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Methamphetamine. Administration during Gestation. Impairs Maternal Behavior. Romana S˘lamberovб. Petra Charousovб. Marie Pometlovб. Department of ...
Romana S˘lamberova´ Petra Charousova´ Marie Pometlova´ Department of Normal Pathological and Clinical Physiology 3rd Faculty of Medicine Charles University, Prague Czech Republic E-mail: [email protected] E-mail: [email protected] E-mail: [email protected]

Methamphetamine Administration during Gestation Impairs Maternal Behavior ABSTRACT: Previous studies demonstrated that repeated drug exposure, such as opiates or cocaine, during the gestation period attenuates maternal behavior of rats; however, it is not known whether methamphetamine (MA), a drug whose usage has increased recently, negatively affects maternal behavior as well. Therefore, the present study tested the hypothesis that repeated subcutaneous administration of MA (5 mg/kg daily) throughout the entire gestation period alters maternal behavior. Dams (control, saline-, and MA-treated) were observed with their pups in two types of tests. In the observation test, 11 types of activities and three types of nursing positions of mothers were recorded 10 times during each 50-min session for the 22-day lactation period. A decrease in nursing and active maternal behavior was found in MA-treated mothers relative to control rats. In addition, stereotypic behavior such as rearing and sniffing was increased in MA- as well as in salinetreated mothers relative to controls. All mothers, regardless of the treatment, displayed significantly less maternal behavior and more nonmaternal activities as postpartum time progressed. In the retrieval test, mothers also were tested for pup retrieval from postpartum Days 1 through 12. MA-treated mothers were slower in retrieving the first pup, returning the first pup into the nest, and returning all pups into the nest relative to controls or saline-treated mothers. Interestingly, the latency to return all pups to the nest was longer in saline-treated mothers relative to controls. In conclusion, the present study demonstrates a novel finding that MA administered during the gestation period has a negative effect on maternal behavior. ß 2004 Wiley Periodicals, Inc. Dev Psychobiol 46: 57–65, 2005. Keywords: methamphetamine; pregnancy; maternal behavior; nursing; retrieval test

INTRODUCTION The negative effects of drugs on the health of drugabusing women has been shown many times (Dickenson, 2001; Weinrieb & O’Brien, 1993); however, when the drug-abusing women become pregnant and do not stop Received 5 May 2004; Accepted 7 September 2004 Correspondence to: R. Sˇlamberova´ Contract grant sponsor: Czech Republic Grant Agency Contract grant number: 305/03/0774 Contract grant sponsor: Czech Republic Ministry of Health Contract grant number: NF/7456-3/2003 Contract grant sponsor: Czech Republic Ministry of Education, Youth and Sports Contract grant numbers: MSM 111200005 and LN00B122 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/dev.20042 ß 2004 Wiley Periodicals, Inc.

using drugs, they continue to expose to danger not only themselves but also their fetuses. Additionally, drugabusing mothers were shown to neglect prenatal and postnatal care of their offspring (Inaba & Cohen, 1993; Larsson, Eriksson, & Zetterstrom, 1979). Some studies (Bridges & Grimm, 1982; Grimm & Bridges, 1983; Mayer, Faris, Komisaruk, & Rosenblatt, 1985; Stafisso-Sandoz, Polley, Holt, Lambert, & Kinsley, 1998), including our own (Sˇlamberova´, Szilagyi, & Vathy, 2001), have shown the disruptive effect of opiates injected during gestation on maternal behavior; however, the situation is not so clear in the research of stimulants such as amphetamines and cocaine. While cocaine was shown to have negative effects on maternal behavior when injected during the lactation period (Elliott, Lubin, Walker, & Johns, 2001; Kinsley et al., 1994; Vernotica, Lisciotto, Rosenblatt, & Morrell, 1996), contrary data have been published on the effects of cocaine administered during

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gestation. Studies have shown impairing effects (Johns, Noonan, Zimmerman, Li, & Pedersen, 1994), minimal negative effects (Nelson, Meter, Walker, Ayers, & Johns, 1998), no effects (Vernotica et al., 1996), or even positive effects (Peeke, Dark, Salamy, Salfi, & Shah, 1994) on postpartum maternal care. Furthermore, the effects of drugs on maternal behavior have been shown to depend on the dose and the timing of drug exposure. Thus, only a handful of studies available have assessed the effects of gestational cocaine administration on maternal behavior, and the results vary from study to study. Moreover, we did not find any data on the effects of gestational methamphetamine (MA) or amphetamine administration on maternal behavior. Only a few older studies have noted that amphetamine administered during lactation decreases maternal behavior (Franˇkova´, 1977; Piccirillo, Alpert, Cohen, & Shaywitz, 1980). Specifically, Piccirilo et al. (1980) found that repeated amphetamine injection several times during lactation decreases the mother–pup intercontact interval, the retrieval latency, and the number of pups retrieved while increasing the interretrieval interval and the number of corners to which the pups were retrieved. It also decreases the time of nest building and nursing while increasing the time of locomotor activities of the mothers. All of these amphetamine effects are dose dependent (Piccirillo et al., 1980). Similarly, Franˇkova´ (1977) demonstrated that mothers treated with amphetamine during the first 10 days of lactation have decreased latencies in the retrieval test and other maternal activities such as interaction with pups, grooming pups, or nest building. To our knowledge, there are no studies testing the effect of MA on maternal behavior. Note that the mechanism(s) of action of MA is not the same as the mechanism(s) of amphetamine (Sabol, Richards, Layton, & Seiden, 1995). MA releases dopamine and serotonin equally while amphetamine is more specific to dopamine (Sabol et al., 1995). The different action of both drugs of dopamine and serotonin release also may account for the different effects of them on maternal behavior. In recent years, MA has become a more popular ‘‘street’’ drug in many countries because of its relatively uncomplicated production and low price compared to cocaine or heroin (Marwick, 2000). MA is a powerful, addictive stimulant with a high potential for abuse. Women, especially during pregnancy, take MA because it decreases appetite and therefore helps control weight, and increases energy. Statistics show that only 17% of women abusers in the United States were primary MA users, but 38% had used it during pregnancy (Marwick, 2000). The impact of MA on the mother and her child is, therefore, even more important; however, this research is still in its infancy and the findings are inconclusive.

Some studies have shown that repeated administration of MA (50 mg/kg) to pregnant rats results in higher incidence of delivery failure and mother’s death relative to controls (see Acuff-Smith, George, Lorens, & Vorhees, 1992). Additionally, MA-injected mothers (1, 3, and 5 mg/ kg) have shorter gestation periods and smaller litters relative to controls, and their weight gain also is lower (Martin, 1975; Martin, Martin, Radow, & Sigman, 1976). Moreover, the effect of MA exposure during gestation on maternal behavior has not been investigated. Therefore, the present study examined the effect of MA (5 mg/kg daily) throughout the entire gestation period on maternal behaviors tested in two different types of tests. A modified method, as used in our previous works (Sˇlamberova´, Bar, & Vathy, 2003; Sˇlamberova´, et al., 2001), is used in the present study.

METHODS Adult female Wistar rats (250–300 g) were purchased from Anlab farms (Prague, Czech Republic). Animals were housed 4 to 5 animals per 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 12:12 hr light:dark cycle, with lights on at 0600 hr. One week after arrival, females were smeared by vaginal lavage to determine the phase of estrous cycle. The smear was examined by light microscopy using 20 magnification. To ensure successful conception, at the onset of the estrus phase of the estrous cycle (Turner & Bagnara, 1976), female rats were housed with sexually mature stimulus males overnight (1 female and 1 male per cage). The next morning (Day 1 of gestation), females were smeared again for the presence of sperm and returned to their previous home cages. The females were randomly assigned to the MAtreated, saline-treated, or control group; thus, all treatment conditions were present in each cage. d-Methamphetamine HCl was provided from the Faculty of Pharmacy of Charles University in Hradec Kra´love´ (Czech Republic). MA was injected sc in a dose of 5 mg/kg/day through the entire gestation period (i.e., from the first to the last day of gestation). While the number of gestation days is not always the same in each female rat, we chose to administer an additional one to two drug injections rather than induce withdrawal in pregnant dams prior to delivery. The solution concentration was 10 mg/ml; thus, each animal received 0.5 ml/kg of the solution. The 5-mg/kg dose was chosen based on the findings of Weissman and Caldecott-Hazard (1995) that this dose alters locomotor and exploratory behaviors. Further, other studies (Acuff-Smith, Schilling, Fisher, & Vorhees, 1996; Cho, Lyu, Lee, Kim, & Chin, 1991; Martin et al., 1976) have shown that MA in dose of 5 or 10 mg/kg administered to pregnant rodents results in fetal brain drug concentrations, which approximate those reported in human infants whose mothers abused MA. Saline was injected sc at the same time and volume as MA. Control females were left undisturbed and not injected. All pregnant rats were weighted daily to note weight gain. On Day 21 of gestation, females were

Methamphetamine and Maternal Behavior removed from the group cages and placed into maternity cages. Expected day of delivery in our colony is 22 days. The day of delivery was counted as postpartum day (PD) 0. The number of pups in the litter and the percentage of males and females in each litter was recorded and compared between groups. On PD1, MA-exposed pups were injected intradermally with black India ink in the left foot pad and saline-exposed pups in the right foot pad for future identification. Pups from controlled mothers were not tattooed. Pups were then crossfostered so that each mother received some of her own and some of the pups of the mother with the other two treatments. Whenever possible, the same number of male and female pups and the same number of different drug exposures were kept in each litter. The number of pups in each litter was adjusted to 10. Mothers (8 per treatment group) with their pups were observed in two series of tests. The observer in both tests was blind to the treatment of the animals.

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11 other activities of mothers were recorded during each session: (1) mother in or out of the nest, (2) mother in contact with any of her pups, (3) mother licking or 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 selfgrooming also were counted together as a single measure of selfcare.), (10) mother rearing, and (11) mother sniffing with head raised. Data analysis. 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). The Bonferroni test was used for post hoc comparison. Differences were considered significant if p < 0.05. Retrieval Test

Data Analysis 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). A one-way ANOVA (drug exposure) was conducted to analyze the number of pups in each litter and the percentage of males and females in each litter. A two-way ANOVA (Drug Exposure  Sex) was used to analyze differences in birth weight. A threeway ANOVA (Drug Exposure  Raising Mother  Sex of Pups) was used to analyze differences in weight gain during the lactation period. The average of animals of the same sex and prenatal drug exposure in each litter was used as the unit in the analyses of birth weight and weight gain. The Bonferroni test was used for post hoc comparison in all statistical analyses. Differences were considered significant if p < 0.05.

Observation Test Maternal behavior was observed daily for 50 min in the home cage of each mother and her litter between PD1 and PD22. Observations were made during the light phase of the light:dark cycle between 0800 to 0900 hr. Similar methods were used as in our previous studies (Sˇlamberova´ et al., 2003; Sˇlamberova´ et al., 2001). 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 exhibited by the mothers and three nursing positions (discussed later) 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 a mother was nursing (total nursing). Three different positions were recognized as nursing: (a) Arched-nursing is when the mother is arched over her pups with legs splayed; b) blanket-nursing is when the mother is over her litter, but did not have her back arched and there was no obvious extension of her legs; (c) passive-nursing is when the mother is lying on her side or back with one or more suckling pups. The first two nursing positions were designated as active and the third one as passive nursing. In addition to nursing,

The same mothers and pups were tested for the retrieval test after the behavioral observations for the observation test were completed. The retrieval test was conducted daily between 0900 to 1000 hr on PD1 to PD12, so each mother and litter was tested 12 times. The same method was used in our previous studies (Sˇlamberova´ et al., 2003; Sˇlamberova´ et al., 2001). All pups were removed from their mother and placed in a separate cage for 5 min. After this brief separation, the entire litter was returned to their mother into the maternity cage, and the pups were spread all around the cage. The mother was then observed for 10 min, and the following measures were recorded: (a) the latency to carry the first pup, (b) the latency to return the first pup into the nest, and (c) the latency to return all the pups into the nest. Any unusual behaviors such as (a) removing a previously returned pup from the nest, (b) intensive caring for the pups around the cage before placing them in the nest, and (c) 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. It also was noted whether the mother carried first one of her own pups or one of the adopted pups into the nest. Data Analysis. Latencies were analyzed by one-way ANOVA (drug treatment) with repeated measure (12 days of testing). Bonferroni test was used for post hoc comparison. The incidence whether the mother carried one of her own or an adopted pup first to the nest was analyzed by w2 test. Differences were considered significant if p < 0.05.

RESULTS There were no significant differences between groups in weight gain during the gestation period or in the incidence of a successful and/or a healthy pregnancy. The length of a 22-day gestation period differed between groups, w2 ¼ 6.57; p < 0.05. Five of 8 females in the control group, 2 of 8 saline-treated females, and 0 of 8 MA-treated

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females had 22-day gestation periods. In the MA-treated group, the length of the gestation period was the longest (23–24 days). There were no females with a gestation less than 22 days. The number of pups and the percentage of males and females in litters were not significantly altered by the drug treatment (see Table 1). The birth weight in prenatally MA-exposed pups was lower than controls or salineexposed pups regardless of sex, F(2, 41) ¼ 53.46, p < 0.0001 (Table 1). Further, males were heavier than females on PD1 regardless of their prenatal drug exposure, F(1, 41) ¼ 14.27, p < 0.001 (Table 1). There were no differences in weight gain during lactation between groups as a function of drug treatment or raising mothers. As expected, males gained more weight, F(1, 125) ¼ 3.68, p < 0.05, during lactation than female pups.

Observation Test Nursing. There were no differences in total nursing between groups; however, when different types of nursing positions were examined, there were differences between groups of mothers having different drug treatments. MA-treated mothers nursed generally less by arched, F(2, 21) ¼ 5.54, p < 0.05 (Figure 1A), or blanket, F(2, 21) ¼ 8.35, p < 0.05 (Figure 1B), position of active nursing than controls or saline-exposed mothers. There were no changes induced by drug treatment in passive nursing (Figure 1C). Both types of active nursing, arched position, F(21, 441) ¼ 8.36, p < 0.0001, and blanket position, F(21, 441) ¼ 4.85, p < 0.0001, decreased whereas passive nursing, F(21, 441) ¼ 5.13, p < 0.0001, increased as the postpartum time progressed (Data not shown.) All of these time-dependent changes in nursing occurred independently of drug treatment. Maternal Activities. MA-treated mothers were less often in the nest, F(2, 21) ¼ 3.31, p < 0.05, in contact with their pups, F(2, 21) ¼ 2.89, p < 0.05, and grooming pups, F(2, 21) ¼ 12.69, p < 0.0001, than controls or saline-treated mothers (see Table 2). Additionally, mothers—regardless Table 1.

FIGURE 1 Effects of MA administration during the gestation period on average nursing score during the lactation period (PD1–22). Values are presented as averages for all 22 sessions (means  SEM; n ¼ 8). (A) Arched position of active nursing, (B) Blanket position of active nursing, (C) Passive nursing.  p < 0.05 (ANOVA; Bonferroni post hoc test).

of their drug treatment—were less often in the nest, F(21, 441) ¼ 5.55, p < 0.0001, in contact with their pups, F(21, 441) ¼ 5.35, p < 0.0001, and grooming pups, F(21, 441) ¼ 5.32, p < 0.0001, as the postpartum time progressed (Data not shown.) Occurrences of carrying the pups by mother and mother manipulating the shavings during the sessions were too low to be able to run the statistic. Nonmaternal Activities. There were no changes in resting with eyes closed or in self-care (eating, drinking,

Effects of MA Administration on Litter Sizes and Birth Weight of Pups Litters

Control Saline MA

Birth Weight

Males (average in litter)

Females (average in litter)

Males (average in litter)

Females (average in litter)

7  0.8 5  0.6 5  0.7

6  0.9 5  0.8 6  0.9

7.15  0.13 7.48  0.15 6.35  0.11

6.83  0.15 7.13  0.14 5.79  0.11

Note. Values are mean  SEM; n ¼ 8.  p < 0.001 versus females regardless of prenatal drug exposure.  p < 0.0001 versus controls and saline-exposed pups of the same sex.

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Table 2. Effects of MA Administration during Gestation on Maternal and Nonmaternal Activities of Rats

Maternal activities

Nonmaternal activities

In the nest In contact with pups Grooming pups Carrying pups Manipulating nest shavings Self-care Eating Drinking Self-grooming Resting with eyes closed Rearing Sniffing

Control

Saline

MA

6.84  0.34 7.17  0.37 1.86  0.06 NA NA

6.5  0.35 7.08  0.28 1.81  0.05 NA NA

4.99  0.39 5.82  0.32 0.4  0.04 NA NA

2.2  0.32 0.89  0.19 0.75  0.15 0.56  0.06 2.29  0.29

2.21  0.31 0.83  0.13 0.66  0.16 0.72  0.1 1.67  0.47

1.85  0.35 0.89  0.19 0.44  0.14 0.52  0.12 1.7  0.4

0.87  0.17 0.77  0.17

2.39  0.29{ 2.27  0.27{

2.45  0.25{ 2.52  0.22{

Note. Values are averages from all 22 days of testing (mean  SEM); n ¼ 8. 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.  p < 0.0001 versus controls and saline-treated mothers. { p < 0.001 versus controls (ANOVA).

or self-grooming) between groups (Table 2). As the postpartum time progressed and regardless of their treatment, mothers exhibited more eating, F(21, 441) ¼ 3.28, p < 0.0001, drinking, F(21, 441) ¼ 3.95, p < 0.0001, and sleeping, F(21, 441) ¼ 4.54, p < 0.0001. Self-grooming, F(21, 441) ¼ 2.00, p < 0.01, in all rats fluctuated during postpartum, and was at a maximum on PD4 and decreased during the last third of lactation period (Data not shown.) As shown in Table 2, rearing, F(2, 21) ¼ 3.54, p < 0.05, and sniffing, F(2, 21) ¼ 3.83, p < 0.05, was increased in MA-treated mothers as well as in saline-treated mothers relative to the controls. These activities did not change as postpartum progressed (Data not shown.)

Retrieval Test There was a main effect of drug treatment on the latency to carry the first pup, F(2, 21) ¼ 5.14, p < 0.05, to return the first pup, F(2, 21) ¼ 3.93, p < 0.05, and return all pups into the nest, F(2, 21) ¼ 2.86, p < 0.05. Specifically, pregnant dams treated with MA showed increased latency to carry the first pup (Figure 2A), to return the first pup (Figure 2B), and return all pups into the nest (Figure 2C) relative to controls. Further, saline-treated mothers showed increased latency to return all pups into the nest (Figure 2C) relative to controls. The latency to return the first pup into the nest, F(11, 231) ¼ 2.95, p < 0.001 (Figure 2B), and to return all pups into the nest, F(11, 231) ¼ 3.5, p < 0.0001 (Figure 2C), fluctuated as postpartum progressed, but the general trend was a decreased latency relative to PD1. There was no interaction between

FIGURE 2 Effects of MA administration during the gestation period on latencies in the retrieval test. Values are means  SEM (n ¼ 8) in all 12 sessions. (A) Latency to carry first pup, (B) latency to return first pup into the nest, and (C) latency to return all pups into the nest. Main effect of drug treatment in all measures (ANOVA). (A) MA > Salines or Controls ¼ p < 0.05 (Bonferroni post hoc test). (B) MA > Salines or Controls ¼ p < 0.05 (Bonferroni post hoc test). (C) MA > Controls ¼ p < 0.01; Salines > Controls ¼ p < 0.05 (Bonferroni post-hoc test).

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prenatal drug treatment and days of testing. The latency to carry the first pup, F(11, 231) ¼ 1.02, p ¼ 0.42 (Figure 2A), did not change during the study. No unusual behaviors, as defined in the Methods section, were exhibited by any of the mothers during the 12 days of testing. Even the incidence to carry first their own or adopted pup into the nest did not differ between mothers of different treatment groups.

DISCUSSION In contrast to the work of Martin and colleagues (Martin, 1975; Martin et al., 1976) showing shorter a gestation period in MA-treated mothers, we have shown the opposite. MA-treated mothers had the longest gestation period relative to controls and saline-treated mothers. In addition, there is no decrease in weight gain during the gestation period as shown by Martin and colleagues (Martin, 1975; Martin et al., 1976). Further, we did not see the decreasing effect of maternal MA injections on litter size found by Martin (1975). The reason why we did not see the negative effect of MA on the length of gestation period or weight gain during gestation and the litter size might be due to different frequencies of injections. While we injected 5 mg/kg of MA once daily, Martin et al. (Martin, 1975; Martin et al., 1976) used the same dose administered twice daily. The finding that MA administered during the gestational period decreases birth weight is in agreement with clinical evidence showing decreased body weight, length, and head circumference in neonates born to mothers who abused MA during pregnancy (Little, Snell, & Gilstrap, 1988). Similarly, Martin et al. (1976) demonstrated decreased birth weight after MA administration (5 mg/ kg twice daily). Additionally, our data show no changes in weight gain during lactation and thus correlate with the studies of others (Acuff-Smith et al., 1992; Cabrera, Yracheta, Li, Levy, Van de Kar, & Battaglia, 1993). In maternal behavior tests, our data demonstrate that MA exposure during gestation attenuates active nursing as well as other maternal activities. To our knowledge, there are no studies showing the effect of MA exposure on maternal behavior. Therefore, we can compare our results only to the effects of other drugs. When comparing to amphetamine (Franˇkova´, 1977; Piccirillo et al., 1980), it has been shown that repeated amphetamine injection in several time periods during lactation dose-dependently disrupts maternal behavior, such as decreases the mother– pup intercontact interval, the retrieval latency, and the number of pups retrieved. On the other hand, it increases the interretrieval interval and the number of corners to which the pups were retrieved. Nest-building and nursing times also are decreased by amphetamine administration

in their study (Franˇkova´, 1977; Piccirillo et al., 1980). Note that those rats (Franˇkova´, 1977; Piccirillo et al., 1980) are injected with amphetamine during lactation while rats in the present work receive MA during the gestation period; however, the similar results of the present study and the work of Franˇkova´ (1977) and Piccirillo et al. (1980) demonstrate that the effect of the chronic injections of MA on maternal behavior persists and is comparable to the acute effect of other amphetamines. When comparing our data to studies examining the effect of cocaine administration during gestation on maternal behavior, the data are in agreement with those showing impaired effects of cocaine (Johns et al., 1994). Johns et al. (1994) demonstrated that chronic cocaine administration (30 mg/kg) on gestation Days 8 to 20 delays or diminishes the postpartum onset of maternal behavior, increases the latency to crouch over pups, and decreases the duration of crouching during a 30-min observation period that immediately followed parturition. Latencies to nest building also are longer in cocaine-treated dams than in saline controls (Johns et al., 1994). However, other studies investigating the effect of cocaine administered during gestation on maternal behavior have shown no results or the opposite results (Peeke et al., 1994; Vernotica et al., 1996). Vernotica et al. (1996) found that during the first 4 hr after cocaine injection (20 or 40 mg/kg), there are significant deficits in maternal behavior while 16 hr after cocaine injection, when the plasma level of cocaine falls to nondetectable levels, the drug-injected females show normal maternal behavior comparable to saline-injected controls. Vernotica et al. concluded that cocaine impairs maternal behavior only when circulating and does not have a residual effect in the transiently drug-free, chronically drug-treated dam. While the cocaine data are inconsistent for maternal behavior, there are no studies examining the effect of MA or other amphetamines administered during gestation on maternal behavior. Thus, the present study brings novel data into the research of drugs of abuse on maternal behaviors. Why MA-treated mothers show reduced maternal behavior toward their pups is not clear. It was shown that MA administration alters the dopaminergic and serotoninergic systems in several brain regions (Akiyama, Ishihara, & Kashihara, 1996; Amano, Matsubayashi, & Sasa, 1996; Sabol, Roach, Broom, Ferreira, & Preau, 2001). Repeated MA 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). Moreover, Segal and Kuczenski (1997) demonstrated a correlation between MA-induced behavioral changes and changes in

Methamphetamine and Maternal Behavior

dopamine and serotonin levels in areas closely connected with locomotion, such as caudate putamen and nucleus accumbens. It is, therefore, possible that similarly to Segal and Kuczenski’s (1997) work, MA in the present study may negatively affect the levels of dopamine and serotonin in brain regions that govern maternal behaviors. It already has been demonstrated that MA administration alters the dopaminergic and serotoninergic systems (Akiyama et al., 1996; Amano et al., 1996; Sabol et al., 2001) in brain regions that were shown to be involved in maternal behavior such as the amygdala and/or the nucleus accumbens (Fleming & Walsh, 1994; Numan, 1974). The effect of chronic MA treatment on these systems in the preoptic area, another important brain region that is involved in nursing and maternal behavior (Fleming & Walsh, 1994; Numan, 1974) has not been investigated. This might be a future step of our effort to clarify the mechanism(s) of the effect of MA on maternal behavior. Our hypothesis is supported by the findings of Vernotica, Rosenblatt, and Morrell (1999), demonstrating that bilateral microinfusion of cocaine (50 mg/0.5 ml/side) that affects the same neurotransmitter systems as MA (Rothman & Baumann, 2003) into the medial preoptic area or nucleus accumbens impairs maternal behavior. Further, Elliott et al. (2001) suggested that cocaine may disrupt maternal behavior and maternal aggression through its action on the oxytocinergic system because cocaine alters oxytocin levels in the medial preoptic area and amygdala. When comparing nonmaternal activities, there are no differences in self-care in general, or eating, drinking, and self-grooming separately. There also are no differences in time that mother spent with eyes closed between groups. The present study, however, demonstrates that MA as well as saline injections during gestation increase rearing and sniffing activities relative to controls. These activities might be defined as stereotypic behavior (Decsi, Gacs, Zambo, & Nagy, 1979; Tzschentke & Schmidt, 1998). There are no differences between MA- and salineinduced effects on stereotypic behavior. Thus, it is possible that not the drug, per se, but the mild stress that is induced by the repeated injections during gestation increases stereotypic behavior even when injections are no longer administered. There is a steady decrease in active nursing and maternal activities with the progression of the postpartum period in both experiments. This may be explained by the maturation and increased activity of pups during the third week of lactation. Pups are moving all around the maternal cage, and learn to eat pellets and drink water on their own. Simultaneously to the decrease of active nursing and maternal activities, nonmaternal activities such as mother eating, drinking, and resting with eyes closed increases as postpartum days progress.

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Thus, the present study demonstrates that MA administered daily during the entire gestation period impairs maternal behaviors tested by observation (without disturbing either mother or pups) and retrieval (short separation of pups and mothers) tests. To our knowledge, this is the first study showing such an effect of MA. Even though similar studies were published with other drugs investigating maternal behaviors, the present results bring new insights to the abusing effects of MA on maternal behaviors. The mechanisms(s) of action of MA on maternal behavior will be examined in future studies.

NOTES This study was supported by Grant 305/03/0774 from the Grant Agency of the Czech Republic, Grant NF/7456-3/2003 from the Internal Grant Agency of Ministry of Health of the Czech Republic, and Research Goal MSM 111200005 to R. Sˇ, and by Project LN00B122 from the Ministry of Education, Youth and Sports to M.P. 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. The procedures for animal experimentation utilized in this report were 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 (246/ 1992) and with the regulations of the Ministry of Agriculture of the Czech Republic (311/1997).

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