The effect of methamphetamine exposure during pregnancy and lactation on hippocampal doublecortin expression, learning and memory of rat offspring
Zahra Jalayeri‑Darbandi1 · Aliakbar Rajabzadeh1,2 · Mahmoud Hosseini3 · Farimah Beheshti4 · Alireza Ebrahimzadeh‑bideskan1,2
Abstract
The aim of this study was to evaluate the effect of methamphetamine (MA) exposure during pregnancy and lactation on doublecortin (DCX) expression in the hippocampus of rat offspring and also on learning/memory. Thirty-five pregnant Wistar rats were randomly divided into seven groups of 5 rats each: three experimental groups, each receiving 5 mg/kg body weight (BW) intraperitoneal (i.p.) injections of MA during pregnancy or/and lactation; three sham groups, each receiving saline injections; one control group, receiving no injection. After the interventions, two male pups (1 and 22 days old) were randomly selected from each mother, sacrificed and their brains subjected to DCX immunohistochemistry. One additional male pup from each mother was randomly selected and maintained for 60 days for testing in the Morris water maze and passive avoidance tests. MA administration during pregnancy was found to have significantly decreased the number of DCXpositive cells in the CA1, CA3 and DG regions of the hippocampus in the 1-day pups (P ≤ 0.05) and to have significantly decreased the number of DCX-positive cells in only two regions of the hippocampus, the CA1 and DG regions, in 22-day old pups. In comparison, exposure to MA during lactation was only associated with a significant decrease in the number of DCX-positive cells in the DG. Exposure to MA during pregnancy had significant impact on the intensity of DCX expression in the hippocampus of 1- and 22-day pups (P ≤ 0.05). There was no significant difference in memory/learning among the study groups. Our results indicate the administration of MA during pregnancy had a greater effect that during the lactation period on DCX expression in the hippocampus of rat offspring.
Keywords Doublecortin · Hippocampus · Lactation · Learning · Memory · Methamphetamine · Pregnancy
Introduction
Methamphetamine (MA) is a member of the amphetamine group of sympathomimetic drugs which stimulate the central nervous system (CNS). It is therefore similar to amphetamines. MA has many common street names, including “meth”, “glass”, “ice”, “crystal”, and “speed” (Ijomone et al. 2011; Baptista 2015). It as a low-molecular-weight lipophilic substance that can pass through the placenta and blood–brain barrier; consequently, exposure to this drug during pregnancy may detrimentally affect the fetus (Smith et al. 2003). However, the most important side effect of MA exposure is damage to the CNS, possibly due to the higher sensitivity of the CNS to the drug during brain development (Thompson et al. 2004).
The hippocampus is more vulnerable to the effects of MA than other regions of the CNS (Thompson et al. 2004; Turowski and Kenny 2015). As a result, neurobehavioral disorders are commonly observed in children with prenatal MA exposure (Kwiatkowski et al. 2014). Various studies have led to the conclusion that drug use by parents has a significant impact on the factors involved in hippocampal neurogenesis (Tian et al. 2009; Elibol‐Can et al. 2014; Moszczynska et al. 2015). Given the key role of neurogenesis in learning and memory, impaired hippocampal neuron maturation may be associated with decreased spatial cognitive performance (Deng et al. 2009). Accordingly, it is possible that MA exposure during pregnancy can adversely affect subcortical parts of the brain (e.g., hippocampus) in neonates (Smith and Chen 2009; Yuan et al. 2011). The organization and development of the nervous system occur within 8–22 days of gestation, with hippocampal formation continuing after birth and 70% of neurogenesis in the dentate gyrus occurring during the first 2 weeks after birth with the initiation of lactation. Therefore, exposure to toxic substances during these sensitive periods could significantly affect the memory, learning abilities and performance of infants (Gao et al. 2011). However, studies conducted on the effect of MA exposure during pregnancy and lactation on learning and memory are in their earlier stages (Behnke et al. 2013), and most of such studies conducted to date have reported conflicting results (Schutová et al. 2008; Deng et al. 2009; Cao et al. 2013).
Doublecortin (DCX) is a microtubule-associated protein (Walker et al. 2007; Klempin et al. 2011) that is expressed in post-mitotic migrating, and differentiating neurons and plays an essential role in the migration and differentiation of these nerve cells. DCX is an indicator/marker of neurogenesis, which is an ongoing dynamic process during embryonic and perinatal periods of mammalian life (Klempin et al. 2011). It is expressed at high levels during brain development, especially in those regions that undergo continuous neurogenesis, such as the subventricular zone, olfactory bulb, striatum, and hippocampus (Francis et al. 1999; Gleeson et al. 1999).
Hippocampal neurogenesis is a multistep process whereby neural stem cells (NSCs) proceed through a wellestablished sequence of stages as they move from proliferating precursors (Ki67 and BrdU positive cells) to post-mitotic cells (DCX-positive cells) that ultimately become mature neurons [NeuN (a neuron-specific nuclear protein)-positive cells) and integrate into hippocampal networks(Zhang and Jiao 2015).
The aim of this study was to evaluate the effect of MA use during pregnancy and lactation on the distribution and expression of DCX in the hippocampus of the offspring. In addition, as the hippocampus plays an essential role in the formation of spatial memory and cognitive map of the environment, based the movements of the animal, we also assessed the impact of prenatal MA exposure on memory and learning of 2-month-old rat offspring using the Morris water maze (MWM) and passive avoidance (PA) tests.
Materials and methods
This study was conducted at Mashhad University of Medical Sciences, Mashhad, Iran based on the guidelines of the ethics committee of Mashhad University. Experimental protocols were approved by the Animal Care and Use Committee of the Institution.
Chemicals and preparation
The MA used in this study was obtained from SigmaAldrich Co. (St. Louis, MO; cat. nr. M8750), Prior to being injected intraperitoneally (i.p.) at a daily dose of 5 mg/kg body weight (BW) it was dissolved in normal saline (0.9% sodium chloride, 1 mg MA per 1 ml normal saline) (Macúchová et al. 2013).
Animals and treatments
Adult Wistar rats (40 females; 20 males; weight range 200–250 g) were provided by the research unit of the laboratory of animal housing of Mashhad University of Medical Sciences. The rats were housed in metal cages under standard conditions (20 ± 2 °C, 12/12 (light/dark)-h photoperiod) and had ad libitum access to food and water. After mating, followed by performing vaginal smear, sperm-positive female rats were considered to be pregnant (gestation day: 0). Thereafter, we randomly selected 35 pregnant rats and divided these into seven groups of five rats each: three experimental groups (receiving MA injections), three sham controls (receiving normal saline injections) groups, and one control group. The treatments for each group are as follows:
1. Methamphetamine/pregnancy (MA/P): pregnant rats were injected (i.p) with 5 mg/kg BW of MA during the prenatal period, gestational day seven 7 (GD7) until the end of pregnancy period (GD21).
2. Methamphetamine/lactation (MA/L): pregnant rats were injected (i.p) with 5 mg/kg BW of MA during lactation, Postnatal day 1 to 22, (PND1-22).
3. Methamphetamine/pregnancy and lactation (MA/P&L): pregnant rats were injected (i.p) with 5 mg/kg BW MA during both pregnancy and lactation.
4. Sham control/pregnancy (saline/P): pregnant rats were injected (i.p) with normal saline at the same volume as given to the experimental groups (5 ml/kg) during pregnancy (day 7 up to the end of pregnancy).
5. Sham control/lactation (saline/L): pregnant rats were injected (i.p) with normal saline at the same volume as given to the experimental groups (5 ml BW/kg) during lactation (PND1-22).
6. Sham control/pregnancy and lactation (saline/P&L): pregnant rats were injected (i.p) with normal saline at the same volume given to the experimental groups (5 ml/kg) during pregnancy and lactation.
7. Control group (control): pregnant rats received no interventions during pregnancy and lactation.
From day 20 of gestation onwards, female rats were kept in separate cages and observed twice a day to confirm delivery. Date of birth was determined to be postnatal day zero.
Sampling and tissue processing
After delivery, five 1-day and five 22-day male pups were randomly selected from each group (one pup from each mother). Under anesthesia using chloroform, the brain of the rat pups was completely removed, washed with normal saline, and placed in 10% formalin as a fixative. Following routine tissue processing, the samples were embedded in paraffin and cut into 5-µm-thick sections using a rotary microtome (Leitz model 1512; Leica Biosystems, Wetzlar, Germany) in a coronal direction. From each block, ten sections containing the hippocampus were prepared and transferred onto polylysine slides for use in the immunohistochemistry studies (Rajabzadeh et al. 2011).
Immunohistochemistry
Immunohistochemical staining of DCX was performed to detect differentiated cells. Specimens were first deparaffinized in xylene, then rehydrated in a series of descending ethanol concentrations, and finally washed in 0.1 M phosphate-buffered saline (PBS; pH: 6.8) for 15 min (three times, 5 min each time). Antigen retrieval was performed at room temperature for 20 min using 0.01% protein kinase (10 μL of protein kinase in 1000 mL of 0.1 M PBS). Sections were then immersed in 3% hydrogen peroxide ( H2O2)/methanol solution in a dark chamber at room temperature for 10 min and then rinsed in 0.1 M PBS for 15 min (three times, 5 min each time). In addition, the sections were incubated in 10% normal goat serum/PBS (ab7481; Abcam, Cambridge, UK) for 30 min to reduce the background color, followed by exposure of the sections to the primary anti-DCX antibody (ab1823; Abcam) at 1:500 dilution in PBS in a moist compartment at 4 °C overnight. After rinsing with PBS, tissue sections were incubated with the secondary antibody (immunoglobulin goat anti-rabbit) conjugated with horseradish peroxidase (ab6721; Abcam) at 1:300 dilution (in PBS) for 60 min at room temperature. After being rinsed with PBS (three times, 5 min each time), diaminobenzidine solution (Sigma-Aldrich) was added to the samples (0.03 g DAB in 100 mL PBS and 200 µL H 2O2/100 mL PBS) and the solution kept at room temperature for 15 min. As a last step, the sections were washed with tap water and used to perform hematoxylin counterstaining (Mohammadipour et al. 2014).
Quantification of DCX‑positive cells
The sections containing the hippocampus of 1-day and 22-day-old rat pups (five sections per hippocampus) were selected using randomized systematic sampling. Stained sections of the hippocampal cornu ammonis 1 and 3 regions (CA1, CA3) and of the hippocampal dentate gyrus (DG) region were evaluated and photographed using a light microscope, with the highest magnification of 100× (Olympus model BX51; Olympus Corp., Tokyo, Japan) (Fig. 1).
In addition, a counting frame was used to calculate the number of DCX-positive cells, which was estimated on a per unit area basis using the following formula: where NA is the number of DCX-positive cells per unit area, ΣQ represents the number of counted DCX-positive cells, a/f represents the surface area of each counting frame, and ΣP is the total number of frame counts colliding with the sections (Rajabzadeh et al. 2011).
DCX immunoreactivity score
In this study, the immunoreactivity with anti-DCX antibody was determined based on the reports of three observers who were blind to the groups. Immunoreactivity was graded as very weak (+), weak (++), moderate (+++), and strong (++++) (Rajabzadeh et al. 2012).
Behavioral tests
At the end of the lactation period, one male rat offspring was randomly selected from each mother of each group for participation in the behavioral tests. These offspring were kept under standard conditions for 2 months, and the MWM and PA tests were performed on postnatal day 60.
MWM apparatus and procedures
The MWM apparatus used in this study consisted of a black round tank (diameter 136 cm, height: 60 cm, depth 30 cm) filled with water (24–26 °C), with a circular platform (diameter 10 cm, height 28 cm) located approximately two cm under the water surface in the center of the southwest quarter of the tank. The tests were performed in a relatively dark room with four visible corners, which enabled the rats to find the location of the hidden platform. Each sampled rat was randomly evaluated once a day for 5 days (4 experiments at each trial) from the four quarters of the tank (north, east, south and west).
At each trial, the rat was released at one of the starting points in the maze, facing the tank wall. The released rat was allowed 60 s to find the platform, and if it failed to do so within this time period a researcher guided it to the platform. Once on the platform it was allowed to stay there for 15 s at the end of each trial to allow for assessment of the spatial situation of the platform. The rat was then taken out of the pool/off the platform, dried, and kept in the cage for 5 min. During the trial, the place navigation test was used to record the latency period to reach the platform and path length. On the sixth day, the platform was removed and rats were allowed to swim in the pool for 60 s (probe test). Total distance traveled and the times spent in each quadrant were recorded by a computer. All measurements were conducted during the second half of the light period (Hosseini et al. 2010, 2011).
PA apparatus and procedures
The PA test was used to evaluate the memory of animals based on negative reinforcement. This test was conducted in a shuttle box consisting of two light and dark chambers (20 × 20 × 30 cm) separated by a guillotine door. The floor of the dark chamber consisted of steel rods set 1 cm apart and wired to a shock generator. The rat was placed in the light room for 2 consecutive days (5 min/day) to allow adaption to the situation. On the third day, the rats were separately placed in the light room, and latency to enter the dark chamber was recorded for each sample. During the training course, the animals were kept in the light room faced toward the dark chamber. When the rat entered the dark chamber, the guillotine door was closed and electric shock (2 mA, duration 2 s) was activated. In the next stage, all of the rats tested samples were returned to their cages and then placed in the light chamber for 1, 24, and 72 h after training. Latency to enter the dark chamber and total duration of staying in the light and dark chambers, respectively, were recorded for 5 min, which was described as the retention trial (Pourmotabbed et al. 2011).
Statistical analysis
Data analysis was performed using SPSS version 16 (IBM Corp., Armonk, NY) to analyze the data. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to compare the mean number of DCX-positive cells. The data obtained from the acquisition part of MWM and PA test were evaluated and compared using two-way repeated measures ANOVA. All data were expressed as mean ± standard error of the mean. In addition, non-parametric Kruskal–Wallis and Mann–Whitney U-tests were used to analyze the obtained staining intensity data. A P value of ≤ 0.05 was considered to be statistically significant.
Results
Effects of MA on DCX immunoreactivity
In this study, neurons of the CA1, CA3 and DG regions of the hippocampus of 1- and 22-day-old rat pups were stained with anti-DCX antibody. Those neurons in rat pups in the control/saline groups showed more intense staining that those in rat pups in the experimental groups. The Kruskal–Wallis and Mann–Whitney U statistical tests revealed that this reaction was significantly decreased in 1- and 22-day-old rat pups in the MA/P group and 22-day-old rats in the MA/P&L group compared to those in the control and saline groups (P ≤ 0.05). Nevertheless, in terms of anti-DCX staining intensity, there was no significant difference between the hippocampal neurons of the 22-day samples of the MA/L group and those of the control/saline groups (Figs. 2, 3, 5).
DCX-positive cells
DCX‑positive cells assessment
The immunohistochemical test was used to evaluate the effect of MA exposure during pregnancy and/or lactation on the distribution of DCX-positive cells in the hippocampus of rat pups. DCX-positive cells were visible in all the three regions of the hippocampus tested (CA1, CA3, DG) in the MA, saline (sham control), and control groups, but the frequency of these cells was lower in the groups exposed to MA. In addition, evaluation of the hippocampus sections obtained from 1-day-old pups in the MA/P group revealed a significant reduction in the number of DCX-positive cells in the CA1 (P = 0.001), CA3 (P = 0.003), and DG (P = 0.01) regions compared to the control groups (Figs. 2, 4). Evaluation of the hippocampal sections of 22-day-old pups revealed a significant reduction in the number of DCX-positive cells in the CA1 (P = 0.01) and DG (P = 0.001) regions in the MA/P group compared to the control group. In contrast, no significant difference was observed in the number of DCXpositive cells in the CA3 region of the hippocampus in the MA/P group compared to the control group (Figs. 5, 6a). In addition, there was a significant difference in the number of DCX-positive cells in the DG region of the MA/L group compared to the control group (P = 0.001) (Figs. 5, 6b). In the MA/P&L group the number of DCX-positive cells was significantly decreased in the CA1 (P = 0.001) and DG (P = 0.001) regions compared to the control group (Figs. 5, 6c).
Effects of MA on MWM test results
Place navigation test
Two-way ANOVA with repeated measures revealed that the administration of MA was a significant main effect during pregnancy on both latency [f (1,270) = 15.87; P < 0.001] and distance traveled [f (1,270) = 19.02; P < 0.001] to reach the platform. There were also significant effects for days on latency [f (4,270) = 18.64; P < 0.001] and distance traveled [f (4,270) = 11.80; P < 0.001] to reach the platform. There was no significant interaction between treatment and day on the latency [f (4,270) = 0. 352; P > 0.05] and the distance traveled [f (4,270) = 0.137; P > 0.05] to reach the platform. In addition, the results of the post hoc test indicated no significant differences between MA/P exposure and saline/P groups in terms of time latency and distance traveled to find the hidden platform (Fig. 7a). Two-way repeated measures ANOVA revealed that the administration of MA during lactation was a significant main effect on the latency [f (1,290) = 46.27; P < 0.001] and distance traveled [f (1,290) = 50.80; P < 0.001] to reach the platform. There were also significant effects for days on latency [f (4,290) = 16.56; P < 0.001] and distance traveled [f (4,290) = 22.96; P < 0.001] to reach the platform. There was no significant interaction between treatment and the day on latency [f (4,290) = 1.18; P > 0.05] and distance traveled [f (4,290) = 2.24; P > 0.05] to reach the platform. In addition, the results of the post hoc test indicated a significant difference in both the latency and the distance traveled between the MA/L and saline/L groups on days 1, 2, 4, and 5 (P < 0.05–P < 0.001), (Fig. 7b).
Two-way repeated measures ANOVA showed no significant main effect for MA during pregnancy and lactation on the latency [f (1,310) = 1.92; P > 0.05] and the distance traveled [f (1,310) = 5.13; P > 0.05] to reach the platform. There was a significant effect for days on the latency [f (4,310) = 25.193; P < 0.001] and distance traveled [f (4,310) = 16.55; P < 0.001] to reach the platform. There was no significant interaction between treatment and the day on latency [f (4,310) = 0.223; P > 0.05] and distance traveled [f (4,310) = 0. 223; P > 0.05] to reach the platform [f (4,310) = 0.593; P > 0.05]. In addition, the results of the post hoc test indicated no significant differences between the MA/P&L and saline/P&L groups in terms of time latency and distance traveled to find the hidden platform (Fig. 7c).
The probe test
This test was conducted to assess the spatial memory of offspring. The results demonstrate that exposure of the mother to MA during pregnancy and/or lactation had no significant effect on time latency and distance traveled in the target quadrant (Fig. 8).
PA test
Two-way repeated measures ANOVA revealed that MA had no significant main effect during pregnancy on the latency to enter the dark room [f (1,36) = 1.42; P > 0.05]. There were also no significant effects for time after the shock on latency to enter the dark room [f (2,36) = 0.94; P > 0.05]. There was no significant interaction between treatment and time after the shock on the latency to enter the dark room [f (2,36) = 0.24; P > 0.05]. In addition, results of the post hoc test indicated that there were no significant differences between the MA/P and saline/P groups in terms of latency to enter the dark room (Fig. 9a).
Two-way repeated measures ANOVA showed that MA had no significant main effect during pregnancy on the latency to enter the dark room [f (1,42) = 5.02; P > 0.05]. There were also no significant effects for the time after the shock on the latency to enter the dark room [f (2,42) = 0.46; P > 0.05]. There was no significant interaction between treatment and time after the shock on the latency to enter the dark room [f (2,42) = 0.05; P > 0.05]. In addition, results of the post hoc test indicated no significant differences between the MA/L and saline/L groups in terms of latency to enter the dark room (Fig. 9b).
Two-way repeated measures ANOVA demonstrated that MA had no significant main effect during pregnancy and lactation on latency to enter the dark room [f (1,36) = 1.42; P > 0.05]. There were no significant effects for time after the shock on latency to enter the dark room [f (2,36) = 0.94; P > 0.05]. There was no significant interaction between treatment and time after the shock on the latency to enter the dark room [f (2,36) = 0.24; P > 0.05]. In addition, results of the post hoc test indicated no significant differences between the MA/P&L and saline/P&L groups in terms of latency to enter the dark room (Fig. 9c).
Discussion
The present study was conducted to evaluate the effect of MA exposure during pregnancy and lactation on both the immunoreactivity and the number of DCX-positive cells in the male offspring. The effect of prenatal MA exposure on the memory and learning of 2-month-old offspring was also assessed using the MWM and PA tests.
Studies on the effect of exposure to MA before and after birth are in their primary stages (Behnke et al. 2013) and, in addition, these studies have yielded contradictory results. It has been demonstrated that a single dose of MA (50 mg/kg) decreased cell proliferation in rodents (Hildebrandt et al. 1999). However, chronic administration of this compound (1 mg/kg, i.p.) to rats had no significant effect on cell proliferation in the sub-granular area (Mandyam et al. 2008; Recinto et al. 2012). According to the literature, sensitivity of the hippocampus depends on the MA dose administered, as well as the timing and number of injections. It is possible that the contradictory results obtained in previous studies may be due to the differences in these factors (Baptista et al. 2012).
In our study, the data on staining intensity demonstrated that exposure to MA during pregnancy was associated with reduced immunoreactivity of DCX-positive cells in all three of the hippocampal regions studied (CA1, CA3, DG) in the 1- and 22-day-old rat pups. Exposure to MA during pregnancy also reduced the number of DCX-positive cells in the hippocampal regions in 1-day-old rat pups. Moreover, a significant reduction was observed in the number of DCX-positive cells in the CA1 and DG regions of the hippocampus of 22-day old rat offspring whose mothers had been exposed to MA. These results indicate that the DG and CA1 regions of the hippocampus are more sensitive and vulnerable to MA than the CA3 region. Consequently, prenatal MA exposure had various impacts on different hippocampal regions. A previous study showed that the number of DCX-positive cells significantly decreased only in the DG region after 30 days (Elibol‐Can et al. 2014). However, reduction in the number of DCX-positive cells in the CA3 region was not significant; therefore, this region was considered to be less sensitive to MA compared to the other hippocampal regions.
In our study, MA exposure during lactation had no significant impact on the number of DCX-positive cells in the CA1 and CA3 regions in 22-day-old pups. Nevertheless, a significant decrease was found in the number of these cells in the DG region, which confirms the high sensitivity of this hippocampal region to exposure to MA.
While the effects of MA exposure during pregnancy and/ or lactation on neural cell differentiation in rat pups have not previously been studied, research on adults indicates that MA use has a functional association with reduced number of DCX-positive cells and neurogenesis (Mandyam et al. 2008; Kochman et al. 2009; Yuan et al. 2011). In previous studies conducted by the authors of this present study, the administration of MA during pregnancy decreased the expression of polysialylated neuronal cell adhesion molecule (PSA-NCAM) (Baei et al. 2017; Bagheri et al. 2017). In contrast, the administration of MA during lactation had no such effect. MA injection during the prenatal or postnatal period also increased apoptosis (Baei et al. 2017; Bagheri et al. 2017).
Although the probable mechanisms of the effect of MA on neurogenesis are not completely understood, several possible mechanisms have been proposed, including impaired function of proteins of NPCs, induced cell death through mitochondrial fragmentation and oxidative stress, delay in the cellular cycle of stem cells from G0/G1 to S phase, and decreased expression of factors involved in neurogenesis, such as epidermal growth factor receptor (pEGFR) and 1.2 extracellular kinase signal (ERK1.2) (Yuan et al. 2011; Baptista et al. 2014; Venkatesan et al. 2011).
Since the expression of DCX molecules in hippocampal neurons significantly affects memory and learning, a reduced number of DCX-positive cells could result in memory and learning impairments in neonates. Evaluation of the behavioral test results did not confirm this possibility.
The results of the place navigation test revealed that injection of 5 mg/kg BW of MA during pregnancy and lactation significantly reduced latency and distance traveled to reach the hidden platform, compared to the saline group. While the mentioned variables decreased in the MA/P group, this reduction was not significant. Our findings are in agreement with those of previous studies demonstrating that the daily administration of MA (5 mg/kg BW) during pregnancy had no impact on finding the hidden platform and distance traveled. In addition, exposure to MA did not cause any learning impairments. Nevertheless, it has been shown that acute consumption of MA undermines memory performance (Schutová et al. 2008).
In contrast, a study by Cao et al. (2013) revealed that MA exposure could improve cognitive impairments and spatial memory. In addition, Macúchová et al. (2013) demonstrated that the administration of 5 mg/kg BW daily dose before pregnancy had no effect on finding the hidden platform and distance traveled. However, it did increase the swimming speed of the samples. In this same study, the authors noted that the administration of MA was associated with increased dopamine levels and led to stress and anxiety in neonates. As a result, it was assumed that physical activity increased in neonates, shortening both the time of reaching the platform and duration of traveled distance (Macúchová et al. 2013). Acuff-Smith et al. (1996) reported that the administration of low doses of MA during pregnancy had no effect on the spatial memory of neonates. However, high doses of this compound impaired the memory of infants (Acuff-Smith et al. 1996). Nonetheless, Williams et al. (2003) affirmed that repeated MA injection after birth decreased spatial memory during adulthood (Williams et al. 2003). Moreover, Hrubá et al. (2009) concluded that prenatal administration of 5 mg/kg BW MA had no impact on memory and learning performance in the MWM, whereas postnatal administration of this compound decreased cognitive function. These authors also noted that neonates receiving MA during the prenatal period had a slower pace of swimming (Hrubá et al. 2009) compared to the samples of the saline group. Taking all of these studies into account, it may be concluded that the timing of administration, administration dose and method, and the animal model could affect the results of behavioral tests (Williams et al. 2003; Hrubá et al. 2009; Cao et al. 2013). In the PA test, no significant difference was observed in the latency time to enter the dark chamber at 1, 24 and 72 h after the shock, compared to the saline group. According to the results of this test, daily administration of 5 mg/kg BW of MA during pregnancy and/or lactation had no significant impact on the memory/learning of offspring, and a reduced level of latency and distance traveled to reach the hidden platform, which was observed in MA/P&L group compared to saline group during the place navigation test, might be due to anxiety like behaviors however, it need to be more investigated in the future.
The absence of any significant change in learning and memory in the current study may be due to some mechanism by which MA exposure during pregnancy and/or lactation is prevented from affecting the production of new nerve cells up to the age of 2 months, the period in which learning and memory abilities were examined. In the studies conducted by Aeghtedari and Chou, it was noted that discontinuing of MA exposure could be associated with improved cognitive function and memory (Chou et al. 2007; Eghtedari et al. 2012).
There is a scarcity of studies on the side effects of longterm MA use during pregnancy and lactation, and studies conducted on this issue have yielded conflicting results. According to our findings, MA exposure during lactation had no significant effect on the learning ability of offspring. In this regard, Smith et al. (2003) concluded that MA abuse during brain development results in no impairments in spatial learning during adulthood (Smith and Chen 2009). In addition, results obtained by Moenk et al. (2012) are indicative of improved spatial memory in adults following exposure to MA in adolescence (Moenk and Matuszewich 2012), which is not in line with the results obtained by Hrubá et al. (2009). According to the results of this study, MA exposure during lactation is associated with impaired spatial memory in offspring compared to the saline group (Hrubá et al. 2009).
The results of former studies on the formation of fetal nervous systems in MA abusive mothers during lactation are inconsistent. Our results revealed that the i.p. administration of MA (5 mg/kg BW) during lactation had no significant impact on the number of DCX-positive cells, severity of immunoreactivity of DCX molecules, and memory and learning ability of the offspring. It would appear that the dose of drug received by the rat pups through the breast milk is significantly lower than the amount reaching the fetus through the placenta, which justifies the fewer side effects of MA exposure during lactation (Rambousek et al. 2014).
McDonnell-Dowling and Kelly (2016) reported that the selected method of administration (subcutaneous or by gavage) had a significant impact on neonatal outcomes. These authors concluded that subcutaneous injection was associated with more severe neonatal outcomes compared to the gavage technique (McDonnell-Dowling and Kelly 2016).
We confirmed that MA passed easier through the placenta that through the breast milk. Considering the limited scale of the present study, we recommend that further studies with larger sample sizes using other techniques to accurately determine the effects of MA exposure during pregnancy and lactation are needed.
Conclusion
According to our study, MA exposure during pregnancy as well as pregnancy/lactation could be associated with decreased expression of DCX in the hippocampus. However, drug abuse during lactation resulted in no significant decrease in DCX expression in the hippocampus of rat pups. Moreover, MA exposure during pregnancy and/or lactation had no significant impact on learning ability and memory of 2-month-old offspring.
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