Effect of methamphetamine exposure and cross ... - Wiley Online Library

Lenka Hruba´ Barbora Schutova´ Romana ˇSlamberova´* Marie Pometlova´ Richard Rokyta Department of Normal Pathological and Clinical Physiology Third Faculty of Medicine Charles University in Prague Ke Karlovu 4, 120 00 Praha 2 Prague, Czech Republic E-mail: [email protected]

Effect of Methamphetamine Exposure and Cross-Fostering on Sensorimotor Development of Male and Female Rat Pups ABSTRACT: The present study tested the hypothesis that cross-fostering influences the development of rat pups. Mothers were exposed daily to injection of methamphetamine (M) (5 mg/kg) or saline for 9 weeks: 3 weeks prior to impregnation, throughout gestation and lactation periods. Control females animals without any injections were used. On postnatal day (PD) 1, pups were cross-fostered so that each mother received four pups of her own and eight pups from the mothers with the other two treatments. Offspring were tested for sensorimotor development in preweaning period by using tests of: negative geotaxis, tail pull, righting reflexes, rotarod and bar-holding. Further, the pups were weighed daily. Our results showed that birth weight in prenatally M-exposed pups was lower than in control or saline-exposed pups. Prenatally M-exposed pups gained less weight than control or saline-exposed pups regardless of postnatal treatment and sex. Further, our data demonstrated that prenatal and postnatal M exposure impairs sensorimotor functions in most of the tests. On the other hand, the negative effect of prenatal M exposure was partially suppressed in prenatally M-exposed pups by cross-fostering to control dams. Our hypothesis that cross-fostering may affect postnatal development of pups was confirmed. ß 2008 Wiley Periodicals, Inc. Dev Psychobiol 51: 73–83, 2009. Keywords: methamphetamine; sensorimotor development; cross-fostering; drug abuse

INTRODUCTION Postnatal care plays an important role in the emotional and cognitive development of the children. Physical and emotional deprivation and family conflicts may induce long-term consequences in postnatal development of a child (Meaney, 2001). Children who have been exposed to

Received 17 April 2008; Accepted 9 September 2008 *Associate Professor. Correspondence to: R. Sˇlamberova´ Contract grant sponsor: Ministry of Education, Youth and Sports of the Czech Republic Contract grant numbers: CN LC554, IGA 1A8610-5/2005, MSM 0021620816 Published online 7 October 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/dev.20346 ß 2008 Wiley Periodicals, Inc.

abuse or neglect are more likely to develop numerous psychopathologies such as anxiety, mood disorders and psychosis that might persist throughout their life (Bebbington et al., 2004). In mammals, the mother is the principal caregiver, providing both nutritional resources and behavioral stimulation to offspring. Further, maternal licking/ grooming (LG) is a major source of tactile stimulation for the developing pup, which affects somatic growth and neural development (Schanberg & Field, 1987) and during the first week of postnatal life influence hippocampal development and function (Liu, Diorio, Day, Francis, & Meaney, 2000). It has been investigated that the offspring of mothers who exhibit a higher frequency of LG (high-LG mothers) over the first week of postnatal life show increased hippocampal synaptic density and enhanced spatial learning and memory (Liu et al., 2000). Our previous study (Sˇlamberova´, Charousova´, & Pometlova´, 2005a) demonstrated that administration

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of methamphetamine (M) (5 mg/kg) in gestation and/ or lactation periods affects maternal behavior in rats. Specifically, M attenuates active nursing and other maternal activities, such as mother being in the nest, in contact with pups carrying and grooming pups and nest building (Sˇlamberova´ et al., 2005a). Similarly, maternal behavior has been shown to be impaired by other psychostimulants such as amphetamine and cocaine (Franˇkova´, 1972; Johns, Noonan, Zimmerman, Li, & Pedersen, 1994) or opioids such as morphine (Sˇlamberova´, Szilagyi, & Vathy, 2001). One of the most serious problems of the current and last century is drug-, alcohol-, and nicotine-abuse. Most notably, drug-abuse has been getting more serious during the last few decades. M is a powerfully addictive stimulant drug with a high potential for abuse. It is one of the most frequently used ‘‘hard’’ drugs in the Czech Republic (Vavrˇ´ınkova´, Binder, & Zˇivny´, 2001) and due to its anorectic effects it is one of the most common drugs abused by pregnant women addicted to drugs (Marwick, 2000). M crosses the placental barrier easily (Dattel, 1990) and therefore it may affect the development of the fetus. Furthermore, the newborns may receive this drug postnatally in mothers’ breast milk (Hutchings, 1982). Clinical data suggest that transplacental exposure to M may be detrimental to the developing embryo, particularly the central nervous system (Acuff-Smith, Schilling, Fisher, & Vorhees, 1996). Further, it was shown that M administration alters the dopaminergic and serotoninergic systems in several brain regions (Akiyama, Ishihara, & Kashihara, 1996; Sabol, Roach, Broom, Ferreira, & Preau, 2001). Repeated M administration decreases dopamine and serotonin transporter function (Fleckenstein et al., 1999) and produces a long-term decrease in dopamine levels and in the number of dopamine uptake sites in the rat striatum (Wagner et al., 1980). In addition, our former studies (Sˇlamberova´, Pometlova´, & Charousova´, 2006; Sˇlamberova´, Pometlova´, & Rokyta, 2007) and the work of others (Acuff-Smith et al., 1996) demonstrated that M administered during prenatal and/or preweaning periods alters functional development of rat pups and impairs postural reflexes and sensorimotor functions. There are no available studies examining what the extent of the effect of prenatal M exposure is per se and what the extent of cross-fostering is. To distinguish that the present study tested the hypothesis that cross-fostering of rat pups (both male and female) after birth affects their postnatal development in the following way: the impaired sensorimotor development of prenatally M-exposed pups will be improved by fostering of control mothers and control pup development will be impaired by fostering of postnatally M-treated mother.

Developmental Psychobiology

METHODS 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).

Mothers Adult female albino rats (250–300 g) were purchased from Anlab Farms (Praque, Czech Republic). Animals were housed in groups (4–5/cage) and left undisturbed for a 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. Females were randomly assigned to M-treated (M), saline-treated (S) and control (C) groups. Subcutaneous (s.c.) injection of M (5 mg/kg) was administered daily approximately for 9 weeks: about 3 weeks prior to impregnation, throughout the entire gestation period and for 23 days of lactation period (until the weaning) (for details see Sˇlamberova´, Charousova´, & Pometlova´, 2005b). The dose of 5 mg/kg was chosen based on the findings (Weissman & Caldecott-Hazard, 1995) that this dose alters locomotor and exploratory behaviors. Further, another study (Acuff-Smith et al., 1996) has shown that M in dose of 5 mg/kg administered to pregnant female rats induces changes that are comparable with those found in fetuses of drug-abusing women. Similarly, in other studies (Peachey, Rogers, Brien, Maclean, & Rogers, 1976; Sˇlamberova´ et al., 2005a) this dose of M induced stereotypical behavior in injected female rats. Saline was injected s.c. at the same time and volume as M. Control females did not receive any injections. All females were weighed daily to see possible effects of M treatment on weight gain during the period prior to impregnation and gestation.

Fertilization Approximately 3 weeks after the drug administration, females were smeared by vaginal lavage to determine the phase of estrous cycle. At the onset of the estrus phase of the estrous cycle female rats were housed overnight with sexually mature stimulus males. There was always one female and one male in each cage. The next morning females were smeared again for the presence of sperm and returned to their previous home cages. The day after impregnation was counted as Day 1 of gestation (see Sˇlamberova´ et al., 2005b). On Day 21 of gestation, females were separated to maternity cages. The day of delivery was counted as postnatal day (PD) 0. Pups and Cross-Fostering On PD 1, litter sizes were adjusted to 12 pups. Pups were crossfostered so that four pups (usually two males and two females) remained with their biological mother and the other (usually four males and four females) were assigned to the mothers with the other two treatments. As a result, one mother usually raised four


Effect of Methamphetamine and Cross-Fostering

control pups, four saline-exposed pups and four M-exposed pups. Whenever possible the same number of males and females was kept in each litter. We obtained nine experimental groups based on biological and fostering mother (see Tab. 1). The pups fostered by M-treated mothers were exposed to the effect of M also postnatally from mother’s breast milk. Prenatally M-exposed pups were injected intradermally with black India ink in left foot pad and prenatally saline-exposed pups in right foot pad for identification. Pups from control mothers were not tattooed. On PD 23, pups fostered by M-exposed mothers were ear punched in the left ear and pups fostered by saline-exposed mothers in the right ear. Pups raised by control mothers were not ear punched. Average of animals of the same sex and drug exposure in each litter was used as a unit of analyses to prevent litter effect. During the time while the pups were out of their home cages they were kept warm on a heating pad.

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Righting Reflex on Surface Righting reflex on surface was tested on PD 12 based on studies of Altman and Sudarshan (1975) and Meek et al. (2000) and our previous studies (Sˇlamberova´ et al., 2006, 2007). Each pup was turned on its back (supine position), and the time that it took for the pup to right with all four paws contacting the surface of the testing table was recorded. Righting Reflex in Mid-Air The righting reflex in mid air was tested on PD 17 based on study of Altman and Sudarshan (1975) and our previous studies (Sˇlamberova´ et al., 2006, 2007). Each pup was held on its back 40 cm above a soft pad, then released and position when reaching the soft pad was observed. A score of ‘‘1’’ was given when a pup reached the ground at once with all four paws and ‘‘0’’ when it did not.

Litter Characteristics and Maturation of the Pups Number of pups in the litter and percentage of males and females in each litter was recorded and compared among groups. Birth weight and weight gain during the 23 days of testing were used for statistical analysis. The pups were examined for eye opening, ear opening, startle response and tooth eruption based on study of Meek et al. (Meek, Burda, & Paster, 2000). The startle response was tested by sound of a tuning fork near the ear. Negative Geotaxis Negative geotaxis was tested on PD 9 and PD 11 (Altman & Sudarshan, 1975; Meek et al., 2000). Each pup was placed facing downward on a screen inclined at 30 angle. The latency of turning the face upward (180 ) was recorded. Tail Pull Tail pull was tested on PD 10 on cellulose cotton wool and on PD 12 and PD 14 on a grid (Meek et al., 2000). Pups were pulled by their tail and the ability to resist the pulling by holding on to the pad was recorded. A score of ‘‘1’’ was given when the pups caught with both paws on one of the pads and ‘‘0’’ when they did not. Results for each type of pad were analyzed separately. Table 1.

Rotarod Rotarod was used based on our previous studies (Sˇlamberova´ et al., 2006, 2007). On PD 23 rotarod performance was examined to test the sensorimotor coordination with necessity of active moving to hold the balance on the rotating cylinder. 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 successfully accomplished the task. The maximal number of trials was 6. Number of falls was recorded.

Bar-Holding The bar-holding test was used based on study of Clifton et al. (1991) and our previous studies (Sˇlamberova´ et al., 2006, 2007). The bar-holding test on PD 23 was used to examine vestibular function and sensorimotor coordination with the necessity of no moving to hold the balance on the narrow bar. A wooden bar 40 cm long with a diameter of 1 cm 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 animal’s behavior was rated during 60 s and was assigned a score (see Tab. 2).

Experimental Groups

Group

Biological Mother

Foster Mother

Prenatal and Postnatal Factors

M/M M/S M/C S/M S/S S/C C/M C/S C/C

Methamphetamine Methamphetamine Methamphetamine Saline Saline Saline Control Control Control

Methamphetamine Saline Control Methamphetamine Saline Control Methamphetamine Saline Control

Prenatal and postnatal drug Prenatal drug and postnatal stress Prenatal drug and postnatal care Prenatal stress and postnatal drug Prenatal and postnatal stress Prenatal stress and postnatal care Postnatal drug Postnatal stress Absolute control

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Table 2.

Ratings of Animal Behavior (Clifton et al., 1991)

Score 1 2 3 4 5 6


Behavior Balances with steady posture; paws on top of the beam Grasps sides of beam and/or has shaky movement One or more paw(s) slip off beam Attempts to balance on beam but falls off Drapes over beam and/or hangs on beam and falls off Falls off beam with no attempt to balance or hang on

Statistical Analyses The number of pups in each litter and the percentage of males and females in each litter was analyzed using a one-way ANOVA (prenatal drug exposure). A two-way ANOVA (prenatal drug exposure sex) was used to analyze differences in birth weight. A three-way ANOVA (prenatal drug exposure postnatal drug exposure sex) was used to analyze differences in the negative geotaxis test, in the righting reflex on surface test, in the rotarod test for the best performance and in the rotarod and bar holding tests for the number of falls. A three-way ANOVA (prenatal drug exposure postnatal drug exposure sex) with repeated measure was used to analyze for weight gained during PD 1–23, for trials in rotarod test and for trials and a score in bar holding test. The Bonferroni test was used for post hoc comparisons. For eye opening, startle response, tooth eruption, tail pull test, righting reflex in mid-air test a w2 test was used in specific postnatal days. Differences were considered significant if p < .05 in all analyzes.

RESULTS Impregnation and Delivery

(mean SEM) of prenatally M-exposed pups (5.65 g .07) was lower than that of controls (7.07 g .08) or saline-exposed pups (6.58 g .08) regardless of sex. Further, males (6.52 g .06) were heavier than females (6.34 g .06) on PD 1 regardless of their prenatal drug exposure. Figure 1 shows that prenatally M-exposed pups gained less weight during lactation than controls or saline-exposed pups regardless of sex and postnatal drug treatment [F(2,164) ¼ 37.16; p < .0001]. All pups regardless of prenatal drug exposure that were fostered by M-exposed dams gained less weight during lactation than pups of the same prenatal drug treatment fostered by control or saline-exposed dams [F(2,164) ¼ 21.02; p < .0001]. There were no sex differences in weight gain during lactation [F(2,178) ¼ .3; p ¼ .58]. In tooth eruption, there were no significant sex differences on PD 10 (w2 ¼ .025; p ¼ .87) and PD 11 (w2 ¼ .27; p ¼ .6). As shown in Table 3, the number of animals with tooth eruption was smaller in M/M relative to C/C pups on PD 10 (w2 ¼ 7.033; p < .005) and PD 11 (w2 ¼ 5.71; p < .05). In the ears opening, there were no significant sex differences on PD 11 (w2 ¼ 2.89; p ¼ .067) and PD 12 (w2 ¼ .025; p ¼ .87). As shown in Table 3, the number of animals with ears opened was smaller in groups prenatally exposed to M than in prenatal control or saline-exposed groups with the same postnatal treatment on PD 11 (w2 ¼ 55.61; p < .0001) and on PD 12 (w2 ¼ 39.63; p < .0001). Further, postnatal M exposure impaired the ears opening in C/M and S/M pups relative to C/C and S/C pups, respectively, on PD 11 (w2 ¼ 10.42; p < .005)

Prenatal treatment did not influence the length of gestation in any of the groups [F(2,23) ¼ 2.93; p ¼ .07]. There were no significant differences among groups in weight gain of dams during the period prior to impregnation and throughout the entire gestation period. All groups regardless of the drug treatment gained around 9–16 g during the period prior to impregnation and 130–140 g in gestation.

Litter Characteristics and Maturation of the Pups There were no differences in litter characteristics, such as number of alive pups [F(2,23) ¼ 2.51; p ¼ .1] or male/ female ratio [F(4,44) ¼ 1.16; p ¼ .34] in the litter. The final number of the litters was: nine controls, seven M and nine salines. The final number of tested pups in a group was 20 pups (10 males and 10 females). There was a main effect of prenatal drug exposure [F(2,184) ¼ 88.76; p < .0001] and sex [F(1,184) ¼ 4.23, p < .05] for birth weight. Specifically, birth weight

FIGURE 1 Effect of M administration on weight gained during lactation period (PD 1–PD 23). Values are means SEM for males and females together; n ¼ 20; p < .0001 versus prenatal controls fostered by the same mother; þp < .0001 versus pups fostered by control mother (ANOVA; Bonferroni post hoc test).

Developmental Psychobiology Table 3.

Differences in Characteristics of Maturation Tooth Eruption

Groups C/C S/C M/C C/S S/S M/S C/M S/M M/M

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Ear Opening

Startle Response

Eye Opening

PD 10

PD 11

PD 11

PD 12

PD 11

PD 12

PD 15

PD 16

85 70 60 75 85 60 70 60 45*

100 100 85 100 100 90 95 85 75*

85 60 0* 70 60 0* 35þ 5þ 0*

100 80 35* 100 100 45* 60þ 75 10*

75 25 0* 45 20 0* 20þ 0þ 0*

95 65 5* 100 95 15* 50þ 45 0*

60 70 25* 95 80 30* 25þ 25þ 10

95 100 90 100 100 70 80 100 70

Values are percents of all pups from group (n ¼ 20). p < .05 versus prenatal controls fostered by the same mother. þ p < .05 versus pups of the same prenatal exposure fostered by control mother.

and in C/M relative to C/C pups on PD 12 (w2 ¼ 10.01; p < .005). In a startle response, there were no significant sex differences on PD 11 (w2 ¼ 1.67; p ¼ .19) and PD 12 (w2 ¼ .356; p ¼ .55). As shown in Table 3, the number of animals exhibiting a startle response was smaller in groups prenatally exposed to M than in prenatal control or saline-exposed groups on PD 11 (w2 ¼ 36.52; p < .0001) and 12 (w2 ¼ 68.43; p < .0001). Further, postnatal M exposure impaired the startle response in C/M relative to C/C pups on PD 11 (w2 ¼ 12.13; p < .0005) and PD 12 (w2 ¼ 10.16; p < .005) and in S/M relative to S/C on PD 11 (w2 ¼ 5.7; p < .05). In the eyes opening, there were no significant sex differences on PD 15 (w2 ¼ 1.43; p ¼ .23) and on PD 16 (w2 ¼ .19; p ¼ .66). The eyes opening on PD 15 (w2 ¼ 18.25; p < .001) and 16 (w2 ¼ 22.57; p < .001), respectively, was delayed in prenatally M-exposed pups when compared to controls regardless of postnatal drug exposure. Further, on PD 15 postnatal M exposure delayed the eyes opening in C/M (w2 ¼ 10.42; p < .005) and S/M (w2 ¼ 12.36; p < .005) pups relative to C/C and S/C pups, respectively (see Tab. 3).

Negative Geotaxis There were no significant sex differences either on PD 9 [F(1,162) ¼ .03; p ¼ .85] or on PD 11 [F(1,162) ¼ .25; p ¼ .61]. On PD 9, there was a main effect of prenatal drug exposure [F(2,162) ¼ 31.17; p < .00001]. As shown in Figure 2A (PD 9), prenatally M-exposed pups had longer latency of turning than controls or prenatally saline-exposed pups regardless of postnatal drug treatment. On PD 11, there was a main effect of postnatal M exposure [F(2,162) ¼ 16.56; p < .00001].

FIGURE 2 Effect of M administration tested in negative geotaxis. Data are latencies to turn upwards in negative geotaxis test. Values are means SEM; n ¼ 20. (A) Negative geotaxis test on PD 9. (B) Negative geotaxis test on PD 11. p < .0001 versus prenatal controls or prenatally saline-exposed pups fostered by the same mother; þp < .05 versus pups of the same prenatal exposure fostered by control mother; þþp < .001 versus pups of the same prenatal exposure fostered by control mother (ANOVA; Bonferroni post hoc test).

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Tail Pull There were no significant sex differences either on PD 10 (w2 ¼ 2.36; p ¼ .1) when testing tail pull on cellulose cotton wool or on PD 12 (w2 ¼ 1.82; p ¼ .18) and on PD 14 (w2 ¼ 2.7; p < .1) when tail pull was examined on a grid. As shown in Figure 3A, the number of M/C pups that were successful in the tail pull on cellulose cotton wool on PD 10 was higher than the number of M/S pups (w2 ¼ 7.06; p < .05) or M/M pups (w2 ¼ 17.14; p < .0001). In addition, postnatal M exposure decreased the successfulness in the


tail pull on cellulose cotton wool relative to postnatal control groups (w2 ¼ 8.49; p < .005). In the tail pull on a grid on PD 12 (Fig. 3B), the C/C group of pups was more successful than S/C (w2 ¼ 5.23; p < .05) or M/C (w2 ¼ 10.1; p < .005) group and the C/S group succeeded better in the test than M/S (w2 ¼ 16.94; p < .0001) group. Additionally, postnatal M exposure in control pups (C/M) (w2 ¼ 9.58; p < .005) made them worse relative to their controls (C/C). On PD 14 some of the differences between groups already disappeared. However, as shown in Figure 3C, group of C/C pups remained still more successful when compared to M/C (w2 ¼ 8.53; p < .005) and C/M (w2 ¼ 10.16; p < .005) pups.

Righting Reflex on Surface There were no significant sex differences [F(2,162) ¼ .81; p ¼ .37]. There were main effects of prenatal [F(2,162) ¼ 18.11; p < .0001] and postnatal drug exposure [F(2,162) ¼ 12.27; p < .0001] and an interaction between both, prenatal and postnatal drug exposure [F(4,162) ¼ 4.19; p < .005]. As shown in Figure 4A, foster care of control mother improved the impairing effect of prenatal M exposure pups to the level of control pups (C/C or S/C) as could be seen in M/S and M/M groups. On the other hand, postnatal care by M-treated mother prolonged the latency to right in prenatally control pups (C/M > C/C).

Righting Reflex in Mid-Air There were no significant sex differences (w2 ¼ .53; p ¼ .47). As shown in Figure 4B, the number of pups that were able to successfully right in the righting reflex in mid-air was higher in C/C group relative to M/C (w2 ¼ 4.29; p < .05) and C/M (w2 ¼ 8.53; p < .05) groups of pups and lower in M/S (w2 ¼ 6.14; p < .05) when compared to C/S pups. FIGURE 3 Effect of M administration tested in tail pull test. Data are presented as a percentage of successful/unsuccessful animals. (A) Tail pull test on cellulose cotton wool on PD 10. (B) Tail pull test on grid on PD 12. (C) Tail pull test on grid on PD 14. p < .05 versus prenatal controls fostered by the same mother; þp < .005 versus pups of the same prenatal exposure fostered by control mother (w2 test). Groups: C/C ¼ control pup fostered by control mother, S/C ¼ prenatally saline-exposed pup fostered by control mother, M/C ¼ prenatally M-exposed pup fostered by control mother, C/S ¼ control pup fostered by saline-treated mother, S/S ¼ prenatally saline-exposed pup fostered by saline-treated mother, M/S ¼ prenatally M-exposed pup fostered by saline-treated mother, C/M ¼ control pup fostered by M-treated mother, S/M ¼ prenatally saline-exposed pup fostered by M-treated mother, MM ¼ prenatally M-exposed pup fostered by M-treated mother.


FIGURE 4 Effect of M administration tested in righting reflexes. Values are means SEM; n ¼ 20. (A) Latencies to straighten up in righting reflex on surface test (on PD 12). (B) Data are presented as a percentage of successful/ unsuccessful animals in righting reflex in mid-air on PD 17. p < .05 prenatal controls fostered by the same mother (ANOVA; Bonferroni post hoc test); þp < .005 versus pups of the same prenatal exposure fostered by control mother (w2 test).

Rotarod There were no significant sex differences [F(1,162) ¼ .04; p ¼ .84]. In the time spent on rotating cylinder, there was a main effect of prenatal drug exposure [F(2,162) ¼ 6.66; p < .005] and an interaction between both, prenatal and postnatal drug exposure [F(4,162) ¼ 3.19; p < .05]. As shown in Figure 5A, post hoc test showed that M/M pups spent the least time on the rotating cylinder from all of the groups. All pups, regardless of the prenatal and postnatal drug treatment improved with repeated trials [F(5,810) ¼ 240.07; p < .0001]. As shown in Figure 5B, the M/M pups displayed the most falls from all groups; even more than C/M (p < .05) and M/C (p < .001) groups.


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FIGURE 5 Effect of M administration tested in the rotarod test. Values are means SEM; n ¼ 20. (A) The average time during which the animal stays on rotating cylinder. (B) Number of falls from rotating cylinder during the entire experiment. p < .05 versus prenatal controls fostered by the same mother; þ p < .05 versus pups of the same prenatal exposure fostered by control mother (ANOVA; Bonferroni post hoc test).

Bar-Holding There were no significant sex differences in the length of pup remaining on the bar [F(1,162) ¼ 1.5; p ¼ .22] and in awarded score [F(1,162) ¼ 1.02; p ¼ .12]. The number of falls from the bar was significantly higher in males than females [F(1,162) ¼ 2.59; p < .05]. Neither prenatal nor postnatal drug exposure have shown any significant differences in the bar-holding latency or the number of falls from the bar.

DISCUSSION The aim of the present study was to determine the effect of cross-fostering on birth weight, weight gain, maturation and sensorimotor function in rat pups prenatally exposed to M. Our results indicate that prenatal and postnatal M exposure impair postnatal development of rat pups, especially sensorimotor functions and postural reflexes. It is in agreement with the study of Sˇlamberova´ et al.

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(2006) showing that M administered during prenatal and/ or preweaning periods alters functional development of rat pups and that it alters two generations of offspring (Sˇlamberova´ et al., 2007). There are other studies examining the effect of other stimulant drugs such as amphetamine and cocaine on development of rat pups (Henderson & McMillen, 1990; Sobrian, Ali, Slikker, & Holson, 1995). Study of Henderson and McMillen (1990) demonstrated that the development of righting on surface is delayed in pups, who were prenatally exposed to cocaine (15 mg/kg). Similarly, Sobrian et al. (1995) showed that cocaine (20 mg/kg) produces the delayed effect of righting on surface. On the other hand, there are studies (Smith, Mattran, Kurkjian, & Kurtz, 1989; Vorhees, 1985) with different results. Vorhees (1985) demonstrated that the amphetamine (.5 and 2.0 mg/kg) does not produce any significant effects on measures of behavioral performance, such as surface righting and negative geotaxis. Similarly, Smith et al. (1989) showed that the cocaine dosing produces no significant effects on early postnatal behavioral tests, as was negative geotaxis and righting reflex. These discrepancies between our study and the work of others (Smith et al., 1989; Vorhees, 1985) might be explained by different dosing; while amphetamine was administered in a dose of .5 or 2.0 mg/kg on Days 12–15 of gestation (Vorhees, 1985) and cocaine in a dose of 10 mg/kg from gestation Day 4 through gestation until Day 18 (Smith et al., 1989), we injected M in a dose of 5 mg/kg for 9 weeks: 3 weeks prior to impregnation and during gestation and lactation periods. The finding that M administered during gestation decreases birth weight is in agreement with clinical evidence showing decreased body weight in neonates born to mothers who abused M during pregnancy (Little, Snell, & Gilstrap, 1988). Similarly, Martin, Martin, Radow, and Sigman (1976) demonstrated decreased birth weight after M administration (5 mg/kg twice daily). Additionally, our data show changes in weight gain during lactation in rat pups. We found that prenatally M-exposed pups fostered by control or saline-exposed dams gained more weight during lactation than pups exposed to M both prenatally and postnatally. On the other hand, pups regardless of prenatal drug exposure that were fostered by M-exposed dams gained less weight during lactation than pups fostered by control or saline-exposed dams. This might be partly attributable to worse maternal care induced by administration of M in gestational and/or lactation periods (Sˇlamberova´ et al., 2005a,b). As shown in our previous studies it may affect prenatal and/or postnatal development of the pups (Sˇlamberova´ et al., 2006, 2007). Another possibility how to explain decreased weight gain in pups fostered by M mothers is that M is


known as an anorectic drug (Bittner, Wagner, Aigner, & Seiden, 1981; Suzuki, Fan Chiang, Abe, Ohtani, & Yanaura, 1983). Further, the lower body weights could be caused by the neurochemical alternations in the brain affected prenatally (Nasif, Cuadra, & Ramirez, 1999; Silva-Araujo & Tavares, 1995). Tan (2003) showed that the prenatal amphetamine exposure (10 or 5 mg/kg) results in decreased birth weights and the pup body weights in amphetamine groups are lower than weights in controls at PD 22 and PD 60 (Tan, 2003). However, the decreased birth weight was not the result of malnutrition caused by amphetamine exposure, because pup of pair-fed dams in the saline group had similar body weights to those in the ad lib group (Tan, 2003). Our results further demonstrate delay in tooth eruption only in pups prenatally exposed to M fostered by M-treated mothers, which is in disagreement with the data of Vorhees (1985) investigating the effect of amphetamine. On the other hand, the present study demonstrates that pups prenatally exposed to M opened their eyes later than controls or saline-exposed pups. This finding is in agreement with the work of Cho, Lyu, Lee, Kim, and Chin (1991) and Martin (1975), while it is in contrast with the data of Weissman and Caldecott-Hazard (1993). Further, Vorhees (1985) showed that the .5 mg/kg amphetamine group showed delayed eye opening, but the 2.0 mg/kg group did not. Similarly, Henderson and McMillen (1990) showed that in cocaine-exposed group there are no changes in the time of eyes opening. There are many other studies showing eye defects (i.e., anophthalmia, microphthalmia and folded retina) after prenatal M exposure (Acuff-Smith, George, Lorens, & Vorhees, 1992; Vorhees & Acuff-Smith, 1990). The type of defect was shown to be dependent on the developmental stage at the time of dosing (Acuff-Smith et al., 1996). Because our study does not show any visible eye defects, it seems that not only the stage at the time of dosing, but also the dose per se plays a role in the seriousness of the eye defects. On the other hand, the delay of eye opening when the dose of 5 mg/kg of M was used in the present study may be due to the same mechanism(s) as the induction of eye defects after the dose of 20 mg/kg of M. The other physical maturation, such as ear opening and startle response, has been shown to be affected by prenatal and postnatal M exposure in the present work. The finding of Acuff-Smith et al. (1992) demonstrating that the offspring exposed prenatally to M have significantly lower olfactory orientation scores (PD 9, 11, 13) to their home cage scent supports our findings. On the other hand, Vorhees (1985) demonstrated that auditory startle and olfactory orientation are not affected by prenatal amphetamine exposure. The dosing of the drug seems to play a role in the inconsistency of these results again. Further, it has to be noted that in the present study Wistar


rats were tested, in contrast to the study of Vorhees (1985) where Sprague–Dawley rats were used. In addition, the present study is the first to show the effects of crossfostering on postnatal development of pre- and postnatally M-exposed pups. The physical maturation, such as eye and ear opening as well as the startle response is delayed in control or prenatally saline exposed pups postnatally exposed to M relative to absolute controls (i.e., control pups fostered by control mothers). Similarly, control pups postnatally exposed to M are significantly worse in the following tests: negative geotaxis on PD 11, tail pull, righting reflex on surface and in mid-air than absolute controls (i.e., control pups fostered by control mothers). There are no studies showing the effect of crossfostering on the development of rat pups exposed to M. Therefore, we can compare our results only to the data showing the effect of cross-fostering on ethanol abuse. The ethanol study of Lancaster, Phillips, Patsalos, and Wiggins (1984) demonstrated that the effect of lactational ethanol exposure is worse than the effect of ethanol exposure during gestation. Lancaster et al. (1984) demonstrated that the body growth was affected more severely by gestational exposure, and gestational effects were generally less severe with adequate nutrition. Specifically, offspring of ethanol-treated dams that were cross-fostered to pair-fed and well nourished dams during lactation had delayed eye opening, persistent lag in body growth and slightly lower brain myelin concentrations. Offspring of dams, who were either pair-fed or well nourished during gestation, but cross-fostered during lactation to ethanol-treated dams, had abnormal organ weights, abnormal brain weights and severely depressed brain myelin concentrations persisting through adulthood. That might explain why pups exposed to ethanol both in utero and during lactation, are not statistically different from those exposed during lactation only. This might be also an explanation of our results showing that the pups exposed both prenatally and postnatally to M are not significantly different from the control pups postnatally exposed to M in the following tests: negative geotaxis on PD 11, tail pull on a grid (PD 12 and 14) and righting reflex in mid air (PD 17). On the other hand, in other tests (negative geotaxis on PD 11, righting reflex on surface (PD 12), tail pull on cellulose cotton wool (PD 10) and rotarod on PD 23) better results in pups prenatally exposed to M fostered by control mothers were found. This might be explained by improving effect of cross-fostering. The reasons may be two: (1) it may be due to no postnatal M administration during lactation. While M/M animals received M both, pre- and postnatally, the M/C pups only prenatally. (2) It may be that the maternal care by control mother partially improves the impairing effect of M. We have data showing that M (5 mg/kg) in gestation and/or lactation periods impairs


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maternal behavior. Specifically, it attenuates active nursing and other maternal activities, such as mother being in the nest, in contact with pups, carrying and grooming pups and a nest building (Sˇlamberova´ et al., 2005a,b). In contrast, we showed that control mothers cared about the pups more than M-treated mothers and this ‘‘better care’’ was independent on the fact that some of the pups were their own and some were adoptive (Sˇlamberova´ et al., 2005a). The fact that better maternal care may improve the development of pups is supported by studies of others (Gonzalez, Lovic, Ward, Wainwright, & Fleming, 2001; Levine, 1994; Liu et al., 1997, 2000). Maternal licking and grooming is a major source of tactile stimulation for the developing pup, which affects somatic growth and neural development (Levine, 1994). In rats, offspring born to mothers who display high levels of LG during the first week postpartum are less fearful, have an attenuated corticosterone response to stress, increased levels of hippocampal glucocorticoid receptor expression and enhanced spatial learning and memory compared with offspring of mothers, who displayed low levels of LG (Liu et al., 1997, 2000). Further, provision of licking-like tactile stimulation to rat pups reduced behavioral indices of anxiety and improved social learning (Gonzalez et al., 2001). In conclusion, this is the first study showing such an effect of M and cross-fostering. The present study demonstrates that M administered daily during the impregnation, throughout the entire gestation and lactation periods impairs the postural reflexes and sensorimotor coordination in rat pups. The postnatal care of control mothers partially improves the development of rat pups prenatally exposed to M. Our hypothesis, that the cross-fostering may affect postnatal development of pups, was confirmed. Our results support the hypothesis that variation in rat maternal care could serve as a mechanism for a nongenomic behavioral mode of transmission of traits.

NOTES This study was supported by project CN LC554, grant IGA 1A8610-5/2005, and Research Goal # MSM 0021620816 from Ministry of Education, Youth and Sports of the Czech Republic. 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 Czech Government Requirements under the Policy of Humans Care of Laboratory Animals (No. 246/1992) and with the regulations of the Ministry of Agriculture of the Czech Republic (No. 311/1997).

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