Effect of methamphetamine exposure during ... - Wiley Online Library

Romana Slamberova´ Marie Pometlova´ Richard Rokyta Department of Normal Pathological and Clinical Physiology Third Faculty of Medicine Charles University in Prague Prague, Czech Republic E-mail: [email protected]

Effect of Methamphetamine Exposure During Prenatal and Preweaning Periods Lasts for Generations in Rats ABSTRACT: Our previous studies demonstrated that methamphetamine (MA) administration during gestation and/or lactation affects maternal behavior in rats and that birth weight and sensory-motor coordination of their pups are also influenced. The present study tested the hypothesis that the effect of MA induces long-term changes affecting second generation of rats that were not exposed to the drug. Adult females exposed during prenatal and preweaning periods to 5 mg/kg MA daily, were examined for regularity of estrous cycle and mated with stimulus, unexposed males. Dams (nontreated absolute control, saline- and MA-exposed) were observed with their pups in two tests of maternal behavior (observational and retrieval tests). Their pups were further tested throughout the preweaning period to examine their development. Our data demonstrate that MA-exposed mothers displayed more nursing, were more often in the nest and in contact with their pups, and were faster in retrieving their pups than saline-exposed and/or control mothers. There were no differences in litter characteristics, birth weight and weight gain of pups between groups. Interestingly, pups from mothers exposed to MA during prenatal and preweaning period had impaired sensory-motor coordination. They achieved righting reflex in mid-air later than both control groups. Additionally, they had more falls in rotarod and bar-holding tests than pups from both control and saline-exposed mothers. In homing performance, pups from MA- and salineexposed dams learned slower to return to the home box than pups from control dams. Thus, the present study demonstrates that MA abused by mothers may affect two generations of their offspring. ß 2007 Wiley Periodicals, Inc. Dev Psychobiol 49: 312–322, 2007. Keywords: methamphetamine; generation; maternal behavior; development; sensory-motor coordination; homing

INTRODUCTION Family life affects development, behavior, and predisposition to some diseases of each individual is considerable. Physical and emotional deprivation and family Received 1 June 2006; Accepted 5 November 2006 Correspondence to: Romana Sˇlamberova´ Contract grant sponsor: Internal Grant Agency of the Ministry of Health of the Czech Republic Contract grant number: 1A8610-5/2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/dev.20203 ß 2007 Wiley Periodicals, Inc.

conflicts may induce long-term consequences in postnatal development of a child (Bifulco, Brown, & Adler, 1991; Meaney, 2001). Preclinical experiments demonstrated that female pups’ experiences during prenatal and preweaning periods could significantly affect their future maternal behavior toward their own offspring (Fleming, O’Day, & Kraemer, 1999). Meaney and colleagues (Francis, Diorio, Liu, & Meaney, 1999; Meaney, 2001) repeatedly showed that a trans-generational rule of ‘‘like mother–like daughter’’ exists. They (Francis et al., 1999; Meaney, 2001) found that female pups of rat mothers that received increased maternal care were also ‘‘better mothers’’ in their adulthood than female rats from mothers exhibiting neglected maternal behavior. Thus, it seems

Developmental Psychobiology. DOI 10.1002/dev

Methamphetamine Affects Two Generations

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that the ability of maternal behavior is transmitted from generation to generation. Our previous studies (Sˇlamberova´, Charousova´, & Pometlova´, 2005a,b) demonstrated that methamphetamine (MA) administration during gestation and/or lactation affects maternal behavior in rats. Further, we found (Sˇlamberova´, Pometlova´, & Charousova´, 2006) reduction in birth weight in pups exposed to MA during prenatal and/or preweaning periods. Sensory-motor coordination examined by righting reflex, rotarod and bar-holding tests was also affected (Sˇlamberova´ et al., 2006). There are few studies (Becker & Randall, 1987; Friedler, 1978; Lam, Homewood, Taylor, & Mazurski, 2000) available including our own (Sˇlamberova´, Riley, & Vathy, 2005d) showing that prenatal drug or alcohol abuse may affect two generations. Friedler (1978) demonstrated that there is a transgenerational effect of morphine on body weight and motor coordination when administered to female mice prior to gestation period. Similarly, in our previous work (Sˇlamberova´ et al., 2005d) offspring of prenatally morphineexposed dams exhibited a longer latency to right than controls. Additionally, Becker and Randall (1987) showed that female mice prenatally exposed to alcohol were more likely to have offspring with lower fetal weight. Also, more recent work by Lam et al. (2000) showed lower weight gain and increased latency to right in pups from prenatally alcohol-exposed mice than controls. Thus, there is evidence that alcohol and drug exposure may affect even second-generation animals. There are no studies examining the effect of MA or other stimulant drugs like amphetamine or cocaine on development of second generation of drug-abused women or drug-exposed animals. Therefore, the present study tested the hypothesis that MA induces long-term changes lasting for two generations in rats. That is, mothers exposed to the drug during prenatal and preweaning periods (first generation) and their pups (second generation) that were not exposed to the drug at all.

week in a temperature-controlled (22 –24 C) colony room with free access to food and water on a 12 hr (light): 12 hr (dark) cycle with lights on at 0600 hr. One week after arrival, females were randomly assigned to MA-treated, saline-treated, or control group. MA was injected daily subcutaneously (s.c.) in a dose of 5 mg/kg (the total of the d-Methamphetamine HCl) for approximately 9 weeks: about three weeks prior to impregnation, throughout the entire gestation period and for 21 days of lactation (for details see Sˇlamberova´ et al., 2005a). This timing of drug administration (during premating, gestation, and lactation) better mimic the situation in drug abuse women. The dose of 5 mg/kg of MA was chosen based on the findings of Weissman and Caldecott-Hazard (1995) showing that this dose alters locomotor and exploratory behaviors. Moreover, our previous finding demonstrating that rat pups exposed prenatally to daily dose of 5 mg/kg of MA impairs their locomotor activity (Sˇlamberova´ et al., 2006). There are studies showing that this dose results in fetal brain drug concentrations, which approximate those reported in human infants whose mothers abuse MA (Acuff-Smith, Schilling, Fisher, & Vorhees, 1996; Cho, Lyu, Lee, Kim, & Chin, 1991; Martin, Martin, Radow, & Sigman, 1976; Won, Bubula, McCoy, & Heller, 2001; Yamamoto, Yamamoto, Fukui, & Kurishita, 1992). The concentration of the solution was 10 mg/ml, thus, each animal received the amount of .5 ml/kg of the solution. Saline was injected s.c. at the same time and volume as MA. Control females did not receive any injections. On day 21 of gestation, females were removed from the group cages and placed into maternity cages. The day of delivery was counted as postnatal day (PND) 0. Number of pups in each litter was adjusted to 10. Whenever possible the same number of male and female pups was kept in each litter. On PND 21 pups were weaned, ear-punched for identification, and housed in groups by sex. Animals were left undisturbed until adulthood. Only one female from each prenatal treatment was used from each litter to avoid litter effects. The remaining animals were assigned to other studies. Thus, 30 prenatally exposed females were tested for maternal behavior (as described below); 10 from each drug exposure (control, saline, MA). The procedures for animal experimentation utilized in this report was reviewed and approved by the Institutional Animal Care and Use Committee and is in agreement with the European Communities Council Directive No. 86/609/EEC.

METHODS

Experiment 1: Mothers (First Generation)

Drugs Physiological saline (.9% NaCl) was purchased from Sigma (Prague, Czech Republic), D-Methamphetamine HCl was provided from Faculty of Pharmacy of Charles University in Hradec Kra´love´ (Czech Republic). Prenatal and Preweaning Treatment Adult female albino Wistar rats (n ¼ 30; 250–300 g) were purchased from Anlab farms (Prague, Czech Republic). Animals were housed in groups (4–5/cage) and left undisturbed for a

Estrous Cycle. For 3 weeks prior to impregnation, adult (PND 60–80) female rats (n ¼ 10) were weighed daily and smeared by vaginal lavage to see whether or not prenatal MA-exposure altered their weight and/or the regularity of their estrous cycle. The smear was examined by light microscopy using 20 magnification. The regularity of the estrous cycle and the duration of each cycle were recorded. Estrous cycle was considered regular when it was of 4–5 days duration and reoccurred regularly during the period of 3 weeks. Estrous cycle was considered irregular when it was about 4 days long, but reoccurred irregularly during the period of 3 weeks, or when it was longer than 5 days (Aiello, 1998; Sˇlamberova´ et al., 2005a).

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Impregnation and Delivery. At the onset of the estrus phase of the estrous cycle, when the probability of conception is the highest (Sˇlamberova´ et al., 2005a; Turner & Bagnara, 1976) female rat was housed with a sexually mature, stimulus male overnight. There was always one female and one male in a cage. The next morning females were smeared again for the presence of sperm and returned to their home cages. This was counted as day 1 of gestation. All pregnant rats were weighed daily to see possible effects of MA treatment on weight gain during gestation period. On day 21 of gestation, females were removed from the group cages and placed into maternity cages. Expected day of delivery in our colony is the 22nd day of gestation. There were no females with a gestation period shorter than 22 days. The day of delivery was counted as postpartum day (PPD) 0. Observational Test. Maternal behavior was observed daily for 50 min in the home cage of each mother (n ¼ 8–10) and her litter between PPD 1–22. The time of observation was during the light phase of the light/dark cycle between 0800–0900 hr. A similar method was used as in our previous studies (Sˇlamberova´, Bar, & Vathy, 2003; Sˇlamberova´ et al., 2005a). During each 50-min session, each mother and her litter were observed 10 times for 5 s at 5-min intervals. Eleven types of activities and two nursing positions (see below) of mothers were recorded during each session. Thus, each mother and litter was observed 220 times (22 days 10 observations/session). During each observation ‘‘1’’ was given if a behavior occurred and a ‘‘0’’ if it did not. First, it was noted whether or not a mother was nursing. Two types of nursing were recognized: (a) active nursing: when the mother was actively positioned over her litter; (b) passivenursing: when the mother was lying on her side or on her back with one or more suckling pups. In addition to nursing, 11 other maternal activities were recorded during each session: (1) mother in or out of the nest; (2) mother in contact with any of her pups; (3) mother grooming any of her pups; (4) mother carrying pups; (5) mother manipulating nest shavings; (6) mother resting with eyes closed; (7) mother eating; (8) mother drinking; (9) mother self-grooming [eating, drinking, and self-grooming were also counted together as a single measure of self-care]; (10) mother sniffing with head raised; and (11) mother rearing. Retrieval Test. The same mothers and pups were tested for Retrieval test always after the Observational test ended. The retrieval test was conducted daily from PPD 1 to PPD 12 between 0900 and 1000 hr, so each mother (n ¼ 8–10) and litter was tested 12 times. The same method was used as in our previous studies (Sˇlamberova´ et al., 2003, 2005a). All pups were removed from their mothers and placed in a separate cage for 5 min. This short separation is not considered to be stressful for the pups or mother (Myers, Brunelli, Squire, Shindeldecker, & Hofer, 1989). The cage with pups was placed on a heating pad to prevent chilling. After this brief separation, the entire litter was returned to their mothers and the pups were scattered all around the cage. The mother was then observed for 10 min, and the following latencies were recorded: (1) to carry the first pup; (2) to return the first pup into the nest; and (3) to return all pups into the nest. Any unusual behaviors such as: (1) removing a previously returned pup from the nest; (2) intensive

Developmental Psychobiology. DOI 10.1002/dev caring of the pups around the cage before placing them into the nest; and (3) extensive disruption of the nest shavings, were evaluated as well. During each observation ‘‘1’’ was given if a behavior occurred and a ‘‘0’’ if it did not. Data Analyses. The incidence of regular or irregular estrous cycle and the incidence of successful and/or healthy pregnancy were analyzed by w2 test. The weight changes during the 3 weeks of injections prior to impregnation and the weight gained during the gestation period were analyzed using a one-way ANOVA (drug treatment). In the Observational test, the occurrence of each activity (maximum 10 in each session) was counted in each of 22 sessions. All activities were tested using a one-way ANOVA (drug treatment) with Repeated Measure (22 days of testing). In the retrieval test, latencies were analyzed by one-way ANOVA (drug treatment) with Repeated Measure (12 days of testing). Bonferroni test was used for post hoc comparisons in ANOVA analyses. Differences were considered significant if p < .05. Experiment 2: Pups (Second Generation) Day of birth was counted as PND 0. Number of pups in each litter was adjusted to 10. The final number of litters was: 10 controls, 8 salines, and 10 MA. Whenever possible the same number of male and female pups was kept in each litter; in most cases it was 5:5, but in very few cases it was 4:6. Even though all animals from each litter participated in all the tests, for statistical analysis the average number of animals in each sex and drug exposure of each litter was used as the unit. Thus, in each litter for statistical analyses six means were used that represent: control male, control female, saline male, saline female, MA male, MA female. The same animals were used in all tests. During the time while the pups were out of their home cage they were kept warm on a heating pad. Litter Characteristics and Maturation of the Pups. The same method was used as in our previous studies (Sˇlamberova´ et al., 2005a, 2006). Number of pups in the litter, number of dead pups, and percentage of males and females in each litter was recorded and compared between groups. Ano-genital distance (AGD) was measured on PND 1 for possible masculinizing or feminizing effects of MA. Pups were weighted daily between PND 1 and 22. The birth weight and weight gain for the 22 days of testing were used for statistical analysis. The days of eye opening, testes descend in males, and vagina opening in females were recorded. A one-way ANOVA (mother’s drug exposure) was conducted to analyze the number of pups in each litter, the number of stillborn and the percentage of males and females in each litter. A two-way ANOVA (mother’s drug exposure sex) was used to analyze differences in AGD, birth weight, and weight gain during PND 1–22. The Bonferroni test was used for post hoc comparisons. For eye opening and sexual maturation (in males ¼ testes descend, in females ¼ vagina opening) a w2 test was used on specific postnatal days. Differences were considered significant if p < .05 in all analyzes. Righting Reflex in Mid-Air. As in our previous work (Sˇlamberova´ et al., 2006) righting reflex in mid-air was checked



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daily after PND 12 until it successfully displayed. Each pup was held on its back 40 cm above a soft pad, then released and observed the position it reached the soft pad. A score of ‘‘1’’ was given when a pup reached the ground at once with all four paws and a ‘‘0’’ when it did not. A w2 test was used for statistical analyses of the righting reflex in mid-air in specific days. Differences were considered significant if p < .05.

The numbers of jumps were compared by w2 test. The animals that chose the ‘‘strategy of jumping’’ were removed from the rest of the statistical analyses. Trials were analyzed by a two-way ANOVA (mother’s drug exposure sex) with Repeated Measure (trials) to assess the pups’ ability to learn new task. The best performance and the number of falls were analyzed using a twoway ANOVA (mother’s drug exposure sex). For post hoc comparisons, the Bonferroni test was conducted. Differences were considered significant if p < .05.

Homing. Homing test is a test of pups’ nest-seeking behavior that was used on PND 15. The same method was used as in our previous work (Sˇlamberova´ et al., 2006) and was adopted from the studies of Franˇkova´ and Tikal (1989) and Bulut and Altman (1974). The apparatus contains two transparent Plexiglas boxes, which are crossable through a hole, 4 cm in diameter, located 1.5 cm above the floor at the midline of the dividing wall of the box. One box is defined as a ‘‘home box’’ and is divided to two equal compartments by a transparent Plexiglas wall, and the other box is defined as ‘‘starting box.’’ Pups were placed from their home cage to the half of the home box located farther from the starting box, so they were not able to cross over from the home box. This compartment of the home box was filled with bedding material. They were left undisturbed to habituate (approx. 10 min). After habituation, the tested animal was placed into the middle of the starting box facing away from the home box. The time that the animal needed to enter the home box was recorded. If the animal entered the home box with all four paws within 60 s the trial was counted as correct. If the animal did not enter the home box within 60 s the trial was marked as incorrect and the pup was placed with its littermates. The test was repeated at 1-min intervals until the pup succeeds at least four times in five consecutive trials (criteria for learning). The maximal number of trials was 10. A two-way ANOVA (mother’s drug exposure sex) with Repeated Measure (trials) was used to analyze differences in the latencies of homing. For post hoc comparisons, the Bonferroni test was conducted. The number of trials needed to finish the task and the number of correct and incorrect trials were compared between groups using a w2 test. Differences were considered significant if p < .05 in all measures.

Bar-Holding. The bar-holding test on PND 23 was used to examine vestibular function and sensory-motor coordination with the necessity to balance on the narrow bar as in our previous work (Sˇlamberova´ et al., 2006). A 40 cm long, 1 cm in diameter wooden bar was suspended 80 cm above a padded soft surface. The rat was held by the nape of its neck and its forepaws were allowed to touch the bar. The time of bar-holding was recorded with a limit of 120 s. Rats were subjected to three consecutive trials. Dropped boluses were counted after each trial. A two-way ANOVA (mother’s drug exposure sex) with Repeated Measure (trials) was used to analyze differences in the bar-holding test. For post hoc comparisons, the Bonferroni test was conducted. Number of falls was compared by w2 test. Differences were considered significant if p < .05.

Rotarod. On PND 23 rotarod performance was examined to test the sensory-motor coordination with necessity of active moving to hold the balance on the rotating cylinder (see Sˇlamberova´ et al., 2006). Pups were positioned on a rugged cylinder (11.5 cm in diameter; rotating at a constant speed of 6 rpm) in the opposite direction of cylinder rotation, so they were able to walk forward. The duration of balance on the rotarod was determined during 120 s. Rats were subjected to trials until they successfully accomplished the task. The maximal number of trials was 10. There were animals that chose to jump from the rotating cylinder rather than to stay there for the testing period. The choice of jumping looked clearly different than the falls from the cylinder. While the ‘‘falling animals’’ fell from the cylinder in the direction of the cylinder rotation, the ‘‘jumping animals’’ jumped actively in the forward direction to a specifically chosen place.

RESULTS Experiment 1: Mothers Estrous Cycle. There were no differences between groups in the regularity or length of the estrous cycle. Impregnation and Delivery. The incidence of successful and/or a healthy gestation and the length of gestation did not significantly differ between groups. The final number of litters was: 10 controls, 8 salines, and 10 MA. There were no significant differences between groups in weight gain during gestation period. All groups regardless of the drug treatment gained around 140–160 g in gestation. Even during lactation there was no difference in weight between groups. Observational Test. There was a main effect of drug treatment in active [F(2, 25) ¼ 3.53, p < .05] and passive [F(2, 25) ¼ 7.55, p < .01] nursing. As shown in Table 1, mothers that were exposed to MA in prenatal and preweaning periods displayed active nursing more than saline-exposed rats (p < .05) and passive nursing more than saline-exposed (p < .05) and control rats (p < .01). Additionally, there was a main effect of drug treatment in number of mother being in nest [F(2, 25) ¼ 4.24, p < .05] and in contact with her pups [F(2, 25) ¼ 8.23, p < .01]. MA-exposed mothers were more often in the nest (p < .05) and in contact with their pups (p < .01) than saline-exposed rats. In nonmaternal activities, there were

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Table 1. Effects of Prenatal and Pre-weaning Exposure to MA on Maternal and Non-maternal Activities of Rat Mothers Control

Saline

MA

Observational test Maternal activities Active nursing Passive nursing In nest In contact with pups Grooming pups Carrying pups Manipulating shavings

3.41 0.28 0.49 0.13 4.00 0.37 5.18 0.25 0.78 0.07 NA NA

2.75 0.31 0.36 0.14 3.20 0.41 4.10 0.28 0.67 0.08 NA NA

3.85 0.28 1.04 0.13 4.79 0.37 5.57 0.25 0.70 0.07 NA NA

þ *þ þ þ

Nonmaternal activities Sleeping Eating Drinking Self-grooming Sniffing Rearing

1.57 0.22 1.32 0.13 1.02 0.10 0.65 0.11 0.77 0.24 0.87 0.19

1.13 0.25 1.27 0.15 0.97 0.11 1.07 0.13 0.86 0.27 1.18 0.21

2.01 0.22 0.85 0.13 0.88 0.10 0.71 0.11 0.74 0.24 1.04 0.19

þ *þ

Retrieval test Carry 1st pup 1st pup in the nest All pups in the nest

231.65 43.56 238.47 46.07 336.33 42.89

108.92 49.04 126.07 51.50 266.20 47.95

35.35 43.86 39.47 46.07 227.50 42.89

* *

Values are averages of all 22 testing days in the Observational test and of all 12 testing days in the Retrieval test (means SEM); n ¼ 8–10. Numbers in Observational test is number of observed activity (max. 10) per day; numbers in Retrieval test is the time in seconds. NA ¼ not analyzed measures (low incidence of these activities could not be analyzed by the one-way ANOVA with repeated measure). *p < 0.05 vs control and þ p < 0.05 vs saline-exposed mothers (One-way ANOVA with repeated measure).

main effects in drug treatment in sleeping [F(2, 25) ¼ 3.55, p < .05] and eating [F(2, 25) ¼ 3.68, p < .05] of mothers. MA-exposed mothers relaxed more with their eyes closed than saline-exposed mothers (p < .05). There were no differences in other maternal and nonmaternal activities between groups. While maternal activities including active nursing decreased (data not shown), nonmaternal activities and the incidence of passive nursing increased as the postpartum time progressed regardless of drug exposure (data not shown). Retrieval Test. There was a main effect of drug treatment in latencies to carry the first pup [F(2, 25) ¼ 5.10, p < .05] and to return the first pup into the nest [F(2, 25) ¼ 4.69, p < .05]. As shown in Table 1, mothers that were exposed in prenatal and preweaning periods to MA were faster in carrying the first pup (p < .05) and in returning the first pup into the nest (p < .05) than control mothers. Their latency to carry the first pup [F(22, 275) ¼ 2.61, p < .001] and to return the first pup into the nest [F(22, 275) ¼ 2.79, p < .001] increased as the postpartum time progressed in control mothers, while it did not change in MA- and

saline-exposed rats. There were no differences between groups in the latency to return all the pups into the nest. No unusual behavior as described in Methods was observed.

Experiment 2: Pups Litter Characteristics and Maturation of the Pups. There were no differences between groups in litter characteristics, birth weight, weight gain, or maturation (data not shown). Righting Reflexes. As shown in Figure 1, the number of female pups from MA-exposed dams that successfully righted in mid-air on PND 15 was lower than the number of females from control or saline-exposed dams [w2 ¼ 7.74; p < .05]. Further, the number of female pups from saline-exposed dams that successfully righted in mid-air on PND 15 was higher than the number of females from control dams [w2 ¼ 7.74; p < .05]. The w2 test showed similar effect of MA and saline exposure on PND 14, PND 16, and PND 17 (data not shown). There



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FIGURE 1 Effect of MA exposure on righting reflex in midair in second generation. Values are percentages of pups that successfully accomplished the test on PND 15. n ¼ 46–48. p < .05 versus female pups from control (w2 test). þ p < .05 versus female pups from saline-exposed dams (w2 test).

were no significant differences in righting reflex in mid-air in male pups in any of these days. Homing. The number of pups that were not able to accomplish the criteria of 4 trials under 60 s in 5 consecutive trials did not differ between groups (data not shown). When the unsuccessful animals were removed from the data statistical analysis showed differences in the latencies of pups’ homing. There was a main effect of drug treatment in the latency of homing; in first five consecutive trials that were successfully accomplished [F(2, 53) ¼ 3.49, p < .05]. Specifically, as shown in Figure 2A pups from MA- (p < .05) and saline-exposed (p < .05) dams learned slower to return to the home box than pups from control dams regardless of sex. Even though the figure indicate drug differences only in male pups, the statistical results show main effect of mother’s drug exposure and no interaction [F(2, 53) ¼ .59, p ¼ .56] or effect of sex [F(1, 53) ¼ .67, p ¼ .42]. There was also main effect of drug treatment in the total time of the best five consecutive trials [F(2, 53) ¼ 14.93, p < .0001]. This measure indicates, additionally to the first five accomplished trials, the quickness of nestseeking after learning the task. Pups from MA- (p < .001) and saline-exposed (p < .0001) dams had higher total time of the best five consecutive trials than pups from controls regardless of sex (Fig. 2B). There was no effect of sex [F(1, 53) ¼ 1.32, p ¼ .26] and no interaction between mothers’ drug exposure and sex on the best trials [F(2, 53) ¼ 1.29, p ¼ .28]. Rotarod. There were significant differences between groups in ‘‘jumping’’ [w2 ¼ 6.8; p < .05]. Females of

FIGURE 2 Effect of MA exposure on homing performance in second generation. (A) Latencies of first five performances. (B) Summary of latencies of the five best consecutive trials. Values are means SEM. n ¼ average of animals of the same drug exposure and sex (8–10). (A) p < .05, main effect of prenatal drug exposure; MA < Control (two-way ANOVA with repeated measure). (B) p < .05 versus pups from control dams (two-way ANOVA with repeated measure).

MA- or saline-exposed dams jumped less often from the rotarod than controls (24/46 controls, 13/48 salines, 9/47 MA). There were no differences between groups of males in ‘‘jumping’’ (17/46 controls, 15/47 salines, 16/46 MA). The animals that ‘‘jumped’’ were not included in the statistical analyses. As shown in Figure 3A, there were no differences in time spent on the rotating cylinder during the fist three trials. However, the number of falls differed between groups. While there was no main effect of mothers’ drug exposure [F(2, 53) ¼ 2.54, p < .13] or sex [F(1, 53) ¼ 2.28, p < .11], there was an interaction between them [F(2, 53) ¼ 2.52, p < .05]. As shown in Figure 3B, female rats of MA-exposed dams fell more from the rotarod than female rats of control dams in the 120-s interval (p < .05). Bar-Holding. As shown in Figure 4A, there was a difference in the incidence of falls from the bar

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FIGURE 3 Effect of MA exposure on rotarod performance in second generation. (A) Times during which the animal stays on the rotating cylinder in first three trials. (B) Number of falls from the rotating cylinder during the entire experiment. Values are means SEM. n ¼ average of animals of the same drug exposure and sex (8–10). p < .05 versus female pups from control dams (two-way ANOVA).

[w2 ¼ 17.36; p < .01]. In both sexes pups of MA-exposed dams had a higher incidence of falling from the bar than pups of controls or saline-exposed dams. The difference was most apparent in male pups. Additionally, there was a main effect in the number of boluses dropped in trials [F(2, 53) ¼ 5.48, p < .01]. Pups of MA-exposed dams dropped significantly more boluses in each trial than pups of control (p < .05) or saline-exposed (p < .01) dams. As shown in Figure 4B boluses dropped was especially apparent in females.

DISCUSSION Our previous studies (Sˇlamberova´, 2005; Sˇlamberova´ et al., 2006; Sˇlamberova´, Pometlova´, Syllabova´, & Mancˇusˇkova´, 2005c; Sˇlamberova´ & Rokyta, 2005a,b;


FIGURE 4 Effect of MA exposure on bar-holding test in second generation. (A) Percentage of pups that were able to hold on the bar for the time of 120 s and pups that felt down, respectively. (B) Number of boluses dropped by the pups during one trial. Values are means SEM. n ¼ average of animals of the same drug exposure and sex (8–10). p < .01 versus pups from control or saline-exposed dams of the same sex [(A), w2 test; (B), two-way ANOVA].

Yamamotova´, Sˇlamberova´, Jedlicˇka, & Jakub, 2004) and the work of others (Acuff-Smith et al., 1996; Vorhees & Pu, 1995; Weissman & Caldecott-Hazard, 1995; Williams, Moran, & Vorhees, 2003) demonstrated that MA administered during prenatal and/or preweaning periods alter functional development of rat pups, impairs learning in adulthood, and alters pain and seizure thresholds in adult male and female rats. Further, we (Sˇlamberova´ et al., 2005a,b) demonstrated that administration of MA in gestational and/or lactation periods impairs maternal behavior that may affect prenatal and/or postnatal development of their pups. The finding of impaired maternal behavior after administration of excitatory drugs of mothers (compared to this study— generation 0) was shown also in studies of others (Elliott, Lubin, Walker, & Johns, 2001; Franˇkova´, 1977; Kinsley et al., 1994; Piccirillo, Alpert, Cohen, & Shaywitz, 1980;


Vernotica, Lisciotto, Rosenblatt, & Morrell, 1996). The present study extends these findings for test of maternal behavior and examination development of next generation rats that were not exposed to the effect of the drug at all. The present data show that MA exposure affects maternal behavior of female rats that were exposed to the drug during prenatal and preweaning periods (Experiment 1). Interestingly, dams exposed to MA displayed more nursing activities and were more often in the nest and in contact with their pups than saline-exposed or control rats. Based on the increased nursing and increased presence of mother in the nest it seems that MA-exposed dams are ‘‘better mothers’’ than saline-exposed or control dams. However, there may be other explanations, because the main difference observed was in passive nursing, which suggests an active role of pups not mothers. Moreover, the increased incidence of sleeping suggests that the presence of MA-exposed mothers in the nest and in contact with their pups was not an active maternal activity but passive activity such as sleeping. Interestingly, there were more differences between MA-exposed and saline-exposed mothers than between MA-exposed and control mothers in the observational test. Note that maternal injections may induce stress in MA- and saline-exposed animals that subsequently could have caused long-term changes in their behavior (Drago, Di Leo, & Giardina, 1999; Schindler, Sˇlamberova´, & Vathy, 2004). Thus, it seems that it is not the differences between MA and control group, but more likely the difference between MA and saline groups that is the one showing the effect of the drug per se. Already in our previous study (Sˇlamberova´ et al., 2005a) we demonstrated that administration of saline during gestation and lactation affects maternal activities of injected rat mothers. Nursing score of active nursing was smaller in saline-treated dams than controls without injection, but did not significantly differ from the score in MA-treated mothers. There are very few studies testing maternal behavior and how is it affected by stimulant drugs that would use two control groups (group with saline injection and absolute control without any injection). Only Peeke, Dark, Salamy, Salfi, and Shah (1994) demonstrated that dams injected with cocaine exhibited more nursing relative to both saline-exposed and control dams. Thus, they did not show any effect of saline injection on maternal behavior as we did in the present study. However, when they examined the effect of cocaine and saline injection on progeny of those dams they found that cocaine- as well as saline-exposed pups were more active in locomotion than control pups. In the retrieval test, the present study demonstrates that MA-exposed mothers are faster than controls in carrying the first pup and placing it into the nest, but that the latency


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to return all pups into the nest does not differ between groups. Interestingly, the difference between control and MA-exposed dams increases as the postpartum time progresses. It seems that control dams are less anxious about their pups after few days, while MA-exposed dams remain anxious to grip the first pup(s) during the entire experiment. When compared to our previous study (Sˇlamberova´ et al., 2005a) demonstrating increased latencies in retrieval test of mothers that were given MA during gestation and lactation, the present study examining the effect of MA on the next generation shows the opposite effect. Those generational differences are the same as in our previous work (Sˇlamberova´ et al., 2003; Sˇlamberova´, Szilagyi, & Vathy, 2001) examining the effect of morphine on maternal behavior of two generations. In those studies (Sˇlamberova´ et al., 2001, 2003) we demonstrated that morphine when administered to female rats during their gestation increased the latency to return all the pups into the nest. However, when mothers were exposed to morphine prenatally latencies in the retrieval test were decreased. Thus, we demonstrated that morphine has opposite effect on the retrieval test in two generations of female rats (Sˇlamberova´ et al., 2001, 2003). The question remains then: Are these generational differences induced by some breeding experiences and/ or stress or are prenatally drug-exposed females just faster than females exposed to drug during gestation? Information about the effect of MA on oxytocin levels would help to resolve this problem. Unfortunately, we found no such studies investigating MA effects on oxytocin levels. However, there are studies (Elliott et al., 2001; Johns et al., 2004; Johns, Lubin, Walker, Meter, & Mason, 1997) showing that acute or chronic cocaine administration alters oxytocin levels and the number and binding affinity of oxytocin receptors in brain areas related to nursing and maternal behavior. Thus, it might be that MA has similar effects on oxytocin as cocaine, because of their similar pharmacokinetics. Future studies would be necessary to examine such a hypothesis. When discussing the results of Experiment 2, the effect of MA on second generation is very interesting and brings novel evidence to the research of MA abuse. Similarly as in our previous work (Sˇlamberova´ et al., 2005d) and the study of others (Becker & Randall, 1987; Friedler, 1978; Lam et al., 2000) the present data show that MA exposure affects even generation that was not at all exposed directly to the effect of addictive substance. An obvious question arises here, is there a relation between changes in pups’ development and changes in maternal behavior. Based on the findings that MAexposed mothers spend more time with their pups, nurse more, and are faster in retrieving pups, one would expect improved development in their pups. However, our findings show the opposite to be true. Further, because

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MA-exposed dams eat less than control or salineexposed dams, expectation of lower birth weight of their pups would be expected. However, there are no changes in birth weight, weight gain during preweaning period or other litter or maturational characteristics. Thus, the current findings in maternal behavior do not seem to be responsible for any of those impairments observed in the development of pups discussed below. Despite all of these it should be noted that all measures of maternal behavior in the current study are crude and do not cover the complexity of behavior that could have impacted the development of pups. Similar to our previous work (Sˇlamberova´ et al., 2006) showing that MA impairs sensory-motor coordination in the first generation; the present data demonstrate impairing effects of MA on the performance of righting reflex in mid-air, rotarod, and bar-holding tests in the pups of second generation. These findings support our previous results as well as the data of others showing impairment in righting reflexes in second generation of mothers, who were receiving morphine or ethanol (Friedler, 1978; Lam et al., 2000; Sˇlamberova´ et al., 2005d). The reason why the second generation of pups are affected by drugs injected to their ‘‘grandparents’’ is still unresolved. There are studies reporting possible changes in genetics in two generations of rats exposed prenatally to alcohol or drugs (Jeng, Wong, Ting, & Wells, 2005; Lam et al., 2000). However, both genetic and epigenetic issues have to be taken into consideration to resolve this problem. Therefore, more studies are necessary to investigate maternal drugs of abuse on several generations of their offspring to assess possible genetic changes Interestingly, our results show that pups from mothers that were exposed to saline during prenatal and preweaning periods are slower in homing similarly to pups from MA-exposed mothers. It might be that daily saline injection to rat mothers during gestation and lactation acts as a stressor not only to injected mothers but also to their offspring. There are studies (Drago et al., 1999; Schindler et al., 2004; Sˇlamberova´ & Rokyta, 2005a) showing that prenatal saline exposure may cause mild prenatal stress and affect behavior of adult rats. However, no one examined whether this mild stressor-induced effect persists to the next generation that was not exposed to the stress. On the other hand, in performance of righting reflex in mid-air the result of saline and MA exposure relative to control was the opposite in female rats. This is another interesting finding suggesting that stress to the mother enhances her pups’ functional maturation relative to controls. The question however remains, why in some cases the stress induced by repeated maternal injections has the same effect as MA exposure and in some cases their effects are the opposite. It seems that stress affects differently learning, that requires a deliberate behavior,


while righting reflex does not. Further, it is possible that that MA effect results from a synergy between both the stress of injection and the drug itself. Our finding that pups (especially females) from MAexposed mothers dropped more boluses during the barholding test demonstrates an increased fear response relative to other pups. If one would think about homing test that it might be, at least partially, a task of ‘‘neophobia or anxiety,’’ our results showing increased latency of homing in pups of both MA- and saline-exposed dams would support our idea even further. Thus, it might be that pups of mothers exposed to MA or saline during prenatal and preweaning period respond to new environment with increased stress reaction. This hypothesis, however, would have to be verified in future experiments examining MA effects on the hypothalamic-pituitary-adrenal (HPA) axis in the second generation rats. Studies (McCormick, Smythe, Sharma, & Meaney, 1995; Richardson, Zorrilla, Mandyam, & Rivier, 2006; Vallee et al., 1997; Weinstock, Matlina, Maor, Rosen, & McEwen, 1992) showing that prenatal stress induces long-term changes in HPA axis support this hypothesis. The present results show also gender differences in specific tests. Interestingly, while sensory-motor coordination, such as righting reflex in mid-air, rotarod test or bar-holding test, drug-induced differences are apparent mostly in females, differences in the homing test are more obvious in male rats. Thus, it may be concluded that the impairment of MA on second generation is sex-specific and that male and female rats are differently sensitive to challenges presented to them in different tests. All, sensory-motor coordination, learning abilities, and stress response is known to be sex-dimorphic (Bardin & Catterall, 1981; Homo-Delarche et al., 1991). There are studies (Bisagno, Ferguson, & Luine, 2003; Bowman et al., 2004; Lehmann, Pryce, Bettschen, & Feldon, 1999) showing that prenatal stress and/or postnatal separation influences males and females differently. While females show greater differences induced by prenatal stress or postnatal separation in locomotion and responses to stress, males mostly demonstrate differences in learning abilities. In conclusion, the present study shows novel evidence that MA abused during gestation and lactation periods alters two generations of offspring. Thus, the present work extends previous evidence (Acuff-Smith et al., 1996; Sˇlamberova´ et al., 2005c, 2006; Vorhees & Pu, 1995; Weissman & Caldecott-Hazard, 1995; Williams et al., 2003) demonstrating several effects of prenatal MA exposure on first generation, and as such it took them to the next level. However, the mechanism(s) of how MA induces behavioral changes in the generation that is not exposed to the drug remains to be answered. To examine possible factors, such as breeding or genomic


transmission, may be the way to answer these MAinduced long-term effects.

NOTES This study was supported by grants: # 1A8610-5/2005 from the Internal Grant Agency of the Ministry of Health of the Czech Republic to R.Sˇ. The authors express their appreciation to Dr. Ilona Vathy for critical reading and editing of the manuscript and to Jarmila Kourˇilova´, Helena Smetanova´, and Zuzana Jezˇdı´kova´ for their excellent technical assistance.

REFERENCES Acuff-Smith, K. D., Schilling, M. A., Fisher, J. E., & Vorhees, C. V. (1996). Stage-specific effects of prenatal D-Methamphetamine exposure on behavioral and eye development in rats. Neurotoxicol Teratol, 18(2), 199–215. Aiello, S. E. (1998). Merck veterinary manual (8th ed.) Whitehouse Station, NJ, USA: Merck & Company, Incorporated. Bardin, C. W., & Catterall, J. F. (1981). Testosterone: A major determinant of extragenital sexual dimorphism. Science, 211, 1285–1294. Becker, H. C., & Randall, C. L. (1987). Two generations of maternal alcohol consumption in mice: Effect on pregnancy outcome. Alcohol Clin Exp Res, 11(3), 240–242. Bifulco, A., Brown, G. W., & Adler, Z. (1991). Early sexual abuse and clinical depression in adult life. Br J Psychiatry, 159, 115–122. Bisagno, V., Ferguson, D., & Luine, V. N. (2003). Chronic D-amphetamine induces sexually dimorphic effects on locomotion, recognition memory, and brain monoamines. Pharmacol Biochem Behav, 74(4), 859–867. Bowman, R. E., MacLusky, N. J., Sarmiento, Y., Frankfurt, M., Gordon, M., & Luine, V. N. (2004). Sexually dimorphic effects of prenatal stress on cognition, hormonal responses, and central neurotransmitters. Endocrinology, 145(8), 3778– 3787. Bulut, F., & Altman, J. (1974). Spatial and tactile discrimination learning in infant rats motivated by homing. Dev Psychobiol, 7(5), 465–473. Cho, D. H., Lyu, H. M., Lee, H. B., Kim, P. Y., & Chin, K. (1991). Behavioral teratogenicity of methamphetamine. J Toxicol Sci, 16 Suppl 1,37–49. Drago, F., Di Leo, F., & Giardina, L. (1999). Prenatal stress induces body weight deficit and behavioural alterations in rats: The effect of diazepam. Eur Neuropsychopharmacol, 9(3), 239–245. Elliott, J. C., Lubin, D. A., Walker, C. H., & Johns, J. M. (2001). Acute cocaine alters oxytocin levels in the medial preoptic area and amygdala in lactating rat dams: Implications for cocaine-induced changes in maternal behavior and maternal aggression. Neuropeptides, 35(2), 127–134. Fleming, A. S., O’Day, D. H., & Kraemer, G. W. (1999). Neurobiology of mother-infant interactions: Experience and


321

central nervous system plasticity across development and generations. Neurosci Biobehav Rev, 23(5), 673–685. Francis, D., Diorio, J., Liu, D., & Meaney, M. J. (1999). Nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science, 286(5442), 1155–1158. Franˇkova´, S. (1977). Drug-induced changes in the maternal behavior of rats. Psychopharmacology (Berl), 53(1), 83–87. Franˇkova´, S., & Tikal, K. (1989). Responses to the change in the environment in pairs of male rats genetically selected for activity level. Act Nerv Super (Praha), 31(4), 241–247. Friedler, G. (1978). Pregestational administration of morphine sulfate to female mice: Longterm effects on the development of subsequent progeny. J Pharmacol Exp Ther, 205(1), 33–39. Homo-Delarche, F., Fitzpatrick, F., Christeff, N., Nunez, E. A., Bach, J. F., & Dardenne, M. (1991). Sex steroids, glucocorticoids, stress and autoimmunity. J Steroid Biochem Mol Biol, 40(4-6), 619–637. Jeng, W., Wong, A. W., Ting, A. K. R., & Wells, P. G. (2005). Methamphetamine-enhanced embryonic oxidative DNA damage and neurodevelopmental deficits. Free Radic Biol Med, 39(3), 317–326. Johns, J. M., Lubin, D. A., Walker, C. H., Joyner, P., Middleton, C., Hofler, V., et al. (2004). Gestational treatment with cocaine and fluoxetine alters oxytocin receptor number and binding affinity in lactating rat dams. Int J Dev Neurosci, 22(5-6), 321–328. Johns, J. M., Lubin, D. A., Walker, C. H., Meter, K. E., & Mason, G. A. (1997). Chronic gestational cocaine treatment decreases oxytocin levels in the medial preoptic area, ventral tegmental area and hippocampus in Sprague–Dawley rats. Neuropeptides, 31(5), 439–443. Kinsley, C. H., Turco, D., Bauer, A., Beverly, M., Wellman, J., & Graham, A. L. (1994). Cocaine alters the onset and maintenance of maternal behavior in lactating rats. Pharmacol Biochem Behav, 47(4), 857–864. Lam, M. K., Homewood, J., Taylor, A. J., & Mazurski, E. J. (2000). Second generation effects of maternal alcohol consumption during pregnancy in rats. Prog Neuropsychopharmacol Biol Psychiatry, 24(4), 619–631. Lehmann, J., Pryce, C. R., Bettschen, D., & Feldon, J. (1999). The maternal separation paradigm and adult emotionality and cognition in male and female Wistar rats. Pharmacol Biochem Behav, 64(4), 705–715. Martin, J. C., Martin, D. C., Radow, B., & Sigman, G. (1976). Growth, development and activity in rat offspring following maternal drug exposure. Exp Aging Res, 2(3), 235–251. McCormick, C. M., Smythe, J. W., Sharma, S., & Meaney, M. J. (1995). Sex-specific effects of prenatal stress on hypothalamic-pituitary-adrenal responses to stress and brain glucocorticoid receptor density in adult rats. Brain Res Dev Brain Res, 84(1), 55–61. Meaney, M. J. (2001). Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations. Annu Rev Neurosci, 24, 1161–1192. Myers, M. M., Brunelli, S. A., Squire, J. M., Shindeldecker, R. D., & Hofer, M. A. (1989). Maternal behavior of SHR rats

322


and its relationship to offspring blood pressures. Dev Psychobiol, 22(1), 29–53. Peeke, H. V., Dark, K. A., Salamy, A., Salfi, M., & Shah, S. N. (1994). Cocaine exposure prebreeding to weaning: Maternal and offspring effects. Pharmacol Biochem Behav, 48(2), 403–410. Piccirillo, M., Alpert, J. E., Cohen, D. J., & Shaywitz, B. A. (1980). Amphetamine and maternal behavior: Dose response relationships. Psychopharmacology (Berl), 70(2), 195– 199. Richardson, H. N., Zorrilla, E. P., Mandyam, C. D., & Rivier, C. L. (2006). Exposure to repetitive versus varied stress during prenatal development generates two distinct anxiogenic and neuroendocrine profiles in adulthood. Endocrinology, 147(5), 2506–2517. Schindler, C. J., Sˇlamberova´, R., & Vathy, I. (2004). Cholera toxin B decreases bicuculline seizures in prenatally morphine- and saline-exposed male rats. Pharmacol Biochem Behav, 77(3), 509–515. Sˇlamberova´, R. (2005). Flurothyl seizures susceptibility is increased in prenatally methamphetamine-exposed adult male and female rats. Epilepsy Res, 65(1-2), 121–124. Sˇlamberova´, R., Bar, N., & Vathy, I. (2003). Long-term effects of prenatal morphine exposure on maternal behaviors differ from the effects of direct chronic morphine treatment. Dev Psychobiol, 43(4), 281–289. Sˇlamberova´, R., Charousova´, P., & Pometlova´, M. (2005a). Maternal behavior is impaired by methamphetamine administered during pre-mating, gestation and lactation. Reprod Toxicol, 20(1), 103–110. Sˇlamberova´, R., Charousova´, P., & Pometlova´, M. (2005b). Methamphetamine administration during gestation impairs maternal behavior. Dev Psychobiol, 46(1), 57–65. Sˇlamberova´, R., Pometlova´, M., & Charousova´, P. (2006). Postnatal development of rat pups is altered by prenatal methamphetamine exposure. Prog Neuropsychopharmacol Biol Psychiatry, 30(1), 82–88. Sˇlamberova´, R., Pometlova´, M., Syllabova´, L., & Mancˇusˇkova´, M. (2005c). Learning in the place navigation task, not the new-learning task, is altered by prenatal methamphetamine exposure. Brain Res Dev Brain Res, 157(2), 217– 219. Sˇlamberova´, R., Riley, M. A., & Vathy, I. (2005d). Crossgenerational effect of prenatal morphine exposure on neurobehavioral development of rat pups. Physiol Res, 54(6), 655–660.

Developmental Psychobiology. DOI 10.1002/dev Sˇlamberova´, R., & Rokyta, R. (2005a). Occurrence of bicuculline-, NMDA- and kainic acid-induced seizures in prenatally methamphetamine-exposed adult male rats. Naunyn Schmiedebergs Arch Pharmacol, 372(3), 236–241. Sˇlamberova´, R., & Rokyta, R. (2005b). Seizure susceptibility in prenatally methamphetamine-exposed adult female rats. Brain Res, 1060(1–2), 193–197. Sˇlamberova´, R., Szilagyi, B., & Vathy, I. (2001). Repeated morphine administration during pregnancy attenuates maternal behavior. Psychoneuroendocrinology, 26(6), 565–576. Turner, C. D., & Bagnara, J. T. (1976). Endocrinology of the ovary. In C. D. Turner & J. T. Bagnara(Eds.), General endocrionology (pp. 450–495). Philadelphia: W. B. Saunders Company. Vallee, M., Mayo, W., Dellu, F., Le Moal, M., Simon, H., & Maccari, S. (1997). Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: Correlation with stress-induced corticosterone secretion. J Neurosci, 17(7), 2626–2636. Vernotica, E. M., Lisciotto, C. A., Rosenblatt, J. S., & Morrell, J. I. (1996). Cocaine transiently impairs maternal behavior in the rat. Behav Neurosci, 110(2), 315–323. Vorhees, C. V., & Pu, C. (1995). Ontogeny of methamphetamine-induced neurotoxicity in the rat model. NIDA Res Monogr, 158, 149–171. Weinstock, M., Matlina, E., Maor, G. I., Rosen, H., & McEwen, B. S. (1992). Prenatal stress selectively alters the reactivity of the hypothalamic-pituitary adrenal system in the female rat. Brain Res, 595(2), 195–200. Weissman, A. D., & Caldecott-Hazard, S. (1995). Developmental neurotoxicity to methamphetamines. Clin Exp Pharmacol Physiol, 22(5), 372–374. Williams, M. T., Moran, M. S., & Vorhees, C. V. (2003). Refining the critical period for methamphetamine-induced spatial deficits in the Morris water maze. Psychopharmacology (Berl), 168(3), 329–338. Won, L., Bubula, N., McCoy, H., & Heller, A. (2001). Methamphetamine concentrations in fetal and maternal brain following prenatal exposure. Neurotoxicol Teratol, 23(4), 349–354. Yamamoto, Y., Yamamoto, K., Fukui, Y., & Kurishita, A. (1992). Teratogenic effects of methamphetamine in mice. Nippon Hoigaku Zasshi, 46(2), 126–131. Yamamotova´, A., Sˇlamberova´, R., Jedlicˇka, M., & Jakub, T. (2004). Gender differences in nociception in adult rats prenatally treated with methamphetamine. Homeostasis, 43(2), 99–101.