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Effect of methamphetamine exposure and cross-fostering on cognitive function in adult male rats
Behavioural Brain Research 208 (2010) 63–71
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Effect of methamphetamine exposure and cross-fostering on cognitive function in adult male rats ∗ ˇ Lenka Hrubá, Barbora Schutová, Marie Pometlová, Richard Rokyta, Romana Slamberová Charles University in Prague, Third Faculty of Medicine, Department of Normal, Pathological and Clinical Physiology, Ke Karlovu 4, Prague, Czech Republic
a r t i c l e
i n f o
Article history: Received 19 June 2009 Received in revised form 15 October 2009 Accepted 2 November 2009 Available online 10 November 2009 Keywords: Methamphetamine Cross-fostering Cognitive functions
a b s t r a c t The aim of our study was to examine the effect of prenatal methamphetamine (MA) exposure and crossfostering on cognitive functions of adult male rats tested in Morris water maze (MWM). Rat mothers were exposed daily to injection of MA (5 mg/kg) or saline for 9 weeks: prior to impregnation, throughout gestation and lactation periods. Females without any injections were used as an absolute control. On postnatal day 1, pups were cross-fostered so that each mother raised 4 pups of her own and 8 pups from the mothers with the other two treatments. Four types of tests were used: (1) Place navigation test (Learning), (2) Probe test (Probe), (3) Retention memory test (Memory) and (4) Visible platform task. Our results demonstrate that the prenatal exposure to MA does not impact learning and memory, while postnatal exposure to MA shows impairments in cognition. In the test of learning, all animals fostered to MA-treated dams had longer latencies, bigger search error and used lower spatial strategies than the animals fostered to control or saline-treated mother, regardless of prenatal exposure. Regardless of postnatal exposure, the animals prenatally exposed to saline swam faster in all the tests than the animals prenatally exposed to MA and controls, respectively. This study indicates that postnatal but not prenatal exposure to MA affects learning in adult male rats. However, it is still not clear whether these impairments are due to a direct effect of MA on neuronal structure or due to an indirect effect of MA mediated by impaired maternal care. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The abuse of methamphetamine (MA) is a worldwide problem because of its ease of production, low cost, stimulatory effect, and addictive properties [26]. Approximately half of MA users are woman, mostly of reproductive age, and consequently some percentage of these become pregnant while using the drug [46]. MA crosses the placental barrier easily [8] and therefore it may affect the development of the fetus. There are studies suggesting that MA exposure during pregnancy can impair the development of the neonatal central nervous system [38,50]. Smith et al. [34] found increased creatine in the striatum in children exposed to MA and these findings suggest an abnormality in energy metabolism in the brains of children exposed to MA in a utero. Further, Struthers and Hansen [36] demonstrated deficiencies in visual recognition task after prenatal MA exposure in humans, which are thought to rely upon hippocampal function [7]. Both hippocampus and striatum are regions important in spatial learning and memory in humans and rodents [5,15].
∗ Corresponding author. Tel.: +420 224902713; fax: +420 224902750. ˇ E-mail address: [email protected] (R. Slamberová). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.11.001
In rats, Acuff-Smith et al. [1] investigated the effect of MA administered at different times during gestation on cognitive functions of the progeny. They found that high doses of MA (15 and 20 mg/kg) administered in early days of gestation impair spatial memory in the Morris water maze (MWM), while lower doses (5 and 10 mg/kg) did not have any effect on cognition in adult offspring. Postnatally, the lactating pups may receive this drug in mother’s breast milk since amphetamines are concentrated and secreted in milk [35]. Study of Bayer et al. [6] showed that the hippocampus in rats is still developing during the postnatal days (PD) 11–20 and that this development is analogous to human hippocampal development during the third trimester of pregnancy. In addition, study of Vorhees et al. [44] demonstrated that neonatal MA exposure affects cognitive functions in rats. Rat pups injected with MA on PD 11–20 exhibited spatial learning and memory impairments in the Morris water maze (MWM), whereas rats exposed to MA on PD 1–10 were unaffected on the cognitive task [44]. Besides that, MA administered to mothers during lactational periods impairs their maternal behavior toward pups [37]. Maternal licking/grooming is a major source of tactile stimulation which play an important role in somatic growth and neural development in pups [32]. Study of Francis et al. [10] showed that the maternal care during the first week of postnatal life influences behavioral responses to stress in offspring. Specifically, it has been shown that
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the offspring of mothers, who exhibit a higher frequency of licking and grooming and arched back nursing (LG–ABN) over the first week of postnatal life, are behaviorally less fearful and show more modest hypothalamic–pituitary–adrenal (HPA) responses to stress than do the offspring of low LG–ABN mothers [10]. Cross-fostering is a method whereby offspring are removed from their biological mothers at birth and raised by surrogate mothers (some of the pups from one mother are fostered to the other, and vice versa). It allows to distinguish the contribution of prenatal and postnatal drug exposure in offspring. Concerning postnatal exposure, however, it is not possible to differentiate between the direct effect of a given compound on pups via mother’s milk and indirect effect of impaired maternal behavior [14,37]. Our previous studies showed that prenatal and postnatal MA exposure has both short- and long-term effects on rat development and behavior. Specifically, MA exposure impairs postural reflexes and sensorimotor function during pre-weaning period [14] and the effect of prenatal MA exposure lasts until adulthood [39,40]. There are no studies investigating the prenatal and early postnatal (via breast milk) MA exposure on cognitive functions in adulthood. There are several studies, in which rat pups were exposed directly to injection of MA (2, 5, 10 mg/kg, s.c.) from PD 11 to 15, from PD 16 to 20 or from PD 21 to 35 [21,42,43,46,48,49]. To better simulate the situation in humans, we chose another design of MA administration. Because drug abusing human mothers do not inject drug to their children, but to themselves, and therefore their children are exposed to the effect of the drug during lactation indirectly via breast milk, we do the same in the rats. Furthermore, there are studies showing different responses of pups to adoption. Early adoption on PD 1 prevents the stressinduced secretion of corticosterone, reduces locomotor activity in a novel environment, and improves spatial recognitive function [4]. Later adoption (PD 5 and 12) prolongs stress-induced secretion of corticosterone, increases locomotor response to novelty, and disrupts spatial recognition [4]. Lu et al. [19] determined that the cross-fostered mice showed normal memory function in the Y-maze and novel object recognition test compared to biological groups. Study from Liu et al. [18] investigated the effect of cross-fostering on learning and memory caused by differences in maternal care. Spatial learning and memory of animals born to high LG–ABN dams but reared by low LG–ABN mothers was indistinguishable from those of high LG–ABN pups reared by high LG–ABN mothers. However, animals born to low LG–ABN dams and reared by low LG–ABN displayed the worst cognitive functions than animals from all the other groups (including animals born to low LG–ABN, but reared by high LG–ABN dams). Those results suggest that the cross-fostering from low to high LG–ABN dams improved the cognition in offsprings, while the cross-fostering did not affect the performance animals born to high LG–ABN regardless of the rearing mother (biological × adoptive) [18]. Therefore, the present study is the first to test the hypothesis that cross-fostering and/or postnatal MA exposure via maternal breast milk modifies the prenatal effect of MA on cognitive function of adult male rats. 2. Methods 2.1. Drugs Physiological saline (0.9% NaCl) was purchased from Sigma (Prague, Czech Republic), d-Methamphetamine HCl was provided from Faculty of Pharmacy of Charles University in Hradec Králové (the Czech Republic). 2.2. Mothers Adult female albino Wistar rats (250–300 g) were delivered by Anlab (Praque, the Czech Republic) from Charles River Laboratories International, Inc. Animals were housed in groups (4–5/cage) and left undisturbed for a week in a temperature-
controlled (22–24 ◦ C) colony room with free access to food and water on a 12 h (light):12 h (dark) cycle with lights on at 06:00 h. Females were randomly assigned to MA-treated (MA), saline-treated (S) and control (C) groups. To best to simulate the situation in humans MA was injected daily for approximately 9 weeks: about 3 weeks prior to impregnation, throughout the entire gestation period and 3 weeks in lactation period (until the weaning). The dose of injections was 5 mg/kg and was administered subcutaneously (s.c.). The dose was chosen based on the findings that MA in dose of 5 mg/kg administered to pregnant female rats induces changes that are comparable with those found in fetuses of drug-abusing women [1]. Saline was injected s.c. at the same time and volume as MA. Control females did not receive any injections. 2.3. Fertilization Approximately 3 weeks after the drug administration, females were smeared by vaginal lavage to determine the phase of the estrous cycle. At the onset of the estrous phase of the estrous cycle female rats were housed overnight with sexually mature males. There was always one female and one male in each cage. The next morning females were smeared again for the presence of sperm and returned to their previous home cages. The day after impregnation was counted as day 1 of gestation (see [37]). On day 21 of gestation, females were separated to maternity cages. The day of delivery was counted as PD 0. 2.4. Pups and cross-fostering On PD 1, litter sizes were adjusted to 12 pups. Pups were cross-fostered so that 4 pups (usually 2 males and 2 females) remained with their biological mother and the other (usually 4 males and 4 females) were assigned to the mothers with the other two treatments. As a result, one mother usually raised 4 control pups, 4 salineexposed pups and 4 MA-exposed pups. Whenever possible the same number of males and females was kept in each litter. We obtained nine experimental groups based on biological and fostering mother (see Table 1). The pups fostered by MAtreated mothers were exposed to the effect of MA also postnatally from mother’s breast milk [35]. Prenatally MA-exposed pups were injected intradermally with black India ink in left foot pad and prenatally saline-exposed pups in right foot pad for identification. Pups from control mothers were not tattooed. On PD 21, pups fostered to MA-exposed mothers were ear-punched in the left ear and pups fostered to saline-exposed mothers in the right ear. Pups fostered to control mothers were not ear-punched. Always one male of the same drug exposure was used from each litter to prevent litter effect. The rest of the animals were used in different experiments. A total of 24 litters were used in the experiment. There were no significant differences in the incidence of a successful and/or a healthy pregnancy. The number of alive pups and death pups in litters were not significantly altered by the prenatal drug exposure. 2.4.1. Morris water maze In total 72 male offspring (8 per each treatment group) were tested in adulthood (PD 85–90) for learning and memory in the MWM (blue circular tank, 2 m in diameter) filled with opaque water. On the rim of the pool, four starting positions were marked north (N), south (S), east (E), west (W), thus dividing the pool into four quadrants. A transparent circle platform (13 cm in diameter) 1 cm below the water surface was used for learning and memory tasks. The platform (placed in the N–E quadrant of the pool) was invisible for the swimming rats. Various pictures hanging on the walls were available to the rats as extra-maze cues. Rats’ performance was tracked automatically using a video tracking system EthoVision 3.1 (Noldus Information Technology, Netherlands). Rats were tested over a 15-day period. Four types of tests were used in the present study: Place navigation test (Learning), Probe test, Retention memory test (Memory) and Visible platform task. In the Place navigation test, animals were allowed to learn to find the hidden platform in the first 5 consecutive days. If the rats were unable to find the platform within the limit of 60 s, they were guided to the platform manually. Each rat was exposed to 8 trials daily starting from 4 different positions. The position of the platform was the same in all trials. The rat remained on the platform for 30 s prior to next trial to have a chance to orient itself in the room. After finishing all trials in the experimental day, animal was dried by towel, placed in a dry holding cage for approximately 2 min and finally returned to its home cage. In the Probe test, which was performed on the 6th day, the platform was removed and the animal was allowed to swim in the pool for 60 s. The start position was north (N) for the Probe test. The Retention memory test was performed on the 12th day. The rat was allowed to find the Hidden platform located in the same position as in the learning test within 60 s. Each rat was exposed to 8 trials starting from 4 different positions with intertrial interval of 30 s. On the 15th day, the rats performed a visible platform task to assess motor ability. The water level was lowered to 1–2 cm below to height of the platform and a black sock was placed on the platform to further distinguish the platform from the water. The platform was moved to a novel quadrant in the pool and 8 trials were conducted with the visible platform, following the same procedure as for learning and memory as describe above.
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Table 1 Experimental groups. Group
Biological mother
Foster mother
Prenatal and postnatal factors
MA/MA MA/S MA/C S/MA S/S S/C C/MA C/S C/C
Methamphetamine Methamphetamine Methamphetamine Saline Saline Saline Control Control Control
Methamphetamine Saline Control Methamphetamine Saline Control Methamphetamine Saline Control
Prenatal and postnatal drug Prenatal drug and postnatal care of the saline control mothers Prenatal drug and postnatal care of the control mothers Prenatal saline control mothers and postnatal drug Prenatal and postnatal saline control mothers Prenatal saline control mothers and postnatal care of the control mothers Postnatal drug Postnatal care of saline control mothers Absolute control
2.5. Data analyzed In the tests of learning and memory, (A) the latency to reach the hidden platform, (B) the length of the trajectory (the length of the swim path [cm]), (C) the search error (a measure of proximity to the escape platform) and (D) the speed of swimming were recorded. In addition to search error, we analyzed search strategies to further confirm the effect of prenatal and postnatal methamphetamine exposure. Swimming pathways (search strategies) for each rat were manually analyzed by two laboratory technicians, so that predominating strategy in each trail was always identified and frequency of the following search strategies per day was recorded: (1) thigmotaxis (wall-hugging)—a persistent swim along the wall of the pool that could include sporadic swims towards the centre of the pool, (2) random search—swimming over the entire area of the pool in straight swims or in wide circular swims, (3) scanning—swimming over the central area of the pool, (4) chaining—circular swimming at a fixed distance from the wall, in which the platform was located, (5) focal search in an incorrect quadrant—direct swim to an incorrect quadrant of the pool followed by loops and turns there, (6) focal search in the target quadrant—direct swim to the correct quadrant of the pool followed by loops and turns there, (7) spatial search—a direct swim path to the platform. This method was previously used for analyzing search strategies in mice [16]. In the Probe test the following measures were recorded: speed of swimming; frequency and duration of presence in the quadrant where the platform was located in the learning and memory tests. The measure of the search error in Probe test cannot be examine because the platform is not present [21,42,43,46,48,49]. In the visible platform task the following measures were recorded: latency to reach visible platform, speed of swimming and the length of the trajectory (the length of the swim path [cm]).
There was no main effect of postnatal exposure [F(2, 63) = 0.49; p = 0.61] or interaction between pre- and postnatal drug exposure [F(2, 63) = 0.49; p = 0.61] in the speed of swimming. The swimming speed increased with the days of learning [F(4, 252) = 7.32; p < 0.0001] in all animals regardless of pre- or postnatal drug exposure (Fig. 2C). In the search error (Fig. 3), a main effect of postnatal exposure was demonstrated [F(2, 63) = 3.41; p < 0.05], such that all animals fostered to MA-treated dams, regardless of prenatal exposure, had bigger search error than the animals fostered to control or saline-treated mothers. No main effect of prenatal exposure [F(2,
2.5.1. Statistical methods The following statistics were used in the present study: in the Place navigation test—two-way ANOVA (prenatal exposure × postnatal exposure) with multilevel repeated measure (days × trials/day); in the Probe test—two-way ANOVA (prenatal exposure × postnatal exposure); in the Retention memory test and in the visible platform task—two-way ANOVA (prenatal exposure × postnatal exposure) with repeated measure (trials). Bonferroni test was used for post hoc comparisons. In addition, Chi2 test was used to analyze the frequency of the search strategies. In all tests, the differences were considered significant if p < 0.05.
3. Results 3.1. Place navigation test In the latency to reach the hidden platform (Fig. 1), effect of postnatal exposure was found [F(2, 63) = 3.87; p < 0.05]; on average the animals fostered to MA-treated dams (prenatal control, saline or MA) had longer latencies than the animals fostered to control or saline-treated mothers, regardless of prenatal exposure (Fig. 1B). There was no main effect of prenatal exposure [F(2, 63) = 0.16; p = 0.85] and no interaction between pre- and postnatal exposure [F(4, 63) = 1.96; p = 0.11] in the latencies. All animals, regardless of the drug exposure, demonstrated the same learning ability over the 5-day test period as represented by a decrease in the latency to find the hidden platform [F(4, 252) = 253.01; p < 0.00001] (Fig. 1C). In the speed of swimming (Fig. 2), significant main effect of prenatal exposure was demonstrated [F(2, 63) = 5.46; p < 0.01], such that animals prenatally exposed to saline regardless of postnatal exposure swam faster than the animals prenatally exposed to MA (p < 0.01) or control animals (p < 0.05) (Fig. 2B). There were no differences in the speed of swimming between animals prenatally exposed to MA and prenatally control animals (p = 0.51).
Fig. 1. Latency to reach the hidden platform in Place navigation task. (A) Effect of prenatal and postnatal MA exposure. Results are presented as average of all trials in 5 days. (B) Effect of postnatal MA exposure. + p < 0.05 main effect of postnatal exposure; rats postnatally MA-exposed had longer latencies than postnatally control or saline-exposed rats. Results are presented as average of all trials in 5 days. (C) Effect of postnatal MA exposure. Results are presented as averages of 8 trials per day in training days 1–5. Values are means ± SEM (n = 8) (ANOVA; Bonferroni post hoc test).
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63) = 0.11; p = 0.89] or interaction between pre- and postnatal drug exposure [F(2, 63) = 2.13; p = 0.08] was found in the search error (Fig. 3B). All animals, regardless of the drug exposure, demonstrated the same learning ability over the 5-day test period as represented by a decrease in search error [F(4, 252) = 200.54; p < 0.00001] (Fig. 3C). In the length of the trajectory (Fig. 4), no effect of prenatal [F(2, 63) = 0.23; p = 0.80] or postnatal [F(2, 63) = 0.79; p = 0.46] exposure in adulthood was found. All animals, regardless of the drug exposure, demonstrated the same ability of learning over the 5-day test period as represented by a shortened trajectory [F(4, 252) = 223.46; p < 0.00001] (Fig. 4C). Table 2 displays an overview of incidence in the search strategies used by the rats in the 5 days in the Place navigation test. Analysis of the search strategies in the test of learning showed significant effects of postnatal exposure in days 1–5 for random searching (Fig. 5A), scanning pool (Fig. 5B), direct swim (Fig. 5C) and searching in correct quadrant (Fig. 5D). The prenatal drug exposure did not induce any significant changes in the search strategies. In the testing day 1, main effect of postnatal exposure in adulthood was demonstrated [2 = 72.00; p < 0.0001], such that all
Fig. 3. Search error in Place navigation task. (A) Effect of prenatal and postnatal MA exposure. Results are presented as average of all trials in 5 days. (B) Effect of postnatal MA exposure. + p < 0.05 main effect of postnatal exposure; rats postnatally MA-exposed had bigger search errors than postnatally control or saline-exposed rats. Results are presented as average of all trials in 5 days. (C) Effect of postnatal MA exposure. Results are presented as averages of 8 trials per day in training days 1–5. Values are means ± SEM (n = 8) (ANOVA; Bonferroni post hoc test).
animals fostered to MA-treated dams, regardless of prenatal exposure, displayed more random searching (Fig. 5A) and scanned more the pool (Fig. 5B) than rats fostered to control mothers. Further, rats fostered to MA-treated mothers displayed less direct swim (Fig. 5C) and swam less in the correct target quadrant (Fig. 5D) than rats fostered to control mothers. In the testing day 2, significant effect of postnatal exposure for the frequency of used search strategies was shown [2 = 140.04; p < 0.0001], such that all animals fostered to MA-treated dams, regardless of prenatal exposure, displayed more random searchTable 2 Percentage of search strategies used in groups in the test Place navigation task in days 1–5. Strategy
Fig. 2. The speed of swimming in Place navigation task. (A) Effect of prenatal and postnatal MA exposure. Results are presented as average of all trials in 5 days. (B) Effect of prenatal MA exposure. Main effect of prenatal exposure; rats prenatally control (* p < 0.05) or prenatally MA-exposed rats (# p < 0.01) swam slower than prenatally saline-exposed rats. Results are presented as average of all trials in 5 days. (C) Effect of prenatal MA exposure. Results are presented as averages of 8 trials per day in training days 1–5. Values are means ± SEM (n = 8) (ANOVA; Bonferroni post hoc test).
Thigmotaxis Random Scanning Chaining Incorrect Q Correct TQ Spatial search
Experimental groups C/C
C/MA
C/S
S/C
S/MA
S/S
MA/C
MA/MA
MA/S
2 6 1 1 33 15 42
2 14+ 7+ 2 32 9+ 34+
4 10 4 2 31 13 36
3 8 3 2 36 12 36
2 17+ 9+ 4 39 6+ 23+
3 6 4 3 36 8 40
4 8 2 1 28 15 42
4 18+ 4 2 30 10+ 32+
4 9 3 2 32 11 39
Statistics: Chi2 test. Values are percent, n = 8. + p < 0.0001 vs. C/C or S/C. Q = quadrant, TG = target quadrant.
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Table 3 Percentage of search strategies used in groups in the test Retention memory task. Strategy
Thigmotaxis Random Scanning Chaining Incorrect Q Correct TQ Spatial search
Experimental groups C/C
C/MA
C/S
S/C
S/MA
S/S
MA/C
MA/MA
MA/S
0 0 3 0 25 3 69
0 6 8 3 31 4 48+
0 0 0 1 36 8 55
0 0 0 1 33 0 66
0 0 1 3 53+ 0 43+
0 0 0 0 39 8 53
0 0 1 0 24 17 58
0 0 0 0 33 1 66
0 0 0 0 33 9 58
Statistics: Chi2 test. Values are percent, n = 8. + p < 0.001 vs. C/C or S/C. Q = quadrant, TG = target quadrant.
exposure [F(2, 63) = 0.00; p = 1]} or in the duration of presence in the quadrant where the hidden platform was placed during learning {prenatal exposure [F(2, 63) = 0.7; p = 0.5], postnatal exposure [F(2, 63) = 0.37; p = 0.69]}. For the speed of swimming, there was main effect of prenatal treatment [F(2, 63) = 8.19; p < 0.05]; animals prenatally exposed to MA swam slower than control (p < 0.05) or prenatally saline-exposed (p < 0.001) animals (Fig. 6A). There was no main effect of postnatal exposure [F(2, 63) = 1.36; p = 0.26] or interaction between pre- and postnatal drug exposure [F(4, 63) = 2.32; p = 0.07] in the speed of swimming in the Probe test. 3.3. Retention memory test
Fig. 4. The length of trajectory in Place navigation task. (A) Effect of prenatal and postnatal MA exposure. Results are presented as average of all trials in 5 days. (B) Effect of postnatal MA exposure. Results are presented as average of all trials in 5 days. (C) Effect of postnatal MA exposure. Results are presented as averages of 8 trials per day in training days 1–5. Values are means ± SEM (n = 8) (ANOVA; Bonferroni post hoc test).
ing (Fig. 5A) than rats fostered to control mothers. Further, rats fostered to MA-treated mothers used less direct swim (Fig. 5C) and swam less in the correct target quadrant (Fig. 5D) than rats fostered to control mothers. In the testing day 3, main effect of postnatal exposure was demonstrated [2 = 64.06; p < 0.0001], such that all animals fostered to MA-treated dams, regardless of prenatal exposure, scanned more the pool (Fig. 5B) and displayed less direct swim (Fig. 5C) than rats fostered to control mothers. In the testing day 4, main effect of postnatal exposure was shown [2 = 37.8; p < 0.0001], such that all animals fostered to MAtreated dams, regardless of prenatal exposure, scanned more the pool (Fig. 5B) than rats fostered to control mothers. In the testing day 5, no main effect of postnatal exposure was found [2 = 5.12; p = 0.53]. No main effect of prenatal exposure was found on day 1 [2 = 11.02; p = 0.08], day 2 [2 = 10.69; p = 0.1], day 3 [2 = 3.85; p = 0.7], day 4 [2 = 11.82; p = 0.06], or day 5 [2 = 3.15; p = 0.79] of learning.
Statistical analysis showed no significant main effects in the latencies to find the hidden platform {prenatal exposure [F(2, 63) = 1.70; p = 0.19], postnatal exposure [F(2, 63) = 0.90; p = 0.41]}, in the search errors {prenatal exposure [F(2, 63) = 2.52; p = 0.09], postnatal exposure [F(2, 63) = 1.22; p = 0.31]}, or in the lengths of the trajectories {prenatal exposure [F(2, 63) = 1.32; p = 0.27], postnatal exposure [F(2, 63) = 1.30; p = 0.28]}. In the speed of swimming (Fig. 6B), significant main effect of prenatal exposure was demonstrated [F(2, 63) = 4.00; p < 0.05], such that all animals prenatally exposed to MA, regardless of postnatal exposure, swam slower than the animals prenatally exposed to saline (p < 0.01). There were no differences in the speed of swimming in animals prenatally exposed to MA and prenatal controls (p = 0.25). There was no main effect of postnatal exposure [F(2, 63) = 0.51; p = 0.60] or interaction between pre- and postnatal drug exposure [F(4, 63) = 1.2; p = 0.32] in the speed of swimming. In the search strategies (Table 3), main effect of postnatal drug exposure [2 = 29.51; p < 0.0001] was found. Rats fostered to MAtreated mothers used less direct swim and swam less in the correct quadrant than rats fostered to control dams, regardless of prenatal exposure. Further, animals fostered to MA-treated mothers swam more in the incorrect quadrant than rats fostered to control mothers. No main effect of prenatal drug exposure was found [2 = 10.02; p = 0.06]. 3.4. Visible platform task Statistical analysis showed no significant main effects in the latencies to find the visible platform {prenatal exposure [F(2, 62) = 1.60; p = 0.21], postnatal exposure [F(2, 62) = 0.65; p = 0.52]}, in speed of swimming {prenatal exposure [F(2, 62) = 0.17; p = 0.84], postnatal exposure [F(2, 62) = 0.68; p = 0.51]}, or in the lengths of the trajectories {prenatal exposure [F(2, 62) = 2.25; p = 0.11], postnatal exposure [F(2, 62) = 0.69; p = 0.50]}.
3.2. Probe test
4. Discussion
Statistical analysis showed no significant main effects in the frequency {prenatal exposure [F(2, 63) = 0.75; p = 0.47], postnatal
The aim of the present study was to determine the effect of cross-fostering and/or postnatal MA exposure via maternal breast
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Fig. 5. Effect of postnatal MA exposure on the frequency of strategies used in Place navigation task in training days 1–5. (A) Random search strategy—main effect of postnatal exposure in days 1 and 2; rats postnatally MA-exposed > postnatally control animals, (B) scanning—main effect of postnatal exposure in days 1, 3 and 4; rats postnatally MA-exposed > postnatally control animals, (C) spatial search strategy—main effect of postnatal exposure in days 1, 2 and 3; rats postnatally MA-exposed < postnatally control animals, (D) search in correct quadrant; main effect of postnatal exposure in days 1 and 2; rats postnatally MA-exposed < postnatally control animals. Statistics: Chi2 test; values are sums of frequencies in days 1–5, n = 8. + p < 0.0001 rats postnatally MA-exposed vs. postnatally control animals.
milk on cognitive functions in adult male rats prenatally exposed to MA. Our results demonstrate that prenatal exposure to MA does not impact learning and memory, while postnatal exposure to MA show impairments in cognitive functions, mainly in learning. One limited factor should be noted at the beginning of the discussion. In theory, it would be possible that early postnatal and/or preweaning stress caused by tattooing and ear-punching, respectively, might influence the performance in the MWM of adult rats, because only prenatally saline- and MA-exposed animals not controls were exposed to this stressor. However, no differences were found in cognitive functions between prenatally saline-exposed animals and prenatal controls, which make this theoretical possibility improbable. The effects of prenatal and postnatal MA exposure are discussed in details. First, our results show that prenatal MA exposure at a dose of 5 mg/kg did not affect the latency, search error, length of the trajectory and search strategies used in Place navigation task. This finding is in agreement with the work of Schutová et al. [33], who showed that the dose of MA (5 mg/kg) administered prenatally did not impair learning in the MWM. There is another study showing that prenatal exposure to low doses of MA (5 or 10 mg/kg) does not affect spatial memory in the MWM, while higher doses of MA (15, 20 mg/kg) did induce impairments of spatial memory in the MWM tested in adulthood [1]. Thus, it seems
that the effect of prenatal MA exposure on cognition depends on the dose used and that higher doses would be required to induce changes in cognition that would last until adulthood. However note, that the dose was chosen based of the finding that such a dose induces similar behavioral changes as in drug-abusing humans [45]. Second, the present data demonstrate that the postnatal MA exposure from mothers’ breast milk in preweaning period (PD 1–21) increases the deficit in spatial learning when assessed in adulthood by using the MWM. While MA/MA, S/MA and C/MA animals received MA postnatally, the MA/C and MA/S animals got only prenatal drug exposure. One possible explanation for this is the direct effect of MA administration via mother’s breast milk. There is a study showing that neonatal administration of MA at doses of 5, 10 or 15 mg/kg administered four times daily on PD 11–20 produce lasting spatial learning and memory deficits [50]. As the hippocampus in rats is still developing during the PD 11–20 and this development is analogous to human hippocampal development during the third trimester of pregnancy [6], the neonatal period may be more critical for the effects of MA on cognitive functions in rats than the prenatal period. The hippocampus is important for several types of learning (i.e. spatial learning) and undergoes neurogenesis and neural pruning during the first few weeks of life in rats [13].
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Fig. 6. Effect of prenatal and postnatal MA exposure on speed of swimming in the Probe test (A) and Retention memory test (B). Results are presented as averages of 8 trials per day. Values are means ± SEM (n = 8). (A) In the Probe test: main effect of prenatal exposure; prenatally MA-exposed animals swam slower than prenatal controls (* p < 0.05) or saline-exposed rats (# p < 0.001), regardless of postnatal exposure. (B) In the Retention memory test: main effect of prenatal exposure; rats prenatally MA-exposed swam slower than animals prenatally exposed to saline (# p < 0.05) (ANOVA; Bonferroni post hoc test).
In our study, only cumulative distance (search error) from the platform and the latencies in reaching the platform are affected by postnatal exposure to MA. Animals fostered to MA-treated dams (prenatal control, saline or MA) have longer latencies and have bigger search error than the animals fostered to control or salinetreated mothers. Based of the works of Gallagher et al. [12] and Lindner [17], cumulative distance from the platform and the length of trajectory are better indicators of spatial learning ability than the latency. However, the length of trajectory is not affected by postnatal exposure to MA in the present work. Thus, we used analysis of search strategies to explain in detail the behavioral nature of cognitive impairments of male rats. Although for two individual rats the length trajectories can be almost identical, their performance (search strategies) can differ markedly. While one of the animals searches for the platform relatively close to it, the other searches in quadrants distant to the platform randomly and finds the platform accidentally. The results of the search strategy analysis in the Place navigation test demonstrates that the incidence of random search and scaning is significantly greater and the incidence of direct swim and searching in correct quadrant is significantly lower in all animals fostered to MA-treated dams relative to rats fostered to control mothers. Random search and scanning are non-spatial strategies and are used mainly in the beginning of the learning period [16]. Rats fostered to MA-treated dams began to search the whole surface area of the pool randomly in the testing days 1 and 2 and later on they scanned the inner area of the pool more selectively (days 1, 3 and 4). Moreover, rats fostered to MA-treated mothers used lower spatial search and searching in correct quadrant in testing days 1–3. The develop-
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ment of spatial learning for the platform location is reflected by a focal search of a target quadrant or by a direct swim to the platform and are used if an animal remembers the exact location of the platform [16]. The aforementioned findings suggest that rats fostered to MA-treated mothers have weaker spatial learning at least in the testing days 1–3 than rats fostered to control mothers. However, by the end of learning, animals fostered to MA-treasted mothers are able to find the location of the hidden platform similarly to control animals. Further, we found no differences between experimental groups in the Probe test. The measures commonly used to assess performance in the Probe test are designed to reflect the spatial bias of an animal’s search pattern. These data suggest that all experimental groups had comparable navigational abilities. The second possibility of how to explain the effect of postnatal MA exposure is that the MA/C and MA/S animals are fostered to control or saline-treated control mothers and as such they were not exposed to the impaired maternal behavior of MA-treated dams. Changes in maternal behavior (impaired maternal care, such as active nursing, mother being in the nest or in contact with pups, carrying and grooming pups and a nest building, mother-pup intercontact interval, retrieval latency, inter-retrieval interval, number of pup retrieved, grooming pups and nest building) have been described by many studies after application of psychostimulants [11,25,37]. Since it is generally accepted that maternal care is essential for the somatic growth and neural development of the pups [32], lack of the care acts as a stressful stimulus for the pups. There is a study showing that differences in maternal care during the first week of postnatal life influences hippocampal development and function [18]. It has been investigated that the offspring of mothers, who exhibit a higher frequency of LG–ABN over the first week of postnatal life, show increased hippocampal synaptic density and enhanced spatial learning and memory [18]. The mechanism by which MA or differences in maternal care may prevent proper spatial learning ability is, however, unknown. It is possible that changes in stress reactivity may play a role in these effects, because worse maternal care can cause stress for pups in preweaning period that may persist until adulthood. Extended periods of stress and elevated levels of glucocorticoids can alter brain function and increase vulnerability to neurological impacts [28,29]. There are studies showing that neonatal MA produces increase in corticosterone that impairs excitability of nerve cells in the hippocampus [9] and induces neuronal atrophy of the hippocampus [22] that further result in spatial learning deficits in the MWM [30,31]. MA was also shown to protract activation of the HPA system during the neonatal period, which may induce long-term changes in the stress response when the animals are adult [47]. Others have demonstrated long-term learning and memory deficits in novel object recognition and spatial memory following neonatal maternal separation, a stressor that also causes corticosterone release [2]. Moreover, already Morris [23] noted that swimming in the MWM may cause some stress for the tested animals, which was further supported by the finding of Akirav et al. [3] and Roozendaal et al. [27] showing adrenal activation and release of corticosterone following training in the MWM. However, future studies examining the effect of MA on cognitive functions in respect of stress would be necessary to examine this hypothesis. Third, we found that all animals prenatally exposed to MA, regardless of postnatal exposure, swam slower than the animals prenatally exposed to saline in Place navigation task and Memory retention task. The swimming velocity can be used to measure motivation to find the hidden platform [20]. Therefore, the decreased swimming velocity may suggest decreased motivation of prenatally MA-exposed animals. However, because the present study does not show any differences between prenatally MA-exposed rats and prenatally control rats in the speed of swimming, this hypothesis does not seem to be relevant. Similarly, these
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data cannot be attributed to impaired motor function. The visible platform task assessed the ability of rat to swim onto a visible platform, without relying on spatial memory. There were no differences between rats exposed (prenatally or postnatally) to MA and salineexposed or control rats on swim speeds of the visible platform task. These findings are in agreement with previous studies that found no main effect between rats exposed to MA and control rats on swimming ability when measured in the straight channel task [48] or to reach a visible platform in MWM [21]. On the other hand, because the present study show increase in the speed of swimming in prenatally saline-exposed rats when compared to controls, the effect of prenatal stress seems to play more important role in this measure. It was shown [24,41] that placebo injection of saline administered to pregnant mothers may induce mild stress for the rat and that way indirectly affect the development of her pups. Fourth, neither prenatal nor postnatal MA exposure affected latencies, search error and the length of trajectory in the Retention memory test. This is in agreement with study of Acuff-Smith et al. [1] showing that MA 5 mg/kg does not influence memory in the MWM. The results of search strategy analysis demonstrates that rats postnatally exposed to MA search the pool by using spatial strategies (focal search in correct target quadrant, direct swim) less, than prenatally control animals. Further, animals fostered to MA-treated mothers swam more in the incorrect quadrant than rats fostered to control mothers. These results suggest that prenatally and postnatally MA-exposed animals memorized the location of the platform similarly to control animals, however, they used different strategies. In conclusion, the present study investigated the effect of MA exposure and cross-fostering on learning and memory of adult male rats tested in the MWM. Taken together, our results show that prenatal exposure to MA at dose as low as 5 mg/kg does not impair learning in the MWM, while postnatal exposure to MA from mother’s breast milk and/or their worse maternal care impair learning of adult male rats. On the other hand, “better” maternal care of control mothers does not affect learning and memory of rat pups prenatally exposed to MA. In contrast to our previous study [14] the role of maternal care on learning and memory abilities in adulthood remains unclear. More studies would be necessary to examine the long-term effects of cross-fostering on offspring of drug abusing mothers. Acknowledgements This study was supported by project CN LC554, grant IGA 1A8610-5/2005, and Research Goal #MSM 0021620816 from Ministry of Education, Youth and Sports of the Czech Republic. The procedures for animal experimentation utilized in this report was reviewed and approved by the Institutional Animal Care and Use Committee and is in agreement with the Czech Government Requirements under the Policy of Humans Care of Laboratory Animals (No. 246/1992) and with the regulations of the Ministry of Agriculture of the Czech Republic (No. 311/1997). References [1] Acuff-Smith KD, Schilling MA, Fisher JE, Vorhees CV. Stage-specific effects of prenatal d-methamphetamine exposure on behavioral and eye development in rats. Neurotoxicol Teratol 1996;18(2):199–215. [2] Aisa B, Tordera R, Lasheras B, Del Rio J, Ramirez MJ. Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats. Psychoneuroendocrinology 2007;32(3):256–66. [3] Akirav I, Sandi C, Richter-Levin G. Differential activation of hippocampus and amygdala following spatial learning under stress. Eur J Neurosci 2001;14(4):719–25. [4] Barbazanges A, Vallee M, Mayo W, Day J, Simon H, Le Moal M, et al. Early and later adoptions have different long-term effects on male rat offspring. J Neurosci 1996;16(23):7783–90.
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Effect of methamphetamine exposure and cross-fostering on cognitive function in adult male rats ∗ ˇ Lenka Hrubá, Barbora Schutová, Marie Pometlová, Richard Rokyta, Romana Slamberová Charles University in Prague, Third Faculty of Medicine, Department of Normal, Pathological and Clinical Physiology, Ke Karlovu 4, Prague, Czech Republic
a r t i c l e
i n f o
Article history: Received 19 June 2009 Received in revised form 15 October 2009 Accepted 2 November 2009 Available online 10 November 2009 Keywords: Methamphetamine Cross-fostering Cognitive functions
a b s t r a c t The aim of our study was to examine the effect of prenatal methamphetamine (MA) exposure and crossfostering on cognitive functions of adult male rats tested in Morris water maze (MWM). Rat mothers were exposed daily to injection of MA (5 mg/kg) or saline for 9 weeks: prior to impregnation, throughout gestation and lactation periods. Females without any injections were used as an absolute control. On postnatal day 1, pups were cross-fostered so that each mother raised 4 pups of her own and 8 pups from the mothers with the other two treatments. Four types of tests were used: (1) Place navigation test (Learning), (2) Probe test (Probe), (3) Retention memory test (Memory) and (4) Visible platform task. Our results demonstrate that the prenatal exposure to MA does not impact learning and memory, while postnatal exposure to MA shows impairments in cognition. In the test of learning, all animals fostered to MA-treated dams had longer latencies, bigger search error and used lower spatial strategies than the animals fostered to control or saline-treated mother, regardless of prenatal exposure. Regardless of postnatal exposure, the animals prenatally exposed to saline swam faster in all the tests than the animals prenatally exposed to MA and controls, respectively. This study indicates that postnatal but not prenatal exposure to MA affects learning in adult male rats. However, it is still not clear whether these impairments are due to a direct effect of MA on neuronal structure or due to an indirect effect of MA mediated by impaired maternal care. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The abuse of methamphetamine (MA) is a worldwide problem because of its ease of production, low cost, stimulatory effect, and addictive properties [26]. Approximately half of MA users are woman, mostly of reproductive age, and consequently some percentage of these become pregnant while using the drug [46]. MA crosses the placental barrier easily [8] and therefore it may affect the development of the fetus. There are studies suggesting that MA exposure during pregnancy can impair the development of the neonatal central nervous system [38,50]. Smith et al. [34] found increased creatine in the striatum in children exposed to MA and these findings suggest an abnormality in energy metabolism in the brains of children exposed to MA in a utero. Further, Struthers and Hansen [36] demonstrated deficiencies in visual recognition task after prenatal MA exposure in humans, which are thought to rely upon hippocampal function [7]. Both hippocampus and striatum are regions important in spatial learning and memory in humans and rodents [5,15].
∗ Corresponding author. Tel.: +420 224902713; fax: +420 224902750. ˇ E-mail address: [email protected] (R. Slamberová). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.11.001
In rats, Acuff-Smith et al. [1] investigated the effect of MA administered at different times during gestation on cognitive functions of the progeny. They found that high doses of MA (15 and 20 mg/kg) administered in early days of gestation impair spatial memory in the Morris water maze (MWM), while lower doses (5 and 10 mg/kg) did not have any effect on cognition in adult offspring. Postnatally, the lactating pups may receive this drug in mother’s breast milk since amphetamines are concentrated and secreted in milk [35]. Study of Bayer et al. [6] showed that the hippocampus in rats is still developing during the postnatal days (PD) 11–20 and that this development is analogous to human hippocampal development during the third trimester of pregnancy. In addition, study of Vorhees et al. [44] demonstrated that neonatal MA exposure affects cognitive functions in rats. Rat pups injected with MA on PD 11–20 exhibited spatial learning and memory impairments in the Morris water maze (MWM), whereas rats exposed to MA on PD 1–10 were unaffected on the cognitive task [44]. Besides that, MA administered to mothers during lactational periods impairs their maternal behavior toward pups [37]. Maternal licking/grooming is a major source of tactile stimulation which play an important role in somatic growth and neural development in pups [32]. Study of Francis et al. [10] showed that the maternal care during the first week of postnatal life influences behavioral responses to stress in offspring. Specifically, it has been shown that
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the offspring of mothers, who exhibit a higher frequency of licking and grooming and arched back nursing (LG–ABN) over the first week of postnatal life, are behaviorally less fearful and show more modest hypothalamic–pituitary–adrenal (HPA) responses to stress than do the offspring of low LG–ABN mothers [10]. Cross-fostering is a method whereby offspring are removed from their biological mothers at birth and raised by surrogate mothers (some of the pups from one mother are fostered to the other, and vice versa). It allows to distinguish the contribution of prenatal and postnatal drug exposure in offspring. Concerning postnatal exposure, however, it is not possible to differentiate between the direct effect of a given compound on pups via mother’s milk and indirect effect of impaired maternal behavior [14,37]. Our previous studies showed that prenatal and postnatal MA exposure has both short- and long-term effects on rat development and behavior. Specifically, MA exposure impairs postural reflexes and sensorimotor function during pre-weaning period [14] and the effect of prenatal MA exposure lasts until adulthood [39,40]. There are no studies investigating the prenatal and early postnatal (via breast milk) MA exposure on cognitive functions in adulthood. There are several studies, in which rat pups were exposed directly to injection of MA (2, 5, 10 mg/kg, s.c.) from PD 11 to 15, from PD 16 to 20 or from PD 21 to 35 [21,42,43,46,48,49]. To better simulate the situation in humans, we chose another design of MA administration. Because drug abusing human mothers do not inject drug to their children, but to themselves, and therefore their children are exposed to the effect of the drug during lactation indirectly via breast milk, we do the same in the rats. Furthermore, there are studies showing different responses of pups to adoption. Early adoption on PD 1 prevents the stressinduced secretion of corticosterone, reduces locomotor activity in a novel environment, and improves spatial recognitive function [4]. Later adoption (PD 5 and 12) prolongs stress-induced secretion of corticosterone, increases locomotor response to novelty, and disrupts spatial recognition [4]. Lu et al. [19] determined that the cross-fostered mice showed normal memory function in the Y-maze and novel object recognition test compared to biological groups. Study from Liu et al. [18] investigated the effect of cross-fostering on learning and memory caused by differences in maternal care. Spatial learning and memory of animals born to high LG–ABN dams but reared by low LG–ABN mothers was indistinguishable from those of high LG–ABN pups reared by high LG–ABN mothers. However, animals born to low LG–ABN dams and reared by low LG–ABN displayed the worst cognitive functions than animals from all the other groups (including animals born to low LG–ABN, but reared by high LG–ABN dams). Those results suggest that the cross-fostering from low to high LG–ABN dams improved the cognition in offsprings, while the cross-fostering did not affect the performance animals born to high LG–ABN regardless of the rearing mother (biological × adoptive) [18]. Therefore, the present study is the first to test the hypothesis that cross-fostering and/or postnatal MA exposure via maternal breast milk modifies the prenatal effect of MA on cognitive function of adult male rats. 2. Methods 2.1. Drugs Physiological saline (0.9% NaCl) was purchased from Sigma (Prague, Czech Republic), d-Methamphetamine HCl was provided from Faculty of Pharmacy of Charles University in Hradec Králové (the Czech Republic). 2.2. Mothers Adult female albino Wistar rats (250–300 g) were delivered by Anlab (Praque, the Czech Republic) from Charles River Laboratories International, Inc. Animals were housed in groups (4–5/cage) and left undisturbed for a week in a temperature-
controlled (22–24 ◦ C) colony room with free access to food and water on a 12 h (light):12 h (dark) cycle with lights on at 06:00 h. Females were randomly assigned to MA-treated (MA), saline-treated (S) and control (C) groups. To best to simulate the situation in humans MA was injected daily for approximately 9 weeks: about 3 weeks prior to impregnation, throughout the entire gestation period and 3 weeks in lactation period (until the weaning). The dose of injections was 5 mg/kg and was administered subcutaneously (s.c.). The dose was chosen based on the findings that MA in dose of 5 mg/kg administered to pregnant female rats induces changes that are comparable with those found in fetuses of drug-abusing women [1]. Saline was injected s.c. at the same time and volume as MA. Control females did not receive any injections. 2.3. Fertilization Approximately 3 weeks after the drug administration, females were smeared by vaginal lavage to determine the phase of the estrous cycle. At the onset of the estrous phase of the estrous cycle female rats were housed overnight with sexually mature males. There was always one female and one male in each cage. The next morning females were smeared again for the presence of sperm and returned to their previous home cages. The day after impregnation was counted as day 1 of gestation (see [37]). On day 21 of gestation, females were separated to maternity cages. The day of delivery was counted as PD 0. 2.4. Pups and cross-fostering On PD 1, litter sizes were adjusted to 12 pups. Pups were cross-fostered so that 4 pups (usually 2 males and 2 females) remained with their biological mother and the other (usually 4 males and 4 females) were assigned to the mothers with the other two treatments. As a result, one mother usually raised 4 control pups, 4 salineexposed pups and 4 MA-exposed pups. Whenever possible the same number of males and females was kept in each litter. We obtained nine experimental groups based on biological and fostering mother (see Table 1). The pups fostered by MAtreated mothers were exposed to the effect of MA also postnatally from mother’s breast milk [35]. Prenatally MA-exposed pups were injected intradermally with black India ink in left foot pad and prenatally saline-exposed pups in right foot pad for identification. Pups from control mothers were not tattooed. On PD 21, pups fostered to MA-exposed mothers were ear-punched in the left ear and pups fostered to saline-exposed mothers in the right ear. Pups fostered to control mothers were not ear-punched. Always one male of the same drug exposure was used from each litter to prevent litter effect. The rest of the animals were used in different experiments. A total of 24 litters were used in the experiment. There were no significant differences in the incidence of a successful and/or a healthy pregnancy. The number of alive pups and death pups in litters were not significantly altered by the prenatal drug exposure. 2.4.1. Morris water maze In total 72 male offspring (8 per each treatment group) were tested in adulthood (PD 85–90) for learning and memory in the MWM (blue circular tank, 2 m in diameter) filled with opaque water. On the rim of the pool, four starting positions were marked north (N), south (S), east (E), west (W), thus dividing the pool into four quadrants. A transparent circle platform (13 cm in diameter) 1 cm below the water surface was used for learning and memory tasks. The platform (placed in the N–E quadrant of the pool) was invisible for the swimming rats. Various pictures hanging on the walls were available to the rats as extra-maze cues. Rats’ performance was tracked automatically using a video tracking system EthoVision 3.1 (Noldus Information Technology, Netherlands). Rats were tested over a 15-day period. Four types of tests were used in the present study: Place navigation test (Learning), Probe test, Retention memory test (Memory) and Visible platform task. In the Place navigation test, animals were allowed to learn to find the hidden platform in the first 5 consecutive days. If the rats were unable to find the platform within the limit of 60 s, they were guided to the platform manually. Each rat was exposed to 8 trials daily starting from 4 different positions. The position of the platform was the same in all trials. The rat remained on the platform for 30 s prior to next trial to have a chance to orient itself in the room. After finishing all trials in the experimental day, animal was dried by towel, placed in a dry holding cage for approximately 2 min and finally returned to its home cage. In the Probe test, which was performed on the 6th day, the platform was removed and the animal was allowed to swim in the pool for 60 s. The start position was north (N) for the Probe test. The Retention memory test was performed on the 12th day. The rat was allowed to find the Hidden platform located in the same position as in the learning test within 60 s. Each rat was exposed to 8 trials starting from 4 different positions with intertrial interval of 30 s. On the 15th day, the rats performed a visible platform task to assess motor ability. The water level was lowered to 1–2 cm below to height of the platform and a black sock was placed on the platform to further distinguish the platform from the water. The platform was moved to a novel quadrant in the pool and 8 trials were conducted with the visible platform, following the same procedure as for learning and memory as describe above.
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Table 1 Experimental groups. Group
Biological mother
Foster mother
Prenatal and postnatal factors
MA/MA MA/S MA/C S/MA S/S S/C C/MA C/S C/C
Methamphetamine Methamphetamine Methamphetamine Saline Saline Saline Control Control Control
Methamphetamine Saline Control Methamphetamine Saline Control Methamphetamine Saline Control
Prenatal and postnatal drug Prenatal drug and postnatal care of the saline control mothers Prenatal drug and postnatal care of the control mothers Prenatal saline control mothers and postnatal drug Prenatal and postnatal saline control mothers Prenatal saline control mothers and postnatal care of the control mothers Postnatal drug Postnatal care of saline control mothers Absolute control
2.5. Data analyzed In the tests of learning and memory, (A) the latency to reach the hidden platform, (B) the length of the trajectory (the length of the swim path [cm]), (C) the search error (a measure of proximity to the escape platform) and (D) the speed of swimming were recorded. In addition to search error, we analyzed search strategies to further confirm the effect of prenatal and postnatal methamphetamine exposure. Swimming pathways (search strategies) for each rat were manually analyzed by two laboratory technicians, so that predominating strategy in each trail was always identified and frequency of the following search strategies per day was recorded: (1) thigmotaxis (wall-hugging)—a persistent swim along the wall of the pool that could include sporadic swims towards the centre of the pool, (2) random search—swimming over the entire area of the pool in straight swims or in wide circular swims, (3) scanning—swimming over the central area of the pool, (4) chaining—circular swimming at a fixed distance from the wall, in which the platform was located, (5) focal search in an incorrect quadrant—direct swim to an incorrect quadrant of the pool followed by loops and turns there, (6) focal search in the target quadrant—direct swim to the correct quadrant of the pool followed by loops and turns there, (7) spatial search—a direct swim path to the platform. This method was previously used for analyzing search strategies in mice [16]. In the Probe test the following measures were recorded: speed of swimming; frequency and duration of presence in the quadrant where the platform was located in the learning and memory tests. The measure of the search error in Probe test cannot be examine because the platform is not present [21,42,43,46,48,49]. In the visible platform task the following measures were recorded: latency to reach visible platform, speed of swimming and the length of the trajectory (the length of the swim path [cm]).
There was no main effect of postnatal exposure [F(2, 63) = 0.49; p = 0.61] or interaction between pre- and postnatal drug exposure [F(2, 63) = 0.49; p = 0.61] in the speed of swimming. The swimming speed increased with the days of learning [F(4, 252) = 7.32; p < 0.0001] in all animals regardless of pre- or postnatal drug exposure (Fig. 2C). In the search error (Fig. 3), a main effect of postnatal exposure was demonstrated [F(2, 63) = 3.41; p < 0.05], such that all animals fostered to MA-treated dams, regardless of prenatal exposure, had bigger search error than the animals fostered to control or saline-treated mothers. No main effect of prenatal exposure [F(2,
2.5.1. Statistical methods The following statistics were used in the present study: in the Place navigation test—two-way ANOVA (prenatal exposure × postnatal exposure) with multilevel repeated measure (days × trials/day); in the Probe test—two-way ANOVA (prenatal exposure × postnatal exposure); in the Retention memory test and in the visible platform task—two-way ANOVA (prenatal exposure × postnatal exposure) with repeated measure (trials). Bonferroni test was used for post hoc comparisons. In addition, Chi2 test was used to analyze the frequency of the search strategies. In all tests, the differences were considered significant if p < 0.05.
3. Results 3.1. Place navigation test In the latency to reach the hidden platform (Fig. 1), effect of postnatal exposure was found [F(2, 63) = 3.87; p < 0.05]; on average the animals fostered to MA-treated dams (prenatal control, saline or MA) had longer latencies than the animals fostered to control or saline-treated mothers, regardless of prenatal exposure (Fig. 1B). There was no main effect of prenatal exposure [F(2, 63) = 0.16; p = 0.85] and no interaction between pre- and postnatal exposure [F(4, 63) = 1.96; p = 0.11] in the latencies. All animals, regardless of the drug exposure, demonstrated the same learning ability over the 5-day test period as represented by a decrease in the latency to find the hidden platform [F(4, 252) = 253.01; p < 0.00001] (Fig. 1C). In the speed of swimming (Fig. 2), significant main effect of prenatal exposure was demonstrated [F(2, 63) = 5.46; p < 0.01], such that animals prenatally exposed to saline regardless of postnatal exposure swam faster than the animals prenatally exposed to MA (p < 0.01) or control animals (p < 0.05) (Fig. 2B). There were no differences in the speed of swimming between animals prenatally exposed to MA and prenatally control animals (p = 0.51).
Fig. 1. Latency to reach the hidden platform in Place navigation task. (A) Effect of prenatal and postnatal MA exposure. Results are presented as average of all trials in 5 days. (B) Effect of postnatal MA exposure. + p < 0.05 main effect of postnatal exposure; rats postnatally MA-exposed had longer latencies than postnatally control or saline-exposed rats. Results are presented as average of all trials in 5 days. (C) Effect of postnatal MA exposure. Results are presented as averages of 8 trials per day in training days 1–5. Values are means ± SEM (n = 8) (ANOVA; Bonferroni post hoc test).
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63) = 0.11; p = 0.89] or interaction between pre- and postnatal drug exposure [F(2, 63) = 2.13; p = 0.08] was found in the search error (Fig. 3B). All animals, regardless of the drug exposure, demonstrated the same learning ability over the 5-day test period as represented by a decrease in search error [F(4, 252) = 200.54; p < 0.00001] (Fig. 3C). In the length of the trajectory (Fig. 4), no effect of prenatal [F(2, 63) = 0.23; p = 0.80] or postnatal [F(2, 63) = 0.79; p = 0.46] exposure in adulthood was found. All animals, regardless of the drug exposure, demonstrated the same ability of learning over the 5-day test period as represented by a shortened trajectory [F(4, 252) = 223.46; p < 0.00001] (Fig. 4C). Table 2 displays an overview of incidence in the search strategies used by the rats in the 5 days in the Place navigation test. Analysis of the search strategies in the test of learning showed significant effects of postnatal exposure in days 1–5 for random searching (Fig. 5A), scanning pool (Fig. 5B), direct swim (Fig. 5C) and searching in correct quadrant (Fig. 5D). The prenatal drug exposure did not induce any significant changes in the search strategies. In the testing day 1, main effect of postnatal exposure in adulthood was demonstrated [2 = 72.00; p < 0.0001], such that all
Fig. 3. Search error in Place navigation task. (A) Effect of prenatal and postnatal MA exposure. Results are presented as average of all trials in 5 days. (B) Effect of postnatal MA exposure. + p < 0.05 main effect of postnatal exposure; rats postnatally MA-exposed had bigger search errors than postnatally control or saline-exposed rats. Results are presented as average of all trials in 5 days. (C) Effect of postnatal MA exposure. Results are presented as averages of 8 trials per day in training days 1–5. Values are means ± SEM (n = 8) (ANOVA; Bonferroni post hoc test).
animals fostered to MA-treated dams, regardless of prenatal exposure, displayed more random searching (Fig. 5A) and scanned more the pool (Fig. 5B) than rats fostered to control mothers. Further, rats fostered to MA-treated mothers displayed less direct swim (Fig. 5C) and swam less in the correct target quadrant (Fig. 5D) than rats fostered to control mothers. In the testing day 2, significant effect of postnatal exposure for the frequency of used search strategies was shown [2 = 140.04; p < 0.0001], such that all animals fostered to MA-treated dams, regardless of prenatal exposure, displayed more random searchTable 2 Percentage of search strategies used in groups in the test Place navigation task in days 1–5. Strategy
Fig. 2. The speed of swimming in Place navigation task. (A) Effect of prenatal and postnatal MA exposure. Results are presented as average of all trials in 5 days. (B) Effect of prenatal MA exposure. Main effect of prenatal exposure; rats prenatally control (* p < 0.05) or prenatally MA-exposed rats (# p < 0.01) swam slower than prenatally saline-exposed rats. Results are presented as average of all trials in 5 days. (C) Effect of prenatal MA exposure. Results are presented as averages of 8 trials per day in training days 1–5. Values are means ± SEM (n = 8) (ANOVA; Bonferroni post hoc test).
Thigmotaxis Random Scanning Chaining Incorrect Q Correct TQ Spatial search
Experimental groups C/C
C/MA
C/S
S/C
S/MA
S/S
MA/C
MA/MA
MA/S
2 6 1 1 33 15 42
2 14+ 7+ 2 32 9+ 34+
4 10 4 2 31 13 36
3 8 3 2 36 12 36
2 17+ 9+ 4 39 6+ 23+
3 6 4 3 36 8 40
4 8 2 1 28 15 42
4 18+ 4 2 30 10+ 32+
4 9 3 2 32 11 39
Statistics: Chi2 test. Values are percent, n = 8. + p < 0.0001 vs. C/C or S/C. Q = quadrant, TG = target quadrant.
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Table 3 Percentage of search strategies used in groups in the test Retention memory task. Strategy
Thigmotaxis Random Scanning Chaining Incorrect Q Correct TQ Spatial search
Experimental groups C/C
C/MA
C/S
S/C
S/MA
S/S
MA/C
MA/MA
MA/S
0 0 3 0 25 3 69
0 6 8 3 31 4 48+
0 0 0 1 36 8 55
0 0 0 1 33 0 66
0 0 1 3 53+ 0 43+
0 0 0 0 39 8 53
0 0 1 0 24 17 58
0 0 0 0 33 1 66
0 0 0 0 33 9 58
Statistics: Chi2 test. Values are percent, n = 8. + p < 0.001 vs. C/C or S/C. Q = quadrant, TG = target quadrant.
exposure [F(2, 63) = 0.00; p = 1]} or in the duration of presence in the quadrant where the hidden platform was placed during learning {prenatal exposure [F(2, 63) = 0.7; p = 0.5], postnatal exposure [F(2, 63) = 0.37; p = 0.69]}. For the speed of swimming, there was main effect of prenatal treatment [F(2, 63) = 8.19; p < 0.05]; animals prenatally exposed to MA swam slower than control (p < 0.05) or prenatally saline-exposed (p < 0.001) animals (Fig. 6A). There was no main effect of postnatal exposure [F(2, 63) = 1.36; p = 0.26] or interaction between pre- and postnatal drug exposure [F(4, 63) = 2.32; p = 0.07] in the speed of swimming in the Probe test. 3.3. Retention memory test
Fig. 4. The length of trajectory in Place navigation task. (A) Effect of prenatal and postnatal MA exposure. Results are presented as average of all trials in 5 days. (B) Effect of postnatal MA exposure. Results are presented as average of all trials in 5 days. (C) Effect of postnatal MA exposure. Results are presented as averages of 8 trials per day in training days 1–5. Values are means ± SEM (n = 8) (ANOVA; Bonferroni post hoc test).
ing (Fig. 5A) than rats fostered to control mothers. Further, rats fostered to MA-treated mothers used less direct swim (Fig. 5C) and swam less in the correct target quadrant (Fig. 5D) than rats fostered to control mothers. In the testing day 3, main effect of postnatal exposure was demonstrated [2 = 64.06; p < 0.0001], such that all animals fostered to MA-treated dams, regardless of prenatal exposure, scanned more the pool (Fig. 5B) and displayed less direct swim (Fig. 5C) than rats fostered to control mothers. In the testing day 4, main effect of postnatal exposure was shown [2 = 37.8; p < 0.0001], such that all animals fostered to MAtreated dams, regardless of prenatal exposure, scanned more the pool (Fig. 5B) than rats fostered to control mothers. In the testing day 5, no main effect of postnatal exposure was found [2 = 5.12; p = 0.53]. No main effect of prenatal exposure was found on day 1 [2 = 11.02; p = 0.08], day 2 [2 = 10.69; p = 0.1], day 3 [2 = 3.85; p = 0.7], day 4 [2 = 11.82; p = 0.06], or day 5 [2 = 3.15; p = 0.79] of learning.
Statistical analysis showed no significant main effects in the latencies to find the hidden platform {prenatal exposure [F(2, 63) = 1.70; p = 0.19], postnatal exposure [F(2, 63) = 0.90; p = 0.41]}, in the search errors {prenatal exposure [F(2, 63) = 2.52; p = 0.09], postnatal exposure [F(2, 63) = 1.22; p = 0.31]}, or in the lengths of the trajectories {prenatal exposure [F(2, 63) = 1.32; p = 0.27], postnatal exposure [F(2, 63) = 1.30; p = 0.28]}. In the speed of swimming (Fig. 6B), significant main effect of prenatal exposure was demonstrated [F(2, 63) = 4.00; p < 0.05], such that all animals prenatally exposed to MA, regardless of postnatal exposure, swam slower than the animals prenatally exposed to saline (p < 0.01). There were no differences in the speed of swimming in animals prenatally exposed to MA and prenatal controls (p = 0.25). There was no main effect of postnatal exposure [F(2, 63) = 0.51; p = 0.60] or interaction between pre- and postnatal drug exposure [F(4, 63) = 1.2; p = 0.32] in the speed of swimming. In the search strategies (Table 3), main effect of postnatal drug exposure [2 = 29.51; p < 0.0001] was found. Rats fostered to MAtreated mothers used less direct swim and swam less in the correct quadrant than rats fostered to control dams, regardless of prenatal exposure. Further, animals fostered to MA-treated mothers swam more in the incorrect quadrant than rats fostered to control mothers. No main effect of prenatal drug exposure was found [2 = 10.02; p = 0.06]. 3.4. Visible platform task Statistical analysis showed no significant main effects in the latencies to find the visible platform {prenatal exposure [F(2, 62) = 1.60; p = 0.21], postnatal exposure [F(2, 62) = 0.65; p = 0.52]}, in speed of swimming {prenatal exposure [F(2, 62) = 0.17; p = 0.84], postnatal exposure [F(2, 62) = 0.68; p = 0.51]}, or in the lengths of the trajectories {prenatal exposure [F(2, 62) = 2.25; p = 0.11], postnatal exposure [F(2, 62) = 0.69; p = 0.50]}.
3.2. Probe test
4. Discussion
Statistical analysis showed no significant main effects in the frequency {prenatal exposure [F(2, 63) = 0.75; p = 0.47], postnatal
The aim of the present study was to determine the effect of cross-fostering and/or postnatal MA exposure via maternal breast
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Fig. 5. Effect of postnatal MA exposure on the frequency of strategies used in Place navigation task in training days 1–5. (A) Random search strategy—main effect of postnatal exposure in days 1 and 2; rats postnatally MA-exposed > postnatally control animals, (B) scanning—main effect of postnatal exposure in days 1, 3 and 4; rats postnatally MA-exposed > postnatally control animals, (C) spatial search strategy—main effect of postnatal exposure in days 1, 2 and 3; rats postnatally MA-exposed < postnatally control animals, (D) search in correct quadrant; main effect of postnatal exposure in days 1 and 2; rats postnatally MA-exposed < postnatally control animals. Statistics: Chi2 test; values are sums of frequencies in days 1–5, n = 8. + p < 0.0001 rats postnatally MA-exposed vs. postnatally control animals.
milk on cognitive functions in adult male rats prenatally exposed to MA. Our results demonstrate that prenatal exposure to MA does not impact learning and memory, while postnatal exposure to MA show impairments in cognitive functions, mainly in learning. One limited factor should be noted at the beginning of the discussion. In theory, it would be possible that early postnatal and/or preweaning stress caused by tattooing and ear-punching, respectively, might influence the performance in the MWM of adult rats, because only prenatally saline- and MA-exposed animals not controls were exposed to this stressor. However, no differences were found in cognitive functions between prenatally saline-exposed animals and prenatal controls, which make this theoretical possibility improbable. The effects of prenatal and postnatal MA exposure are discussed in details. First, our results show that prenatal MA exposure at a dose of 5 mg/kg did not affect the latency, search error, length of the trajectory and search strategies used in Place navigation task. This finding is in agreement with the work of Schutová et al. [33], who showed that the dose of MA (5 mg/kg) administered prenatally did not impair learning in the MWM. There is another study showing that prenatal exposure to low doses of MA (5 or 10 mg/kg) does not affect spatial memory in the MWM, while higher doses of MA (15, 20 mg/kg) did induce impairments of spatial memory in the MWM tested in adulthood [1]. Thus, it seems
that the effect of prenatal MA exposure on cognition depends on the dose used and that higher doses would be required to induce changes in cognition that would last until adulthood. However note, that the dose was chosen based of the finding that such a dose induces similar behavioral changes as in drug-abusing humans [45]. Second, the present data demonstrate that the postnatal MA exposure from mothers’ breast milk in preweaning period (PD 1–21) increases the deficit in spatial learning when assessed in adulthood by using the MWM. While MA/MA, S/MA and C/MA animals received MA postnatally, the MA/C and MA/S animals got only prenatal drug exposure. One possible explanation for this is the direct effect of MA administration via mother’s breast milk. There is a study showing that neonatal administration of MA at doses of 5, 10 or 15 mg/kg administered four times daily on PD 11–20 produce lasting spatial learning and memory deficits [50]. As the hippocampus in rats is still developing during the PD 11–20 and this development is analogous to human hippocampal development during the third trimester of pregnancy [6], the neonatal period may be more critical for the effects of MA on cognitive functions in rats than the prenatal period. The hippocampus is important for several types of learning (i.e. spatial learning) and undergoes neurogenesis and neural pruning during the first few weeks of life in rats [13].
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Fig. 6. Effect of prenatal and postnatal MA exposure on speed of swimming in the Probe test (A) and Retention memory test (B). Results are presented as averages of 8 trials per day. Values are means ± SEM (n = 8). (A) In the Probe test: main effect of prenatal exposure; prenatally MA-exposed animals swam slower than prenatal controls (* p < 0.05) or saline-exposed rats (# p < 0.001), regardless of postnatal exposure. (B) In the Retention memory test: main effect of prenatal exposure; rats prenatally MA-exposed swam slower than animals prenatally exposed to saline (# p < 0.05) (ANOVA; Bonferroni post hoc test).
In our study, only cumulative distance (search error) from the platform and the latencies in reaching the platform are affected by postnatal exposure to MA. Animals fostered to MA-treated dams (prenatal control, saline or MA) have longer latencies and have bigger search error than the animals fostered to control or salinetreated mothers. Based of the works of Gallagher et al. [12] and Lindner [17], cumulative distance from the platform and the length of trajectory are better indicators of spatial learning ability than the latency. However, the length of trajectory is not affected by postnatal exposure to MA in the present work. Thus, we used analysis of search strategies to explain in detail the behavioral nature of cognitive impairments of male rats. Although for two individual rats the length trajectories can be almost identical, their performance (search strategies) can differ markedly. While one of the animals searches for the platform relatively close to it, the other searches in quadrants distant to the platform randomly and finds the platform accidentally. The results of the search strategy analysis in the Place navigation test demonstrates that the incidence of random search and scaning is significantly greater and the incidence of direct swim and searching in correct quadrant is significantly lower in all animals fostered to MA-treated dams relative to rats fostered to control mothers. Random search and scanning are non-spatial strategies and are used mainly in the beginning of the learning period [16]. Rats fostered to MA-treated dams began to search the whole surface area of the pool randomly in the testing days 1 and 2 and later on they scanned the inner area of the pool more selectively (days 1, 3 and 4). Moreover, rats fostered to MA-treated mothers used lower spatial search and searching in correct quadrant in testing days 1–3. The develop-
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ment of spatial learning for the platform location is reflected by a focal search of a target quadrant or by a direct swim to the platform and are used if an animal remembers the exact location of the platform [16]. The aforementioned findings suggest that rats fostered to MA-treated mothers have weaker spatial learning at least in the testing days 1–3 than rats fostered to control mothers. However, by the end of learning, animals fostered to MA-treasted mothers are able to find the location of the hidden platform similarly to control animals. Further, we found no differences between experimental groups in the Probe test. The measures commonly used to assess performance in the Probe test are designed to reflect the spatial bias of an animal’s search pattern. These data suggest that all experimental groups had comparable navigational abilities. The second possibility of how to explain the effect of postnatal MA exposure is that the MA/C and MA/S animals are fostered to control or saline-treated control mothers and as such they were not exposed to the impaired maternal behavior of MA-treated dams. Changes in maternal behavior (impaired maternal care, such as active nursing, mother being in the nest or in contact with pups, carrying and grooming pups and a nest building, mother-pup intercontact interval, retrieval latency, inter-retrieval interval, number of pup retrieved, grooming pups and nest building) have been described by many studies after application of psychostimulants [11,25,37]. Since it is generally accepted that maternal care is essential for the somatic growth and neural development of the pups [32], lack of the care acts as a stressful stimulus for the pups. There is a study showing that differences in maternal care during the first week of postnatal life influences hippocampal development and function [18]. It has been investigated that the offspring of mothers, who exhibit a higher frequency of LG–ABN over the first week of postnatal life, show increased hippocampal synaptic density and enhanced spatial learning and memory [18]. The mechanism by which MA or differences in maternal care may prevent proper spatial learning ability is, however, unknown. It is possible that changes in stress reactivity may play a role in these effects, because worse maternal care can cause stress for pups in preweaning period that may persist until adulthood. Extended periods of stress and elevated levels of glucocorticoids can alter brain function and increase vulnerability to neurological impacts [28,29]. There are studies showing that neonatal MA produces increase in corticosterone that impairs excitability of nerve cells in the hippocampus [9] and induces neuronal atrophy of the hippocampus [22] that further result in spatial learning deficits in the MWM [30,31]. MA was also shown to protract activation of the HPA system during the neonatal period, which may induce long-term changes in the stress response when the animals are adult [47]. Others have demonstrated long-term learning and memory deficits in novel object recognition and spatial memory following neonatal maternal separation, a stressor that also causes corticosterone release [2]. Moreover, already Morris [23] noted that swimming in the MWM may cause some stress for the tested animals, which was further supported by the finding of Akirav et al. [3] and Roozendaal et al. [27] showing adrenal activation and release of corticosterone following training in the MWM. However, future studies examining the effect of MA on cognitive functions in respect of stress would be necessary to examine this hypothesis. Third, we found that all animals prenatally exposed to MA, regardless of postnatal exposure, swam slower than the animals prenatally exposed to saline in Place navigation task and Memory retention task. The swimming velocity can be used to measure motivation to find the hidden platform [20]. Therefore, the decreased swimming velocity may suggest decreased motivation of prenatally MA-exposed animals. However, because the present study does not show any differences between prenatally MA-exposed rats and prenatally control rats in the speed of swimming, this hypothesis does not seem to be relevant. Similarly, these
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data cannot be attributed to impaired motor function. The visible platform task assessed the ability of rat to swim onto a visible platform, without relying on spatial memory. There were no differences between rats exposed (prenatally or postnatally) to MA and salineexposed or control rats on swim speeds of the visible platform task. These findings are in agreement with previous studies that found no main effect between rats exposed to MA and control rats on swimming ability when measured in the straight channel task [48] or to reach a visible platform in MWM [21]. On the other hand, because the present study show increase in the speed of swimming in prenatally saline-exposed rats when compared to controls, the effect of prenatal stress seems to play more important role in this measure. It was shown [24,41] that placebo injection of saline administered to pregnant mothers may induce mild stress for the rat and that way indirectly affect the development of her pups. Fourth, neither prenatal nor postnatal MA exposure affected latencies, search error and the length of trajectory in the Retention memory test. This is in agreement with study of Acuff-Smith et al. [1] showing that MA 5 mg/kg does not influence memory in the MWM. The results of search strategy analysis demonstrates that rats postnatally exposed to MA search the pool by using spatial strategies (focal search in correct target quadrant, direct swim) less, than prenatally control animals. Further, animals fostered to MA-treated mothers swam more in the incorrect quadrant than rats fostered to control mothers. These results suggest that prenatally and postnatally MA-exposed animals memorized the location of the platform similarly to control animals, however, they used different strategies. In conclusion, the present study investigated the effect of MA exposure and cross-fostering on learning and memory of adult male rats tested in the MWM. Taken together, our results show that prenatal exposure to MA at dose as low as 5 mg/kg does not impair learning in the MWM, while postnatal exposure to MA from mother’s breast milk and/or their worse maternal care impair learning of adult male rats. On the other hand, “better” maternal care of control mothers does not affect learning and memory of rat pups prenatally exposed to MA. In contrast to our previous study [14] the role of maternal care on learning and memory abilities in adulthood remains unclear. More studies would be necessary to examine the long-term effects of cross-fostering on offspring of drug abusing mothers. Acknowledgements This study was supported by project CN LC554, grant IGA 1A8610-5/2005, and Research Goal #MSM 0021620816 from Ministry of Education, Youth and Sports of the Czech Republic. The procedures for animal experimentation utilized in this report was reviewed and approved by the Institutional Animal Care and Use Committee and is in agreement with the Czech Government Requirements under the Policy of Humans Care of Laboratory Animals (No. 246/1992) and with the regulations of the Ministry of Agriculture of the Czech Republic (No. 311/1997). References [1] Acuff-Smith KD, Schilling MA, Fisher JE, Vorhees CV. Stage-specific effects of prenatal d-methamphetamine exposure on behavioral and eye development in rats. Neurotoxicol Teratol 1996;18(2):199–215. [2] Aisa B, Tordera R, Lasheras B, Del Rio J, Ramirez MJ. Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats. Psychoneuroendocrinology 2007;32(3):256–66. [3] Akirav I, Sandi C, Richter-Levin G. Differential activation of hippocampus and amygdala following spatial learning under stress. Eur J Neurosci 2001;14(4):719–25. [4] Barbazanges A, Vallee M, Mayo W, Day J, Simon H, Le Moal M, et al. Early and later adoptions have different long-term effects on male rat offspring. J Neurosci 1996;16(23):7783–90.
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