Prenatal cocaine dampened behavioral responses to methylphenidate in male and female adolescent rats

Neurotoxicology and Teratology 28 (2006) 165 – 172 www.elsevier.com/locate/neutera

Prenatal cocaine dampened behavioral responses to methylphenidate in male and female adolescent rats Annelyn Torres-Reveron 1 , Diana L. Dow-Edwards ⁎ Program in Neural and Behavioral Sciences and Department of Physiology and Pharmacology, SUNY Health Sciences Center at Brooklyn, Brooklyn, New York, United States Received 2 June 2005; received in revised form 30 November 2005; accepted 5 December 2005 Available online 10 February 2006

Abstract Clinical and animal data point toward deficits in attention and arousal after prenatal cocaine exposure. Since methylphenidate (MPD) is widely used to treat attention disorders, we wanted to determine whether prenatal cocaine (PC) exposure affects the behavioral response to MPD in young rats of both sexes. Pregnant dams received 60 mg/kg of cocaine or vehicle from gestational days 8–22 by intragastric intubations. After delivery, litters were culled to 10 (5 males, 5 females) and fostered. On a single day between PND 41–44 locomotion was recorded in a Plexiglas box within an Accuscan activity monitor after receiving a single injection of 10 mg/kg intraperitoneally of MPD or saline. Rats were also videotaped for analysis of stereotyped behavior. Results showed that MPD administration enhanced locomotion compared to saline injected groups. PC exposure in male rats did not have any effect on the locomotor response to MPD compared to prenatal controls. However, PC-exposed males showed a lower amount of time spent in low intensity stereotypy compared to prenatal control males and both groups of females that received MPD. PC exposure in female rats that received MPD dampened the locomotor response compared to prenatal control females that also received MPD. In conclusion PC exposure dampens the behavioral response to MPD differentially in males and females with an apparent selectivity of locomotion in females and stereotyped behavior in males. © 2006 Elsevier Inc. All rights reserved. Keywords: Locomotor activity; Stereotyped behavior; Adolescence; Sex differences

1. Introduction Animal studies that evaluate the effects of prenatal cocaine exposure have been very helpful in understanding the neurological and behavioral consequences of drug exposure in the developing brain. Rats exposed to cocaine during the prenatal period are more likely to exhibit sensitivity to environmental demands and stressors and show abnormal inhibitory control, response initiation and information processing speed [18,56]. More

Abbreviations: MPD, Methylphenidate; PND, Postnatal day; G, Gestational day; ADHD, Attention Deficit Hyperactivity Disorder. ⁎ Corresponding author. 450 Clarkson Ave, Box 29, Brooklyn NY 11209, United States. Tel.: +1 718 270 3987; fax: +1 718 270 2241. E-mail addresses: [email protected] (A. Torres-Reveron), [email protected] (D.L. Dow-Edwards). 1 Current address: Weill Medical College of Cornell University, Dept. of Neurology and Neuroscience, Division of Neurobiology 411 E 69th St. Room 410 NY, NY 10021, United States. 0892-0362/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2005.12.005

recently, Strupp and collaborators demonstrated that prenatal exposure to cocaine impairs selective attention by increasing the susceptibility to distractors [19]. Our laboratory has also shown that prenatal cocaine can decrease glucose metabolism in selected areas of the brain [15] and that these effects can be sexually dimorphic [16]. Therefore, prenatal cocaine exposure can produce alterations in the brain observable at both the behavioral and functional levels. It is well stated in the pre-clinical literature that prenatal cocaine exposure produces selective alterations in the dopaminergic systems [21,25]. Methylphenidate (MPD) has a similar mechanism of action as cocaine and other psychostimulants by inhibiting the reuptake of dopamine and norepinephrine. However MPD acts on the serotonin system to a lesser extent compared to cocaine. Inhibition of reuptake enhances the neurotransmitter concentrations available in the synapse [23,44]. MPD is widely used in the clinical setting to treat children and adults with attention deficit [30]. Given that both prenatal cocaine and MPD can produce effects on the

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catecholaminergic systems, it is expected that developmental exposure to cocaine will have enduring effects on the behavioral response to a subsequent exposure to a drug with a similar mechanism of action such as MPD. Several studies have examined the behavioral responses to psychostimulant administration in animals with prenatal cocaine exposure. In rodents, prenatal cocaine has been shown to both increase [10,45], decrease [36,54] or have no effects [27] on the behavioral response to a subsequent cocaine or dopaminergic challenge in adult animals. A cocaine challenge in preweanling pups exposed to cocaine prenatally has been shown to decrease locomotor activity specifically in female animals [35]. Amphetamine challenges in prenatally cocaine exposed adult and young animals have been shown to have no effects on locomotor activity [5,59] while others have found effects of amphetamine challenge during the preweanling period but not at postnatal day 30 [53]. Cocaine self-administration in adult rats was reduced in animals with prenatal exposure [26]. In rabbits, prenatal cocaine exposure has been shown to consistently reduce the motor and head-bobbing behavior in response to a subsequent amphetamine administration during adulthood [50,57]. However, preweaning rabbits pre-exposed to cocaine did not show a diminished motor response to amphetamine [50]. Thus the age of testing can influence the response to subsequent psychostimulant administration. To our knowledge, there is no literature on the response to MPD after prenatal cocaine exposure. The present study evaluates the effects of prenatal cocaine on the locomotor and stereotypic responses to MPD when administered during the adolescent period in rats of both sexes. Our results demonstrate that adolescent female rats exposed to cocaine during the prenatal period showed a dampened locomotor response to MPD compared to prenatal controls, while adolescent male rats exposed to cocaine during the prenatal period showed a dampened stereotypic behavioral response compared to prenatal controls. 2. Materials and methods 2.1. Subjects Nulliparous Sprague–Dawley rats (Charles River, VAF strain, Charles River Laboratories, Wilmington, MA) in proestrous were placed with males of the same strain at 4:00 PM. The next morning, females were checked for the presence of sperm by vaginal lavage. If present, that day was designated as gestational day 1 (G1). Pregnant dams were individually housed in plastic cages with bedding and assigned to receive either 60 mg/kg/day of cocaine or vehicle (sterile water) by intragastric intubations. Dams in the vehicle control group were pair-fed and watered with the cocaine group beginning on G8 (see prenatal cocaine dosing section below). All dams except those in the pair-fed group had free access to food and water. The rats were kept under a 12 h light–dark cycle (lights on at 7:00 h) and at a temperature of 20–22 °C. All procedures were approved by SUNY Institutional Animal Care and Use Committee.

2.2. Prenatal cocaine dosing Prenatal cocaine administration was carried out as previously described in Ref. [16]. Briefly, beginning on G8 and continuing daily until G22 (inclusive) rats received either 60 mg/kg/day cocaine HCl (NIDA Research Triangle Institute, Research Triangle Park, NC) or vehicle (sterile water, Baxter, Deerfield, IL) administered by intragastric intubation using a 16 gauge straight feeding needle in a volume corresponding to 5 ml/kg of body weight. We have previously reported that a dose of 60 mg/ kg of cocaine by intragastric intubation produces a peak fetal level of the drug of 3000 ng/ml in plasma at approximately 15 min after maternal administration [13], similar to plasma drug levels seen in human fetuses [1,37]. Each dam in the vehicle control group was paired to a dam that belonged to the cocaine group weighing within ± 5 g on G1. The pair-fed dam was only given the amount of food and water consumed by the cocaine-treated rat for the same gestational day. On the day of birth (usually G23) and designated as postnatal day 1 (PND1) all pups were sexed, weighed, culled to 10 pups (5 males, 5 females) and surrogate fostered to non-treated dams delivering within the preceding 48 h. Animals were weighed every 7 days until PND 28. At PND 21 animals were ear punched and separated into same sex cages containing 5 pups until the day designated for behavioral testing. 2.3. Behavioral procedure On a single day between PND 41–44 rats were tested for the behavioral response to MPD or vehicle. From each litter, 2 males and 2 females were randomly assigned to receive saline, and similarly other pairs of males and females were assigned to receive MPD. A total of 8 animals from each litter was used such that no more than 2 animals, and in some instances just 1 animal from a particular litter contributed to a single sex/challenge drug group. We chose PND 41–44 rather than an earlier age (PND 30 or 35) because at this age females have reached sexual maturity (vaginal opening) and it is close to the onset of puberty (preputial separation) for males [34]. Before receiving any drug, rats were placed for 20 min in a Plexiglas box (42 × 42 × 30 cm with no bedding) equipped with a Versamax activity monitor (VMRXYZ16; Accuscan Instruments, Columbus, OH) to record baseline locomotor activity. After 20 min rats were injected with either 10 mg/kg of methylphenidate HCl (NIDA Research Triangle Institute, Research Triangle Park, NC) or 0.9% NaCl solution (Baxter, Deerfield, IL) intraperitoneally (i.p.) at a volume of 1 ml/kg. The dose of MPD chosen for this study was based on the findings from a previous study from our laboratory [62] in which we did not find gender differences in behavioral responses to MPD for adolescent (PND 45) animals, thereby providing a stable behavioral background to measure changes induced by prenatal cocaine. Although the dose selected was higher than doses used clinically, 10 mg/kg of MPD produces robust behavioral responses in rats and is in the high range of MPD doses used by humans for recreational purposes [33,41]. Following the injection, locomotor activity was recorded for 1 h in 12–5 min bins. Rats were

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also videotaped using a VHS RCA camcorder (CC4352) for later analysis of 7 different categories of behavior: time spent quiet, non-stereotyped sniffing, grooming, rearing and 3 intensities of stereotyped behavior: low, medium and high intensity stereotypy using the computer program Noldus Observer 5.0 (Noldus, Wageningen, The Netherlands) as previously described [62]. Behavioral categories measured were defined as follows: (1) Quiet: sitting or sleeping without any observable movements. (2) Sniffing: movement of the whiskers, but without stereotyped head movements. (3) Grooming: repetitive movements of the frontal paws around head or other parts of the body. (4) Rearing: lifting both front paws from the floor for greater than 1 s. (5) Low intensity stereotypy: repetitive movements of the head side to side usually combined with locomotion. (6) Medium intensity stereotypy: faster movements of the head, with or without locomotion. (7) High intensity stereotypy: occurs only when the animal is not locomoting and is characterized by big and fast circular movements of the head. An observer blind to the treatment status measured a total of 19 min of behavior from the videotapes distributed as follows: 2 min during baseline, 1 min at 12 min after drug injection, 15 continuous min at 20 min after injection which corresponds to the peak in behavioral activity [62] and 1 min at 30 min after injection. Female rats were subjected to vaginal smears after behavioral assessments to determine the phase of estrous cycle. 2.4. Statistical analyses Maternal weight gain during pregnancy, litter size and litter sex ratio were analyzed using a Student's t-test. Since more than one pup from a litter was used in the experimental design and this has been shown to affect α adversely [28], the statistical analysis was designed to control for significant litter effects. Weight gain of pups across development was analyzed with a generalized mixed linear model with litter as covariate in a 2 sex × 2 treatment × 5 day design. Baseline locomotor activity was cube transformed and analyzed using a 2 prenatal treatment × 2 sex generalized mixed linear model with litter as covariate. Locomotor activity after MPD injection was also cube transformed and analyzed using a 2 prenatal treatment × 2 sex generalized mixed linear model with litter as covariate but

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only for the MPD-injected rats due to the high incidence of 0 values in the data of saline-injected animals. Time spent in each of the observed behaviors was analyzed separately for each category of behavior. Stereotyped behaviors were analyzed only for the MPD injected animals (since these behaviors were not observed in saline-injected rats) using the generalized mixed linear model. Pre-planned simple effects analyses of prenatal treatment within gender were also conducted. Given that the generalized mixed linear model assumes that the standard deviation is the same in every group, the data were transformed to comply with this requirement. Statistical analyses were carried out using SAS software (SAS Institute, Cary, NC). Stages of estrous cycle were assigned a numeric value and were analyzed using the Kruskal–Wallis non-parametric test within SYSTAT (SPSS, Chicago, IL). A value of p b 0.05 was used as the acceptable level of significance. 3. Results A total of 88 pups belonging to 12 different litters was used in this study. The final number of animals per group was 10–12. 3.1. Physiological measurements There was no difference between the weight gain during gestation of cocaine pre-treated dams and water pair-fed dams for either the period between G1 and G22 or during the treatment period itself, G8 to G22 (Table 1). In addition, prenatal cocaine treatment did not produce any difference in the litter size or the sex ratio of litters compared to control litters (p N 0.05 for all comparisons using t-test; see Table 1). The body weight of pups during development measured every 7 days showed a significant main effect of sex (F(1, 402) = 32.35, p b 0.001) for which the females weighed less than the males. No main effect of prenatal treatment was found for the body weight of pups on PND 1, 7, 14, 21 or 28. However, we observed a significant prenatal treatment by day interaction (F(4, 402) = 9.68, p b 0.001). Analysis of simple effects of the treatment on each day revealed that there were no effects of prenatal treatment on the weights for each individual day. 3.2. Locomotor activity Baseline locomotion and locomotion after either saline or MPD injections in male and female rats are illustrated in Fig. 1. Baseline locomotor activity collected during 20 min before injection was averaged and analyzed as a single value. For baseline activity, we observed a significant main effect of sex (F(1, 75) = 7.73, p = 0.006). Female rats showed a 16% lower

Table 1 Physiological measurements of litters Pair fed controls

Cocaine treated

Litter

1

2

3

4

5

6

Mean ± SE

7

8

9

10

11

12

Mean ± SE

Weight gain G1-22 Weight gain G8-22 Litter size Male to Female ratio

72.5 64.7 14 0.75

154.8 123.4 19 1.38

160.2 142.1 17 1.43

115 97.9 16 0.6

103.2 82.9 15 1.5

102.1 89.2 14 1.33

118.0 ± 13.8 100.0 ± 11.5 15.8 ± 0.8 1.2 ± 0.2

153.7 113.2 16 1

105.4 98.7 12 2

134.3 114.9 17 0.88

117.5 90.1 14 0.75

106.4 93.7 15 2

126 92.7 13 1.6

123.9 ± 7.5 100.6 ± 4.4 14.5 ± 0.8 1.4 ± 0.2

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A M-Wat-Sal M-Coc-Sal M-Wat-MPD M-Coc-MPD

300

3000 250

2500 200

2000

150

12

9 10 11

8

7

6

1

5

0 4

500

0

3

50

2

1000

B 3500

*

300

3000 250

*

2500

*

12

10 11

9

0 8

0

7

500

6

50

5

1000

4

100

3

1500

2

150

F-Wat-Sal F-Coc-Sal F-Wat-MPD F-Coc-MPD

*

2000

1

200

Ba se

Total distance traveled (cm)

these illustrate the effects of prenatal cocaine administration. High intensity stereotypy was not observed in any of the groups. Other observed behaviors will only be discussed in the text. For reliability, a subset of the sessions was re-scored and compared to the first measurement. A correlation coefficient of r = 0.9601 was achieved.

1500

100

Ba se

Total distance traveled (cm)

3500

Fig. 1. Locomotor activity during baseline and after injection of either saline or MPD collected for 1 h and illustrated in 5 min intervals. (A) Adolescent male rats and (B) adolescent female rats that were exposed to prenatal cocaine or water (controls). Baseline activity was collected during 20 min and represented as an average for that period in a separate panel to illustrate sex differences in behavior. ● = prenatal water and saline controls, ○ = prenatal cocaine treatment and salineinjected animals, ▴ = prenatal water and MPD injected animals, ▵ = prenatal cocaine treatment and MPD injected animals. N = 10–12 rats per group. Bars represent SEM and ⁎ represents significantly different from all other groups by test of simple effects.

locomotor activity during baseline compared to male rats. This was mainly observed for the first 15 min of baseline activity collection because by 20 min both sexes showed minimal activity. After the injection of MPD we found a significant three way interaction of prenatal treatment by sex by intervals of locomotor activity (F(11, 431) = 2.28, p = 0.01). The saline injected subjects were not included in this analysis. Tests of simple effects revealed that female rats of the prenatal control group injected with MPD showed a significantly higher locomotion than all the other groups that received MPD for intervals 2–5 corresponding to 10–25 min after the injection (interval 2: p = 0.003; interval 3: p = 0.002; interval 4: p = 0.009; interval 5: p = 0.04). The patterns of locomotor activity in both groups of males that received MPD and the females in the prenatal cocaine group were very similar (Fig 1, A and B). 3.3. Observed behaviors For simplicity purposes, we will only illustrate changes produced in sniffing and the 2 different categories of ste reotyped behaviors: low and medium intensity stereotypy since

3.3.1. Sniffing There were no effects of prenatal treatment or sex for sniffing during the baseline activity period (not shown). Total sniffing after the injection of either saline or MPD is shown on Fig. 2, A and B. We found a significant main effect of drug administration (saline or MPD) (F(1, 94) = 69.05, p b 0.001) but no effect of prenatal treatment or sex. Preplanned comparisons showed that both male and female rats that received MPD spent significantly more time sniffing compared to rats that received saline regardless of their prenatal treatment. 3.3.2. Low intensity stereotypy There was no low intensity stereotypy for any of the groups during the baseline activity period; therefore it was not analyzed. Similarly, saline injected animals did not show low intensity stereotypy after injection and were excluded from the analysis. Total time spent in low intensity stereotypy for MPD injected animals is shown in Fig 2, C and D. For MPD injected animals we found a significant main effect of prenatal treatment (F(1, 41) = 5.01, p = 0.03). Preplanned evaluation of simple effects of prenatal treatment within each sex revealed that male rats that received cocaine during the prenatal period showed a significant decrease in the amount of time spent in low intensity stereotypy compared to the control group (p = 0.043). The amount of low intensity stereotypy in female rats that received MPD and were prenatally exposed to either cocaine or water was similar. 3.3.3. Medium intensity stereotypy Similar to low intensity stereotypy, medium intensity stereotypy was absent in all groups during baseline and therefore was not analyzed. Saline injected animals were also excluded since this behavior was not observed in these animals. Total time spent in medium intensity stereotypy for MPD injected animals is shown in Fig 2, E and F. In general, medium stereotypy rarely occurred in both groups of MPD injected animals compared to the time these animals spent in sniffing or low-intensity stereotypy. There were no significant effects of either prenatal treatment or sex observed for medium stereotypy. However, both male and female rats prenatally exposed to cocaine showed a 92% reduction in the time spent in medium intensity stereotypy compared to MPD-injected water controls. This suggests that the reduction observed in low intensity stereotypy for prenatally cocaine-exposed males was not due to a shift toward higher intensities of stereotyped behavior. 3.3.4. Other observed behaviors During the baseline activity period we did not observe any effects of sex or prenatal treatment for the amount of time spent in quiet, rearing or grooming behavior. In fact, rearing and grooming rarely occurred during the baseline activity period.

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treatment in MPD injected animals. Saline animals were excluded from the analysis due to the high incidence of zero values in these behaviors. 3.4. Estrous cyclicity Analysis of the estrous cycle revealed that the cycles of female rats were evenly distributed among all the stages for rats grouped by saline or MPD treatment (Kruskal–Wallis: p = 0.96) as well as for rats grouped by water or cocaine prenatal treatment (p = 0.149). Therefore it is unlikely that behavioral results are due to the clustering of rats in one stage of the estrous cycle. 4. Discussion

Fig. 2. Total time spent in sniffing and two different intensities of stereotyped behavior during 1 h after either MPD or saline injections. (A) Sniffing in male rats, (B) sniffing in female rats, (C) low intensity stereotypy in male rats, (D) low intensity stereotypy in female rats, (E) medium intensity stereotypy in male rats, (F) medium intensity stereotypy in female rats. White bars illustrate water pretreated rats and gray bars represent cocaine pre-treated rats. Empty bars illustrate saline injected animals and hatched bars illustrate MPD injected animals. Data for group means illustrated with bars representing SEM. ⁎ represents MPD injected groups significantly different from saline injected animals regardless of prenatal treatment. # represents significantly different from prenatal control males that also received MPD.

Time spent in quiet behavior was larger for saline-injected rats than for MPD injected rats (not shown) but this is a reflection of the increase in other active behaviors as expected following psychostimulant administration. For rearing and grooming behaviors, we did not observe any effects of sex or prenatal

Our study is the first to demonstrate that prenatal cocaine exposure produces a sex and behavior-specific reduction in responses to a high dose of methylphenidate administration during the adolescent period. Prenatal cocaine exposed rats that were not challenged with MPD showed a normal pattern of activity. In addition, baseline activity of all groups was similar, supporting the notion that prenatal cocaine exposure does not produce abnormal exploratory behavior or alter baseline activity in adolescent rats [48]. Several previous studies have evaluated the effects of prenatal cocaine exposure in rodents on the response to psychostimulants such as cocaine or amphetamine [10,20,27,36,45], but the results are conflicting. The major difference between our study and previous studies is the route of administration for cocaine. All previous studies with the exception of one [45] used subcutaneous injections of cocaine, compared to intragastric administration in the present study. The plasma levels of cocaine that are produced after subcutaneous injections of the drug peak more slowly [9] compared to intragastric administration [13] which may result in different neurochemical effects. Other factors such as the use of different rat strains (Long–Evans vs. Sprague–Dawley) could also contribute to the differences observed among studies. In addition, the length of cocaine exposure during the rat pregnancy was also different among the previous studies. This might have been a major factor which contributed to the differences observed since each region undergoes a unique pattern of development throughout the pre- and postnatal period in the rat (for a review see Ref. [2]). Finally, most of the previous studies evaluated psychostimulant responses in prenatal cocaine exposed animals during adulthood and there is ample evidence that adolescents respond differently to psychostimulants compared to adults [55]. We believe that studying the responses to psychostimulants during the adolescent period is important since during this period many behavioral abnormalities such as attention deficits become evident clinically [24]. 4.1. Possible mechanism by which prenatal cocaine decreases the behavioral response to methylphenidate during adolescence Our data suggest that the mechanism by which prenatal cocaine decreases the behavioral response to MPD is different

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in males versus females since prenatal cocaine diminishes the locomotor response only in females and stereotyped behavior was diminished only in the males. Since different neuronal circuits mediate locomotor activity and stereotyped behavior, the data suggest that prenatal cocaine has selective effects on the development of these circuits in male and female rats. The first factor to consider is that mesencephalic neurons from males show a maturational delay compared to neurons from females in cell cultures [6]. Rats from embryonic days 16–21 showed sexspecific differences in the density of GABAergic and tyrosine hydroxylase immuno-reactive fibers in the striatum. Specifically, striata from female rats showed higher densities of tyrosine hydroxylase-positive axons and GABA immuno-reactive cell bodies compared to male rats of the same embryonic age [43]. Moreover, the dopamine uptake capacity of sex specific cell cultures from embryonic days 14 and 17 was always higher in the female compared to male cultures [46] suggesting an earlier functional maturation of dopaminergic fibers in females compared to males. Consequently a similar prenatal cocaine exposure period in males and females will result in a longer exposure for striatal cells in females since the maturation is earlier than that in the males. On the other hand, an earlier maturation of dopaminergic activity in the female may also reflect the greater concentration of cells that contain the dopamine transporter (DAT) upon which cocaine acts [7,38– 40,49]. Both of these factors might render females more susceptible to the neurotoxic effects of cocaine. However we propose that there are additional factors that interact to produce the abnormal behavior following prenatal cocaine exposure observed in both males and females. It was recently published that male rats exposed to cocaine prenatally show deficits in striatal dopamine release while no difference was observed in female rats [22]. Prenatal cocaine exposure can also produce a deficit in function of dopamine D1 receptors in the striatum, specifically by uncoupling the D1 receptor from its second messenger [17,63,64]. In addition, the uncoupling of D1 receptors appears to be sex-specific, as assessed by behavioral responses to selective D1 or D2/3 agonists in rats exposed to cocaine during the preweaning period [14] or prenatally [29]. Since the induction of stereotyped behavior has been shown to require striatal D1 activation [8] and D1 receptor activity is dampened in cocaine pre-exposed male rats, we believe that the ability of methylphenidate to induce stereotypy in cocaine pre-exposed male rats compared to control animals is hindered due to the decrease in dopaminergic D1 response system in the striatum. Female rats appear to be somewhat protected from this deficit due to intrinsic developmental differences outlined above regarding the striatum and perhaps due to direct effects of estrogen on striatal dopamine release [3]. On the other hand, locomotor activity in response to MPD was only dampened in cocaine pre-exposed females as compared to controls and no differences in locomotor activity were observed between the prenatal exposure groups of males. Normal female rats show increases in behavioral responses to psychostimulants compared to males regardless of differences in drug metabolism [4], as observed in the present study for

locomotor activity. In addition to sex specific effects of prenatal cocaine in the dopaminergic system, it has been reported that prenatal cocaine produces a reduction in the serotonergic 5-HT 1A receptors, specifically at PND 30 and only in female prenatally-exposed rats [31] compared to controls. Serotonin 1A agonists have been shown to potentiate the locomotor responses to psychostimulant administration [42]. Therefore, since prenatal cocaine selectively reduces serotonergic activity in adolescent females and serotonin contributes to the locomotor response to psychostimulants, these two factors might have contributed to the reduced locomotor response to MPD observed in prenatally cocaine-exposed females in the present study. However, it is possible that if behavior is measured during adulthood, the same pattern of differences might not be seen since in adulthood serotonergic activity reaches normal levels in prenatally cocaine exposed female rats compared to controls and in turn, prenatally cocaine exposed male rats exhibit a deficiency during adulthood (see Ref. [31]). The effects of prenatal cocaine on noradrenergic mechanisms can influence the locomotor enhancing properties of psychostimulants such as amphetamine [11] and MPD might also contribute to the behavioral outcome observed in female rats. In fact, prenatal cocaine exposure affects the neural outgrowth and connectivity of locus coeruleus neurons (which are the majority of the noradrenergic cells) more so in females than in males [52]. This suggests that decreased noradrenergic innervation of the female brain might also contribute to the decreased behavioral response to MPD observed in prenatally cocaine treated animals. 4.2. Clinical relevance One of the major problems in children prenatally exposed to cocaine is their deficit in the regulation of attention and arousal, deficits similar to those observed in children diagnosed with Attention Deficit Hyperactivity Disorder (ADHD). Clinical studies have suggested that children with ADHD may have deficits within dopamine pathways involving the frontal cortex and basal ganglia [61]. It is tempting to hypothesize that some of these children diagnosed with ADHD may have been exposed to cocaine prenatally. However it is difficult to test this hypothesis since parents are reluctant to disclose prenatal drug histories and this information is rarely included in birth records [60]. In addition, there is no available prospective clinical study in prenatally cocaine-exposed individuals that has specifically reported the number of patients diagnosed with ADHD. However, there is clinical evidence suggestive of similar behavioral disturbances in prenatally cocaine exposed children and those diagnosed with ADHD [12,32,47,51,58]. Thus future clinical studies in children with attention and arousal problems should consider studying, whenever possible, if the individual was exposed to prenatal cocaine and/or other prenatal neuroteratogens that might affect the behavior of the individual later in life. In addition, prenatal exposure to neuroteratogens such as cocaine should be assessed in the medical history of children treated for attention and arousal problems in order to better design an appropriate treatment when psychostimulants such as MPD are prescribed.

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