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The influence of methamphetamine on maternal behavior and development of the pups during the neonatal period
International Journal of Developmental Neuroscience 59 (2017) 37–46
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The influence of methamphetamine on maternal behavior and development of the pups during the neonatal period Mária Ševčíková, Ivana Hrebíčková, Eva Macúchová, Romana Šlamberová
MARK
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Charles University, Third Faculty of Medicine, Department of Normal, Pathological and Clinical Physiology, Prague, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords: Psychostimulants Methamphetamine Maternal behavior Sensorimotor development Neonatal period Wistar
Since it enters breast milk, methamphetamine (MA) abuse during lactation can not only affect the quality of maternal behavior but also postnatal development of pups. The aim of the present study was to examine the effect of injected MA (5 mg/kg) on maternal behavior of rats and the differences in postnatal development, during postnatal days (PD) 1–11, of pups when the pups were directly exposed (i.e., injected) to MA or received MA indirectly via breast milk. Maternal behavior was examined using observation test (PD 1–22) and pup retrieval test (PD 1–12). The following developmental tests were also used: surface righting reflex (PD 1–12), negative geotaxis (PD 9), mid-air righting reflex (PD 17), and the rotarod and beam-balance test (PD 23). The weight of the pups was recorded during the entire testing period and the day of eye opening was also recorded. MA-treated mothers groomed their pups less and returned the pups to the nest slower than control dams. The weight gain of pups indirectly exposed to MA was significantly slower. In addition, pups indirectly exposed to MA were slower on the surface righting reflex (on PD 1 and PD 2) and the negative geotaxis test. In females, indirect exposure to MA led to earlier eye opening compared to controls. At the end of lactation, males who received MA indirectly via breast milk performed worse on the balance beam test compared to males who received MA directly. However, direct exposure to MA improved performance on rotarod relative to controls. Our results suggest that indirect MA exposure, via breast milk, has a greater impact than direct MA exposure.
1. Introduction Methamphetamine (MA) is the most widely used synthetic stimulant in the world. In many countries across the globe it is reportedly the second most prevalent illicit drug after cannabis (EMCDDA, 2009). In Europe, countries with the highest production and consumption of MA are the Czech and Slovak Republic (EMCDDA, 2013). Due to its effects, such as euphoria, increased energy, and suppressed appetite, together with its low cost and relatively easy production, MA is commonly abused among women (Smeriglio and Wilcox, 1999; Smith et al., 2008). Woman may have a greater risk of more severe MA-dependence than men. In a study by Maxwell (2014), women became dependent on MA faster than men. When abused during pregnancy, MA crosses the placental barrier (Burchfield et al., 1991; Dattel, 1990) and during lactation, MA can enter breast milk (Rambousek et al., 2014); these represent mechanisms by which MA can impair the development of the fetus both prenatally and postnatally. Experimental studies in our laboratory have demonstrated that the
administration of MA to female rats during gestation and/or lactation impairs maternal behavior. MA-exposed mothers provided less care for their pups, while showing more self-care activities (Šlamberová et al., 2005a,b). Their pups, prenatally exposed to MA, displayed delayed sensorimotor development on the surface righting reflex and on the mid-air righting reflex, beam balance, and rotarod tests (Hrubá et al., 2009; Šlamberová et al., 2006). This impairment on pup development could be modulated by postnatal fostering using control dams (Hrubá et al., 2009). In our previous study (Malinová-Ševčíková et al., 2014), pups were exposed to MA during either the first half of embryonic development (embryonic day (ED) 1–11) or the second half of embryonic development (ED 12–22); results showed accelerated eye opening and impaired surface righting reflex in pups exposed to MA during first half of prenatal development. MA exposure during the second half of prenatal development led to decreased birth weight and reduced weight gain as well as impaired performance on the beam balance test (Malinová-Ševčíková et al., 2014). The present study continues to examine the effects of MA on maternal behavior and sensorimotor development of pups during the next developmental
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Corresponding author at: Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Ke Karlovu 4, 120 00 Praha 2, Czech Republic. E-mail addresses: [email protected] (M. Ševčíková), [email protected] (I. Hrebíčková), [email protected] (E. Macúchová), [email protected] (R. Šlamberová). http://dx.doi.org/10.1016/j.ijdevneu.2017.03.005 Received 11 November 2016; Received in revised form 12 March 2017; Accepted 13 March 2017 Available online 19 March 2017 0736-5748/ © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.
International Journal of Developmental Neuroscience 59 (2017) 37–46
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2.2. Organization of experimental groups
stage, i.e., after parturition. The neonatal period covered in scientific papers lies on the timeline from postnatal day (PD) 1–21, or shorter (for review see Jablonski et al., 2016). In a recent paper, we defined the neonatal period as PD 1–11 to compare our results with the result of our previous research (Malinová-Ševčíková et al., 2014). The brain develops and matures over a longer period than most other organs. Its development begins during embryogenesis and continues through fetogenesis and the neonatal period. The normal ontogeny of neural development in rodents is different from humans because rodents undergo considerable postnatal development and humans have considerably more prenatal maturation of their nervous systems. These differences may be confounded with differences in exposure methods during critical periods of nervous system development and thus differences in vulnerability between developing animals and humans (e.g., lactation transfer during the first postnatal week in rodents and transplacental transfer during the third trimester in humans) (Benešová et al., 1984; Clancy et al., 2007; Rice and Barone 2000). Along these lines, the present study compared direct and indirect MA exposure during the neonatal period. Since drug abusing human mothers do not inject drugs into their children, children are only exposed to MA during lactation, i.e., indirectly via maternal breast milk. MA is metabolized in the body of mother (the half-life of MA in rats and humans is 70 min and 12 h, respectively) (Cho et al., 2001) therefore, pups might experience transplacental exposure to lesser amounts of the drug. There is little research reporting on the transfer of MA into human or rat breast milk. There is one clinical study that reported the presence of MA in breast milk 24 h after MA injection (Bartu et al., 2009). Another amphetamine drug class, dexamphetamine, which is common pharmacotherapy for attention deficit disorders and attention deficit hyperactivity disorder (ADHD), was confirmed to be present in maternal breast milk at 5.7% of the adjusted infant dose (Ilett et al., 2007). The presence of MA in rat breast milk, which was collected from the stomach of the pups 1 h after ingestion, was confirmed in a study by Rambousek et al. (2014). Our interest here, was to examine the effect of MA on rat pups, independent of maternal exposure. We hypothesized that:
One day after parturition, females were randomly divided into the following groups: 1) Group with direct MA exposure − Mothers were left intact for the entire lactation period. In each cage, the pups were assigned to those, who were injected subcutaneously (s.c.) with MA at 5 mg/kg/ day (volume 1 ml/kg/day) on PD 1–11 and the control group. The control pups received needle prick (not saline) at the same time the MA group got their s.c. injection. We used sham controls because our previous unpublished experience showed that newborn pups injected with saline died at higher rates than MA injected pups. 2) Group with indirect MA exposure − Mothers were exposed to MA or SA. MA exposed mothers were injected s.c. with MA at 5 mg/kg/day in PD 1–11, SA females received a s.c. injection of saline at the same time and same volume (1 ml/kg/day) as the MA group. All females were weighted daily (PD 1–11). The pups were exposed to the effect of SA or MA, via the maternal breast milk, depending on the mother they were assigned to. The dose of MA at 5 mg/kg/day exposed to pregnant laboratory rats leads to such drug concentrations in the brain of fetuses that correspond to the amount of drug observed in the fetuses of drug-dependent human mothers (Acuff-Smith et al., 1996; Cho et al., 1991; Martin et al., 1976). This dose is therefore served as an experimental model for determination of potential risk related to in utero drug exposure in humans. 2.3. Data analyzed 2.3.1. Litter characteristics On PD 1, the number of pups and percentage of males and females in each litter was counted. Thereafter, the number of pups in each litter was adjusted to 12. Whenever possible, the same numbers of male and female pups were kept in each litter. For identification, neonatally MAexposed pups were injected intradermally with black India ink in the left foot and control pups in right foot. Pups underwent the same manipulation throughout the testing period, i.e., weighing and rewriting the testing number on their backs. The day of eye opening was recorded. The eyes were considered open when both eyes of the pup were fully opened. Three-way ANOVA (Drug x Sex x Injection period) was used to analyze birth weight and weight gain of the pups. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. The Chi-square test was used for analysis of eye opening. Differences were considered significant, if p < 0.05.
1) Exposure to MA during PD 1–11 would affect neurogenesis in the neocortex, hippocampus, and cerebellum, which are still undergoing neurogenesis (Bayer, 1980; Ignacio et al., 1995; Rice and Barone, 2000); therefore, it was expected that MA exposure would affect cerebellar locomotion functions and neocortex decision making functions. In the morphological aspect of the study, defects or delays in the development of eyes were also predicted. 2) Both direct and indirect MA exposure would have adverse effects on the offspring, however, we expected direct effects of MA, after direct injection vs. indirect via maternal breast milk, based on biodegradation of MA within dam, to have the greatest effect.
2.3.2. Maternal behavior 2.3.2.1. Observational test. Maternal behavior was observed daily, PD 1–22, for 50 min in the home cage of the mother and her pups. Observations were made during the light phase of the light-dark cycle between 08:00–09:00 h (Malinová-Ševčíková et al., 2014; Šlamberová et al., 2005a,b). During each 50-min session, each mother and her pups were observed 10 times for 5 s at 5 min intervals. Eleven types of activities exhibited by mothers and three types of nursing positions (see below) were recorded during each session. Thus, each mother and her pups were observed 220 times (22 days x 10 observations per day). During each observation “1” indicated that the behavior occurred and “0” indicated that it was absent. First, it was noted whether a mother was nursing or not. Three different positions were recognized as nursing: a) arched nursing (when the mother is arched over her pups with legs splayed), b) blanket nursing (when the mother is over her litter, but did not have her back arched, plus no obvious extension of her legs), c) passive nursing (when the mother was lying on her side or back with one or more suckling pups). The first two nursing positions were designated as active and the
2. Methods 2.1. Prenatal and postnatal animal care Adult albino Wistar rats were purchased from Velaz (Prague, Czech Republic) raised by Charles River Laboratories International, Inc. Females (250–300 g) were housed 5 per cage and males (300–350 g) were housed 4 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 h (light):12 h (dark) cycle (lights on from 0600 h). After one week, 2 females were housed in a cage with 1 male, for two weeks, for mating. One day before the expected day of birth, females were housed in separate cages. The day of birth was counted as PD 0. The mothers with their pups were not disturbed on PD 0. 38
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The Chi-square test was used to analyze differences in the mid-air righting reflex. Differences were considered significant, if p < 0.05.
third one as passive nursing. In addition to nursing, 11 maternal activities were recorded: 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) eating, 8) drinking, 9) self-care activities (mother eating, drinking or self-grooming), 10) rearing, 11) sniffing with head raised. The occurrence of each activity (maximum of 10 in each session) was counted in each of the 22 sessions. One-way ANOVA (Drug) with Repeated Measure (Days) was used to analyze each maternal activity separately. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
2.3.3.4. Balance beam test. The balance beam test (PD 23) was used to examine vestibular function and sensorimotor coordination needed for maintenance of the balance on the narrow bar (Hrubá et al., 2009; Malinová-Ševčíková et al., 2014; Murphy et al., 1995). A wooden bar 40 cm long with a diameter of 1 cm was suspended 80 cm above a soft, padded surface. The pup, held by the nape of its neck, was placed on the bar in such a way that its forepaws could touch the bar. The time of the fore- and hindlimb grasping reflex was recorded with a limit of 120 s. Three consecutive trials were measured. The three-way ANOVA (Drug x Sex x Type of application) with Repeated Measure (Trials) was used to analyze differences in performance on the balance beam test. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
2.3.2.2. Retrieval test. After the observational test ended, the mother and pups were tested using the Retrieval test (Malinová-Ševčíková et al., 2014; Šlamberová et al., 2005a,b). The Retrieval test was conducted daily (PD 1–12) between 09:00–10:00 h. Each mother and her pups were tested 12 times. All pups were removed from their mothers and placed in a separate cage for 5 min. The cage with pups was placed on a heating pad to prevent chilling. After separation, all pups were returned to their mothers, although the pups were placed in different parts of the cage. The mother was then observed for 10 min and the following latencies were recorded: 1) latency to picking up the first pup, 2) latency of returning the first pup to the nest, 3) latency of returning all pups to the nest. Unusual types of behavior were recorded as well: 1) removing a previously returned pup from the nest, 2) carrying of the pups randomly about the cage before placing them in the nest, and 3) extensive disruption of nest shavings. During each observation “1” indicated that the behavior occurred and “0” indicated that it was absent. Latencies were analyzed using the One-way ANOVA (Drug) with Repeated Measure (Days). The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
2.3.3.5. Rotarod test. Rotarod performance was examined on PD 23 to test the sensorimotor coordination and dynamic postural reactions necessary for active moving to maintain balance on a rotating cylinder (Hrubá et al., 2009; Malinová-Ševčíková et al., 2014; Šlamberová et al., 2006). Pups were positioned on a non-slippery cylinder (11.5 cm in diameter, rotating at a constant speed of 6 rpm) oriented in the direction opposite of cylinder rotation, so they could walk forward, to offset the rotation. The duration of balance on the rotarod was determined for 120 s. Trials were repeated until the pups successfully repeated the task, or until there were 6 failures; the number of falls was recorded. The three-way ANOVA (Drug x Sex x Type of application) with Repeated Measure (Trials) was used to analyze differences in rotarod test. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05. 3. Results
2.3.3. Battery of tests used to assess pup development 3.1. Litter characteristics 2.3.3.1. Surface righting reflex. The surface righting reflex was tested daily (PD 1–12) (Altman and Sudarshan, 1975; Hrubá et al., 2009; Malinová-Ševčíková et al., 2014). Each pup was placed in the supine position and the time that it took for the pup to right itself with all four paws contacting the surface of the testing surface was recorded. The three-way ANOVA (Drug x Sex x Type of application) with Repeated Measure (Days) was used to analyze differences in the surface righting reflex. The Tuckey post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
Drug exposure during the postpartum period resulted lower weights of the dams that received MA [F(1,14) = 6.51; p < 0.05] (Fig. 1A). The weight gain of the pups during lactation was associated with the method of MA exposure. Pups exposed indirectly to MA/SA via breast milk gained less weight vs. pups with direct (injections) exposure [F (1,283) = 104.82; p < 0.001] (Fig. 1B). However, difference between control and MA groups and between sexes were not significant. Regarding day of eye opening, there were significant differences between groups on PD 13 [χ2 = 17.48; p < 0.05] and 14 [χ2 = 29.16; p < 0.0001]. On the PD 13, more females indirectly exposed to MA opened their eyes compared with control females and to the females directly exposed to MA. On PD 14, more indirectly MA exposed males and females, as well as directly MA exposed females opened their eyes compared to control pups. Additionally, more indirectly MA exposed males and females opened their eyes compared with directly MA exposed males and females. No sex differences were observed between any of the groups (Table 1).
2.3.3.2. Negative geotaxis. The negative geotaxis test was performed on PD 9 (Altman and Sudarshan, 1975; Hrubá et al., 2009; MalinováŠevčíková et al., 2014). Each pup was placed with its head inclined downward on a wire mesh surface, inclined at a 30° angle. Each pup was given three trials and the best latency for orienting themselves with their head facing up the incline, i.e., 180° of horizontal rotation, was recorded. If the pup was slid off the surface, it was put back in the head downward orientation. The three-way ANOVA (Drug x Sex x Type of application) was used to analyze differences in negative geotaxis. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
3.2. Maternal behavior 3.2.1. Observational test There were no differences in any of the non-maternal activities between MA- and SA-treated dams. In maternal activities, pup grooming was decreased in MA exposed mothers relative to SA exposed mothers [F(1,14) = 5.23; p < 0.05]. There were no differences in other maternal activities between MA- and SA-treated dams. The incidence of mothers carrying pups and drinking during the observation periods was too low for statistical analysis (Table 2).
2.3.3.3. Mid-air righting reflex. The mid-air righting reflex was tested on PD 17 (Altman and Sudarshan, 1975; Hrubá et al., 2009; MalinováŠevčíková et al., 2014). Each pup was held dorsal surface facing down, 40 cm above a soft pad, then released and the pups position on landing was observed. A score of “1” indicated that pup landed on all four paws and “0” indicated that it did not. 39
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Fig. 1. Effect of neonatal MA exposure on: A) weight of the mothers during the injection schedule − PD 1–11, (n = 8); B) weight gain of the pups over the entire lactation period − PD 1–22, (n = 40–106). Values are means ± SEM. MA = methamphetamine, PD = postnatal day. **p < 0.01 MA vs. control. ***p < 0.001 direct vs. indirect exposure regardless drug.
Table 1 Differences in eye opening times.
PD 13 PD 14
Direct application
Indirect application
Males
Males
Females
Females
C
MA
C
MA
C
MA
C
MA
3.64 20.0
3.7 27.78
9.8 21.57
6.52 43.48*
8.7 42.86
15.0 60.0##
4.55 36.36
30.0*,## 70.0*,#
Values are percent of all pups of the same sex and prenatal drug exposure which had their eyes fully opened on the corresponding day (n = 20–55), MA = methamphetamine, C = control, PD = postnatal day. * p < 0.05 MA vs. control, the same method of drug exposure. # p < 0.05 pups direct vs. indirect exposure, the same sex and drug exposure. ## p < 0.01 pups direct vs. indirect exposure, the same sex and drug exposure.
Fig. 2. Effect of MA exposure on retrieval test of mothers during the lactation period − PD 1–12. Values are means ± SEM (n = 8). MA = methamphetamine, PD = postnatal day. *p < 0.05 MA vs. control.
Table 2 Effect of MA exposure during the postpartum period on maternal and non-maternal activities of rats. Observational test
Control
Maternal activities Nursing Active nursing Passive nursing In nest In contact with pups Manipulating shavings Carrying pups Grooming pups
5.81 4.27 1.55 5.28 6.09 0.32 NA 0.87
± ± ± ± ± ±
Non-maternal activities Self-grooming Drinking Eating Sleeping Sniffing Rearing
0.89 NA 1.38 1.95 1.13 1.01
± 0.10
3.3.1. Surface righting reflex The surface righting reflex showed differences among the experimental groups on PD1 and PD 2 [F(1,283) = 8.75; p < 0.01]. During the rest of testing (PD 3–12), there were no differences among any of the experimental groups (Fig. 3A). As shown in Fig. 3B, indirectly exposed males, regardless the drug, were slower on the righting reflex test on PD 1 compared to directly exposed males (p < 0.001). Indirectly MA exposed males were also slower on the righting reflex test on PD 2 compared to directly MA exposed males (p < 0.05). On PD1, control females indirectly exposed were slower on righting reflex test compared to indirectly MA exposed females (p < 0.001) as well as to directly exposed control females (p < 0.001). On PD 2, indirectly SA exposed females were slower compared with directly exposed control females (p < 0.05) but not to indirectly MA exposed females. The surface righting reflex showed sex differences, but only on PD 1. Indirectly MA exposed females were faster in righting compared with indirectly MA exposed males (p < 0.01).
MA
0.31 0.39 0.45 0.28 0.29 0.06
± 0.08
± ± ± ±
3.3. Battery of tests related to pup development
0.10 0.39 0.18 0.19
6.72 4.19 2.53 5.95 6.59 0.26 NA 0.60
± ± ± ± ± ±
0.31 0.39 0.45 0.28 0.29 0.06
0.61 NA 1.27 3.11 0.86 0.70
± 0.14
± 0.08*
± ± ± ±
0.10 0.39 0.18 0.19
Values are shown as means ± SEM (n = 8) and represent the frequency of behavior during 50 min of observation every day over the lactation period. MA = methamphetamine, NA = not analyzed measures. * p < 0.05 MA vs. control.
3.3.2. Negative geotaxis The main effect of negative geotaxis test was based on method of exposure. Pups directly exposed were able to manage the negative geotaxis test in shorter times compared to pups indirectly exposed via maternal breast milk [F(1,266) = 37.11; p < 0.001] (Fig. 4A). The effect exposure method was observed regardless drug treatment or gender. Males, who received SA via maternal breast milk took longer to pass the test than control males receiving direct exposure, per the Bonferroni post-hoc test (p < 0.001) (Fig. 4B). Females, who received MA via maternal milk took longer to orient facing against gravity compared to direct MA exposed females (p < 0.01) (Fig. 4C). Intersex differences were not confirmed.
3.2.2. Retrieval test We found no significant differences in latency of picking up the pup and returning the first pup to the nest during the 12 days of testing. Latency in returning all pups to the nest was longer in MA injected vs. SA injected mothers [F(1,14) = 5.33; p < 0.05] (Fig. 2). There was no interaction between drug treatment and postpartum days in the observed categories. No unusual behaviors, as defined in the Methods section, were exhibited by any of the mothers during the 12 days of observations.
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Fig. 3. A) Effect of prenatal MA exposure on the surface righting reflex test on PD 1–6 regardless sex (n = 40–106). B) Effect of prenatal MA exposure on surface righting reflex during PD 1 and 2 on males (n = 20–55). C) Effect of prenatal MA exposure on surface righting reflex on during PD 1 and 2 on females (n = 20–51). Values represent the time required for rotating from the supine position to a position with all four paws in contact with the surface (floor) and are shown as means ± SEM. MA = methamphetamine, PD = postnatal day. ***p < 0.001 MA vs. control of the same injection period. #p < 0.05 direct vs. indirect exposure (of the same drug). ###p < 0.001 direct vs. indirect exposure (of the same drug).
3.3.5. Rotarod test On the rotating rod test, the effect of neonatal drug exposure, but not exposure method or sex was observed. MA exposed pups, both directly and indirectly, were able to walk longer on the cylinder om the first trial compared with both directly and indirectly exposed control groups [F(1,282) = 7.52, p < 0.01] (Fig. 6A). The number of boluses of each animal was counted during the rotarod test. Pups directly exposed during the neonatal period, regardless the neonatal drug, dropped fewer boluses than pups indirectly exposed [F(1,282) = 8.26, p < 0.01] (Fig. 6B).
3.3.3. Mid-Air righting reflex Drug administration, type of exposure, and sex did not produce any differences in the mid-air righting reflex.
3.3.4. Balance beam test On the balance beam test, an interaction between the type of exposure and the neonatal drug was found [F(1,282) = 7.17, p < 0.01]. The Bonferroni post-hoc test showed that pups indirectly exposed to MA were less capable of balancing on the beam compared to pups directly exposed to MA (p < 0.05; Fig. 5A). This effect was significant in males (p < 0.01; Fig. 5B) but not females (Fig. 5C). The number of boluses of each animal was also counted during the beam test. Pups directly exposed during the neonatal period, regardless of drug, dropped fewer boluses than pups indirectly exposed [F(1,282) = 57.37, p < 0.001] (Fig. 5D).
4. Discussion The aim of the present study was to examine the effect of MA exposure during the postpartum period on maternal behavior and the postnatal consequences of neonatal exposure to MA on development of pups. We expected that application of MA during neonatal development 41
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Fig. 4. Effect of prenatal MA exposure on the negative geotaxis test on PD 9. (A) regardless the sex (n = 40–98), (B) males (n = 20–52), and (C) females (n = 20–46). Values are means ± SEM and represent the time required for turning from a position of negative geotaxis into position of positive geotaxis. MA = methamphetamine, PD = postnatal day. ##p < 0.01 direct vs. indirect exposure (of the same drug). ###p < 0.001 direct vs. indirect exposure (of the same drug).
Fig. 5. Effect of neonatal MA exposure on the balance beam test on PD 23. (A) regardless the sex (n = 40–105), (B) MA exposed males (n = 20–55), (C) MA exposed females (n = 20–50) {the graphs show the average time that animals stayed on the balance beam during the first trial}, and (D) the number of boluses during the performance on the beam (n = 40–105). Values are means ± SEM. MA = methamphetamine, PD = postnatal day. #p < 0.05 direct vs. indirect exposure (of the same drug). ##p < 0.01 direct vs. indirect exposure (of the same drug). ###p < 0.001 direct vs. indirect exposure.
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Fig. 6. Effect of neonatal MA exposure on the performance on the rotarod on the PD 23 − A) regardless the sex. The graph shows the average time that animals endure to walk on the rotarod during the first trial. B) Number of boluses during the entire performance on the rotarod. Values are means ± SEM (n = 40–105). MA = methamphetamine, PD = postnatal day. **p < 0.01 MA vs. control. ##p < 0.01 direct vs. indirect application.
behavior. In the 1980′s, Baker developed a hypothesis that under-nutrition and other insults or adverse stimuli in utero and during infancy can permanently change the bodýs structure, physiology, and metabolism. Fetuses and neonates that are rapidly growing are more vulnerable to under-nutrition and outcomes depend on the developmental period during which the stress occurs (Barker and Osmond, 1986). While MA exposure throughout gestation is known to alter somatic growth (Hrubá et al., 2009; Smith et al., 2006; Šlamberová et al., 2006), there is an assumption that exposure to certain drugs during specific parts of the development period are equally capable of altering somatic growth (Dobbing and Sands, 1971; Smith and Chen, 2010). Our previous results showed a decreased birth weight and rate of weight gain of pups were different after MA exposure, during the second half of the prenatal period (ED 12–22), compared to MA exposure during the first half of the prenatal period (Malinová-Ševčíková et al., 2014). Studies with neonatal MA exposure confirmed persistent body weight decreases, when MA was administrated, during PD 1–10 at doses of 10 mg/kg, 4times a day (Vorhees et al., 1994a). However, the administration of MA during PD 11–20 or PD 6–15 causes only transient reductions in body weight (Vorhees et al., 2009). The results of this study agree with anorectic effects of MA (Bittner et al., 1981) regarding the weight of mothers, which was significantly lower when MA was injected during the early phase of lactation. Interestingly, MA administration during PD 1–11 produces differences in weight gain relative to the type of administration, but not between MA-exposed pups and controls; however, the data is somewhat difficult to interpret. We can only hypothesize regarding the reasons why indirect application causes lower weight gain. The first possibility is that the stress of daily injections might cause decreased milk production in the mothers that could lead to lower weigh gain of their offspring (Lau and Simpson, 2004). Another possibility is that direct application during PD 1–11 is outside the stress hyporesponsive period (SHRP) (Sapolsky and Meaney, 1986; Williams et al., 2003b) (more details below) and MA exposure during this period might lead to more moderate effects than if it were administered during the SHRP. On the other hand, exposure to MA via maternal breast milk might change the hypothesized mechanism of SHRP and maternal stress hormones may have a greater than anticipated impact on pups. A third possibility is that it may be associated with the limitation of drug administration in the present experiment. Pups direct exposed to MA received the drug once a day, while the pups indirectly exposed to MA via breast had, more or less, continuous exposure to MA. We plan research that will focus on determining the concentration of MA in the breast milk of mothers as well as in the plasma and brains of pups. The developing visual system is extremely vulnerable to exposure to neurotoxic drugs (Dominguez et al., 1991). Since rats are born with relatively undeveloped closed eyes, neonatal exposure to MA might
to cause functional changes that corresponded to brain structures that are developing during specific parts of the neonatal period. Abnormal maternal behavior may affect postnatal development of pups. However, we observed differences in maternal behavior, between MA- and SA-exposed mothers, but only regarding pup grooming. In all other maternal and non-maternal activities, no differences between MA vs. SA exposed mothers were observed. In the retrieval test, MAexposed mothers displayed prolonged latency in returning all pups to the nest. In a study by Piccirillo et al. (1980), the nursing time was decreased after amphetamine exposure at doses of 0.25, 0.50, and 1.50 mg/kg during PD 3–4 and 10–11. Moreover, retrieval latencies and time spent nest building were prolonged; additionally, the number of retrieved pups was decreased (Piccirillo et al., 1980). Maternal behavior was observed 30 min after the drug application, e.g. time of highest amphetamine efficacy, which may have contributed to the effect. The study confirmed impaired maternal behavior immediately following drug intoxication. In contrast, our study tried to focus on the effects of MA on long-term maternal care. A study by Vernotica et al. (1996) demonstrated that cocaine acutely impaired maternal behavior during the intoxication period, while 16 h after the cocaine injection, i.e., after plasma cocaine levels had fallen to non-detectable levels, the drug-treated mothers displayed maternal behavior comparable to saline-treated mothers. Our previous studies demonstrated that active nursing was attenuated, when MA was administered during the gestation period and led to increased passive nursing; this was also true when MA was administered during the 9 weeks associated with pre-mating, gestation, and lactation. In addition, the time spent in contact with pups and pup grooming by MA-exposed mothers has been shown to be decreased and latencies in pup retrieval prolonged (Šlamberová et al., 2005a,b). When MA was administered during the first and second half of gestation, there was no evidence of impaired maternal care. However, some maternal and non-maternal activities, such as active of nursing, time spent in the nest, time spent in contact with pups, and sleeping, were increased in mothers exposed to MA or saline during the second half of gestation compared to mothers exposed during the first half of gestation (Malinová-Ševčíková et al., 2014). The maternal behavior of rats during progression of the postpartum period in not static, but is dynamic. It changes in response to developing behavioral and physiological needs of the pups (Grota and Ader, 1969). Pups are more active during the later phase of the weaning period and can find their mother by themselves (Šlamberová et al., 2001). The medial preoptic area (mPOA), is widely believed to be a brain structure critically involved in postpartum maternal responsiveness, which acts as a primary locus of integration and orchestrates effective expression of maternal behavior to the developmental stage of the pups during postpartum (Pereira and Morrell, 2009). We can only guess whether stress caused by injections during the postpartum period or the needs of pups contributed to changes in neural circuits and patterns in maternal 43
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MA lasted for 3 weeks, i.e., until weaning (PD 23). All the other studies observed juvenile or adult aged animals. Vorhees et al. (1994b) administered MA (30 mg/kg, twice a day) during two pre-weaning periods, i.e. first half, PD 1–10 and second half, PD 11–20. Both early and late MA-exposed offspring exhibited reduced locomotor activity with the most evident effects occurring on PD 30. Additionally, postnatal MA exposure through breast milk during the pre-weaning period, PD 1–21, decreased locomotion and exploratory behavior on the open-field test and increased anxiety-like behavior on the elevated plus maze (Hrubá et al., 2012). In our previous research, using the same administration schedule as in this research, showed increased traveled distances in acute MA-exposed pups after neonatal MA exposure (Hrebíčková et al., in press). Changes in locomotor activity, along with our results, indirectly indicate that dopaminergic functionality may be affected by neonatal MA exposure (Jablonski et al., 2016; Vorhees et al., 1994b; Williams et al., 2003a). In the present study, the mid-air righting reflex test did not produce any significant differences between drug exposure, method of administration, or gender. The same results were observed in our previous study, in which pups were exposed to MA during either the first or second half of their prenatal development (Malinová-Ševčíková et al., 2014). The insignificant results in both of our studies might be explained by an inappropriate choice of testing day. Although two of our studies (Hrubá et al., 2008; Šlamberová et al., 2006) showed differences when pups were tested on PD 17, as in the present study, another study found differences on PD 15, but not earlier nor later (Šlamberová et al., 2007). This explanation is supported by a study by Mesquita et al. (2007), in which pups stressed during the neonatal period displayed differences until PD 12, but not afterwards. A study involving neonatal exposure (PD 2–12) to ethanol found a delay in the mid-air righting reflex when tested on PD 15–18, but no other developmental tests were affected (Diaz et al., 2014). To compare our results with the results of our previous research (Malinová-Ševčíková et al., 2014) we were primarily interested in direct (injected) drug administration. However, drug abusing human mothers do not inject drugs into their children, therefore, postpartum exposure to MA occurs during lactation, consequently we decided to use pups exposed indirectly, i.e., via maternal breast milk. We are aware of the limitations that come from comparing direct and indirect MA exposure; however, this was not our primary intention. However, seeing the results, which led to the rejection of the hypothesis 2), we decided to make the comparison anyway. It is necessary to mention that in the direct exposure we used sham controls because our previous unpublished experience showed that newborn pups injected with saline died at higher rates than MA exposed pups. Moreover, our previous research (Hrubá et al., 2009; Šlamberová et al., 2006) showed that the differences between SA and controls, using our conditions, were negligible. Regarding these findings, we decided to minimalize the quantity of animals used in the study and use sham controls. Since our research shows significant differences in most of the tests between types of exposure, but not between MA exposed and control pups, suggesting that the stress factor needs to be taken into consideration. The neonatal window of PD 4–14 is known as the SHRP. It is an interval during which basal corticosterone levels are low and the adrenal response to stress is attenuated compared to earlier or later periods of development (Sapolsky and Meaney, 1986). Stress during the SHRP leads to increased corticosterone levels that last longer than increases caused later in development (Vazquez, 1998). The SHRP is hypothesized to be neuroprotective against stress-induced overstimulation of glucocorticoid receptors (Sapolsky, 1996; Sapolsky and Meaney, 1986). Considering that MA administration induces the HPA axis, which leads to over-response and release of corticosterone in neonatal rats (Schaefer et al., 2006; Williams et al., 2006), it can be assumed that MA exposure during SHRP would lead to more severe effects than MA exposure outside of SHRP. Vorhees et al. (2009) demonstrated an
affect eye opening. Rodrigues et al. (2006) showed that during the critical periods, in which catecholamines can influence the development of neurons, MA transiently affects the pattern of the dopaminergic system in the developing retina. Our previous studies confirm delayed eye opening after prenatal exposure to MA during the gestation and/or lactation period (Hrubá et al., 2009; Šlamberová et al., 2006). Moreover, prenatally MA exposed pups opened their eyes earlier when the drug was administered during ED 1–11 compared with the pups exposed during ED 12–22 (Malinová-Ševčíková et al., 2014). In a recent paper, the results of eye opening do not fully correspond with the other results and these discrepancies are somewhat difficult to interpret. Indirectly MA exposed pups of both sexes opened their eyes earlier (PD 14) compared with the same sex pups directly exposed to MA. The role of stress should not be ignored. Maternal stress (PD 9) has been confirmed to be a modulator of eye opening (Ellenbroek et al., 2005). Future studies are planned to compare levels of MA in the blood and brains of pups after direct and indirect MA exposure on PD 1–11. The present study moves closer to answering the question as to whether timing of prenatal or neonatal MA administration is an important factor for changes in sensorimotor development (MalinováŠevčíková et al., 2014), which is why we used a battery of tests on different postnatal days. The surface righting reflex, which was tested on PD 1–12, examined tactile maturation, which develops prior to motor skills and is under control of the brain stem (Pellis and Pellis, 1994). The negative geotaxis test is an automatic, stimulus-linked orientation movement considered diagnostic of vestibular and/or proprioceptive function (de Castro et al., 2007). The rotarod and balance beam test were related to sensorimotor development that requires fully developed cerebellar coordination, which occurs near the end of lactation, therefore it was tested on PD 23. The rotarod test requires that the pup keep moving against the direction of the cylinder rotation to prevent falling − this engages dynamic postural reactions whereas the balance beam test the fine motor movements necessary for maintaining balance on narrow bar (Pometlová et al., 2009). From our previous studies, it is known that prenatal exposure to MA affects sensorimotor coordination. Pups exposed to MA during gestation only or during both gestation and lactation were found to have slower surface righting reflex on PD 1–5 and PD 12, respectively (Hrubá et al., 2008; Šlamberová et al., 2006). Moreover, pups exposed to MA during the first half of their prenatal development were slower at righting on PD 1 and 2 than animals exposed during the second half of embryonic development (Malinová-Ševčíková et al., 2014). On the negative geotaxis test, prenatally MA-exposed pups and pups exposed to MA during the second half of embryonic development had longer reorientation times, compared to animals exposed during the first half of embryonic development (Hrubá et al., 2009; Malinová-Ševčíková et al., 2014). Our previous studies also showed impaired performance on the rotarod after prenatal MA exposure, although no differences were seen regarding the balance beam (Hrubá et al., 2009; Pometlová et al., 2009). No differences were observed between pups exposed to MA during first or second half of prenatal development on both tests (Malinová-Ševčíková et al., 2014). The present study revealed significant differences between direct and indirect MA exposure during the neonatal period. Animals that received MA or saline indirectly were slower on the surface righting reflex test on PD 1 and 2 as well as reorienting on the negative geotaxis test conducted on PD 9. At the end of lactation period, males indirectly exposed to MA had poorer performance on the balance beam compared to directly exposed males. However, MA-exposed pups, regardless of exposure method, managed to walk on the rotarod for longer times than control pups. The present study is unique regarding its experiment schedule. There are few articles examining the development of the pups after neonatal exposure to MA. The only study that examined pups after neonatal exposure to MA during the pre-weaning period was one of our previous studied (Hrubá et al., 2009). However, the administration of 44
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2009. Neurogenesis inhibition in the dorsal vagal complex by chronic immobilization stress in the adult rat. Neuroscience 158, 524–536. 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. Cho, A.K., Melega, W.P., Kuczenski, R., Segal, D.S., 2001. Relevance of pharmacokinetic parameters in animal models of methamphetamine abuse. Synapse 39, 161–166. Clancy, B., Finlay, B.L., Darlington, R.B., Anand, K.J., 2007. Extrapolating brain development from experimental species to humans. Neurotoxicology 28, 931–937. de Castro, V.L., Destefani, C.R., Diniz, C., Poli, P., 2007. Evaluation of neurodevelopmental effects on rats exposed prenatally to sulfentrazone. Neurotoxicology 28, 1249–1259. Dattel, B.J., 1990. Substance abuse in pregnancy. Semin. Perinatol. 14, 179–187. Diaz, M.R., Vollmer, C.C., Zamudio-Bulcock, P.A., Vollmer, W., Blomquist, S.L., Morton, R.A., Everett, J.C., Zurek, A.A., Yu, J., Orser, B.A., Valenzuela, C.F., 2014. Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor delta subunit in cerebellar granule neurons and delays motor development in rats. Neuropharmacology 79, 262–274. Dobbing, J., Sands, J., 1971. Vulnerability of developing brain. IX. The effect of nutritional growth retardation on the timing of the brain growth-spurt. Biol. Neonate 19, 363–378. Dominguez, R., Aguirre Vila-Coro, A., Slopis, J.M., Bohan, T.P., 1991. Brain and ocular abnormalities in infants with in utero exposure to cocaine and other street drugs. Am. J. Dis. Child. 145, 688–695. EMCDDA, 2009. Methamphetamine: a European Union Perspective in the Global Context. European Monitoring Center for Drugs and Drug Addiction, Luxembourg. EMCDDA, 2013. European Drug Report: The Trends and Developments. European Monitoring Center for Drugs and Drug Addiction, Lisbon. Ellenbroek, B.A., Derks, N., Park, H.J., 2005. Early maternal deprivation retards neurodevelopment in Wistar rats. Stress 8, 247–257. Grota, L.J., Ader, R., 1969. Continuous recording of maternal behavior of Rattus norvegicus. Anim. Behav. 17, 722–729. Hall, C., 1934. Emotional behavior of rats: I. Defecation and urination as measures of individual differences in emotionality. J. Comp. Psychol. 18, 385–403. Hrebíčková, I., Ševčíková, M., Nohejlová, K., Šlamberová, R., in press. Critical neurodevelopmental period for the effect of methamphetamine on activity in LABORAS cage of adult male and female rats. Neurotoxicology and Teratology. Hrubá, L., Schutová, B., Šlamberová, R., Pometlová, M., 2008. Does cross-fostering modify the impairing effect of methamphetamine on postnatal development of rat pups? Prague Med. Rep. 109, 50–61. Hrubá, L., Schutová, B., Šlamberová, R., Pometlová, M., Rokyta, R., 2009. Effect of methamphetamine exposure and cross-fostering on sensorimotor development of male and female rat pups. Dev. Psychobiol. 51, 73–83. Hrubá, L., Schutová, B., Šlamberová, R., 2012. Sex differences in anxiety-like behavior and locomotor activity following prenatal and postnatal methamphetamine exposure in adult rats. Physiol. Behav. 105, 364–370. Ignacio, M.P., Kimm, E.J., Kageyama, G.H., Yu, J., Robertson, R.T., 1995. Postnatal migration of neurons and formation of laminae in rat cerebral cortex. Anat. Embryol. (Berl.) 191, 89–100. Ilett, K.F., Hackett, L.P., Kristensen, J.H., Kohan, R., 2007. Transfer of dexamphetamine into breast milk during treatment for attention deficit hyperactivity disorder. Br. J. Clin. Pharmacol. 63, 371–375. Jablonski, S.A., Williams, M.T., Vorhees, C.V., 2016. Neurobehavioral effects from developmental methamphetamine exposure. Curr. Top. Behav. Neurosci. 29, 183–230. Lau, C., Simpson, C., 2004. Animal models for the study of the effect of prolonged stress on lactation in rats. Physiol. Behav. 82, 193–197. Lister, R.G., 1990. Ethologically-based animal models of anxiety disorders. Pharmacol. Ther. 46, 321–340. Malinová-Ševčíková, M., Hrebíčková, I., Macúchová, E., Nová, E., Pometlová, M., Šlamberová, R., 2014. Differences in maternal behavior and development of their pups depend on the time of methamphetamine exposure during gestation period. Physiol. Res. 63 (Suppl. 4), S559–572. 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, 235–251. Maxwell, J.C., 2014. A new survey of methamphetamine users in treatment: who they are, why they like meth, and why they need additional services. Subst. Use Misuse 49, 639–644. Mesquita, A.R., Pego, J.M., Summavielle, T., Maciel, P., Almeida, O.F., Sousa, N., 2007. Neurodevelopment milestone abnormalities in rats exposed to stress in early life. Neuroscience 147, 1022–1033. Murphy, M.P., Rick, J.T., Milgram, N.W., Ivy, G.O., 1995. A simple and rapid test of sensorimotor function in the aged rat. Neurobiol. Learn Mem. 64, 181–186. Pellis, S.M., Pellis, V.C., 1994. Development of righting when falling from a bipedal standing posture: evidence for the dissociation of dynamic and static righting reflexes in rats. Physiol. Behav. 56, 659–663. Pereira, M., Morrell, J.I., 2009. The changing role of the medial preoptic area in the regulation of maternal behavior across the postpartum period: facilitation followed by inhibition. Behav. Brain Res. 205, 238–248. Piccirillo, M., Alpert, J.E., Cohen, D.J., Shaywitz, B.A., 1980. Amphetamine and maternal behavior: dose response relationships. Psychopharmacology (Berl.) 70, 195–199. Pometlová, M., Hrubá, L., Šlamberová, R., Rokyta, R., 2009. Cross-fostering effect on postnatal development of rat pups exposed to methamphetamine during gestation and preweaning periods. Int. J. Dev. Neurosci. 27, 149–155. Rambousek, L., Kačer, P., Syslová, K., Bumba, J., Bubeníková-Valešová, V., Šlamberová, R., 2014. Sex differences in methamphetamine pharmacokinetics in adult rats and its
increased number of errors, on cognitive tests, after neonatal exposure to MA during PD 6–15 compared to MA exposure on PD 11–20. However, no effect was shown after MA administration on PD 1–10 (Vorhees et al., 1994a, 2009). The animals were subjected to cognitive tests in adulthood in the mentioned papers, however, we have little knowledge about the research that examined the pups during the preweaning period, or the use of developmental tests after prenatal MA exposure. In a recent paper, our results suggested that the method of drug administration might be of considerable significance during SHRP. Emotionality in rodents has traditionally been tested using the open field test and recording defecation and ambulation as relevant parameters of arousal and anxiety (Archer, 1973; Hall, 1934). Particularly high levels of defecation and low levels of ambulation were interpreted as indicators of high levels of emotionality (Whimbey and Denenberg, 1967). However, a lot of papers have been published since these early studies that suggest that defecation is administration not a particularly reliable measure of anxiety-like behavior (e.g., Lister, 1990; Ramos and Mormede, 1998). In none of our previous studies were we able to confirm that differences in defecation between groups could be used a meaningful measure of anxiety (Hrubá et al., 2009; Malinová-Ševčíková et al., 2014; Pometlová et al., 2009; Šlamberová et al., 2006). Interestingly, in the present study, on the balance beam and rotarod test, we observed increased number of boluses from pups indirectly exposed compared to pups directly exposed. These findings might confirm our assumption that indirectly exposure pups felt, subjectively, more stressed. Increased number of boluses have also been seen in rats, which were stressed through immobilization (Chigr et al., 2009). In conclusion, the present study is unique in comparing the effects of direct and indirect MA exposure on pup development. Based on our previous data (Rambousek et al., 2014), further analyses of MA concentration in the brains, blood of pups, and breast milk after direct and indirect exposure is necessary. Acknowledgments This study was supported by grant # GA 14-03708S from Grant Agency of the Czech Republic, project # Progres Q35, GAUK 88315 and 260388/SVV/2017 from Charles University. The authors express their appreciation to Zuzana Ježdíková for her excellent technical assistance. The procedures for animal experimentation utilized in this study were reviewed and approved by the Institutional Animal Care and Use Committee and comply with the requirements of the Czech Government under the Policy of Humane Care of Laboratory Animals (No. 246/ 1992) and with subsequent regulations from the Ministry of Agriculture of the Czech Republic. 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, 199–215. Altman, J., Sudarshan, K., 1975. Postnatal development of locomotion in the laboratory rat. Anim. Behav. 23, 896–920. Archer, J., 1973. Tests for emotionality in rats and mice: a review. Anim. Behav. 21, 205–235. Benešová, O., Pětová, J., Pavlík, A., 1984. Brain Maldevelopment and Drugs. Avicenum, Czechoslovak Medical Press, Prague. Barker, D.J., Osmond, C., 1986. Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet 1, 1077–1081. Bartu, A., Dusci, L.J., Ilett, K.F., 2009. Transfer of methylamphetamine and amphetamine into breast milk following recreational use of methylamphetamine. Br. J. Clin. Pharmacol. 67, 455–459. Bayer, S.A., 1980. Development of the hippocampal region in the rat: II. Morphogenesis during embryonic and early postnatal life. J. Comp. Neurol. 190, 115–134. Bittner, S.E., Wagner, G.C., Aigner, T.G., Seiden, L.S., 1981. Effects of a high-dose treatment of methamphetamine on caudate dopamine and anorexia in rats. Pharmacol. Biochem. Behav. 14, 481–486. Burchfield, D.J., Lucas, V.W., Abrams, R.M., Miller, R.L., DeVane, C.L., 1991. Disposition and pharmacodynamics of methamphetamine in pregnant sheep. JAMA 265, 1968–1973. Chigr, F., Rachidi, F., Segura, S., Mahaut, S., Tardivel, C., Jean, A., Najimi, M., Moyse, E.,
45
International Journal of Developmental Neuroscience 59 (2017) 37–46
M. Ševčíková et al.
development transiently restricts somatic growth. Life Sci. 86, 482–487. Smith, L.M., LaGasse, L.L., Derauf, C., Grant, P., Shah, R., Arria, A., Huestis, M., Haning, W., Strauss, A., Della Grotta, S., Liu, J., Lester, B.M., 2006. The infant development, environment, and lifestyle study: effects of prenatal methamphetamine exposure, polydrug exposure, and poverty on intrauterine growth. Pediatrics 118, 1149–1156. Smith, L.M., LaGasse, L.L., Derauf, C., Grant, P., Shah, R., Arria, A., Huestis, M., Haning, W., Strauss, A., Della Grotta, S., Fallone, M., Liu, J., Lester, B.M., 2008. Prenatal methamphetamine use and neonatal neurobehavioral outcome. Neurotoxicol. Teratol. 30, 20–28. Vazquez, D.M., 1998. Stress and the developing limbic-hypothalamic-pituitary-adrenal axis. Psychoneuroendocrinology 23, 663–700. Vernotica, E.M., Lisciotto, C.A., Rosenblatt, J.S., Morrell, J.I., 1996. Cocaine transiently impairs maternal behavior in the rat. Behav. Neurosci. 110, 315–323. Vorhees, C.V., Ahrens, K.G., Acuff-Smith, K.D., Schilling, M.A., Fisher, J.E., 1994a. Methamphetamine exposure during early postnatal development in rats: I. Acoustic startle augmentation and spatial learning deficits. Psychopharmacology (Berl.) 114, 392–401. Vorhees, C.V., Ahrens, K.G., Acuff-Smith, K.D., Schilling, M.A., Fisher, J.E., 1994b. Methamphetamine exposure during early postnatal development in rats: II. Hypoactivity and altered responses to pharmacological challenge. Psychopharmacology (Berl.) 114, 402–408. Vorhees, C.V., Skelton, M.R., Grace, C.E., Schaefer, T.L., Graham, D.L., Braun, A.A., Williams, M.T., 2009. Effects of (+)-methamphetamine on path integration and spatial learning, but not locomotor activity or acoustic startle, align with the stress hyporesponsive period in rats. Int. J. Dev. Neurosci. 27, 289–298. Whimbey, A.E., Denenberg, V.H., 1967. Two independent behavioral dimensions in openfield performance. J. Comp. Physiol. Psychol. 63, 500–504. Williams, M.T., Blankenmeyer, T.L., Schaefer, T.L., Brown, C.A., Gudelsky, G.A., Vorhees, C.V., 2003a. Long-term effects of neonatal methamphetamine exposure in rats on spatial learning in the Barnes maze and on cliff avoidance, corticosterone release, and neurotoxicity in adulthood. Brain Res. Dev. Brain Res. 147, 163–175. Williams, M.T., Moran, M.S., Vorhees, C.V., 2003b. Refining the critical period for methamphetamine-induced spatial deficits in the Morris water maze. Psychopharmacology (Berl.) 168, 329–338. Williams, M.T., Schaefer, T.L., Furay, A.R., Ehrman, L.A., Vorhees, C.V., 2006. Ontogeny of the adrenal response to (+)-methamphetamine in neonatal rats: the effect of prior drug exposure. Stress 9, 153–163.
transferal to pups via placental membrane and breast milk. Drug Alcohol Depend. 139, 138–144. Ramos, A., Mormede, P., 1998. Stress and emotionality: a multidimensional and genetic approach. Neurosci. Biobehav. Rev. 22, 33–57. Rice, D., Barone Jr., S., 2000. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ. Health Perspect. 108 (Suppl. 3), 511–533. Rodrigues, L.G., Melo, P., Silva, M.C., Tavares, M.A., 2006. Effects of postnatal exposure to methamphetamine on the development of the rat retina. Ann. N. Y. Acad. Sci. 1074, 604–619. Sapolsky, R.M., Meaney, M.J., 1986. Maturation of the adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Res. 396, 64–76. Sapolsky, R.M., 1996. Stress, glucocorticoids, and damage to the nervous system: the current state of confusion. Stress 1, 1–19. Schaefer, T.L., Ehrman, L.A., Gudelsky, G.A., Vorhees, C.V., Williams, M.T., 2006. Comparison of monoamine and corticosterone levels 24 h following (+) methamphetamine, (+/−)3,4-methylenedioxymethamphetamine, cocaine, (+) fenfluramine or (+/−)methylphenidate administration in the neonatal rat. J. Neurochem. 98, 1369–1378. Šlamberová, R., Szilagyi, B., Vathy, I., 2001. Repeated morphine administration during pregnancy attenuates maternal behavior. Psychoneuroendocrinology 26, 565–576. Šlamberová, R., Charousová, P., Pometlová, M., 2005a. Maternal behavior is impaired by methamphetamine administered during pre-mating, gestation and lactation. Reprod. Toxicol. 20, 103–110. Šlamberová, R., Charousová, P., Pometlová, M., 2005b. Methamphetamine administration during gestation impairs maternal behavior. Dev. Psychobiol. 46, 57–65. Šlamberová, R., Pometlová, M., Charousová, P., 2006. Postnatal development of rat pups is altered by prenatal methamphetamine exposure. Prog. Neuropsychopharmacol. Biol. Psychiatry 30, 82–88. Šlamberová, R., Pometlová, M., Rokyta, R., 2007. Effect of methamphetamine exposure during prenatal and preweaning periods lasts for generations in rats. Dev. Psychobiol. 49, 312–322. Smeriglio, V.L., Wilcox, H.C., 1999. Prenatal drug exposure and child outcome Past, present, future. Clin. Perinatol. 26, 1–16. Smith, A.M., Chen, W.J., 2010. Amphetamine treatment during early postnatal
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
The influence of methamphetamine on maternal behavior and development of the pups during the neonatal period Mária Ševčíková, Ivana Hrebíčková, Eva Macúchová, Romana Šlamberová
MARK
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Charles University, Third Faculty of Medicine, Department of Normal, Pathological and Clinical Physiology, Prague, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords: Psychostimulants Methamphetamine Maternal behavior Sensorimotor development Neonatal period Wistar
Since it enters breast milk, methamphetamine (MA) abuse during lactation can not only affect the quality of maternal behavior but also postnatal development of pups. The aim of the present study was to examine the effect of injected MA (5 mg/kg) on maternal behavior of rats and the differences in postnatal development, during postnatal days (PD) 1–11, of pups when the pups were directly exposed (i.e., injected) to MA or received MA indirectly via breast milk. Maternal behavior was examined using observation test (PD 1–22) and pup retrieval test (PD 1–12). The following developmental tests were also used: surface righting reflex (PD 1–12), negative geotaxis (PD 9), mid-air righting reflex (PD 17), and the rotarod and beam-balance test (PD 23). The weight of the pups was recorded during the entire testing period and the day of eye opening was also recorded. MA-treated mothers groomed their pups less and returned the pups to the nest slower than control dams. The weight gain of pups indirectly exposed to MA was significantly slower. In addition, pups indirectly exposed to MA were slower on the surface righting reflex (on PD 1 and PD 2) and the negative geotaxis test. In females, indirect exposure to MA led to earlier eye opening compared to controls. At the end of lactation, males who received MA indirectly via breast milk performed worse on the balance beam test compared to males who received MA directly. However, direct exposure to MA improved performance on rotarod relative to controls. Our results suggest that indirect MA exposure, via breast milk, has a greater impact than direct MA exposure.
1. Introduction Methamphetamine (MA) is the most widely used synthetic stimulant in the world. In many countries across the globe it is reportedly the second most prevalent illicit drug after cannabis (EMCDDA, 2009). In Europe, countries with the highest production and consumption of MA are the Czech and Slovak Republic (EMCDDA, 2013). Due to its effects, such as euphoria, increased energy, and suppressed appetite, together with its low cost and relatively easy production, MA is commonly abused among women (Smeriglio and Wilcox, 1999; Smith et al., 2008). Woman may have a greater risk of more severe MA-dependence than men. In a study by Maxwell (2014), women became dependent on MA faster than men. When abused during pregnancy, MA crosses the placental barrier (Burchfield et al., 1991; Dattel, 1990) and during lactation, MA can enter breast milk (Rambousek et al., 2014); these represent mechanisms by which MA can impair the development of the fetus both prenatally and postnatally. Experimental studies in our laboratory have demonstrated that the
administration of MA to female rats during gestation and/or lactation impairs maternal behavior. MA-exposed mothers provided less care for their pups, while showing more self-care activities (Šlamberová et al., 2005a,b). Their pups, prenatally exposed to MA, displayed delayed sensorimotor development on the surface righting reflex and on the mid-air righting reflex, beam balance, and rotarod tests (Hrubá et al., 2009; Šlamberová et al., 2006). This impairment on pup development could be modulated by postnatal fostering using control dams (Hrubá et al., 2009). In our previous study (Malinová-Ševčíková et al., 2014), pups were exposed to MA during either the first half of embryonic development (embryonic day (ED) 1–11) or the second half of embryonic development (ED 12–22); results showed accelerated eye opening and impaired surface righting reflex in pups exposed to MA during first half of prenatal development. MA exposure during the second half of prenatal development led to decreased birth weight and reduced weight gain as well as impaired performance on the beam balance test (Malinová-Ševčíková et al., 2014). The present study continues to examine the effects of MA on maternal behavior and sensorimotor development of pups during the next developmental
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Corresponding author at: Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Ke Karlovu 4, 120 00 Praha 2, Czech Republic. E-mail addresses: [email protected] (M. Ševčíková), [email protected] (I. Hrebíčková), [email protected] (E. Macúchová), [email protected] (R. Šlamberová). http://dx.doi.org/10.1016/j.ijdevneu.2017.03.005 Received 11 November 2016; Received in revised form 12 March 2017; Accepted 13 March 2017 Available online 19 March 2017 0736-5748/ © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.
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2.2. Organization of experimental groups
stage, i.e., after parturition. The neonatal period covered in scientific papers lies on the timeline from postnatal day (PD) 1–21, or shorter (for review see Jablonski et al., 2016). In a recent paper, we defined the neonatal period as PD 1–11 to compare our results with the result of our previous research (Malinová-Ševčíková et al., 2014). The brain develops and matures over a longer period than most other organs. Its development begins during embryogenesis and continues through fetogenesis and the neonatal period. The normal ontogeny of neural development in rodents is different from humans because rodents undergo considerable postnatal development and humans have considerably more prenatal maturation of their nervous systems. These differences may be confounded with differences in exposure methods during critical periods of nervous system development and thus differences in vulnerability between developing animals and humans (e.g., lactation transfer during the first postnatal week in rodents and transplacental transfer during the third trimester in humans) (Benešová et al., 1984; Clancy et al., 2007; Rice and Barone 2000). Along these lines, the present study compared direct and indirect MA exposure during the neonatal period. Since drug abusing human mothers do not inject drugs into their children, children are only exposed to MA during lactation, i.e., indirectly via maternal breast milk. MA is metabolized in the body of mother (the half-life of MA in rats and humans is 70 min and 12 h, respectively) (Cho et al., 2001) therefore, pups might experience transplacental exposure to lesser amounts of the drug. There is little research reporting on the transfer of MA into human or rat breast milk. There is one clinical study that reported the presence of MA in breast milk 24 h after MA injection (Bartu et al., 2009). Another amphetamine drug class, dexamphetamine, which is common pharmacotherapy for attention deficit disorders and attention deficit hyperactivity disorder (ADHD), was confirmed to be present in maternal breast milk at 5.7% of the adjusted infant dose (Ilett et al., 2007). The presence of MA in rat breast milk, which was collected from the stomach of the pups 1 h after ingestion, was confirmed in a study by Rambousek et al. (2014). Our interest here, was to examine the effect of MA on rat pups, independent of maternal exposure. We hypothesized that:
One day after parturition, females were randomly divided into the following groups: 1) Group with direct MA exposure − Mothers were left intact for the entire lactation period. In each cage, the pups were assigned to those, who were injected subcutaneously (s.c.) with MA at 5 mg/kg/ day (volume 1 ml/kg/day) on PD 1–11 and the control group. The control pups received needle prick (not saline) at the same time the MA group got their s.c. injection. We used sham controls because our previous unpublished experience showed that newborn pups injected with saline died at higher rates than MA injected pups. 2) Group with indirect MA exposure − Mothers were exposed to MA or SA. MA exposed mothers were injected s.c. with MA at 5 mg/kg/day in PD 1–11, SA females received a s.c. injection of saline at the same time and same volume (1 ml/kg/day) as the MA group. All females were weighted daily (PD 1–11). The pups were exposed to the effect of SA or MA, via the maternal breast milk, depending on the mother they were assigned to. The dose of MA at 5 mg/kg/day exposed to pregnant laboratory rats leads to such drug concentrations in the brain of fetuses that correspond to the amount of drug observed in the fetuses of drug-dependent human mothers (Acuff-Smith et al., 1996; Cho et al., 1991; Martin et al., 1976). This dose is therefore served as an experimental model for determination of potential risk related to in utero drug exposure in humans. 2.3. Data analyzed 2.3.1. Litter characteristics On PD 1, the number of pups and percentage of males and females in each litter was counted. Thereafter, the number of pups in each litter was adjusted to 12. Whenever possible, the same numbers of male and female pups were kept in each litter. For identification, neonatally MAexposed pups were injected intradermally with black India ink in the left foot and control pups in right foot. Pups underwent the same manipulation throughout the testing period, i.e., weighing and rewriting the testing number on their backs. The day of eye opening was recorded. The eyes were considered open when both eyes of the pup were fully opened. Three-way ANOVA (Drug x Sex x Injection period) was used to analyze birth weight and weight gain of the pups. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. The Chi-square test was used for analysis of eye opening. Differences were considered significant, if p < 0.05.
1) Exposure to MA during PD 1–11 would affect neurogenesis in the neocortex, hippocampus, and cerebellum, which are still undergoing neurogenesis (Bayer, 1980; Ignacio et al., 1995; Rice and Barone, 2000); therefore, it was expected that MA exposure would affect cerebellar locomotion functions and neocortex decision making functions. In the morphological aspect of the study, defects or delays in the development of eyes were also predicted. 2) Both direct and indirect MA exposure would have adverse effects on the offspring, however, we expected direct effects of MA, after direct injection vs. indirect via maternal breast milk, based on biodegradation of MA within dam, to have the greatest effect.
2.3.2. Maternal behavior 2.3.2.1. Observational test. Maternal behavior was observed daily, PD 1–22, for 50 min in the home cage of the mother and her pups. Observations were made during the light phase of the light-dark cycle between 08:00–09:00 h (Malinová-Ševčíková et al., 2014; Šlamberová et al., 2005a,b). During each 50-min session, each mother and her pups were observed 10 times for 5 s at 5 min intervals. Eleven types of activities exhibited by mothers and three types of nursing positions (see below) were recorded during each session. Thus, each mother and her pups were observed 220 times (22 days x 10 observations per day). During each observation “1” indicated that the behavior occurred and “0” indicated that it was absent. First, it was noted whether a mother was nursing or not. Three different positions were recognized as nursing: a) arched nursing (when the mother is arched over her pups with legs splayed), b) blanket nursing (when the mother is over her litter, but did not have her back arched, plus no obvious extension of her legs), c) passive nursing (when the mother was lying on her side or back with one or more suckling pups). The first two nursing positions were designated as active and the
2. Methods 2.1. Prenatal and postnatal animal care Adult albino Wistar rats were purchased from Velaz (Prague, Czech Republic) raised by Charles River Laboratories International, Inc. Females (250–300 g) were housed 5 per cage and males (300–350 g) were housed 4 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 h (light):12 h (dark) cycle (lights on from 0600 h). After one week, 2 females were housed in a cage with 1 male, for two weeks, for mating. One day before the expected day of birth, females were housed in separate cages. The day of birth was counted as PD 0. The mothers with their pups were not disturbed on PD 0. 38
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The Chi-square test was used to analyze differences in the mid-air righting reflex. Differences were considered significant, if p < 0.05.
third one as passive nursing. In addition to nursing, 11 maternal activities were recorded: 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) eating, 8) drinking, 9) self-care activities (mother eating, drinking or self-grooming), 10) rearing, 11) sniffing with head raised. The occurrence of each activity (maximum of 10 in each session) was counted in each of the 22 sessions. One-way ANOVA (Drug) with Repeated Measure (Days) was used to analyze each maternal activity separately. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
2.3.3.4. Balance beam test. The balance beam test (PD 23) was used to examine vestibular function and sensorimotor coordination needed for maintenance of the balance on the narrow bar (Hrubá et al., 2009; Malinová-Ševčíková et al., 2014; Murphy et al., 1995). A wooden bar 40 cm long with a diameter of 1 cm was suspended 80 cm above a soft, padded surface. The pup, held by the nape of its neck, was placed on the bar in such a way that its forepaws could touch the bar. The time of the fore- and hindlimb grasping reflex was recorded with a limit of 120 s. Three consecutive trials were measured. The three-way ANOVA (Drug x Sex x Type of application) with Repeated Measure (Trials) was used to analyze differences in performance on the balance beam test. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
2.3.2.2. Retrieval test. After the observational test ended, the mother and pups were tested using the Retrieval test (Malinová-Ševčíková et al., 2014; Šlamberová et al., 2005a,b). The Retrieval test was conducted daily (PD 1–12) between 09:00–10:00 h. Each mother and her pups were tested 12 times. All pups were removed from their mothers and placed in a separate cage for 5 min. The cage with pups was placed on a heating pad to prevent chilling. After separation, all pups were returned to their mothers, although the pups were placed in different parts of the cage. The mother was then observed for 10 min and the following latencies were recorded: 1) latency to picking up the first pup, 2) latency of returning the first pup to the nest, 3) latency of returning all pups to the nest. Unusual types of behavior were recorded as well: 1) removing a previously returned pup from the nest, 2) carrying of the pups randomly about the cage before placing them in the nest, and 3) extensive disruption of nest shavings. During each observation “1” indicated that the behavior occurred and “0” indicated that it was absent. Latencies were analyzed using the One-way ANOVA (Drug) with Repeated Measure (Days). The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
2.3.3.5. Rotarod test. Rotarod performance was examined on PD 23 to test the sensorimotor coordination and dynamic postural reactions necessary for active moving to maintain balance on a rotating cylinder (Hrubá et al., 2009; Malinová-Ševčíková et al., 2014; Šlamberová et al., 2006). Pups were positioned on a non-slippery cylinder (11.5 cm in diameter, rotating at a constant speed of 6 rpm) oriented in the direction opposite of cylinder rotation, so they could walk forward, to offset the rotation. The duration of balance on the rotarod was determined for 120 s. Trials were repeated until the pups successfully repeated the task, or until there were 6 failures; the number of falls was recorded. The three-way ANOVA (Drug x Sex x Type of application) with Repeated Measure (Trials) was used to analyze differences in rotarod test. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05. 3. Results
2.3.3. Battery of tests used to assess pup development 3.1. Litter characteristics 2.3.3.1. Surface righting reflex. The surface righting reflex was tested daily (PD 1–12) (Altman and Sudarshan, 1975; Hrubá et al., 2009; Malinová-Ševčíková et al., 2014). Each pup was placed in the supine position and the time that it took for the pup to right itself with all four paws contacting the surface of the testing surface was recorded. The three-way ANOVA (Drug x Sex x Type of application) with Repeated Measure (Days) was used to analyze differences in the surface righting reflex. The Tuckey post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
Drug exposure during the postpartum period resulted lower weights of the dams that received MA [F(1,14) = 6.51; p < 0.05] (Fig. 1A). The weight gain of the pups during lactation was associated with the method of MA exposure. Pups exposed indirectly to MA/SA via breast milk gained less weight vs. pups with direct (injections) exposure [F (1,283) = 104.82; p < 0.001] (Fig. 1B). However, difference between control and MA groups and between sexes were not significant. Regarding day of eye opening, there were significant differences between groups on PD 13 [χ2 = 17.48; p < 0.05] and 14 [χ2 = 29.16; p < 0.0001]. On the PD 13, more females indirectly exposed to MA opened their eyes compared with control females and to the females directly exposed to MA. On PD 14, more indirectly MA exposed males and females, as well as directly MA exposed females opened their eyes compared to control pups. Additionally, more indirectly MA exposed males and females opened their eyes compared with directly MA exposed males and females. No sex differences were observed between any of the groups (Table 1).
2.3.3.2. Negative geotaxis. The negative geotaxis test was performed on PD 9 (Altman and Sudarshan, 1975; Hrubá et al., 2009; MalinováŠevčíková et al., 2014). Each pup was placed with its head inclined downward on a wire mesh surface, inclined at a 30° angle. Each pup was given three trials and the best latency for orienting themselves with their head facing up the incline, i.e., 180° of horizontal rotation, was recorded. If the pup was slid off the surface, it was put back in the head downward orientation. The three-way ANOVA (Drug x Sex x Type of application) was used to analyze differences in negative geotaxis. The Bonferroni post-hoc test was used for comparisons of ANOVA analyses. Differences were considered significant, if p < 0.05.
3.2. Maternal behavior 3.2.1. Observational test There were no differences in any of the non-maternal activities between MA- and SA-treated dams. In maternal activities, pup grooming was decreased in MA exposed mothers relative to SA exposed mothers [F(1,14) = 5.23; p < 0.05]. There were no differences in other maternal activities between MA- and SA-treated dams. The incidence of mothers carrying pups and drinking during the observation periods was too low for statistical analysis (Table 2).
2.3.3.3. Mid-air righting reflex. The mid-air righting reflex was tested on PD 17 (Altman and Sudarshan, 1975; Hrubá et al., 2009; MalinováŠevčíková et al., 2014). Each pup was held dorsal surface facing down, 40 cm above a soft pad, then released and the pups position on landing was observed. A score of “1” indicated that pup landed on all four paws and “0” indicated that it did not. 39
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Fig. 1. Effect of neonatal MA exposure on: A) weight of the mothers during the injection schedule − PD 1–11, (n = 8); B) weight gain of the pups over the entire lactation period − PD 1–22, (n = 40–106). Values are means ± SEM. MA = methamphetamine, PD = postnatal day. **p < 0.01 MA vs. control. ***p < 0.001 direct vs. indirect exposure regardless drug.
Table 1 Differences in eye opening times.
PD 13 PD 14
Direct application
Indirect application
Males
Males
Females
Females
C
MA
C
MA
C
MA
C
MA
3.64 20.0
3.7 27.78
9.8 21.57
6.52 43.48*
8.7 42.86
15.0 60.0##
4.55 36.36
30.0*,## 70.0*,#
Values are percent of all pups of the same sex and prenatal drug exposure which had their eyes fully opened on the corresponding day (n = 20–55), MA = methamphetamine, C = control, PD = postnatal day. * p < 0.05 MA vs. control, the same method of drug exposure. # p < 0.05 pups direct vs. indirect exposure, the same sex and drug exposure. ## p < 0.01 pups direct vs. indirect exposure, the same sex and drug exposure.
Fig. 2. Effect of MA exposure on retrieval test of mothers during the lactation period − PD 1–12. Values are means ± SEM (n = 8). MA = methamphetamine, PD = postnatal day. *p < 0.05 MA vs. control.
Table 2 Effect of MA exposure during the postpartum period on maternal and non-maternal activities of rats. Observational test
Control
Maternal activities Nursing Active nursing Passive nursing In nest In contact with pups Manipulating shavings Carrying pups Grooming pups
5.81 4.27 1.55 5.28 6.09 0.32 NA 0.87
± ± ± ± ± ±
Non-maternal activities Self-grooming Drinking Eating Sleeping Sniffing Rearing
0.89 NA 1.38 1.95 1.13 1.01
± 0.10
3.3.1. Surface righting reflex The surface righting reflex showed differences among the experimental groups on PD1 and PD 2 [F(1,283) = 8.75; p < 0.01]. During the rest of testing (PD 3–12), there were no differences among any of the experimental groups (Fig. 3A). As shown in Fig. 3B, indirectly exposed males, regardless the drug, were slower on the righting reflex test on PD 1 compared to directly exposed males (p < 0.001). Indirectly MA exposed males were also slower on the righting reflex test on PD 2 compared to directly MA exposed males (p < 0.05). On PD1, control females indirectly exposed were slower on righting reflex test compared to indirectly MA exposed females (p < 0.001) as well as to directly exposed control females (p < 0.001). On PD 2, indirectly SA exposed females were slower compared with directly exposed control females (p < 0.05) but not to indirectly MA exposed females. The surface righting reflex showed sex differences, but only on PD 1. Indirectly MA exposed females were faster in righting compared with indirectly MA exposed males (p < 0.01).
MA
0.31 0.39 0.45 0.28 0.29 0.06
± 0.08
± ± ± ±
3.3. Battery of tests related to pup development
0.10 0.39 0.18 0.19
6.72 4.19 2.53 5.95 6.59 0.26 NA 0.60
± ± ± ± ± ±
0.31 0.39 0.45 0.28 0.29 0.06
0.61 NA 1.27 3.11 0.86 0.70
± 0.14
± 0.08*
± ± ± ±
0.10 0.39 0.18 0.19
Values are shown as means ± SEM (n = 8) and represent the frequency of behavior during 50 min of observation every day over the lactation period. MA = methamphetamine, NA = not analyzed measures. * p < 0.05 MA vs. control.
3.3.2. Negative geotaxis The main effect of negative geotaxis test was based on method of exposure. Pups directly exposed were able to manage the negative geotaxis test in shorter times compared to pups indirectly exposed via maternal breast milk [F(1,266) = 37.11; p < 0.001] (Fig. 4A). The effect exposure method was observed regardless drug treatment or gender. Males, who received SA via maternal breast milk took longer to pass the test than control males receiving direct exposure, per the Bonferroni post-hoc test (p < 0.001) (Fig. 4B). Females, who received MA via maternal milk took longer to orient facing against gravity compared to direct MA exposed females (p < 0.01) (Fig. 4C). Intersex differences were not confirmed.
3.2.2. Retrieval test We found no significant differences in latency of picking up the pup and returning the first pup to the nest during the 12 days of testing. Latency in returning all pups to the nest was longer in MA injected vs. SA injected mothers [F(1,14) = 5.33; p < 0.05] (Fig. 2). There was no interaction between drug treatment and postpartum days in the observed categories. No unusual behaviors, as defined in the Methods section, were exhibited by any of the mothers during the 12 days of observations.
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Fig. 3. A) Effect of prenatal MA exposure on the surface righting reflex test on PD 1–6 regardless sex (n = 40–106). B) Effect of prenatal MA exposure on surface righting reflex during PD 1 and 2 on males (n = 20–55). C) Effect of prenatal MA exposure on surface righting reflex on during PD 1 and 2 on females (n = 20–51). Values represent the time required for rotating from the supine position to a position with all four paws in contact with the surface (floor) and are shown as means ± SEM. MA = methamphetamine, PD = postnatal day. ***p < 0.001 MA vs. control of the same injection period. #p < 0.05 direct vs. indirect exposure (of the same drug). ###p < 0.001 direct vs. indirect exposure (of the same drug).
3.3.5. Rotarod test On the rotating rod test, the effect of neonatal drug exposure, but not exposure method or sex was observed. MA exposed pups, both directly and indirectly, were able to walk longer on the cylinder om the first trial compared with both directly and indirectly exposed control groups [F(1,282) = 7.52, p < 0.01] (Fig. 6A). The number of boluses of each animal was counted during the rotarod test. Pups directly exposed during the neonatal period, regardless the neonatal drug, dropped fewer boluses than pups indirectly exposed [F(1,282) = 8.26, p < 0.01] (Fig. 6B).
3.3.3. Mid-Air righting reflex Drug administration, type of exposure, and sex did not produce any differences in the mid-air righting reflex.
3.3.4. Balance beam test On the balance beam test, an interaction between the type of exposure and the neonatal drug was found [F(1,282) = 7.17, p < 0.01]. The Bonferroni post-hoc test showed that pups indirectly exposed to MA were less capable of balancing on the beam compared to pups directly exposed to MA (p < 0.05; Fig. 5A). This effect was significant in males (p < 0.01; Fig. 5B) but not females (Fig. 5C). The number of boluses of each animal was also counted during the beam test. Pups directly exposed during the neonatal period, regardless of drug, dropped fewer boluses than pups indirectly exposed [F(1,282) = 57.37, p < 0.001] (Fig. 5D).
4. Discussion The aim of the present study was to examine the effect of MA exposure during the postpartum period on maternal behavior and the postnatal consequences of neonatal exposure to MA on development of pups. We expected that application of MA during neonatal development 41
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Fig. 4. Effect of prenatal MA exposure on the negative geotaxis test on PD 9. (A) regardless the sex (n = 40–98), (B) males (n = 20–52), and (C) females (n = 20–46). Values are means ± SEM and represent the time required for turning from a position of negative geotaxis into position of positive geotaxis. MA = methamphetamine, PD = postnatal day. ##p < 0.01 direct vs. indirect exposure (of the same drug). ###p < 0.001 direct vs. indirect exposure (of the same drug).
Fig. 5. Effect of neonatal MA exposure on the balance beam test on PD 23. (A) regardless the sex (n = 40–105), (B) MA exposed males (n = 20–55), (C) MA exposed females (n = 20–50) {the graphs show the average time that animals stayed on the balance beam during the first trial}, and (D) the number of boluses during the performance on the beam (n = 40–105). Values are means ± SEM. MA = methamphetamine, PD = postnatal day. #p < 0.05 direct vs. indirect exposure (of the same drug). ##p < 0.01 direct vs. indirect exposure (of the same drug). ###p < 0.001 direct vs. indirect exposure.
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Fig. 6. Effect of neonatal MA exposure on the performance on the rotarod on the PD 23 − A) regardless the sex. The graph shows the average time that animals endure to walk on the rotarod during the first trial. B) Number of boluses during the entire performance on the rotarod. Values are means ± SEM (n = 40–105). MA = methamphetamine, PD = postnatal day. **p < 0.01 MA vs. control. ##p < 0.01 direct vs. indirect application.
behavior. In the 1980′s, Baker developed a hypothesis that under-nutrition and other insults or adverse stimuli in utero and during infancy can permanently change the bodýs structure, physiology, and metabolism. Fetuses and neonates that are rapidly growing are more vulnerable to under-nutrition and outcomes depend on the developmental period during which the stress occurs (Barker and Osmond, 1986). While MA exposure throughout gestation is known to alter somatic growth (Hrubá et al., 2009; Smith et al., 2006; Šlamberová et al., 2006), there is an assumption that exposure to certain drugs during specific parts of the development period are equally capable of altering somatic growth (Dobbing and Sands, 1971; Smith and Chen, 2010). Our previous results showed a decreased birth weight and rate of weight gain of pups were different after MA exposure, during the second half of the prenatal period (ED 12–22), compared to MA exposure during the first half of the prenatal period (Malinová-Ševčíková et al., 2014). Studies with neonatal MA exposure confirmed persistent body weight decreases, when MA was administrated, during PD 1–10 at doses of 10 mg/kg, 4times a day (Vorhees et al., 1994a). However, the administration of MA during PD 11–20 or PD 6–15 causes only transient reductions in body weight (Vorhees et al., 2009). The results of this study agree with anorectic effects of MA (Bittner et al., 1981) regarding the weight of mothers, which was significantly lower when MA was injected during the early phase of lactation. Interestingly, MA administration during PD 1–11 produces differences in weight gain relative to the type of administration, but not between MA-exposed pups and controls; however, the data is somewhat difficult to interpret. We can only hypothesize regarding the reasons why indirect application causes lower weight gain. The first possibility is that the stress of daily injections might cause decreased milk production in the mothers that could lead to lower weigh gain of their offspring (Lau and Simpson, 2004). Another possibility is that direct application during PD 1–11 is outside the stress hyporesponsive period (SHRP) (Sapolsky and Meaney, 1986; Williams et al., 2003b) (more details below) and MA exposure during this period might lead to more moderate effects than if it were administered during the SHRP. On the other hand, exposure to MA via maternal breast milk might change the hypothesized mechanism of SHRP and maternal stress hormones may have a greater than anticipated impact on pups. A third possibility is that it may be associated with the limitation of drug administration in the present experiment. Pups direct exposed to MA received the drug once a day, while the pups indirectly exposed to MA via breast had, more or less, continuous exposure to MA. We plan research that will focus on determining the concentration of MA in the breast milk of mothers as well as in the plasma and brains of pups. The developing visual system is extremely vulnerable to exposure to neurotoxic drugs (Dominguez et al., 1991). Since rats are born with relatively undeveloped closed eyes, neonatal exposure to MA might
to cause functional changes that corresponded to brain structures that are developing during specific parts of the neonatal period. Abnormal maternal behavior may affect postnatal development of pups. However, we observed differences in maternal behavior, between MA- and SA-exposed mothers, but only regarding pup grooming. In all other maternal and non-maternal activities, no differences between MA vs. SA exposed mothers were observed. In the retrieval test, MAexposed mothers displayed prolonged latency in returning all pups to the nest. In a study by Piccirillo et al. (1980), the nursing time was decreased after amphetamine exposure at doses of 0.25, 0.50, and 1.50 mg/kg during PD 3–4 and 10–11. Moreover, retrieval latencies and time spent nest building were prolonged; additionally, the number of retrieved pups was decreased (Piccirillo et al., 1980). Maternal behavior was observed 30 min after the drug application, e.g. time of highest amphetamine efficacy, which may have contributed to the effect. The study confirmed impaired maternal behavior immediately following drug intoxication. In contrast, our study tried to focus on the effects of MA on long-term maternal care. A study by Vernotica et al. (1996) demonstrated that cocaine acutely impaired maternal behavior during the intoxication period, while 16 h after the cocaine injection, i.e., after plasma cocaine levels had fallen to non-detectable levels, the drug-treated mothers displayed maternal behavior comparable to saline-treated mothers. Our previous studies demonstrated that active nursing was attenuated, when MA was administered during the gestation period and led to increased passive nursing; this was also true when MA was administered during the 9 weeks associated with pre-mating, gestation, and lactation. In addition, the time spent in contact with pups and pup grooming by MA-exposed mothers has been shown to be decreased and latencies in pup retrieval prolonged (Šlamberová et al., 2005a,b). When MA was administered during the first and second half of gestation, there was no evidence of impaired maternal care. However, some maternal and non-maternal activities, such as active of nursing, time spent in the nest, time spent in contact with pups, and sleeping, were increased in mothers exposed to MA or saline during the second half of gestation compared to mothers exposed during the first half of gestation (Malinová-Ševčíková et al., 2014). The maternal behavior of rats during progression of the postpartum period in not static, but is dynamic. It changes in response to developing behavioral and physiological needs of the pups (Grota and Ader, 1969). Pups are more active during the later phase of the weaning period and can find their mother by themselves (Šlamberová et al., 2001). The medial preoptic area (mPOA), is widely believed to be a brain structure critically involved in postpartum maternal responsiveness, which acts as a primary locus of integration and orchestrates effective expression of maternal behavior to the developmental stage of the pups during postpartum (Pereira and Morrell, 2009). We can only guess whether stress caused by injections during the postpartum period or the needs of pups contributed to changes in neural circuits and patterns in maternal 43
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MA lasted for 3 weeks, i.e., until weaning (PD 23). All the other studies observed juvenile or adult aged animals. Vorhees et al. (1994b) administered MA (30 mg/kg, twice a day) during two pre-weaning periods, i.e. first half, PD 1–10 and second half, PD 11–20. Both early and late MA-exposed offspring exhibited reduced locomotor activity with the most evident effects occurring on PD 30. Additionally, postnatal MA exposure through breast milk during the pre-weaning period, PD 1–21, decreased locomotion and exploratory behavior on the open-field test and increased anxiety-like behavior on the elevated plus maze (Hrubá et al., 2012). In our previous research, using the same administration schedule as in this research, showed increased traveled distances in acute MA-exposed pups after neonatal MA exposure (Hrebíčková et al., in press). Changes in locomotor activity, along with our results, indirectly indicate that dopaminergic functionality may be affected by neonatal MA exposure (Jablonski et al., 2016; Vorhees et al., 1994b; Williams et al., 2003a). In the present study, the mid-air righting reflex test did not produce any significant differences between drug exposure, method of administration, or gender. The same results were observed in our previous study, in which pups were exposed to MA during either the first or second half of their prenatal development (Malinová-Ševčíková et al., 2014). The insignificant results in both of our studies might be explained by an inappropriate choice of testing day. Although two of our studies (Hrubá et al., 2008; Šlamberová et al., 2006) showed differences when pups were tested on PD 17, as in the present study, another study found differences on PD 15, but not earlier nor later (Šlamberová et al., 2007). This explanation is supported by a study by Mesquita et al. (2007), in which pups stressed during the neonatal period displayed differences until PD 12, but not afterwards. A study involving neonatal exposure (PD 2–12) to ethanol found a delay in the mid-air righting reflex when tested on PD 15–18, but no other developmental tests were affected (Diaz et al., 2014). To compare our results with the results of our previous research (Malinová-Ševčíková et al., 2014) we were primarily interested in direct (injected) drug administration. However, drug abusing human mothers do not inject drugs into their children, therefore, postpartum exposure to MA occurs during lactation, consequently we decided to use pups exposed indirectly, i.e., via maternal breast milk. We are aware of the limitations that come from comparing direct and indirect MA exposure; however, this was not our primary intention. However, seeing the results, which led to the rejection of the hypothesis 2), we decided to make the comparison anyway. It is necessary to mention that in the direct exposure we used sham controls because our previous unpublished experience showed that newborn pups injected with saline died at higher rates than MA exposed pups. Moreover, our previous research (Hrubá et al., 2009; Šlamberová et al., 2006) showed that the differences between SA and controls, using our conditions, were negligible. Regarding these findings, we decided to minimalize the quantity of animals used in the study and use sham controls. Since our research shows significant differences in most of the tests between types of exposure, but not between MA exposed and control pups, suggesting that the stress factor needs to be taken into consideration. The neonatal window of PD 4–14 is known as the SHRP. It is an interval during which basal corticosterone levels are low and the adrenal response to stress is attenuated compared to earlier or later periods of development (Sapolsky and Meaney, 1986). Stress during the SHRP leads to increased corticosterone levels that last longer than increases caused later in development (Vazquez, 1998). The SHRP is hypothesized to be neuroprotective against stress-induced overstimulation of glucocorticoid receptors (Sapolsky, 1996; Sapolsky and Meaney, 1986). Considering that MA administration induces the HPA axis, which leads to over-response and release of corticosterone in neonatal rats (Schaefer et al., 2006; Williams et al., 2006), it can be assumed that MA exposure during SHRP would lead to more severe effects than MA exposure outside of SHRP. Vorhees et al. (2009) demonstrated an
affect eye opening. Rodrigues et al. (2006) showed that during the critical periods, in which catecholamines can influence the development of neurons, MA transiently affects the pattern of the dopaminergic system in the developing retina. Our previous studies confirm delayed eye opening after prenatal exposure to MA during the gestation and/or lactation period (Hrubá et al., 2009; Šlamberová et al., 2006). Moreover, prenatally MA exposed pups opened their eyes earlier when the drug was administered during ED 1–11 compared with the pups exposed during ED 12–22 (Malinová-Ševčíková et al., 2014). In a recent paper, the results of eye opening do not fully correspond with the other results and these discrepancies are somewhat difficult to interpret. Indirectly MA exposed pups of both sexes opened their eyes earlier (PD 14) compared with the same sex pups directly exposed to MA. The role of stress should not be ignored. Maternal stress (PD 9) has been confirmed to be a modulator of eye opening (Ellenbroek et al., 2005). Future studies are planned to compare levels of MA in the blood and brains of pups after direct and indirect MA exposure on PD 1–11. The present study moves closer to answering the question as to whether timing of prenatal or neonatal MA administration is an important factor for changes in sensorimotor development (MalinováŠevčíková et al., 2014), which is why we used a battery of tests on different postnatal days. The surface righting reflex, which was tested on PD 1–12, examined tactile maturation, which develops prior to motor skills and is under control of the brain stem (Pellis and Pellis, 1994). The negative geotaxis test is an automatic, stimulus-linked orientation movement considered diagnostic of vestibular and/or proprioceptive function (de Castro et al., 2007). The rotarod and balance beam test were related to sensorimotor development that requires fully developed cerebellar coordination, which occurs near the end of lactation, therefore it was tested on PD 23. The rotarod test requires that the pup keep moving against the direction of the cylinder rotation to prevent falling − this engages dynamic postural reactions whereas the balance beam test the fine motor movements necessary for maintaining balance on narrow bar (Pometlová et al., 2009). From our previous studies, it is known that prenatal exposure to MA affects sensorimotor coordination. Pups exposed to MA during gestation only or during both gestation and lactation were found to have slower surface righting reflex on PD 1–5 and PD 12, respectively (Hrubá et al., 2008; Šlamberová et al., 2006). Moreover, pups exposed to MA during the first half of their prenatal development were slower at righting on PD 1 and 2 than animals exposed during the second half of embryonic development (Malinová-Ševčíková et al., 2014). On the negative geotaxis test, prenatally MA-exposed pups and pups exposed to MA during the second half of embryonic development had longer reorientation times, compared to animals exposed during the first half of embryonic development (Hrubá et al., 2009; Malinová-Ševčíková et al., 2014). Our previous studies also showed impaired performance on the rotarod after prenatal MA exposure, although no differences were seen regarding the balance beam (Hrubá et al., 2009; Pometlová et al., 2009). No differences were observed between pups exposed to MA during first or second half of prenatal development on both tests (Malinová-Ševčíková et al., 2014). The present study revealed significant differences between direct and indirect MA exposure during the neonatal period. Animals that received MA or saline indirectly were slower on the surface righting reflex test on PD 1 and 2 as well as reorienting on the negative geotaxis test conducted on PD 9. At the end of lactation period, males indirectly exposed to MA had poorer performance on the balance beam compared to directly exposed males. However, MA-exposed pups, regardless of exposure method, managed to walk on the rotarod for longer times than control pups. The present study is unique regarding its experiment schedule. There are few articles examining the development of the pups after neonatal exposure to MA. The only study that examined pups after neonatal exposure to MA during the pre-weaning period was one of our previous studied (Hrubá et al., 2009). However, the administration of 44
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2009. Neurogenesis inhibition in the dorsal vagal complex by chronic immobilization stress in the adult rat. Neuroscience 158, 524–536. 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. Cho, A.K., Melega, W.P., Kuczenski, R., Segal, D.S., 2001. Relevance of pharmacokinetic parameters in animal models of methamphetamine abuse. Synapse 39, 161–166. Clancy, B., Finlay, B.L., Darlington, R.B., Anand, K.J., 2007. Extrapolating brain development from experimental species to humans. Neurotoxicology 28, 931–937. de Castro, V.L., Destefani, C.R., Diniz, C., Poli, P., 2007. Evaluation of neurodevelopmental effects on rats exposed prenatally to sulfentrazone. Neurotoxicology 28, 1249–1259. Dattel, B.J., 1990. Substance abuse in pregnancy. Semin. Perinatol. 14, 179–187. Diaz, M.R., Vollmer, C.C., Zamudio-Bulcock, P.A., Vollmer, W., Blomquist, S.L., Morton, R.A., Everett, J.C., Zurek, A.A., Yu, J., Orser, B.A., Valenzuela, C.F., 2014. Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor delta subunit in cerebellar granule neurons and delays motor development in rats. Neuropharmacology 79, 262–274. Dobbing, J., Sands, J., 1971. Vulnerability of developing brain. IX. The effect of nutritional growth retardation on the timing of the brain growth-spurt. Biol. Neonate 19, 363–378. Dominguez, R., Aguirre Vila-Coro, A., Slopis, J.M., Bohan, T.P., 1991. Brain and ocular abnormalities in infants with in utero exposure to cocaine and other street drugs. Am. J. Dis. Child. 145, 688–695. EMCDDA, 2009. Methamphetamine: a European Union Perspective in the Global Context. European Monitoring Center for Drugs and Drug Addiction, Luxembourg. EMCDDA, 2013. European Drug Report: The Trends and Developments. European Monitoring Center for Drugs and Drug Addiction, Lisbon. Ellenbroek, B.A., Derks, N., Park, H.J., 2005. Early maternal deprivation retards neurodevelopment in Wistar rats. Stress 8, 247–257. Grota, L.J., Ader, R., 1969. Continuous recording of maternal behavior of Rattus norvegicus. Anim. Behav. 17, 722–729. Hall, C., 1934. Emotional behavior of rats: I. Defecation and urination as measures of individual differences in emotionality. J. Comp. Psychol. 18, 385–403. Hrebíčková, I., Ševčíková, M., Nohejlová, K., Šlamberová, R., in press. Critical neurodevelopmental period for the effect of methamphetamine on activity in LABORAS cage of adult male and female rats. Neurotoxicology and Teratology. Hrubá, L., Schutová, B., Šlamberová, R., Pometlová, M., 2008. Does cross-fostering modify the impairing effect of methamphetamine on postnatal development of rat pups? Prague Med. Rep. 109, 50–61. Hrubá, L., Schutová, B., Šlamberová, R., Pometlová, M., Rokyta, R., 2009. Effect of methamphetamine exposure and cross-fostering on sensorimotor development of male and female rat pups. Dev. Psychobiol. 51, 73–83. Hrubá, L., Schutová, B., Šlamberová, R., 2012. Sex differences in anxiety-like behavior and locomotor activity following prenatal and postnatal methamphetamine exposure in adult rats. Physiol. Behav. 105, 364–370. Ignacio, M.P., Kimm, E.J., Kageyama, G.H., Yu, J., Robertson, R.T., 1995. Postnatal migration of neurons and formation of laminae in rat cerebral cortex. Anat. Embryol. (Berl.) 191, 89–100. Ilett, K.F., Hackett, L.P., Kristensen, J.H., Kohan, R., 2007. Transfer of dexamphetamine into breast milk during treatment for attention deficit hyperactivity disorder. Br. J. Clin. Pharmacol. 63, 371–375. Jablonski, S.A., Williams, M.T., Vorhees, C.V., 2016. Neurobehavioral effects from developmental methamphetamine exposure. Curr. Top. Behav. Neurosci. 29, 183–230. Lau, C., Simpson, C., 2004. Animal models for the study of the effect of prolonged stress on lactation in rats. Physiol. Behav. 82, 193–197. Lister, R.G., 1990. Ethologically-based animal models of anxiety disorders. Pharmacol. Ther. 46, 321–340. Malinová-Ševčíková, M., Hrebíčková, I., Macúchová, E., Nová, E., Pometlová, M., Šlamberová, R., 2014. Differences in maternal behavior and development of their pups depend on the time of methamphetamine exposure during gestation period. Physiol. Res. 63 (Suppl. 4), S559–572. 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, 235–251. Maxwell, J.C., 2014. A new survey of methamphetamine users in treatment: who they are, why they like meth, and why they need additional services. Subst. Use Misuse 49, 639–644. Mesquita, A.R., Pego, J.M., Summavielle, T., Maciel, P., Almeida, O.F., Sousa, N., 2007. Neurodevelopment milestone abnormalities in rats exposed to stress in early life. Neuroscience 147, 1022–1033. Murphy, M.P., Rick, J.T., Milgram, N.W., Ivy, G.O., 1995. A simple and rapid test of sensorimotor function in the aged rat. Neurobiol. Learn Mem. 64, 181–186. Pellis, S.M., Pellis, V.C., 1994. Development of righting when falling from a bipedal standing posture: evidence for the dissociation of dynamic and static righting reflexes in rats. Physiol. Behav. 56, 659–663. Pereira, M., Morrell, J.I., 2009. The changing role of the medial preoptic area in the regulation of maternal behavior across the postpartum period: facilitation followed by inhibition. Behav. Brain Res. 205, 238–248. Piccirillo, M., Alpert, J.E., Cohen, D.J., Shaywitz, B.A., 1980. Amphetamine and maternal behavior: dose response relationships. Psychopharmacology (Berl.) 70, 195–199. Pometlová, M., Hrubá, L., Šlamberová, R., Rokyta, R., 2009. Cross-fostering effect on postnatal development of rat pups exposed to methamphetamine during gestation and preweaning periods. Int. J. Dev. Neurosci. 27, 149–155. Rambousek, L., Kačer, P., Syslová, K., Bumba, J., Bubeníková-Valešová, V., Šlamberová, R., 2014. Sex differences in methamphetamine pharmacokinetics in adult rats and its
increased number of errors, on cognitive tests, after neonatal exposure to MA during PD 6–15 compared to MA exposure on PD 11–20. However, no effect was shown after MA administration on PD 1–10 (Vorhees et al., 1994a, 2009). The animals were subjected to cognitive tests in adulthood in the mentioned papers, however, we have little knowledge about the research that examined the pups during the preweaning period, or the use of developmental tests after prenatal MA exposure. In a recent paper, our results suggested that the method of drug administration might be of considerable significance during SHRP. Emotionality in rodents has traditionally been tested using the open field test and recording defecation and ambulation as relevant parameters of arousal and anxiety (Archer, 1973; Hall, 1934). Particularly high levels of defecation and low levels of ambulation were interpreted as indicators of high levels of emotionality (Whimbey and Denenberg, 1967). However, a lot of papers have been published since these early studies that suggest that defecation is administration not a particularly reliable measure of anxiety-like behavior (e.g., Lister, 1990; Ramos and Mormede, 1998). In none of our previous studies were we able to confirm that differences in defecation between groups could be used a meaningful measure of anxiety (Hrubá et al., 2009; Malinová-Ševčíková et al., 2014; Pometlová et al., 2009; Šlamberová et al., 2006). Interestingly, in the present study, on the balance beam and rotarod test, we observed increased number of boluses from pups indirectly exposed compared to pups directly exposed. These findings might confirm our assumption that indirectly exposure pups felt, subjectively, more stressed. Increased number of boluses have also been seen in rats, which were stressed through immobilization (Chigr et al., 2009). In conclusion, the present study is unique in comparing the effects of direct and indirect MA exposure on pup development. Based on our previous data (Rambousek et al., 2014), further analyses of MA concentration in the brains, blood of pups, and breast milk after direct and indirect exposure is necessary. Acknowledgments This study was supported by grant # GA 14-03708S from Grant Agency of the Czech Republic, project # Progres Q35, GAUK 88315 and 260388/SVV/2017 from Charles University. The authors express their appreciation to Zuzana Ježdíková for her excellent technical assistance. The procedures for animal experimentation utilized in this study were reviewed and approved by the Institutional Animal Care and Use Committee and comply with the requirements of the Czech Government under the Policy of Humane Care of Laboratory Animals (No. 246/ 1992) and with subsequent regulations from the Ministry of Agriculture of the Czech Republic. 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, 199–215. Altman, J., Sudarshan, K., 1975. Postnatal development of locomotion in the laboratory rat. Anim. Behav. 23, 896–920. Archer, J., 1973. Tests for emotionality in rats and mice: a review. Anim. Behav. 21, 205–235. Benešová, O., Pětová, J., Pavlík, A., 1984. Brain Maldevelopment and Drugs. Avicenum, Czechoslovak Medical Press, Prague. Barker, D.J., Osmond, C., 1986. Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet 1, 1077–1081. Bartu, A., Dusci, L.J., Ilett, K.F., 2009. Transfer of methylamphetamine and amphetamine into breast milk following recreational use of methylamphetamine. Br. J. Clin. Pharmacol. 67, 455–459. Bayer, S.A., 1980. Development of the hippocampal region in the rat: II. Morphogenesis during embryonic and early postnatal life. J. Comp. Neurol. 190, 115–134. Bittner, S.E., Wagner, G.C., Aigner, T.G., Seiden, L.S., 1981. Effects of a high-dose treatment of methamphetamine on caudate dopamine and anorexia in rats. Pharmacol. Biochem. Behav. 14, 481–486. Burchfield, D.J., Lucas, V.W., Abrams, R.M., Miller, R.L., DeVane, C.L., 1991. Disposition and pharmacodynamics of methamphetamine in pregnant sheep. JAMA 265, 1968–1973. Chigr, F., Rachidi, F., Segura, S., Mahaut, S., Tardivel, C., Jean, A., Najimi, M., Moyse, E.,
45
International Journal of Developmental Neuroscience 59 (2017) 37–46
M. Ševčíková et al.
development transiently restricts somatic growth. Life Sci. 86, 482–487. Smith, L.M., LaGasse, L.L., Derauf, C., Grant, P., Shah, R., Arria, A., Huestis, M., Haning, W., Strauss, A., Della Grotta, S., Liu, J., Lester, B.M., 2006. The infant development, environment, and lifestyle study: effects of prenatal methamphetamine exposure, polydrug exposure, and poverty on intrauterine growth. Pediatrics 118, 1149–1156. Smith, L.M., LaGasse, L.L., Derauf, C., Grant, P., Shah, R., Arria, A., Huestis, M., Haning, W., Strauss, A., Della Grotta, S., Fallone, M., Liu, J., Lester, B.M., 2008. Prenatal methamphetamine use and neonatal neurobehavioral outcome. Neurotoxicol. Teratol. 30, 20–28. Vazquez, D.M., 1998. Stress and the developing limbic-hypothalamic-pituitary-adrenal axis. Psychoneuroendocrinology 23, 663–700. Vernotica, E.M., Lisciotto, C.A., Rosenblatt, J.S., Morrell, J.I., 1996. Cocaine transiently impairs maternal behavior in the rat. Behav. Neurosci. 110, 315–323. Vorhees, C.V., Ahrens, K.G., Acuff-Smith, K.D., Schilling, M.A., Fisher, J.E., 1994a. Methamphetamine exposure during early postnatal development in rats: I. Acoustic startle augmentation and spatial learning deficits. Psychopharmacology (Berl.) 114, 392–401. Vorhees, C.V., Ahrens, K.G., Acuff-Smith, K.D., Schilling, M.A., Fisher, J.E., 1994b. Methamphetamine exposure during early postnatal development in rats: II. Hypoactivity and altered responses to pharmacological challenge. Psychopharmacology (Berl.) 114, 402–408. Vorhees, C.V., Skelton, M.R., Grace, C.E., Schaefer, T.L., Graham, D.L., Braun, A.A., Williams, M.T., 2009. Effects of (+)-methamphetamine on path integration and spatial learning, but not locomotor activity or acoustic startle, align with the stress hyporesponsive period in rats. Int. J. Dev. Neurosci. 27, 289–298. Whimbey, A.E., Denenberg, V.H., 1967. Two independent behavioral dimensions in openfield performance. J. Comp. Physiol. Psychol. 63, 500–504. Williams, M.T., Blankenmeyer, T.L., Schaefer, T.L., Brown, C.A., Gudelsky, G.A., Vorhees, C.V., 2003a. Long-term effects of neonatal methamphetamine exposure in rats on spatial learning in the Barnes maze and on cliff avoidance, corticosterone release, and neurotoxicity in adulthood. Brain Res. Dev. Brain Res. 147, 163–175. Williams, M.T., Moran, M.S., Vorhees, C.V., 2003b. Refining the critical period for methamphetamine-induced spatial deficits in the Morris water maze. Psychopharmacology (Berl.) 168, 329–338. Williams, M.T., Schaefer, T.L., Furay, A.R., Ehrman, L.A., Vorhees, C.V., 2006. Ontogeny of the adrenal response to (+)-methamphetamine in neonatal rats: the effect of prior drug exposure. Stress 9, 153–163.
transferal to pups via placental membrane and breast milk. Drug Alcohol Depend. 139, 138–144. Ramos, A., Mormede, P., 1998. Stress and emotionality: a multidimensional and genetic approach. Neurosci. Biobehav. Rev. 22, 33–57. Rice, D., Barone Jr., S., 2000. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ. Health Perspect. 108 (Suppl. 3), 511–533. Rodrigues, L.G., Melo, P., Silva, M.C., Tavares, M.A., 2006. Effects of postnatal exposure to methamphetamine on the development of the rat retina. Ann. N. Y. Acad. Sci. 1074, 604–619. Sapolsky, R.M., Meaney, M.J., 1986. Maturation of the adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Res. 396, 64–76. Sapolsky, R.M., 1996. Stress, glucocorticoids, and damage to the nervous system: the current state of confusion. Stress 1, 1–19. Schaefer, T.L., Ehrman, L.A., Gudelsky, G.A., Vorhees, C.V., Williams, M.T., 2006. Comparison of monoamine and corticosterone levels 24 h following (+) methamphetamine, (+/−)3,4-methylenedioxymethamphetamine, cocaine, (+) fenfluramine or (+/−)methylphenidate administration in the neonatal rat. J. Neurochem. 98, 1369–1378. Šlamberová, R., Szilagyi, B., Vathy, I., 2001. Repeated morphine administration during pregnancy attenuates maternal behavior. Psychoneuroendocrinology 26, 565–576. Šlamberová, R., Charousová, P., Pometlová, M., 2005a. Maternal behavior is impaired by methamphetamine administered during pre-mating, gestation and lactation. Reprod. Toxicol. 20, 103–110. Šlamberová, R., Charousová, P., Pometlová, M., 2005b. Methamphetamine administration during gestation impairs maternal behavior. Dev. Psychobiol. 46, 57–65. Šlamberová, R., Pometlová, M., Charousová, P., 2006. Postnatal development of rat pups is altered by prenatal methamphetamine exposure. Prog. Neuropsychopharmacol. Biol. Psychiatry 30, 82–88. Šlamberová, R., Pometlová, M., Rokyta, R., 2007. Effect of methamphetamine exposure during prenatal and preweaning periods lasts for generations in rats. Dev. Psychobiol. 49, 312–322. Smeriglio, V.L., Wilcox, H.C., 1999. Prenatal drug exposure and child outcome Past, present, future. Clin. Perinatol. 26, 1–16. Smith, A.M., Chen, W.J., 2010. Amphetamine treatment during early postnatal
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