Prenatal Exposure to Methamphetamine: Up-Regulation of Brain Receptor Genes

Methamphetamine (METH) is a widespread illicit drug. If it is taken by pregnant women, it passes through the placenta and just as it affects the mother, it can impair the development of the offspring. The aim of our study was to identify candidates to investigate for changes in the gene expression in the specific regions of the brain associated with addiction to METH in rats. We examined the various areas of the central nervous system (striatum, hippocampus, prefrontal cortex) for signs of impairment in postnatal day 80 in experimental rats, whose mothers had been administered METH (5 mg/kg/day) during the entire gestation period. Changes in the gene expression at the mRNA level were determined by two techniques, microarray and real-time PCR. Results of two microarray trials were evaluated by LIMMA analysis. The first microarray trial detected either up-regulated or down-regulated expression of 2189 genes in the striatum; the second microarray trial detected either up-regulated or down-regulated expression of 1344 genes in the hippocampus of prenatally METH-exposed rats. We examined the expression of 10 genes using the real-time PCR technique. Differences in the gene expression were counted by the Mann–Whitney U-test. Significant changes were observed in the cocaine- and amphetamine-regulated transcript prepropeptide, tachykinin receptor 3, dopamine receptor D3 gene expression in the striatum regions, in the glucocorticoid nuclear receptor Nr3c1 gene expression in the prefrontal cortex and in the carboxylesterase 2 gene expression in the hippocampus of prenatally METH-exposed rats. The microarray technique also detected up-regulated expression of trace amine-associated receptor 7 h gene in the hippocampus of prenatally METH-exposed rats. We have identified susceptible genes; candidates for the study of an impairment related to methamphetamine addiction in the specific regions of the brain.


HIGHLIGHTS
-The microarray technique detected either up-regulated or down-regulated expression of 2189 genes in the striatum of prenatally METH-exposed rats. -The microarray technique detected either up-regulated or down-regulated expression of 1344 genes in the hippocampus of prenatally METH-exposed rats. -We examined the expression of 10 genes using the realtime PCR technique. -Prenatally administered METH significantly alters the expression of the glucocorticoid nuclear receptor Nr3c1 mRNA within prefrontal cortex of rats. -Prenatally administered METH significantly alters the expression of the cocaine-and amphetamine-regulated transcript prepropeptide, the tachykinin receptor 3 and dopamine receptor D3 mRNA within striatum of rats. -Prenatally administered METH significantly alters the expression of the carboxylesterase 2 mRNA within the hippocampus of rats. -Prenatally administered METH alters the expression of the trace amine-associated receptor mRNA within hippocampus of rats. -Both techniques, microarray and real-time PCR, showed altered expression of the tachykinin receptor 3 and dopamine receptor D3 mRNA within striatum of rats.

INTRODUCTION
Methamphetamine (METH) is one of the most common "hard" drugs taken by pregnant women (Marwick, 2000). It is also one of the most frequently used illicit drugs in the Czechia, Middle and Eastern Europe (Vavřinková et al., 2001;Šlamberová, 2012). Statistics show that only 17% of female drug abusers in the United States were primary METH users, but 38% had used it during pregnancy, because some drug-abused women replace other drugs for METH during pregnancy for its anorectic effect (Marwick, 2000). METH is a powerfully addictive stimulant that metabolizes slowly resulting in the drug having a long-lasting high (8 to 24 h) (Marwick, 2000). This might be the reason that makes it so popular. The National Institute of Drug Abuse (NIDA; part of NIH) in the United States decided to increase funding for studies testing the effects of METH administration during pregnancy. Research concerning the long-term effects of prenatal METH exposure is still in its infancy. Our laboratory specializes in investigating the effects of drugs (especially METH) on rat mothers and their progeny. METH is an indirect agonist of monoamines, which works by disrupting multiple presynaptic and synaptic processes. The resultant increases in dopamine, norepinephrine, and serotonin contribute to its properties as well as inducing other central and peripheral effects (Thompson et al., 2009;Lloyd et al., 2013). In low doses, METH can elevate mood and increase alertness, concentration and energy. At higher doses, it can induce psychosis, breakdown of skeletal muscle, seizures and bleeding in the brain. Chronic, high-dose use can precipitate unpredictable and rapid mood swings, prominent delusions and violent behavior. Chronic abuse is associated with serious health complications including deficits in attention, memory, and executive functions in humans (Cadet and Krasnova, 2009). METH is known to have a high addiction liability (i.e., compulsive METH use) and dependence liability (i.e., withdrawal symptoms occur when METH use ceases) (ChEBI, 2018;Wikipedia contributors, 2019b). METH travels through the bloodstream to the brain, where it readily crosses the blood-brain barrier (Dattel, 1990;Burchfield et al., 1991). The similarity of the chemical structure of METH to monoamine neurotransmitters (dopamine and noradrenaline) determines its mechanism of action (Sulzer et al., 2005;Heal et al., 2013;Rambousek et al., 2014). METH affects the function and trafficking of DAT. It leads to increased release of dopamine from presynaptic terminals, as well as synaptic vesicles (Sulzer et al., 1995). Methamphetamine interacts with Taar1 receptors and this interaction results in inhibition of dopamine uptake, induction of dopamine efflux, and promotion of dopamine transporter internalization. TAAR1 mediates methamphetamine-induced regulation of dopamine transporter function and trafficking in brain striatal synaptosomes (Xie and Miller, 2009;Rutigliano et al., 2018). In Taar1-knock out mice sensitivity to amphetamine and methamphetamine was exacerbated (Lindemann et al., 2008;Achat-Mendes et al., 2012;Rutigliano et al., 2018). The neuropsychiatric complications might, in part, be related to drug-induced neurotoxic effects, which include damage to dopaminergic and serotonergic terminals, neuronal apoptosis, as well as activated astroglial and microglial cells in the brain (Cadet and Krasnova, 2009).
METH present in a drug-abusing women's bloodstream, can pass through the placenta to a fetus and can also be secreted into breast milk (Winslow et al., 2007). Amphetamines have vasoactive effects, which result in the restrictions of the supply of nutrients to a developing fetus, causing the anorectic symptoms often reported (Winslow et al., 2007). Infants born from METH-abusing mothers were found to have significant growth reductions (Dixon and Bejar, 1989;Plessinger, 1998;Cui et al., 2006), cleft palate, cardiac defects (Plessinger, 1998;Cui et al., 2006), aggressive behavior (Eriksson et al., 1989;Cui et al., 2006), peer-related problems (Eriksson and Zetterstrom, 1994;Cui et al., 2006) and learning problems (Cernerud et al., 1996;Cui et al., 2006). Matera and colleagues reported severe brain malformations in a human infant exposed to amphetamine in utero, such as exencephaly, anomalies of gyria, and hemorrhage (Matera et al., 1968;Cui et al., 2006). Human infants exposed to methamphetamine in utero have tended to show a significant decrease in head circumference (Little et al., 1988;Dixon and Bejar, 1989;Cui et al., 2006;Winslow et al., 2007). Non-invasive studies of the brain using Echo or MRI have revealed intracranial hemorrhage (Dixon and Bejar, 1989;Cui et al., 2006) and volume decrease of subcortical area (Chang et al., 2004;Cui et al., 2006). The effects of METH-administered prenatally in clinical studies are summarized in Table 1.
Experimental studies demonstrated that administration of METH to pregnant rats leads to increased likelihood of abortion and maternal death (Acuff- Smith et al., 1992;

Etiopatogenetic factor Effect Author
Amphetamine administration during pregnancy Brain malformations, such as exencephaly, anomalies of gyria, and hemorrhage Matera et al., 1968;Cui et al., 2006 METH and amphetamine administration during pregnancy At birth, 1, and 4 years: the mean weight, height, and head circumference below average Age 10: females, but not males, significantly shorter and lighter At 14-15 years: achievement in mathematics, Swedish language, and sports were statistically low Eriksson et al., 1978;Cernerud et al., 1996;Wouldes et al., 2004 An increased rate of premature delivery and placental abruption Eriksson et al., 1978Eriksson et al., , 1981Wouldes et al., 2004 METH, amphetamine, cocaine administration during pregnancy Cleft lip and palate, heart defects, low birth weight, small head circumference, dead fetus, biliary atresia, undescended testicles, low body fat, premature birth Eriksson et al., 1978;Oro and Dixon, 1987;Little et al., 1988;Dixon and Bejar, 1989;Cui et al., 2006 Perinatal administration of METH and cocaine Tachycardia, bradycardia, abnormal sleep, tremors Oro and Dixon, 1987 METH administration during pregnancy Decreased fetal-growth, no congenital anomalies Little et al., 1988;Cui et al., 2006 Drug administration during pregnancy Delayed development van Baar et al., 1989 Maternal methamphetamine abuse Fetal and infant death Stewart and Meeker, 1997 METH and amphetamine administration during pregnancy At 8 years: significant correlation between amount and duration of exposure prenatally and aggressive behavior and social adjustment Billing et al., 1994;Eriksson and Zetterstrom, 1994;Wouldes et al., 2004 METH and amphetamine administration during pregnancy Cleft, cardiac anomalies and fetal growth retardation Plessinger, 1998;Wouldes et al., 2004;Cui et al., 2006 METH administration during pregnancy Increased muscle tone, tremors, irritability, irregular sleep, impaired adaptability to stress Wouldes et al., 2004 METH administration during pregnancy Smaller subcortical volumes and associated neurocognitive deficits, neurotoxicity to the developing brain Chang et al., 2004;Cui et al., 2006 Pre Prenatal exposure to METH Significantly higher cognitive problems subscale scores than comparisons and were 2.8 times more likely to have cognitive problems scores that were above average Diaz et al., 2014Matějovská et al., 2014. Pregnant rats who receive METH have a shorter gestation period and have fewer litters of young than control mothers (Martin, 1975;Martin et al., 1976). METHadministered prenatally increases infant mortality, reduces weight gain, delays development and slows down reflexes (Martin et al., 1976;Cho et al., 1991;Acuff-Smith et al., 1996;Matějovská et al., 2014). Studies from our laboratory and others demonstrated that prenatal or neonatal exposure to METH seriously impairs the behavior of adult rats and their sensitivity to drugs (Šlamberová, 2012). Also, our studies demonstrated that maternal addiction during gestation and/or lactation periods impairs maternal behavior, which in turn has adverse effects on pup development (Šlamberová et al., 2005a,b, 2006). Such impairment caused by prenatal METH exposure can be partially suppressed by cross-fostering with control rats, demonstrating the positive effect of good postnatal foster care (Hrubá et al., , 2010Pometlová et al., 2009). On the other hand, maternal separation induces social stress and maternal physical stress, aggravating cognitive impairment and responses to stressors in the exposed offspring (Holubová et al., 2016(Holubová et al., , 2018. Thus, it seems that social factors, such as bad or good maternal care or maternal stress, can have significant influence on the development of rat offspring exposed to METH during gestation. In addition, one of our studies found that drug exposure during gestation may even influence second generation pups (Šlamberová et al., 2005d, 2007). There are several brain structures that are associated with drug addiction. The most important are mesolimbic, and mesocortical pathways (Robinson and Becker, 1986) which includes the striatum, hippocampus and prefrontal cortex. Striatum coordinates multiple aspects of cognition, including both motor and action planning, decision-making, motivation, reinforcement, and reward perception (Yager et al., 2015). The hippocampus plays important roles in the consolidation of information from short-term memory to long-term memory, and in spatial memory that enables navigation (Anderson et al., 2007). The prefrontal cortex has been implicated in planning complex cognitive behavior, personality expression, decision making, and moderating social behavior (Yang and Raine, 2009). Various studies have found that prenatal exposure to psychostimulants results in enhanced responsiveness of mesostriatal monoaminergic projections (Bubeníková-Valešová et al., 2009;Fujáková-Lipski et al., 2017). Given that our previous studies demonstrated changes in locomotor activity, cognition as well as social behavior (Fialová et al., 2015;Šlamberová et al., 2015), types of behavior that are associated with the above mentioned brain structures, the aim of the present study was to identify candidates for changes in the gene expression in the striatum, hippocampus and prefrontal cortex in adult rats in PD 80, whose mothers had been administered METH (5 mg/kg/day) during the entire gestation period.

Animals and Housing
The procedures for animal experimentation utilized in this study were reviewed and approved by the Committee for Protection of Experimental Animals of Third faculty of Medicine and by Departmental Committee of Ministry of Education, Youth, and Sports and are in agreement with the Czech Government Requirements under the Policy of Humans Care of Laboratory Animals (no. 246/1992) and also comply with the regulations of the Ministry of Agriculture of the Czech Republic (no. 311/1997). Adult male (300-400 g) and female (250-300 g) Wistar rats from Charles River Laboratories International, Inc., were delivered by VELAZ (Prague, Czechia) and housed 4-5 per cage in a temperature-controlled (22-24.8 • C) colony room with a standard 12 h light/dark cycle, lights on at 06.00 h. Animals were left undisturbed for 1 week with food and water ad libitum. After the acclimation period, the females were weighed and smeared using vaginal lavage to determine the phase of the estrous cycle. Females at the onset of the estrous phase of the estrous cycle were housed with sexually mature males for overnight (1 pair per cage) (Šlamberová et al., 2005a,b,c,d;Schutová et al., 2012). On the following day, the females were smeared and returned to their previous home cages. The fertilization was assigned as Day 1 of gestation based on the presence of sperm in the vaginal smear. On Day 21 of gestation, the females were removed from the group cages and placed into maternity cages (1 female per cage). The day of the delivery was counted as PD 0. The number of pups in each litter was adjusted to 12. Whenever possible, the same numbers of male and female pups were kept in each litter. To avoid litter bias, pups were cross-fostered on PD 1 so that one mother usually raised three male and three female pups of her own and three male and three female pups from another mother (see below). On PD 21, animals were weaned and housed in groups separated by sex. The light/dark cycle of the animals was reversed with lights on at 18.00 h. Animals were left undisturbed until adulthood (Schutová et al., 2012).

Drug Administration and Experimental Groups
For prenatal exposure, the pregnant dams were divided into two groups: the METH group and the SAL group. The females from the METH group were administered D-methamphetamine HCl (Sigma Aldrich, Czechia) at a dose of 5 mg/kg/day and volume of 1 ml/kg/day s.c. for the duration of the gestation period (i.e., from the first to the last day of gestation) (Šlamberová et al., 2005aSchutová et al., 2012). This METH dose was chosen because it results in similar fetal brain drug concentrations to those found in human infants of METHabusing women (Acuff- Smith et al., 1996;Schutová et al., 2012). This dose is also the standard dose used in our experiments (Šlamberová et al., 2005aSchutová et al., 2012). The females from the SAL group were administered 0.9% NaCl solution s.c. at the same time and in the same volume as METH. On PD 1, the offspring were marked according to the prenatal exposure using the intradermal application of black India ink; the METH group of prenatally affected offspring was marked in the left foot pad, and the SAL group of prenatally affected offspring was marked in the right foot pad.

Study Subjects
Before we started the experiment in PD 80, the experimental animals (8 METH and 8 SAL rats) were weighed as were the parts of the brains from which the RNA was isolated. The isolation of total RNA (mixture of mRNA, tRNA, and rRNA) from the snap-frozen tissue was obtained in accordance with the published procedure by Zhu Shirley: Total RNA isolation and purification for microarray (Gilks et al., 2005). The cerebral parts of experimental rats, striatum, hippocampus and prefrontal cortex, were obtained and stored in TRI reagent (Sigma-Aldrich, United States). The tissue was homogenized, centrifuged, washed with chloroform, and precipitated in isopropanol. Total RNA from the striatum, hippocampus and prefrontal cortex was used in the microarray and realtime PCR techniques.

Microarray Technique
The microarray technique was carried out using Genechip Rat Gene 2.0 ST Array (Affymetrix, United States) in the Genomics and Bioinformatics department, Institute of Molecular Genetics of the ASCR (Prague, Czechia). The procedure was carried out twice. Because of the limited resource only 6 properly isolated samples from striatum (from 3 METH and from 3 SAL rats) were examined to distinguish the mRNA expression level of 29489 genes by the microarray technique in the first trial. Also, because of the limited resource only 6 properly isolated samples from the hippocampus (from 3 METH and from 3 SAL rats) were Brain-derived neurotrophic factor is involved in the METH dependence (Ren et al., 2016(Ren et al., , 2017.

Nr3c1
Nuclear receptor subfamily 3, group C, member 1 Glucocorticoid receptor that binds and activates hormone-dependent transcriptional enhancers Glucocorticoid receptors are involved in METH-induced hyperactivity (Ago et al., 2009).

Esr1
Estrogen receptor 1 Acts as a transcriptional activator when bound to estrogen; may play a role in myocardial regulation The gene ESR1 may play a role in the pathophysiology of methamphetamine induced psychosis patients (Kishi et al., 2009) Igf1r Insulin-like growth factor 1 receptor Involved in induction of cell cycle progression and survival in many cell types Insulin like growth factor binding protein 5 is involved in METH-induced apoptosis (Leung et al., 2014).

Real-Time PCR Technique
Total RNA (70 ng) from 8 METH and 8 SAL rats was processed using TaqMan Gene Expression Assays (ThermoFisher Scientific, United States) and TaqMan Gene Expression Master Mix (ThermoFisher Scientific, United States) by real-time PCR in the 7500 Fast Real-Time PCR System (ThermoFisher Scientific, United States). Together 6 genes, Gapdh, Pgk1, 18sRNA, B2m, Rps6, PPIA (Bernaudin et al., 2002;Hori et al., 2005;Shilling et al., 2006;Romanowski et al., 2007;Langnaese et al., 2008;Gubern et al., 2009) were assessed for their potential as being suitable reference genes in all three types of tissue. Reference genes, Rps6 and B2m were chosen according to the best reproducible results observed for gene expression in all three of tissues (striatum, hippocampus, prefrontal cortex), and taking into account the published results of another scientific group (Shilling et al., 2006). A quantification of gene expression was calculated by the Pfaffl method (Pfaffl, 2001;Šmehilová, 2011); exponential amplifications factors of Assays were equal to 1 (ThermoFisher Scientific, United States). Differences were counted by the Mann-Whitney U-test.

RESULTS
The weights of SAL animals ranged from 285 to 360 g with a median of 310,5 g. The weights of METH animals ranged from 298 to 350 g with the median of 330 g. The animals were divided into boxes of four (1-4, 5-8).

Results of the First Microarray Trial in the Striatum
The mRNA expression (cDNA) of 29489 genes was examined in 6 striatum samples: three samples from the prenatally METHexposed rats and three samples from the prenatally SAL-exposed rats. The expression of approximately 2189 genes was either up-regulated or down-regulated (p < 0.05) in the striatum, however, the differences were very low. The borderline significant difference (p < 0.05 and logFC > 1) between METH and SAM samples was observed in the case of Drd3 and Tacr3 genes ( Table 4) (Zoubková, 2019a). The significant difference (p < 0.05 and logFC > 1) between METH and SAM samples was observed in the case of Cartpt gene, olfactory receptor 522 and 1448 genes ( Table 4) (Zoubková, 2019a). The non-significant difference between METH and SAM samples was observed in the case of Foxp2 and Rps6ka3 genes (Table 4) (Zoubková, 2019a).

Results of the Second Microarray the Trial in the Hippocampus
The mRNA expression (cDNA) of 29489 genes was examined in 6 hippocampus samples: three samples from the prenatally METHexposed rats and three samples from the prenatally SAL-exposed rats. The expression of approximately 1344 genes was either upregulated or down-regulated (p < 0.05) in the hippocampus, however, the differences were very low. The borderline significant difference (p < 0.05 and logFC > 1) between METH and SAM samples was observed in the case of Taar7h gene. The significant difference (p < 0.05 and logFC > 1) between METH and SAM samples was observed in the case of carboxylesterase 2i (Ces2) and olfactory receptor 1726 genes ( Table 5). The non-significant difference between METH and SAM samples was observed in the case of Tacr3 gene (Zoubková, 2019b).

Results of Real-Time PCR
For each of the tissue types (striatum, hippocampus and prefrontal cortex) the expression of chosen genes was examined using real-time PCR with TaqMan R Gene Expression Assays (ThermoFisher Scientific, United States). The measured values were normalized to the measured values of two reference genes, B2m and Rps6, and quantified by the Pfaffl method (calculation of ratio of target to reference gene). Significant differences between METH and SAM samples were evaluated by the Mann-Whitney U-test (Supplementary Table S1).

Results of Real-Time PCR in the Prefrontal Cortex
In the prefrontal cortex of prenatally METH-exposed rats, the mRNA expression of the Nr3c1 gene was significantly (p ≤ 0.01) increased compared to that in prenatally SALexposed rats (Figure 1). The measured values were normalized to the measured values of the reference B2m gene. However, the increase was not detected, when the measured values were normalized to the measured values of the reference Rps6 gene. The mRNA expression of the Foxp2 gene of prenatally METHexposed rats was significantly (p ≤ 0.05) decreased compared to that in prenatally SAL-exposed rats in the prefrontal cortex, when the measured values were normalized to the measured values of the reference Rps6 gene. However, no difference was observed, when the measured values were normalized to the measured values of the reference B2m gene.

Results of Real-Time PCR in the Striatum
In the striatum of prenatally METH-exposed rats, the mRNA expression of Tacr3 gene (p < 0.01) and dopamine receptor D3 gene (p < 0.05) were significantly increased compared to that in prenatally SAL-exposed rats. The measured values were normalized to the measured values of the reference B2m gene (Figures 2, 3). No increase however was observed, when the measured values were normalized to the measured values of the reference Rps6 gene.

Results of Real-Time PCR in the Hippocampus
No differences in the mRNA expression were observed for any of the genes between prenatally METH-exposed rats and prenatally SAL-exposed rats in the hippocampus, even if the measured values were normalized to the measured values of either Rps6 or B2m reference genes.

DISCUSSION
Using a real-time PCR technique we examined changes in gene expression in the striatum, hippocampus, and prefrontal cortex. Using microarray we examined the changes in gene expression in the striatum and hippocampus of prenatally METH-exposed rats. Despite our expectations, we found only three genes in the striatum from approximately 2189 up-regulated or downregulated genes, and found only two genes in the hippocampus from approximately 1344 up-regulated or down-regulated genes, whose mRNA expression were significantly altered (p < 0.05 and logFC > 1). In addition, according to our previous studies, particularly the striatum and hippocampus are structures that

Olfactory Receptors
The microarray technique demonstrated significant decrease in the mRNA expression of the genes encoding the olfactory receptors 522 and 1448 in the striatum of prenatally METHexposed rats. In the hippocampus of prenatally METHexposed rats the microarray technique demonstrated significant decrease in the mRNA expression of the olfactory receptor 1726. Given that the olfactory receptors are only present in the Rattus norvegicus species (NCIB database) we did not further investigate the decreased mRNA of olfactory gene expression in the striatum and hippocampus, as such studies in rats would not contribute to our understanding of addiction to METH in humans.

Ribosomal Protein S6
Ribosomal Protein S6 is a major substrate of protein kinases in eukaryotic ribosomes (NCBI gene database). Recent studies  concluded that cellular Ser/Thr kinase mTOR1 activation and rpS6 phosphorylation interrelated (Volovelsky et al., 2016;Huang et al., 2018). Activated mTORC1 complex initiates phosphorylation of rpS6 protein (Volovelsky et al., 2016;Huang et al., 2018), and METH decreases phosphorylation of mTORC1 and its downstream kinases (Liu et al., 2019). mTOR activation in the complexes with proteins, subsequent activation/phosphorylation of down-stream substrates (RSP6 protein) and METH-induced suppression of mTOR1 phosphorylation was also demonstrated in the nucleus accumbens (Huang et al., 2018). In summary, mTORC1 regulates cell proliferation through Rps6 phosphorylation and METH decreases phosphorylation of mTORC1. Given that RPS6 is related to METH metabolism, the results of analyses, where RPS6 is used as a reference gene, are questionable. Of note, the gene encoding Rps6ka3, which is Rps6 kinase, polypeptide 3, was one of 29489 up-regulated genes in the striatum in the first microarray trial.

Beta-2-Microglobulin
Beta-2-Microglobulin is a serum protein found in association with the major histocompatibility complex class I heavy chain on the surface of nearly all nucleated cells (Gussow et al., 1987;NCBI gene database). No differences in the expression of the B2m gene were observed in the striatum or in the hippocampus during the microarray trials. The B2m gene was therefore considered to be a suitable reference gene for use in the real-time PCR studies, when examining all three types of tissues in the prenatally METH-exposed rats.
The previous studies demonstrated that prenatal METH exposure can impair the development of the neonatal central nervous system (Williams et al., 2003;Šlamberová et al., 2006;Smith et al., 2008;Rambousek et al., 2014) and the developing brain seems to be one of the primary targets of developmental toxicities of amphetamine and methamphetamine (Thompson et al., 2009;Lloyd et al., 2013). There was evidence of oxidative DNA damage in embryonic and fetal brains of mice caused by amphetamines (Jeng et al., 2005;Wong et al., 2008). METH can be activated to free radical intermediates that initiate ROS formation and cause oxidative DNA damage in the brain (Jeng et al., 2006;Wong et al., 2008;Jeng and Wells, 2010;Ramkissoon and Wells, 2011). The damage leads to long-term postnatal neurodevelopmental deficits via a mechanism different from that underlying the neurodegeneration observed in METH-exposed adults (Jeng et al., 2005).

Altered Gene Expression in the Striatum: Cartpt, Drd3, Tacr3
Prenatal METH exposure induces abnormalities in the brain's structure and chemistry, especially in the striatum (Chang et al., 2007;Derauf et al., 2012). Won et al. (2001) described regional dependent distribution of METH in pup brains on gestational day 14 with the highest concentration in the striatum (Won et al., 2001;Rambousek et al., 2014). Cart peptide was detected in the rat nucleus accumbens and due to its distribution in the brain and its modulation of dopamine systems, may be involved in aspects of reward and drug abuse Kuhar et al., 2005). In our project, the microarray technique demonstrated a significant increase in the mRNA expression level of the gene coding Cart prepropeptide in the striatum of prenatally METH-exposed rats. The differences in mRNA expression between prenatally METH-exposed and prenatally SAL-exposed rats using microarray were very low. It appears that the dose 5 mg/kg/day of METH administered to the mothers during the gestation period might be too low to cause strong significant impairment in the development of the striatum, hippocampus and prefrontal cortex of the offspring. This may be the reason why we did not detect significant differences in the Cartpt gene expression using real-time PCR technique. However, when comparing our results to those of older studies, it must be pointed out that also acute administration of methamphetamine increased Cartpt mRNA level in the nucleus accumbens (Ogden et al., 2004;Salinas et al., 2006;Jean et al., 2007;Rogge et al., 2008). Kuhar and colleagues demonstrated that Cart peptides have a role in drug abuse by virtue of the fact that they are modulators of mesolimbic function . We can speculate that the mechanism of METH regulation of addiction is via the CREB phosphorylation pathway. METH self-administration causes enrichment of phosphorylated CREB (pCREB) (Krasnova et al., 2013) and CREB may activate Cartpt expression (Rogge et al., 2008), and biologically active Cart peptide plays a role in reward and addiction (Entrez PubMed, 2019a;GeneCards, 2019a).
It was previously reported that METH self-administration was associated with transcriptional changes in genes that regulate transcription, synaptic transmission, and synaptic plasticity in the dorsal striatum (Krasnova et al., 2013). These molecular changes are associated with the transition from recreational drug use to addiction (Volkow et al., 2001;Krasnova et al., 2013). Dopamine Receptor D3 has the highest binding affinity for endogenous dopamine of all known dopamine receptors (Sokoloff et al., 1990;O'Brien and Gardner, 2005) and shows preferential neuroanatomical localization in the limbic area (Bouthenet et al., 1991;Le'vesque et al., 1992;Levant, 1998;Diaz et al., 2000;O'Brien and Gardner, 2005). In our real-time PCR studies, the expression of the Drd3 gene in the striatum of METH exposed rats appeared to be significantly (p ≤ 0.05) increased compared to that in SAL exposed rats. Borderline significant increased mRNA expression of dopamine receptor D3 gene in the striatum was also observed in the first microarray study. Le Foll et al. (2005) reported that a single administration of METH induces a transient increase in BDNF expression in the prefrontal cortex, associated with a long-lasting elevation in dopamine receptor D3 binding and mRNA levels in the nucleus accumbens (Le Foll et al., 2005;Sokoloff et al., 2006). From cocaine use receptors D3 appeared to be up-regulated in the nucleus accumbens (Staley and Mash, 1996;Segal et al., 1997;Neisewander et al., 2004;O'Brien and Gardner, 2005). Given that the effects of METH are believed to result primarily from the increased release of synthesized dopamine and inhibition of dopamine uptake (O'Brien and Gardner, 2005;Chen et al., 2014) and that elevated extracellular concentrations of dopamine lead to increased stimulation of dopaminergic receptors (Robinson and Becker, 1986;Pierce and Kumaresan, 2006;Rambousek et al., 2014) we could speculate that the basis of Drd3 contribution to the addiction to METH is in its sensitivity. A normalization of dopamine receptor D3 function may reduce vulnerability to relapse in psychostimulant abuse (Le Foll et al., 2005;Chen et al., 2014), and a blockade of the dopamine receptor D3 by an antagonist attenuated the incentive motivational effects of METH in rats and may have pharmacotherapeutic potential in the treatment of methamphetamine addiction (Heidbreder et al., 2005;Chen et al., 2014). Tachykinin Receptor 3 gene (NK-3 receptor) expression was significantly (p ≤ 0.01) increased in the striatum of METH exposed rats compared to that in SAL exposed rats in our real-time PCR studies. Borderline significant increased mRNA expression of the Tacr3 gene in striatum was also detected in the microarrays trial. Of further note, we observed that the expression of the Tacr3 gene was also non-significantly up-regulated in METH hippocampus samples in our second microarray study. This result provides new evidence of METH and Tacr3 relation or interaction. The previous findings described the relation between METH neurotoxicity and neurokinin-1 (NK-1) receptor. The striatal neurokinin-1 receptors modulated METH-induced production of nitric oxide (Wang and Angulo, 2011). METH induces pre-and post-synaptic damage in the striatum and can be prevented with pharmacological blockade of the NK-1 receptor (Yu et al., 2004;Tulloch et al., 2011). It was also previously reported that the NK-3 receptor (Tacr3) controls nucleus accumbens dopamine responses to cocaine in rats (Jocham et al., 2007;Barros et al., 2013) and to alcohol in humans (Foroud et al., 2008). In monkeys, repeated administration of cocaine induces a decrease in methylation of NK3 receptor gene (De Souza Silva et al., 2006;Silva et al., 2008) and the NK3 (Tacr3) antagonist blocks the behavioral effects of cocaine Melamed et al., 2013). We can speculate that prenatal METH exposure, like cocaine, decreases the methylation status of the Tacr3 gene and increases the expression of the Tacr3 gene within striatum of rats. If the pharmacological blockage of tachykinin receptors 1 and 3 prevents the METH effects in the striatum, the Tacr3 and its gene is a suitable target for studying the addiction to METH.

Altered Gene Expression in the Hippocampus: Ces2, Taar7h
Previous studies demonstrated that prenatal METH exposure may be neurotoxic to the developing brain; and prenatally METH-exposed children exhibited smaller hippocampus volumes compared with the control group correlated with poorer performance on sustained attention and delayed verbal memory (Chang et al., 2004). METH exposure early in life of mice caused sex-dependent impairments in object recognition, spatial learning, and memory in adulthood (Acevedo et al., 2007). In experimental rats, prenatal MA exposure caused an impairment of non-spatial memory (Šlamberová et al., 2014). Our microarray technique detected significant differences in the expression of the Ces2 gene in the hippocampus of prenatally METH-and SAL-exposed rats. Ces2 is a member of a large multigene family. The enzymes encoded by these genes are responsible for the hydrolysis of ester-and amide-bond-containing drugs such as cocaine and heroin. They also hydrolyze long-chain fatty acid esters and thioesters (Lian et al., 2018;Entrez PubMed, 2019b;GeneCards, 2019b;Wikipedia contributors, 2019a). The products of hydrolysis are generally more polar than the original ester resulting in an increase in water solubility, promoting renal elimination (Laizure et al., 2013). Mice and rats have plasma carboxylesterase activity, which scavenge many organophosphorus compounds, rendering animals more resistant to organophosphates (Duysen et al., 2011;Marrero-Rosado et al., 2018). The human carboxylesterases are located in the cytoplasm and endoplasmic reticulum of numerous tissues, but the greatest quantities are found in the liver and small intestine where they contribute significantly to the first-pass metabolic hydrolysis of substrate drugs (Imai et al., 2003(Imai et al., , 2006Ross and Crow, 2007;Taketani et al., 2007;Hatfield et al., 2011;Laizure et al., 2013). Our result provides evidence about prenatal METH-induced decrease in the expression of the Ces2 mRNA within the hippocampus of experimental rats. We suspect that the changes in the gene expression level in the hippocampus associated with addiction to METH would be more significant, if the prenatally METH-exposed experimental rats were subjected to stress-inducing conditions, such as social behavior, learning and memory tests. However, we can speculate that decreased expression of the Ces2 enzyme could contribute to such METH-induced impairment of hippocampus in rats. It is interesting to note that there is evidence of a clinically significant drug interaction of a carboxylesterase substrate and ethanol in humans. Ethanol-mediated inhibition of human Ces2 hydrolysis of cocaine was demonstrated in hepatic microsomes (Roberts et al., 1993;Laizure et al., 2013). The finding is an in vitro proof of an existence of metabolic process or signaling pathway, which could inhibit the function of Ces2 in mouse and human cells.
Our microarray technique detected up-regulated expression of Taar 7h gene in the hippocampus of prenatally METH-exposed rats. The specific function of Taar7h and other members of a family of Taars has not been determined yet (Rutigliano et al., 2018). Taars are widely distributed throughout peripheral and brain tissues. Some Taars appear to be olfactory receptors, at least in rodents (Liberles and Buck, 2006;Rutigliano et al., 2018). The most effects have been reported on the Taar1 receptor. The previous experiments revealed Taar1 expression in many distinct rodent CNS regions, namely, nucleus accumbens/olfactory tubercle, as well as limbic and monoaminergic areas, such as hippocampus (Borowsky et al., 2001;Bunzow et al., 2001;Rutigliano et al., 2018). Taar1 is a novel G protein-coupled receptor, which responds to a number of endogenous amines, as well as to number psychoactive drugs, i.e., amphetamines. Such sensitization effects of Taar1 to dopamine, serotonin and norepinephrine could account for its involvement in the rewarding effects of drugs of abuse. Accordingly, Taar1-knock out mice developed an earlier and longer-lasting methamphetamine induced conditioned place preference as compared to wildtype littermates (Achat-Mendes et al., 2012;Rutigliano et al., 2018). In addition, Taar1-knock out mice show increased locomotor response to amphetamine and, in the opposite case, the mice overexpressing Taar1 exhibited a lower response to the stimulating effects of amphetamine in terms of locomotor activity and monoamine release (Rutigliano et al., 2018). These findings are important in the understanding of our result of the increased expression of Taar7 gene and of the previous finding that adult female rats in diestrus and adult males postnatally exposed to METH via breast milk had decreased locomotion and exploratory behavior .

Altered Expression in the Prefrontal Cortex: Foxp2, Nr3c1
Previous studies reported the prenatal METH-induced impairment of prefrontal cortex in experimental rats (Fujáková-Lipski et al., 2017;Hrebíčková et al., 2017) and in humans (Thompson et al., 2009). Mizoguchi and Yamada (2019) reported that patients addicted to methamphetamine exhibit impaired cognitive functions such as executive function, attention, social cognition, flexibility, and working memory (Mizoguchi and Yamada, 2019). From our studies we found that the expression of the Foxp2 gene was significantly (p ≤ 0.05) changed using real-time PCR and non-significantly changed using microarray technique in the prefrontal cortex of prenatally METH-exposed rats compared to the gene expression in prefrontal cortex of prenatally SAL-exposed rats. The data from real-time technique were normalized to the expression of the reference Rps6 gene. Given the fact that RPS6 phosphorylation is related to the metabolism of methamphetamine, the result from real-time PCR is questionable. However, the function of the neural learning system is modulated in the case of Foxp2 mutation (Chandrasekaran et al., 2015) and some cases of speech impairment were observed in prenatally METHexposed children in some clinical trials (Cernerud et al., 1996;Wouldes et al., 2004).
In response to various stimuli, including stress, glucocorticoids coordinate metabolic, endocrine, immune, and nervous system responses (Liu et al., 1997;Tronche et al., 1999). In the brain, the glucocorticoid receptor has been thought to modulate emotional behavior, cognitive functions, and addictive states (Tronche et al., 1999). Liu et al. (1997) studied neuroendocrine responses to stress in rats and observed that adult rats, the offspring of mothers that exhibited more licking and grooming of pups during the first 10 days of life, exhibited increased glucocorticoid receptor mRNA expression in the hippocampus (Liu et al., 1997). From our studies, no apparent significant increases in gene expression were observed in the hippocampus of prenatally METH-exposed rats. Our results from the real-time PCR trials demonstrated that the expression of the Nr3c1 gene was significantly (p ≤ 0.01) increased in the prefrontal cortex of prenatally METH-exposed rats compared to that in prenatally SAL-exposed rats. Champagne et al. (2003) and Weaver et al. (2004) studied neuroendocrine responses to stress and  studied neuroendocrine responses to childhood abuse and both confirmed that variations in the maternal behavior of pup-licking are associated with the methylation status of the Nr3c1 gene (Champagne et al., 2003;Weaver et al., 2004;. Future research should examine if prenatal METH exposure is able to alter the methylation status of the Nr3c1 gene. However, our finding is in accordance with findings reported by other scientific groups, that an increased motor activity and declined adaptability (Middaugh, 1989), change of behavior in an unknown environment, and an improvement of cognitive functions and anxiety (Schutová et al., 2008(Schutová et al., , 2012Šlamberová et al., 2015) are associated to METH in gestation. The glucocorticoid nuclear receptor Nr3c1 and its gene are suitable targets for studying the changes associated with addiction to METH.
The differences in the mRNA expression between prenatally METH-exposed and prenatally SAL-exposed rats using both techniques were very low. It appears that the dose 5 mg/kg/day of METH administered to the mothers during the gestation period might be too low to cause strong significant impairment in the development of the striatum, hippocampus and prefrontal cortex of the offspring. We have not demonstrated robust changes in the gene expression levels of prenatally METH-exposed rats. We isolated total RNA from adult rats on PD 80. It is possible that the changes in the gene expression just after birth could have been more robust since postnatally induced permanent behavioral changes have an impact on gene expression in the brain and can dramatically change prenatally adjusted a gene expression scenario that can disappear in adulthood.

CONCLUSION
The aims of our project were fulfilled. We have identified susceptible genes, candidates to the study of an impairment related to addiction to METH. Cartpt, Drd3, Tacr3 in the striatum and the Ces2 in the hippocampus have been found to be associated with addiction to METH. Future research should continue to confirm or refute our finding about an association between prenatal exposure to METH and the changes in the expression of Nr3c1 and Foxp2 genes in the prefrontal cortex. Further research is needed to confirm and investigate the novel finding of prenatally METH-induced decrease of Ces2 mRNA and prenatally METH-induced increase of Taar7h mRNA in the hippocampus. Our plan is to study the changes in the gene expression level in the striatum, hippocampus and prefrontal cortex of prenatally METH-exposed rats during the different stages of gestation. And in addition, we intend to study what genetic and epigenetic changes occur, when prenatally METHexposed rats are subjected to varying stressful conditions.

ETHICS STATEMENT
The study, the protocols and procedures with experimental animals were reviewed and approved by the Committee for Protection of Experimental Animals of Third faculty of Medicine and by Departmental Committee of Ministry of Education, Youth, and Sports. They are also in agreement with the Czech Government Requirements under the Policy of Humans Care of Laboratory Animals (no. 246/1992) and also comply with the regulations of the Ministry of Agriculture of the Czech Republic (no. 311/1997).

AUTHOR CONTRIBUTIONS
HZ isolated the RNA, chose the second group of the genes, carried out the real-time PCR experiments, analyzed data, and wrote the manuscript. AT participated in the real-time PCR experiments and in the data analysis. KN carried out the experiments with animals. MÈ participated in study design and coordination, chose the first group of the genes, and revised the manuscript. RŠ conceived the study, carried out the experiments with animals, and revised the manuscript. All authors approved the final version of the manuscript for submission.

FUNDING
This work was supported by the grant project 18-03806S from Grant Agency of the Czech Republic, by program PROGRES Q35 and 260388/SVV/2019 from Charles University, and project PharmaBrain CZ.02.1.01/0.0/0.0/16_025/0007444 funded from OP VVV.