amphetamine (middle graph, Fig. 2) [condition effect, F (5 ,42)= 10.29, P<0 .001; and Tukey tests, P < 0.05]. More importantly, there was no evidence of behavioral sensitization, as the stereotyped sniffing of rats given successive treatments of amphetamine was no differ- ent than the sniffing of rats given a single acute injection of the same dose of the drug. On the initial 4 test days, the greatest dose (5.0 mg/kg) of amphetamine significantly increased stereotyped sniffing relative to the control groups [Condition x Day interaction, F (20, 168) = 9.36, P < 0.001; and Tukey tests, P < 0.05].

When assessed after a 2-day interval, the stereotyped sniffing counts of 17-day-olds given successive treatments of NPA were significantly greater than the sniffing counts of rats given an acute injection of NPA on the final test day (see Table 1) It(14) = 3.91, P < 0.01]. Rats receiving successive injections of NPA showed a day-dependent increase in sniffing [Condition x Day interaction, F(4, 56) - 8.43, P < 0.001; and Tukey tests, P < 0.05].

Vertical activity. On the final test day, 17-day-old rats given repeated injections of 2.5 mg/kg amphetamine had significantly more vertical activity counts than rats given an acute injection of 2.5 mg/kg amphetamine for the first time (lower graph, Fig. 2) [condition effect, F(5, 42) = 5.42, P < 0.001; and Tukey tests, P < 0.05]. Repeated treatments with 1.0 or 5.0 mg/kg amphetamine did not increase vertical activity, relative to rats given acute injections of the same dose of amphetamine on the final test day. When assessed across days, only the 17- day-olds given repeated injections of 2.5 mg/kg ampheta- mine showed a day-dependent increase in vertical activity [Condition x Day interaction, F (20, 168) = 4.34, P < 0.001; and Tukey tests, P < 0.05].

Behavioral sensitization was apparent when the verti- cal activity counts of NPA- and saline-treated 17-day-old rats were compared after a 2-day interval (see Table 1). On the final test day, rats given successive NPA treatments had significantly more vertical activity counts than rats given a single injection of NPA It (14) = 4. t4, P < 0.001]. Repeated treatments with NPA produced a progressive increase in vertical activity across test days; whereas the rats treated with saline maintained a stable level of re- sponding [Condition x Day interaction, F(4, 56) = 14.82, P < 0.00i; and Tukey tests, P < 0.05].

Experiment 2. Long-term sensitization: assessment after an 8-day interval

Methods

Behavioral procedure. In the second experiment (n = 8), 11- and 17-day-old rats (age at initial testing) were injected on 4 consecutive days with either saline, NPA (1.0 mg/kg),

487

or amphetamine (ll-day-olds: 5.0mg/kg; 17-day-olds: 2.5 mg/kg). (The amphetamine doses were based on the results of experiment 1 and chosen to maximize the chance of getting behavioral sensitization.) After an 8-day interval (i.e., when the rats were 22 or 28 days old, respectively), saline-treated rats were injected with saline, NPA, or amphetamine; whereas rats previously given agonist treat- ments were given the same agonist again on the final test day. Behavioral testing was the same as described for experiment 1.

Results and discussion

Eleven-day-olds

Line-crosses. On the final test day, there was a significant effect due to condition IF(4, 35)= 11.94, P < 0.001]; however, this only indicated that the groups receiving amphetamine were more active than the rats exclusively given saline (upper graph, Fig. 3) [Tukey tests, P < 0.05]. Importantly, l l-day-olds given successive treatments of either NPA or amphetamine did not differ significantly from controls receiving acute agonist treatment on the last test day. Across the initial 4 test days, 11-day-olds receiv- ing amphetamine had significantly more line-crosses than

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Fig. 3. Behavioral scores of rats given four daily injections of either NPA (1.0 mg/kg), amphetamine (5.0 mg/kg) or saline starting at 11 days of age. At 22 days of age (i.e., after an 8-day interval), rats given successive saline injections were then given a single injection of amphetamine, NPA, or saline; whereas, rats given successive injec- tions of amphetamine or NPA received a single injection of the same agonist again. ( I - - - - I ) NPA-NPA; ( ~ - - C ] ) SAL-NPA; (ik A) AMPH-AMPH; (A A) SAL-AMPH; ( V - - - V ) SAL-SAL

rats from the other groups [Condition x Day interaction, F (16, 140) = 8.54, P < 0.001; and Tukey tests P < 0.051.

Stereotyped sniffing. On the final test day, the stereotyped sniffing of 11-day-olds given successive NPA or ampheta- mine injections did not differ from rats receiving acute injections of NPA or amphetamine (middle graph, Fig. 3). When assessed across the initial test days, both NPA and amphetamine produced a progressive increase in stereotyped sniffing, which then declined by the final test day [Condition x Day interaction, F(16, 140) = 11.18, P < 0.001; and Tukey tests, P < 0.05].

Vertical activity. On the final test day, 11-day-olds given a single acute injection of amphetamine had significantly more vertical activity counts than rats given successive injections of amphetamine (lower graph, Fig. 3) [condi- tion effect, F(4, 35)= 9.15, P < 0.001; and Tukey tests, P < 0.05]. In contrast, the groups given chronic injections of NPA did not differ from their controls. None of the groups differed among themselves on the initial 4 test days.

Seventeen-day-olds

Line-crosses. On the final test day, there was no evidence of behavioral sensitization, as 17-day-olds receiving suc- cessive treatments of NPA or amphetamine did not have more line-crosses than rats given acute treatment with the appropriate drug (upper panel, Fig. 4) [Tukey tests, P > 0.05]. There was a significant effect due to condition, but that only indicated that rats given a DA agonist (either NPA or amphetamine) had more line-crosses than rats given saline on the last test day [condition effect, F(4, 35) = 46.14, P < 0.001; and Tukey tests, P < 0.05]. Across the initial 4 test days, rats receiving successive treatments of NPA and amphetamine showed a progress- ive increase in line-crosses, but the line-crosses of these animals showed a subsequent decline on the final test day [Condition x Day interaction, F(16, 140) = 8.69, P < 0.001; and Tukey tests, P < 0.05].

Stereotyped sniffing. On the final test day, 17-day-olds receiving NPA (either acutely of chronically) had more stereotyped sniffing counts than rats given amphetamine or saline (middle panel, Fig. 4) [condition effect, F(4, 35) = 19.95, P < 0.001; and Tukey tests, P < 0.05]. Importantly, there was no evidence of behavioral sensi- tization, because rats receiving successive drug treatments did not differ from rats given a single acute drug injection. When assessed across test days, both NPA and ampheta- mine increased the sniffing of the rats, but only NPA induced a progressive increase in sniffing across the first 4 test days [Condition x Day interaction, F (16, 140) = 14.01, P < 0.001; and Tukey tests, P < 0.05].

Vertical activity. Once again, analysis of the vertical activ- ity data indicated that NPA and amphetamine did not induce behavioral sensitization after the 8-day testing interval, as chronic treatment with NPA or amphetamine did not induce more vertical activity than acute injections of the same drugs (lower panel, Fig. 4). On the final test day, rats given only saline had fewer vertical activity

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Fig. 4. Behavioral scores of rats given four daily injections of NPA (1.0 mg/kg), amphetamine (2.5 mg/kg) or saline starting at 17 days of age and tested for sensitization at 28 days of age (i.e., after an 8-day interval). Injection schedules were tile same as described for Fig. 3. (m II) NPA-NPA; (U] []) SAL-NPA; (A i ) AMPH-AMPH; (IX A) SAL-AMPH; (V --- V) SAL-SAL

counts than rats given amphetamine (either acutely or chronically) or NPA (chronically) [condition effect, F(4, 35) = 7.16, P < 0.001; and Tukey tests, P < 0.05]. When assessed across the initial 4 test days, rats given repeated injections of NPA or amphetamine showed a progressive increase in vertical activity; however, no additional increases were observed on the final test day [Condition x Day interaction, F(16, 140) = 3.53, P < 0.001; and Tukey tests, P < 0.05].

General discussion

In the present study, the abilities of NPA (a direct DA receptor ago~ist) and amphetamine (an indirect DA re- ceptor agonist) to induce short- and long-term behavioral sensitization were assessed in 11- and 17-day-old rats (age at initial injection). At both ages, rats given repeated injections of NPA showed enhanced line-crosses, stereotyped snitIing, and vertical activity when tested after a 2-day interval. Although there were some differences due to dose, amphetamine-treated 11- and 17-day-old rats also showed behavioral sensitization after 2 days (see Figs 1 and 2). Importantly, when an 8-day testing interval was used, neither NPA nor amphetamine was able to induce behavioral sensitization in the 11- ol: H-day-old rats (see Figs 3 and 4). This was the case, even though a range of amphetamine (1.0, 2,5, and 5 mg/kg) was employed and three different behaviors were assessed. Interestingly, the

older rat pups did show a nonsignificant increase in be- havioral activity after the 8-day interval, perhaps indi- cating that the mechanisms responsible for long-term be- havioral sensitization were beginning to mature by t7 days of age. Therefore, when considered together, these experiments indicate that direct and indirect DA agonists induce sensitization through the same underlying neuro- biological processes, because the behavioral sensitization produced by repeated NPA and amphetamine treatments had similar characteristics. Further, and more impor- tantly, the results showed that DA agonists were able to induce short-term behavioral sensitization in preweanling rats, but were unable to induce long-term sensitization.

The dichotomy between short- and long-term behav- ioral sensitization indicates that the mechanisms underlying these processes differ and can be clearly disso- ciated in the preweanling rat pup. In the adult rat, a sim- ilar dissociation is not immediately apparent because behavioral sensitization is observed for several months after initial agonist treatments (Leith and Kuczenski 1982; Kalivas and Stewart 1991). Even so, there is other evi- dence indicating that the sensitization exhibited by adult rats is governed by separate processes which can be differ- entiated according to time since previous agonist injec- tion. For example, adult rats tested for amphetamine- induced sensitization after a 3- to 4-day interval exhibit a pattern of presynaptic functioning (e.g., autoreceptor sensitivity and extracellular DA levels) which is qualita- tively different from rats tested after a 10- to 11-day interval (Wolf et al. 1993). In addition, direct pharmacological intervention using NMDA and non-NMDA antagonists indicates the mechanisms underlying behavioral sensitiza- tion have at least two separate components. More spe- cifically, it appears that the "induction" or "development" of behavioral sensitization in the adult rat is distinct from its long-term "expression" or '*persistence" (Karler et al. 1991; Stewart and Druhan 1993). Although speculative, it is possible that the inability of DA agonists to induce Iong-term sensitization in preweanting rats may be due to the immaturity of those mechanisms mediating the ex- pression of behavioral sensitization.

At present, the exact neurobiological mechanisms un- derlying behavioral sensitization are uncertain; however, several possibilities have been proposed, including: autoreceptor tolerance, increased DA synthesis, actions at NMDA and non-NMDA receptors, drug-induced changes in G-proteins, and conditioned activity (Robin- son and Becker !986; Vaughn et al. 1990; Kativas and Stewart 1991; Karler et al. 1991; Rowlett et al. 199t; Steketee et al. 1991; Stewart and Druhan 1993). The pres- ent study was not designed to differentiate between these various explanations. In fact, in the latter case, no distinc- tion was made between environment specific sensitization (i.e., conditioned activity) and biochemically induced sen- sitization. Instead, we elected to maximize the probability of inducing behavioral sensitization by pairing the test chamber with drug administration, as environmental con- ditioning can significantly enhance the magnitude of a sensitized response (see Drew and Glick 1989; Mattingly and Gotsick 1989; Hoffman and Wise 1992).

More importantly, the results of the present study indicate that the mechanisms critical for the long-term

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expression of behavioral sensitization are not functionally mature in the preweanling rat pup. This is particularly clear in the l l -day-o ld , as successive injections of DA agonists actually resulted in an at tenuated response to subsequent agonist t reatment (see Fig. 3). The reasons for this tolerance-like effect are unclear; however, these results dramatical ly show that the mechanisms mediat ing behav- ioral sensitization are not mature in the younger animal. Rather than being just an oddity, the differential matura- t ion of these mechanisms might make the preweanling rat an excellent model for determining the neurobiological substrates of behavioral sensitization. More specifically, correlating developmental changes in behavioral sensi- tization with matura t ional changes in possible neuro- biological substrates (e.g., DA synthesis, G-proteins, N M D A receptors, etc.) may provide addit ional informa- tion for determining those factors critical for behavioral sensitization (see Spear 1979 and Zo lman and McDouga l l 1983, for reviews). In addition, because the processes in- volved in the induct ion of behavioral sensitization seem to mature before the processes necessary for long-term ex- pression, an ontogenetic approach could distinguish the neurochemical events that are impor tan t for both.

The present results are generally consistent with other developmental psychopharmacologica l studies showing that adult-like responding often does not occur until after the preweanling period (see Spear 1979). More specifically, previous studies have shown that the behavior-act ivat ing properties of D A agonists are often quantitatively greater in older animals (Shalaby and Spear 1980; Fitzgerald and Hann igan 1989; McDouga l l et al. 1990). Qualitative dif- ferences are also occasionally apparent, as some DA me- diated behaviors (e.g., grooming) emerge at various times during the preweanling period, while other behaviors (e.g., wall climbing) only occur during a restricted period (Shalaby and Spear 1980; M o o d y and Spear 1992). In the present study, examples of both quanti tat ive and qualita- tive ontogenet ic differences were also evident. For example, amphetamine and N P A produced quanti tat ively higher levels of vertical activity in the older of the two age groups. More important ly, the inability of preweanling rats to show long-term behavioral sensitization is an example of a dramat ic qualitative behavioral difference. The l l -day-o lds ' tolerance-like response to repeated am- phetamine t reatment is also qualitatively different from 17-day-old rats and adults (compare Figs 3 and 4). There- fore, the results of the present experiments are consistent with other ontogenetic studies showing that psychophar- macological responses to DA-act ing drugs exhibit both quanti tat ive and qualitative changes across development.

Acknowledgements. This research was partially supported by an ASI research grant (CSUSB),

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