{"paper_id":"21e9d878-ae7d-4288-b3ef-1f720828b136","body_text":"Methamphetamine exposure during the preweaning period alters hippocampal neurogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Methamphetamine exposure during the preweaning period alters hippocampal neurogenesis Barbora Čechová, Kristýna Patková, Ivana Fišerová, Šimon Vaculín, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5742375/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Methamphetamine (MA) is one of the most abused illicit drugs in the world. Abuse of this drug among adolescents, given the still-developing physiological functions of the body, including the central nervous system (CNS), is problematic. The hippocampus (HP) is part of the limbic system and is associated with cognition, as well as the reward system, which is mediated by dopamine (DA). Disruption of this system is the primary mechanism underlying addiction and results in cognition deficiencies. Methods: In this work, we focused on the influence of MA on the development of the rat HP during postnatal days 11–20. Subsequently, after MA application, we studied learning and memory processes using the Morris Water Maze (MWM). After the MWM, we measured the expression of the protein doublecortin (DCX), neurotransmitter levels of DA, GLU, 5HT, NA, and GABA, and glial fibrillary acidic protein (GFAP). Results: MA significantly impairs HP neurogenesis when administered during the third postnatal week, as indicated by reduced expression of DCX, which correlates with increased levels of glial fibrillary acidic protein (GFAP), but these changes were not significantly reflected in learning abilities and memory formation nor the levels of neurotransmitters. Conclusion: We speculate that these mechanisms must be strongly expressed on multiple molecular levels to be able to cause cognitive changes. A significant role in this process is associated with the ability of young organisms to compensate and, to a certain extent, neurotoxic effects. drug addiction development neurogenesis neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 BACKGROUND Methamphetamine (MA) is one of the most popular central nervous system (CNS) stimulants and one of the most abused illicit drugs in the world [ 1 ]. It is well known that MA causes the release of monoamine neurotransmitters, i.e., serotonin (5HT), dopamine (DA), and norepinephrine (noradrenaline-NA), as well as glutamate (GLU) and gamma-aminobutyric acid (GABA) [ 2 , 3 ] and a reversal in the action of their transporters [ 1 , 4 ]. Chronic use of MA leads to a strong physical addiction [ 5 ]. Drug addiction is a chronic disorder of the CNS whose main symptom is behavioral change. Altered neurotransmission of DA as a mediator of the reward pathway is crucial for addiction development as well as aberrant learning and memory [ 6 , 7 ]. These circuits underlie substance-driven rewarding effects, as well as the acquisition of drug-seeking behavior [ 8 ]. A crucial structure for contextual drug-related memory formation is the hippocampus (HP) [ 9 – 11 ]. The volume of HP tissue is decreased in MA users, and these volume deficits have been associated with poorer memory [ 12 ]. In rodents, HP neurons begin to develop on approximately embryonic day (ED) nine. By ED 15–18, all neurons have developed and migrated; however, some development continues until adolescence [ 13 – 15 ]. Neurogenesis in the HP can be downregulated by abuse of various drugs; however, this change can be partially restored by abstinence. Reduced HP neurogenesis may contribute to the development of addiction and may be a vulnerability factor for maintaining addiction-related behaviors [ 6 , 16 , 17 ]. Based on spatial learning and memory tests, the HP in rodents does not show signs of adult-like function until at least the end of the third postnatal week. This phenomenon can be explained by the fact that spatial learning and memory are dependent on well-developed motor and sensory systems [ 18 ]. Decreased HP neurogenesis caused by MA administration has been demonstrated by decreased numbers of doublecortin (DCX)-positive cells and other markers, which have also been reported in adult rats via self-administration, as well as short-term administration [ 19 , 20 ]. DCX is expressed in differentiating neurons and plays a crucial role in their migration and differentiation [ 21 ]. It is highly expressed in the brain during development, especially in structures that undergo continuous neurogenesis, such as the HP [ 21 ]. The administration of certain drugs of abuse has been shown to inhibit HP neurogenesis, which can then lead to cognitive impairments in drug users [ 12 ]. Also, prenatal MA exposure significantly alters the expression of DCX, resulting in decreasing numbers of DCX-positive cells in the HP of 1-day-old, as well as 22-day-old rodent pups [ 21 ]. Glutamatergic NMDA (N-methyl-D-aspartate) receptors mediate learning and memory formation in the HP. These receptors mediate synaptic changes underlying learning and memory and are widely expressed in the HP. Through these receptors, long-term potentiation occurs, which is the basis for the formation and preservation of memories [ 22 ]. During MA exposure, elevated cytosolic DA and oxidative stress can stimulate the synthesis of DA metabolites, such as dopaquinones, which can lead to microglial activation, as well as the secretion of high amounts of GLU. MA causes the activation of microglia and astrocytes in the striatum, cerebral cortex, as well as the HP [ 23 ]. DA metabolites are thought to be major activators of microglia through changes in microglial gene expression. Reactive gliosis induced by GLU release has been previously described in the context of MA mechanisms of action [ 24 , 25 ]. Communication between neurons and microglia is critical in brain development and maturation, as well as synaptic plasticity, learning, and memory [ 26 ]. Chronically activated microglia and astrocytes are linked to learning and memory deficits in various neuronal disorders [ 26 ]. Previous studies suggest that MA activates astrocytes, resulting in increased expression of glial fibrillary protein (GFAP) in both rodents and human brains [ 27 ]. Our study investigated the possible harmful effects of MA exposure during postnatal days (PDs) 11–20 when the HP is still developing. In rodents, this age corresponds to approximately 1–2 years in humans, since 13.8 rat days ≈ 1 human year [ 28 ]. It has been previously reported that MA exposure during this period causes some cognitive deficits and alterations in neurotransmission [ 29 , 30 ]. We assessed MA effects on learning and memory using the Morris Water Maze (MWM). Results of behavioral tests were correlated with neurotransmitter levels associated with cognition and the reward system (especially GLU, but also DA, NA, 5HT, and GABA). In addition, GFAP levels are analyzed to detect possible astrocyte activation resulting from reactive gliosis. Alterations in HP neurogenesis were determined by measuring levels of DCX as a marker of neurogenesis. METHODS Animals Adult female and male Wistar rats, bred by Charles River Laboratories International, Inc. and delivered by Velaz (Prague, Czech Republic), were used in experiments. Rats were housed in a temperature-controlled (22–24°C) room using a standard 12 h light/dark cycle with lights on at 6 am. Animals were left undisturbed for one week before fertility determination. Food and water were available ad libitum during this period. For determination of estrous cycle phase, female rats were smeared using vaginal lavage. During the estrous phase, females were housed overnight with sexually mature males. Determination of fertilization was performed by smearing females for the presence of sperm. On the day after birth, i.e., postnatal day (PD) 1, the number of pups in each litter was adjusted to 12–8 males and four females. Females were used in other experiments. Pups were randomly assigned to MA-treated (M), saline-treated (S), and absolute control (C) groups [ 31 , 32 ]. Finally, 10–12 animals per group were used for this study. Chemicals Physiological saline (0.9% NaCl, SA) and d-Methamphetamine hydrochloride (MA) were purchased from Sigma-Aldrich (Czech Republic). MA was dissolved in distilled water to a concentration of 5 mg/ml. Experimental design After birth, pups were left undisturbed with their mothers in standard cages. During PDs 11–20, pups were subcutaneously injected with MA (5 mg/ml/kg per day), while saline-treated (S) rats were given the same volume of saline. Administration of MA or saline was conducted once a day in the morning. Absolute control animals (C) were left undisturbed. Behavioral testing started twenty-four hours after the last MA exposure (i.e., on PD 20). As previously described in several studies conducted by our laboratory, a maximum of two animals from the original litter were used in any one experimental group. The rest of the animals were used in other experiments. Animals were acclimated (left undisturbed) to the room where Morris Water Maze testing was performed for 30 minutes before the start of each test; Fig. 1 shows the experimental design. Figure 1 shows the timeline of the experiment, respective PDs, and duration of individual procedures. This figure was created with BioRender.com. Morris Water Maze The Morris Water Maze (MWM) test was conducted to evaluate the spatial learning and memory abilities of the animals. The test consisted of three phases: Learning, Probe Test, and Memory Test. The standard protocol used by our laboratory and used in this study as well is described in detail in a study by Petriková et al., 2021 [ 31 ]. Learning (PD 21–26) MWM testing was performed in a pool with a diameter of 80 cm, which was chosen based on the age and size of the pups. The goal was to assess the animal's ability to learn the specific location of a hidden platform. The platform was placed in a fixed position, 1 cm below the water surface, making it invisible to the swimming rat. The maze was divided into four compass directions: north (N), south (S), east (E), and west (W), which created four quadrants. The platform was always located in the N-E quadrant. Animals were started from different random positions around the perimeter of the tank and had to find the hidden platform. Each rat performed six trials per day. The interval between trials was 30 seconds. If the rat could not find the platform within 60 seconds, it was manually guided to the platform and remained there for 30 seconds. Rat performance was tracked using a video-tracking system (EthoVision XT16) [ 31 ]. The Probe Test (PD 28) This test was conducted on the 8th day of the experiment. The platform was removed before the test. All rats started from the north (N) position, closest to the platform's position. The rats were allowed to swim freely in the maze for 60 seconds. This test aimed to assess the quadrant preference and spatial strategy used by the rats and evaluate their memory of the spatial map [ 31 ]. The Memory Test (PD 32) This test took place on the 12th day of the experiment. The hidden platform was placed in the same position as during the learning phase. Each rat performed six trials. Each trial lasted 60 seconds. The memory test determined if the rats could remember the position of the hidden platform [ 31 ]. Strategies monitored We monitored the same behavioral strategies, i.e., scanning and thigmotaxis, as in our previous studies [ 33 , 34 ]. Scanning Successful animals eventually develop a scanning strategy where they explore different areas of the pool methodically. They swim in a way that covers different regions of the pool, efficiently locating the hidden platform. This is a sign of good spatial learning and memory. Thigmotaxis In the context of the MWM, thigmotaxis refers to a behavioral strategy where animals tend to swim close to the walls of the maze rather than exploring the open, central area of the pool. This behavior is often considered a natural response that reduces anxiety or stress in unfamiliar and potentially dangerous situations. Sample collection and processing Twenty-four hours after the end of the MWM testing period, animals were anesthetized with an overdose of chloralhydrate (400 mg/kg, Sigma-Aldrich) and given an intracardial perfusion of heparinized saline. The HP was removed, snap-frozen on dry ice, and stored at − 80°C for further processing. Samples were divided for ELISA and Western Blott analysis. During processing, the samples for ELISA were homogenized in physiological saline containing 1 mM EDTA and 4 mM sodium metabisulfite (Sigma Aldrich) for a final concentration of 100 mg/ml. The homogenates were centrifuged for 10 minutes at 10,000 g in a cooled microcentrifuge; the supernatants were aliquoted and stored at − 80°C until assayed. For the Western Blot analysis, HPs were homogenized in ice-cold T-PER™ Tissue Protein Extraction Reagent (78510, Thermofisher Scientific) mixed with cOmplete™ Protease Inhibitor Cocktail (Roche) using a Bead Raptor 12 (Omni) bead homogenizer. Homogenates were sonicated, using a Q700 Sonicator (QSonica LLC, Newtown, CT, USA), for 5 min (30 s on/off cycle) at 4°C. Samples were centrifuged, and the supernatant was collected [ 35 ]. ELISA Levels of neurotransmitters and GFAP were measured with commercially available ELISA kits (LS-F11532-1 Rat GFAP (Sandwich ELISA) LS BIO; BA E-5500R, BA E-2400R BA E-5900R BA E-2500R, LDN Labor Diagnostika Nord GmbH & Co.KG, Germany); analyses were performed according to the manufacturer's instructions. The absorbance was read at 450 nm on an 800™ TS microplate Absorbance Reader (BioTek) [ 35 ]. Western Blot We used a Western blot protocol previously described by Zloh and Fiserova [ 36 , 37 ]. Following the homogenization process, protein quantification was conducted using a BCA assay. The processed samples were diluted to the desired concentration and combined with dithiothreitol (DTT) and a 4x Laemmli Sample Buffer (#1610747, Bio-Rad laboratories). To denature proteins, samples were heated to 95°C for 5 minutes. Subsequently, proteins were separated using SDS-PAGE on Mini-PROTEAN® TGXTM Precast Protein Gels with 15 wells (#4561086, Bio-Rad Laboratories, Hercules, CA, USA), and then transferred to a polyvinylidene fluoride (PVDF) membranes (Immun-Blot PVDF Membrane, Bio-Rad Laboratories). The membranes were blocked with Every Blot Blocking Buffer (Bio-Rad Laboratories). To detect the proteins of interest, PVDF membranes were incubated overnight at 4°C with the following rabbit monoclonal antibodies, all diluted 1:1000 in Every Blot Blocking buffer: α-Actinin (D6F6) XP® Rabbit mAb #6487 and Doublecortin (E6O6A) Rabbit mAb #91954 (Abcam). The next day, the membranes were washed with Tris-buffered saline containing Tween-20 and subsequently incubated with Goat anti-rabbit IgG H&L (HRP) (ab97051, Abcam) diluted 1:10,000 in Every Blot Blocking Buffer for one hour. Membrane images were captured using an Azure C300 Digital Imager and analyzed using AzureSpot software, version 14.2 (Azure Biosystems, Dublin, CA, USA). The amount of DCX in HP samples was then normalized against the levels of endogenous control α-Actinin [ 36 , 37 ]. Statistical analyses The following statistical tests were used in the three experimental phases of the MWM: Learning, one-way ANOVA with multilevel repeated measure (Days × Trials/day); Probe Test, one-way ANOVA; Memory Test, one-way ANOVA with repeated measure (Trials). The Tukey post-hoc test was used to compare groups. A one-way ANOVA was used for Western Blot and ELISA data, and the Tukey post-hoc test was used to compare groups. RESULTS Morris Water Maze test Learning In terms of swimming distance and velocity, there were no significant differences relative to treatment. In terms of latencies in finding the hidden platform during the learning task, the treatment had a significant impact [F (2, 94) = 6.009, p = 0.0035]. On day two, group S had increased latency compared to group M (p = 0.0026), as well as group C (p < 0.0001) (Fig. 2 ). Treatment also had a significant impact on search errors during the learning task [F (2, 94) = 4.185, p = 0.0181]. On day two, there were significantly more search errors in Group M compared to Group S (p < 0.0001) and Group C (p < 0.0001). Figure 2 A shows the amount of time needed to find the hidden platform over six days of learning. Figure 2 B shows the time spent in the quadrant where the platform was originally located (recall that in the Probe test, the platform was removed from the maze). Figure 2 C shows the time needed to find the hidden platform on the last day of testing, i.e., memory evaluation—differences between MA-treated and control animals: ***p < 0.001 (ANOVA and Tukey post-hoc test). Strategies used during learning Treatment had no significant effect with regard to thigmotaxis; however, there was a significant impact on the time spent using the scanning strategy, which varied during learning days [F (2, 94) = 4.409, p = 0.0148] (Fig. 3 B ) . On the first day, group M spent significantly less time using the scanning strategy compared to group S (p = 0.0002), and on day two, group M spent significantly more time using the scanning strategy compared to group C (p = 0.0001). On day four, group M spent significantly more time using the scanning strategy than group C (p = 0.0072); on day five, group M spent significantly more time using the scanning strategy compared to group S (p = 0.0001) and group C (p = 0.0001), and on day six, group M spent significantly less time using the scanning strategy than group C (p = 0.0001) and group S (p = 0.0121). ( Fig. 3 A ). Figure 3 A shows the time spent using the scanning strategy over six days of learning. Figure 3 B shows the time spent using the thigmotaxic strategy over six days of learning. Figure 3 C shows the time spent using the scanning strategy, and Fig. 3 D shows the time spent using the thigmotaxic strategy during the probe test. Figure 3 E shows the time spent using the scanning strategy, and Figure F shows the time spent using the thigmotaxic strategy during the memory test. Difference between MA-treated and control animals: **p < 0.01 (ANOVA and Tukey post-hoc test). Probe We saw no significant differences between groups during the Probe test on day eight (Fig. 3 C, D ). Memory Treatment had a significant effect on the distance traveled during the memory task [F (2, 141) = 4.760, p = 0.0100]; group S swam significantly further than group M (p = 0.0274) and group C (p = 0.0191). (Fig. 3 E, F ). Treatment also significantly affected swimming velocity during the memory task [F (2, 134) = 6.372, p = 0.0023]. Group M (p = 0.0487) and S (p = 0.0016) swam significantly faster than group C. With regard to latency, we saw no significant differences. Treatment did not significantly affect the use of scanning vs. thigmotaxis strategies (Fig. 3 ). Levels of GFAP in the hippocampus Treatment significantly impacted GFAP levels in the HP [F (2, 26) = 8.037, p = 0.002]. The lowest levels of GFAP were measured in group C relative to group M (p = 0.002) and group S (p = 0.020) ( Fig. 4 ). This chart displays GFAP levels in the HP. Levels of GFAP are expressed per wet weight of the HP. The data are presented as the mean ± SEM (n = 6 animals); *p < 0.05, **p < 0.01 (ANOVA and Tukey post-hoc test). Levels of neurotransmitters in the hippocampus There were no statistical differences in the levels of DA [F (2, 27) = 0.332, p = 0.720] (Fig. 5 A ) , NA [F (2, 27) = 1.040, p = 0.3670] (Fig. 5 B ) , GABA [F (2, 27) = 0.607, p = 0.552] (Fig. 5 C ) , GLU [F (2, 27) = 0.413, p = 0.665] (Fig. 5 D ) , or 5HT [F (2, 26) = 0.135, p = 0.873] (Fig. 5 E ). Figure three shows levels of neurotransmitters in the hippocampus. Levels of neurotransmitters are expressed per wet weight of the HP. The data are presented as the mean ± SEM (n = 6). Levels of doublecortin in the hippocampus Levels of doublecortin were significantly affected by treatment [F (2, 9) = 8.250, p = 0.009]; levels were significantly lower in group M compared to group C (p = 0.007 (Fig. 6 ). Figure 6 A shows the protein expression of DCX in the hippocampus of MA-treated, saline-treated, and control animals, and Fig. 6 B shows the representative Western Blot images. The data are presented as the mean ± SEM (n = 5). The difference between MA-treated and control animals: **p < 0.01 (ANOVA and Tukey post-hoc test). DISCUSSION The present study is focused on the effect of MA exposure during the third postnatal week (PD 11–20), which contrasts with our previous studies that investigated the effect of MA administered during prenatal and early postnatal periods. The third postnatal week is the developmental period when the HP fully matures, making it vulnerable to external influences. Therefore, we hypothesized that MA exposure during this specific period could affect HP neurogenesis, neurotransmitter levels, activation of astrocytes, learning, and memory. Knowing the effect of MA exposure during this time will extend our knowledge of the long-term effects of MA on rat pups during development. Learning and memory impairments Several studies deal with the effect of MA on memory and learning. Previous reports by Vorhees et al. and Daberkow et al. uggest that MA exposure during gestation and postnatal development in rats significantly alters learning and memory abilities [ 38 – 41 ]. MA-induced cognitive impairments found by Vorhees et al. and Ye et al. indicate that at the preclinical level, MA may lead to long-term impairments in a variety of cognitive domains [ 40 , 42 ]. In our study, the MWM test did not uncover any MA-induced differences in memory and learning ability, except for smaller swimming distances in animals injected with MA and SA compared to the absolute controls (C). These results are partially in agreement with a study by Joca et al. (2014), which showed no significant impairment in cognitive functions in MWM and object recognition tests after exposure to MA during early adolescence in mice (4 × 7.5 mg/kg on PD 30 and 31)[ 43 ]. In a study by North et al. (2013), adolescent mice showed impairments in short-term memory and altered HP plasticity after repeated high exposures to MA (24 mg/kg/day for 14 days). North et al. also observed that after 7, 14, and 21 days of abstinence, MA-exposed mice exhibited a deficit in spatial memory and decreased HP plasticity [ 44 ]. These varied and conflicting results indicate the importance of the timing of MA administration as well as the administered dose of the drug. Also of importance are the different results in several parameters between saline-treated and absolute control animals. One of our previous studies suggests that not only the drug but also the actual single injection can affect the behavior of laboratory animals [ 45 ]. This fact is very important and points to the necessity of using absolute controls in these types of studies. Studies of humans addicted to MA revealed that behavioral impairments are replicated in laboratory models, including repeated exposure to high doses of MA and self-administration of the drug over a prolonged period. These experimental paradigms, such as those described by Ito, Rogers ), and Recinto et al., result in cognitive dysfunction and memory impairments linked to hippocampus dysfunction[ 46 , 47 ]. Neurodegeneration and neurotoxicity in the HP are also seen in animal models of binge MA exposure and MA self-administration [ 48 ]. This suggests that there is a direct relationship between the toxic effects of MA and the resulting behavioral deficits caused by MA use [ 49 ]. Further mechanistic investigations demonstrated that exposure to MA induced changes in the adaptability and physical composition of neurons in the hippocampus. As an illustration, the administration of MA in a concentrated and widespread manner decreases synaptic strength in CA1 pyramidal neurons associated with high-frequency stimulation between specific brain cells (known as long-term potentiation). This effect is achieved by activating D1 receptors and simultaneously increasing the initial level of communication between the neurons [ 50 ]. GFAP levels are elevated in MA-exposed animals Significantly higher GFAP levels in the HP of MA-exposed animals in comparison to control animals are present in our study. GFAP is a protein produced by reactive astrocytes and can be a signaling mechanism for neural inflammation [ 51 ]. Our results agree with previous studies, such as the study by McConnell et al. (2015), who reported microglial activation after MA exposure in mice [ 52 ]. A study by Sheng et al. (1994) revealed that MA causes loss of DA neurons and an increase in reactive gliosis, which is indicated by increased numbers of cells stained with GFAP antibodies [ 53 ]. Lau et al. (2000) showed that astrocytes cultured from different brain regions reacted differently to MA-induced oxidative stress formation. The most severe effect was seen in striatum astrocytes, which suggests that these astrocytes are more sensitive to oxidative stress induced by MA [ 54 ]. A comparative study by Thomas et al. (2004) showed that MA as well as MDMA (-3,4-methylenedioxymethamphetamine) administered 4 times at 2-hour intervals significantly increased activation of microglia in rodent brains [ 55 ]. Microglia activation has been previously reported in several rodent and human studies. However, unlike our current study, these studies were performed on adult animals [ 52 ]. A study by Peng et al. (2024) examined the role of TREM2 in MA-induced neuroinflammation using BV2 cells, wild-type C57BL/6J mice, CX3CR1GFP/+ transgenic mice, and TREM2 knockout mice [ 56 ]. Postmortem samples from the frontal cortex of individuals with a history of MA use were analyzed to assess TREM2, TLR4, IBA1, and IL-1β levels. TREM2, TLR4, IBA1, IL-1β, iNOS, and Arg-1 expression levels were assessed in BV2 cells and the frontal cortexes of both mice and human MA users. The findings indicated that expression levels of TREM2, TLR4, IBA1, and IL-1β were significantly increased in the BV2 cells of individuals using MA. Microglia exhibited clear activation in the frontal cortex of WT C57BL/6 mice and CX3CR1GFP/+ transgenic mice. Elevated protein levels of IBA1, TREM2, TLR4, and IL-1β were also observed in METH-induced mouse models. Further, TREM2-KO mice exhibited heightened microglial activation, neuroinflammation, and excitotoxicity resulting from MA exposure. These findings indicate that TREM2 could serve as a target for the regulation of MA-induced neuroinflammation [ 56 ]. Methamphetamine decreases DCX expression in the hippocampus In our study, we saw significantly lower levels of expression of DCX in MA-exposed animals compared to group S and group C (i.e., controls). This phenomenon may be caused by decreased growth of new progenitor neural cells in the HP. This result agrees with previous studies, suggesting decreased HP neurogenesis after various MA administration protocols [ 19 , 20 ]. Mutations in this protein can cause defective neuronal migration; additionally, it has been reported that the absence of DCX causes imminent depletion of the progenitor pool during cortical development [ 57 , 58 ]. DCX is highly expressed on neural axons and may regulate microtubules in response to extracellular signals during development [ 59 , 60 ]. Studies have shown that DCX is expressed in various regions of the developing nervous system and is highly expressed in newly produced HP cells. This protein has been adopted as a marker for neuronal precursors and migrating neuroblasts during adult neurogenesis; it detects only newly generated neurons, hence its specificity [ 61 ]. Several studies also indicate that ablation of neurogenesis during adolescence is associated with impaired MWM performance in adulthood as well as with activation of microglia and neuroinflammation [ 62 , 63 ]. An extensive study by Galinato reported that cognitive impairments observed in individuals who abuse MA could be related to changes in the structure and function of HP neurons caused by MA use [ 48 ]. Human imaging studies have demonstrated that chronic MA users have reduced hippocampal volume, specifically in the gray matter, and decreased responsiveness of the hippocampus. These findings, reported by Thompson et al. (2004), Schwartz et al. (2010), and many others, suggest that MA-exposed individuals have impaired hippocampal networking [ 12 , 64 ]. Analysis of human brain tissue after death demonstrates that long-term MA use causes damage to the hippocampus, as shown in studies by Kitamura in 2009 and Kitamura et al. in 2010 [ 65 , 66 ]. This indicates a link between hippocampal dysfunction and toxicity in individuals addicted to MA. A study by Choi et al. (2018) aimed to examine the impact of MA on structural modifications and gene expression in the HP [ 67 ]. The HP demonstrated notable volumetric shrinkage in response to both acute and chronic MA compared to the HP of control subjects. The genes linked to cytoskeleton organization and phagocytosis were downregulated in the acute MA-treated cohort relative to the control group. Conversely, genes related to synaptic transmission, neuron differentiation regulation, and neurogenesis regulation were downregulated in the chronic MA-treated cohort. Our study verified that the expression patterns of ADM, BMP4, CHRD, PDYN, UBA1, profilin 2 (PFN2), ENO2, and NSE mRNAs were consistent with the findings from RNA-Seq, as determined by quantitative RT-PCR. Specifically, the expression levels of PFN2 mRNA and protein, which are crucial for actin cytoskeleton dynamics, were diminished by both acute and chronic MA treatment [ 67 ]. Neurotransmitter levels were not significantly affected by MA Our study found no significant differences in levels of DA between our test groups; however, it is well established that MA exposure profoundly alters DA transmission [ 1 ], so this result was surprising. Then again, there are insufficient studies examining MA effects in adolescents or developing animals to draw conclusions. The relative resistance of the DA system to MA in the brains of adolescent rodents was previously reported by Kokoshka et al. (2000); however, the mechanism underlying this phenomenon is still unknown [ 68 ]. Furthermore, concentrations of other neurotransmitters, such as 5HT, GABA, and NA, were also not significantly affected by MA. Levels of 5HT slightly decreased in MA-exposed animals, while NA levels slightly increased in MA-exposed animals. In vitro studies indicate that MA is twice as potent at releasing NA as DA, and its effect is 60-fold greater on NA than 5HT release [ 69 ]. Therefore, we speculate that our results are due to the MA dose being too low to evoke significant changes. It might also be that the period of MA exposure was too short to increase or decrease the function of these neurotransmitters. Additionally, in developing animals, there may be several processes, such as brain plasticity, which suppress the neurotoxic effects of MA on neurotransmitter systems. This is, however, our speculation, which needs to be confirmed with additional studies. A tendency towards decreased GLU levels in MA-exposed animals compared to control animals was present in our study. These results correlated with performance in the probe and memory tests, where MA-exposed animals showed signs of decreasing tendency as well. Even though these differences were insignificant, the correlation between lower GLU levels and poorer memory in MA-exposed animals does not appear to be a coincidence. Something similar was seen in a study by Yamamoto et al. (1999) that found that GLU release from the HP was decreased in MA-sensitized rats compared to control; additionally, a study by Hori et al. (2010) showed that MA decreased GLU release from nerve terminals of the Shaeffer collateral [ 70 , 71 ]. However, some aforementioned studies also found increased GLU release after MA exposure [ 23 ]. CONCLUSION Our study revealed that MA exposure during the third postnatal week in laboratory rats significantly induced levels of glial fibrillary acidic protein, which is a marker of reactive gliosis. The expression of doublecortin, a marker of neurogenesis inhibition, was also found to be reduced, although neither the behavioral (MWM) test nor neurotransmitter levels were affected. These results may be explained by the hypothesis that the molecular mechanisms of MA toxicity start much earlier, i.e., at the level of reduced HP neurogenesis and microglial activation. Nonetheless, the process is not strong enough to overcome the mechanisms of neuroplasticity and development and cause significant changes in cognitive functions or neurotransmitter levels. Changes in the markers of neurogenesis and microglial activation may also have multiple developmental causes. Declarations Ethics approval The procedures used in this study were reviewed and approved by the Institutional Animal Care and Use Committee and met the requirements of the Czech Government put forth under the Policy of Humans Care of Laboratory Animals (No. 86/609/EEC) and the subsequent regulations of the Ministry of Agriculture of the Czech Republic. Availability of data and materials The data supporting the findings of this study are available from the corresponding author upon reasonable request. Conflicts of interests The authors declare no conflicts of interest. Funding This research was supported by the research program Cooperatio Neurosciences from Charles University, grant project GA 21-30795S from the Grant Agency of the Czech Republic, and project PharmaBrain CZ.02.1.01/0.0/0.0/16_025/0007444 by OPVVV. Consent to Publish declaration Not applicable Authors’ contributions BČ is a doctoral student responsible for all experimental procedures, statistical analysis, and the manuscript. RŠ is a supervisor of BČ and the head of the department and laboratory where this study was conducted. KP, IF, and ŠV were responsible for drug exposure, laboratory analyses, animal manipulations, and sample collections. All authors were involved in all parts of the present study. All authors contributed to the article and approved the submitted version. Acknowledgments The authors would like to express deep gratitude to Zuzana Ježdíková for her excellent technical assistance. Additionally, we want to thank Mgr. Miloslav Zloh for assistance with Western Blot analyses, Dr. Ivana Petríková, Dr. Anna Mikulecká, and Dr. Kateryna Nohejlová for help with setting up and analyzing data from the Morris water maze. The authors also express their appreciation to Thomas Secrest for editing the manuscript. References Čechová B, Šlamberová R. Methamphetamine, neurotransmitters and neurodevelopment. Physiol Res. 2021;70(S3):S301–15. Der-Ghazarian TS, Charmchi D, Noudali SN, Scott SN, Holter MC, Newbern JM, et al. Neural Circuits Associated with 5-HT(1B) Receptor Agonist Inhibition of Methamphetamine Seeking in the Conditioned Place Preference Model. ACS Chem Neurosci. 2019;10(7):3271–83. Jaehne EJ, Semaan H, Grosman A, Xu X, Schwarz Q, van den Buuse M. Enhanced methamphetamine sensitisation in a rat model of the brain-derived neurotrophic factor Val66Met variant: Sex differences and dopamine receptor gene expression. Neuropharmacology. 2023;240:109719. Buck JM, Morris AS, Weber SJ, Raber J, Siegel JA. Effects of adolescent methamphetamine and nicotine exposure on behavioral performance and MAP-2 immunoreactivity in the nucleus accumbens of adolescent mice. Behav Brain Res. 2017;323:78–85. Courtney KE, Ray LA. Methamphetamine: an update on epidemiology, pharmacology, clinical phenomenology, and treatment literature. Drug Alcohol Depend. 2014;143:11–21. Cleva RM, Gass JT, Widholm JJ, Olive MF. Glutamatergic targets for enhancing extinction learning in drug addiction. Curr Neuropharmacol. 2010;8(4):394–408. Hyman SE. Addiction: a disease of learning and memory. Am J Psychiatry. 2005;162(8):1414–22. Barria A, Malinow R. Subunit-specific NMDA receptor trafficking to synapses. Neuron. 2002;35(2):345–53. Koob GF, Volkow ND. Neurobiology of addiction: a neurocircuitry analysis. Lancet Psychiatry. 2016;3(8):760–73. Sartor GC, Aston-Jones G. Post-retrieval extinction attenuates cocaine memories. Neuropsychopharmacology. 2014;39(5):1059–65. Torregrossa MM, Corlett PR, Taylor JR. Aberrant learning and memory in addiction. Neurobiol Learn Mem. 2011;96(4):609–23. Thompson PM, Hayashi KM, Simon SL, Geaga JA, Hong MS, Sui Y, et al. Structural abnormalities in the brains of human subjects who use methamphetamine. J Neurosci. 2004;24(26):6028–36. Cossart R, Khazipov R. How development sculpts hippocampal circuits and function. Physiol Rev. 2022;102(1):343–78. Semple BD, Blomgren K, Gimlin K, Ferriero DM, Noble-Haeusslein LJ. Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species. Prog Neurobiol. 2013;106–107:1–16. Kozareva DA, Cryan JF, Nolan YM. Born this way: Hippocampal neurogenesis across the lifespan. Aging Cell. 2019;18(5):e13007. Jun H, Mohammed Qasim Hussaini S, Rigby MJ, Jang MH. Functional role of adult hippocampal neurogenesis as a therapeutic strategy for mental disorders. Neural Plast. 2012;2012:854285. Karila L, Petit A, Lowenstein W, Reynaud M. Diagnosis and consequences of cocaine addiction. Curr Med Chem. 2012;19(33):5612–8. Albani SH, McHail DG, Dumas TC. Developmental studies of the hippocampus and hippocampal-dependent behaviors: insights from interdisciplinary studies and tips for new investigators. Neurosci Biobehav Rev. 2014;43:183–90. Mandyam CD, Wee S, Crawford EF, Eisch AJ, Richardson HN, Koob GF. Varied access to intravenous methamphetamine self-administration differentially alters adult hippocampal neurogenesis. Biol Psychiatry. 2008;64(11):958–65. Kochman LJ, Fornal CA, Jacobs BL. Suppression of hippocampal cell proliferation by short-term stimulant drug administration in adult rats. Eur J Neurosci. 2009;29(11):2157–65. Jalayeri-Darbandi Z, Rajabzadeh A, Hosseini M, Beheshti F, Ebrahimzadeh-Bideskan A. The effect of methamphetamine exposure during pregnancy and lactation on hippocampal doublecortin expression, learning and memory of rat offspring. Anat Sci Int. 2018;93(3):351–63. Che X, Bai Y, Cai J, Liu Y, Li Y, Yin M, et al. Hippocampal neurogenesis interferes with extinction and reinstatement of methamphetamine-associated reward memory in mice. Neuropharmacology. 2021;196:108717. Shrestha P, Katila N, Lee S, Seo JH, Jeong JH, Yook S. Methamphetamine induced neurotoxic diseases, molecular mechanism, and current treatment strategies. Biomed Pharmacother. 2022;154:113591. McDonnell-Dowling K, Kelly JP. The Role of Oxidative Stress in Methamphetamine-induced Toxicity and Sources of Variation in the Design of Animal Studies. Curr Neuropharmacol. 2017;15(2):300–14. Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 2009;32(12):638–47. Cornell J, Salinas S, Huang HY, Zhou M. Microglia regulation of synaptic plasticity and learning and memory. Neural Regen Res. 2022;17(4):705–16. Friend DM, Keefe KA. Glial reactivity in resistance to methamphetamine-induced neurotoxicity. J Neurochem. 2013;125(4):566–74. Sengupta P. The Laboratory Rat: Relating Its Age With Human's. Int J Prev Med. 2013;4(6):624–30. Crawford CA, Williams MT, Newman ER, McDougall SA, Vorhees CV. Methamphetamine exposure during the preweanling period causes prolonged changes in dorsal striatal protein kinase A activity, dopamine D2-like binding sites, and dopamine content. Synapse. 2003;48(3):131–7. Graham DL, Amos-Kroohs RM, Braun AA, Grace CE, Schaefer TL, Skelton MR, et al. Neonatal +-methamphetamine exposure in rats alters adult locomotor responses to dopamine D1 and D2 agonists and to a glutamate NMDA receptor antagonist, but not to serotonin agonists. Int J Neuropsychopharmacol. 2013;16(2):377–91. Petríková-Hrebiíčková I, Ševčiková M, Šlamberová R. The Impact of Neonatal Methamphetamine on Spatial Learning and Memory in Adult Female Rats. Front Behav Neurosci. 2021;15:629585. Šlamberová R, Mikulecká A, Pometlová M, Schutová B, Hrubá L, Deykun K. Sex differences in social interaction of methamphetamine-treated rats. Behav Pharmacol. 2011;22(7):617–23. Holubová A, Lukášková I, Tomášová N, Šuhajdová M, Šlamberová R. Early Postnatal Stress Impairs Cognitive Functions of Male Rats Persisting Until Adulthood. Front Behav Neurosci. 2018;12:176. Macuchova E, Nohejlova K, Sevcikova M, Hrebickova I, Slamberova R. Sex differences in the strategies of spatial learning in prenatally-exposed rats treated with various drugs in adulthood. Behav Brain Res. 2017;327:83–93. Čechová B, Mihalčiková L, Vaculín Š, Šandera Š, Šlamberová R. Levels of BDNF and NGF in adolescent rat hippocampus neonatally exposed to methamphetamine along with environmental alterations. Physiol Res. 2023;72(S5):S559–71. Fišerová I, Trinh MD, Elkalaf M, Vacek L, Heide M, Martinková S, et al. Isoprenaline modified the lipidomic profile and reduced β-oxidation in HL-1 cardiomyocytes: In vitro model of takotsubo syndrome. Front Cardiovasc Med. 2022;9:917989. Zloh M, Kutilek P, Štofková A. High-Contrast Stimulation Potentiates the Neurotrophic Properties of Muller Cells and Suppresses Their Pro-Inflammatory Phenotype. Int J Mol Sci. 2022;23(15). Daberkow DP, Kesner RP, Keefe KA. Relation between methamphetamine-induced monoamine depletions in the striatum and sequential motor learning. Pharmacol Biochem Behav. 2005;81(1):198–204. Vorhees CV, Inman-Wood SL, Morford LL, Broening HW, Fukumura M, Moran MS. Adult learning deficits after neonatal exposure to D-methamphetamine: selective effects on spatial navigation and memory. J Neurosci. 2000;20(12):4732–9. Vorhees CV, Reed TM, Morford LL, Fukumura M, Wood SL, Brown CA, et al. Periadolescent rats (P41-50) exhibit increased susceptibility to D-methamphetamine-induced long-term spatial and sequential learning deficits compared to juvenile (P21-30 or P31-40) or adult rats (P51-60). Neurotoxicol Teratol. 2005;27(1):117–34. Vorhees CV, Skelton MR, Williams MT. Age-dependent effects of neonatal methamphetamine exposure on spatial learning. Behav Pharmacol. 2007;18(5–6):549–62. Ye T, Pozos H, Phillips TJ, Izquierdo A. Long-term effects of exposure to methamphetamine in adolescent rats. Drug Alcohol Depend. 2014;138:17–23. Joca L, Zuloaga DG, Raber J, Siegel JA. Long-term effects of early adolescent methamphetamine exposure on depression-like behavior and the hypothalamic vasopressin system in mice. Dev Neurosci. 2014;36(2):108–18. North A, Swant J, Salvatore MF, Gamble-George J, Prins P, Butler B, et al. Chronic methamphetamine exposure produces a delayed, long-lasting memory deficit. Synapse. 2013;67(5):245–57. Šlamberová R, Nohejlová K, Ochozková A, Mihalčíiková L. What is the role of subcutaneous single injections on the behavior of adult male rats exposed to drugs? Physiol Res. 2018;67(Suppl 4):S665–72. Recinto P, Samant AR, Chavez G, Kim A, Yuan CJ, Soleiman M, et al. Levels of neural progenitors in the hippocampus predict memory impairment and relapse to drug seeking as a function of excessive methamphetamine self-administration. Neuropsychopharmacology. 2012;37(5):1275–87. Rogers JL, De Santis S, See RE. Extended methamphetamine self-administration enhances reinstatement of drug seeking and impairs novel object recognition in rats. Psychopharmacology. 2008;199(4):615–24. Galinato MH, Takashima Y, Fannon MJ, Quach LW, Morales Silva RJ, Mysore KK, et al. Neurogenesis during Abstinence Is Necessary for Context-Driven Methamphetamine-Related Memory. J Neurosci. 2018;38(8):2029–42. Mandyam CD, Wee S, Eisch AJ, Richardson HN, Koob GF. Methamphetamine self-administration and voluntary exercise have opposing effects on medial prefrontal cortex gliogenesis. J Neurosci. 2007;27(42):11442–50. Swant J, Chirwa S, Stanwood G, Khoshbouei H. Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus. PLoS ONE. 2010;5(6):e11382. Clark KH, Wiley CA, Bradberry CW. Psychostimulant abuse and neuroinflammation: emerging evidence of their interconnection. Neurotox Res. 2013;23(2):174–88. McConnell SE, O'Banion MK, Cory-Slechta DA, Olschowka JA, Opanashuk LA. Characterization of binge-dosed methamphetamine-induced neurotoxicity and neuroinflammation. Neurotoxicology. 2015;50:131–41. Sheng P, Cerruti C, Cadet JL. Methamphetamine (METH) causes reactive gliosis in vitro: attenuation by the ADP-ribosylation (ADPR) inhibitor, benzamide. Life Sci. 1994;55(3):PL51–4. Lau JW, Senok S, Stadlin A. Methamphetamine-induced oxidative stress in cultured mouse astrocytes. Ann N Y Acad Sci. 2000;914:146–56. Thomas DM, Dowgiert J, Geddes TJ, Francescutti-Verbeem D, Liu X, Kuhn DM. Microglial activation is a pharmacologically specific marker for the neurotoxic amphetamines. Neurosci Lett. 2004;367(3):349–54. Peng Y, Yang G, Wang S, Lin W, Zhu L, Dong W, et al. Triggering Receptor Expressed on Myeloid Cells 2 Deficiency Exacerbates Methamphetamine-Induced Activation of Microglia and Neuroinflammation. Int J Toxicol. 2024;43(2):165–76. Gleeson JG, Lin PT, Flanagan LA, Walsh CA. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron. 1999;23(2):257–71. Ayanlaja AA, Xiong Y, Gao Y, Ji G, Tang C, Abdikani Abdullah Z, et al. Distinct Features of Doublecortin as a Marker of Neuronal Migration and Its Implications in Cancer Cell Mobility. Front Mol Neurosci. 2017;10:199. Tint I, Jean D, Baas PW, Black MM. Doublecortin associates with microtubules preferentially in regions of the axon displaying actin-rich protrusive structures. J Neurosci. 2009;29(35):10995–1010. Weimer JM, Anton ES. Doubling up on microtubule stabilizers: synergistic functions of doublecortin-like kinase and doublecortin in the developing cerebral cortex. Neuron. 2006;49(1):3–4. von Halbach B. Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus. Cell Tissue Res. 2011;345(1):1–19. Achanta P, Fuss M, Martinez JL. Jr. Ionizing radiation impairs the formation of trace fear memories and reduces hippocampal neurogenesis. Behav Neurosci. 2009;123(5):1036–45. Rola R, Raber J, Rizk A, Otsuka S, VandenBerg SR, Morhardt DR, et al. Radiation-induced impairment of hippocampal neurogenesis is associated with cognitive deficits in young mice. Exp Neurol. 2004;188(2):316–30. Schwartz DL, Mitchell AD, Lahna DL, Luber HS, Huckans MS, Mitchell SH, et al. Global and local morphometric differences in recently abstinent methamphetamine-dependent individuals. NeuroImage. 2010;50(4):1392–401. Kitamura O. Detection of methamphetamine neurotoxicity in forensic autopsy cases. Leg Med (Tokyo). 2009;11(Suppl 1):S63–5. Kitamura O, Takeichi T, Wang EL, Tokunaga I, Ishigami A, Kubo S. Microglial and astrocytic changes in the striatum of methamphetamine abusers. Leg Med (Tokyo). 2010;12(2):57–62. Choi MR, Chun JW, Kwak SM, Bang SH, Jin YB, Lee Y, et al. Effects of acute and chronic methamphetamine administration on cynomolgus monkey hippocampus structure and cellular transcriptome. Toxicol Appl Pharmacol. 2018;355:68–79. Kokoshka JM, Fleckenstein AE, Wilkins DG, Hanson GR. Age-dependent differential responses of monoaminergic systems to high doses of methamphetamine. J Neurochem. 2000;75(5):2095–102. Rothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI, et al. Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse. 2001;39(1):32–41. Hori N, Kadota MT, Watanabe M, Ito Y, Akaike N, Carpenter DO. Neurotoxic effects of methamphetamine on rat hippocampus pyramidal neurons. Cell Mol Neurobiol. 2010;30(6):849–56. Yamamoto H, Kitamura N, Lin XH, Ikeuchi Y, Hashimoto T, Shirakawa O, et al. Differential changes in glutamatergic transmission via N-methyl-D-aspartate receptors in the hippocampus and striatum of rats behaviourally sensitized to methamphetamine. Int J Neuropsychopharmacol. 1999;2(3):155–63. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5742375\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":398274589,\"identity\":\"61519262-e3a0-4488-92ac-217624398651\",\"order_by\":0,\"name\":\"Barbora Čechová\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Charles University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Barbora\",\"middleName\":\"\",\"lastName\":\"Čechová\",\"suffix\":\"\"},{\"id\":398274590,\"identity\":\"670e6f57-e3fb-4bf8-a373-7608f514ed94\",\"order_by\":1,\"name\":\"Kristýna 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stimulants and one of the most abused illicit drugs in the world [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. It is well known that MA causes the release of monoamine neurotransmitters, i.e., serotonin (5HT), dopamine (DA), and norepinephrine (noradrenaline-NA), as well as glutamate (GLU) and gamma-aminobutyric acid (GABA) [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e] and a reversal in the action of their transporters [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eChronic use of MA leads to a strong physical addiction [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Drug addiction is a chronic disorder of the CNS whose main symptom is behavioral change. Altered neurotransmission of DA as a mediator of the reward pathway is crucial for addiction development as well as aberrant learning and memory [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. These circuits underlie substance-driven rewarding effects, as well as the acquisition of drug-seeking behavior [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eA crucial structure for contextual drug-related memory formation is the hippocampus (HP) [\\u003cspan additionalcitationids=\\\"CR10\\\" citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. The volume of HP tissue is decreased in MA users, and these volume deficits have been associated with poorer memory [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn rodents, HP neurons begin to develop on approximately embryonic day (ED) nine. By ED 15\\u0026ndash;18, all neurons have developed and migrated; however, some development continues until adolescence [\\u003cspan additionalcitationids=\\\"CR14\\\" citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. Neurogenesis in the HP can be downregulated by abuse of various drugs; however, this change can be partially restored by abstinence. Reduced HP neurogenesis may contribute to the development of addiction and may be a vulnerability factor for maintaining addiction-related behaviors [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eBased on spatial learning and memory tests, the HP in rodents does not show signs of adult-like function until at least the end of the third postnatal week. This phenomenon can be explained by the fact that spatial learning and memory are dependent on well-developed motor and sensory systems [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. Decreased HP neurogenesis caused by MA administration has been demonstrated by decreased numbers of doublecortin (DCX)-positive cells and other markers, which have also been reported in adult rats via self-administration, as well as short-term administration [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. DCX is expressed in differentiating neurons and plays a crucial role in their migration and differentiation [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. It is highly expressed in the brain during development, especially in structures that undergo continuous neurogenesis, such as the HP [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. The administration of certain drugs of abuse has been shown to inhibit HP neurogenesis, which can then lead to cognitive impairments in drug users [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Also, prenatal MA exposure significantly alters the expression of DCX, resulting in decreasing numbers of DCX-positive cells in the HP of 1-day-old, as well as 22-day-old rodent pups [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eGlutamatergic NMDA (N-methyl-D-aspartate) receptors mediate learning and memory formation in the HP. These receptors mediate synaptic changes underlying learning and memory and are widely expressed in the HP. Through these receptors, long-term potentiation occurs, which is the basis for the formation and preservation of memories [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. During MA exposure, elevated cytosolic DA and oxidative stress can stimulate the synthesis of DA metabolites, such as dopaquinones, which can lead to microglial activation, as well as the secretion of high amounts of GLU. MA causes the activation of microglia and astrocytes in the striatum, cerebral cortex, as well as the HP [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. DA metabolites are thought to be major activators of microglia through changes in microglial gene expression. Reactive gliosis induced by GLU release has been previously described in the context of MA mechanisms of action [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Communication between neurons and microglia is critical in brain development and maturation, as well as synaptic plasticity, learning, and memory [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Chronically activated microglia and astrocytes are linked to learning and memory deficits in various neuronal disorders [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Previous studies suggest that MA activates astrocytes, resulting in increased expression of glial fibrillary protein (GFAP) in both rodents and human brains [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eOur study investigated the possible harmful effects of MA exposure during postnatal days (PDs) 11\\u0026ndash;20 when the HP is still developing. In rodents, this age corresponds to approximately 1\\u0026ndash;2 years in humans, since 13.8 rat days\\u0026thinsp;\\u0026asymp;\\u0026thinsp;1 human year [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. It has been previously reported that MA exposure during this period causes some cognitive deficits and alterations in neurotransmission [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eWe assessed MA effects on learning and memory using the Morris Water Maze (MWM). Results of behavioral tests were correlated with neurotransmitter levels associated with cognition and the reward system (especially GLU, but also DA, NA, 5HT, and GABA). In addition, GFAP levels are analyzed to detect possible astrocyte activation resulting from reactive gliosis. Alterations in HP neurogenesis were determined by measuring levels of DCX as a marker of neurogenesis.\\u003c/p\\u003e\"},{\"header\":\"METHODS\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnimals\\u003c/h2\\u003e \\u003cp\\u003eAdult female and male Wistar rats, bred by Charles River Laboratories International, Inc. and delivered by Velaz (Prague, Czech Republic), were used in experiments. Rats were housed in a temperature-controlled (22\\u0026ndash;24\\u0026deg;C) room using a standard 12 h light/dark cycle with lights on at 6 am. Animals were left undisturbed for one week before fertility determination. Food and water were available \\u003cem\\u003ead libitum\\u003c/em\\u003e during this period. For determination of estrous cycle phase, female rats were smeared using vaginal lavage. During the estrous phase, females were housed overnight with sexually mature males. Determination of fertilization was performed by smearing females for the presence of sperm. On the day after birth, i.e., postnatal day (PD) 1, the number of pups in each litter was adjusted to 12\\u0026ndash;8 males and four females. Females were used in other experiments. Pups were randomly assigned to MA-treated (M), saline-treated (S), and absolute control (C) groups [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. Finally, 10\\u0026ndash;12 animals per group were used for this study.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eChemicals\\u003c/h3\\u003e\\n\\u003cp\\u003ePhysiological saline (0.9% NaCl, SA) and d-Methamphetamine hydrochloride (MA) were purchased from Sigma-Aldrich (Czech Republic). MA was dissolved in distilled water to a concentration of 5 mg/ml.\\u003c/p\\u003e\\n\\u003ch3\\u003eExperimental design\\u003c/h3\\u003e\\n\\u003cp\\u003eAfter birth, pups were left undisturbed with their mothers in standard cages. During PDs 11\\u0026ndash;20, pups were subcutaneously injected with MA (5 mg/ml/kg per day), while saline-treated (S) rats were given the same volume of saline. Administration of MA or saline was conducted once a day in the morning. Absolute control animals (C) were left undisturbed. Behavioral testing started twenty-four hours after the last MA exposure (i.e., on PD 20). As previously described in several studies conducted by our laboratory, a maximum of two animals from the original litter were used in any one experimental group. The rest of the animals were used in other experiments. Animals were acclimated (left undisturbed) to the room where Morris Water Maze testing was performed for 30 minutes before the start of each test; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e shows the experimental design.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e shows the timeline of the experiment, respective PDs, and duration of individual procedures. This figure was created with BioRender.com.\\u003c/p\\u003e\\n\\u003ch3\\u003eMorris Water Maze\\u003c/h3\\u003e\\n\\u003cp\\u003eThe Morris Water Maze (MWM) test was conducted to evaluate the spatial learning and memory abilities of the animals. The test consisted of three phases: Learning, Probe Test, and Memory Test. The standard protocol used by our laboratory and used in this study as well is described in detail in a study by Petrikov\\u0026aacute; et al., 2021 [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e].\\u003c/p\\u003e\\n\\u003ch3\\u003eLearning (PD 21–26)\\u003c/h3\\u003e\\n\\u003cp\\u003eMWM testing was performed in a pool with a diameter of 80 cm, which was chosen based on the age and size of the pups. The goal was to assess the animal's ability to learn the specific location of a hidden platform. The platform was placed in a fixed position, 1 cm below the water surface, making it invisible to the swimming rat. The maze was divided into four compass directions: north (N), south (S), east (E), and west (W), which created four quadrants. The platform was always located in the N-E quadrant. Animals were started from different random positions around the perimeter of the tank and had to find the hidden platform. Each rat performed six trials per day. The interval between trials was 30 seconds. If the rat could not find the platform within 60 seconds, it was manually guided to the platform and remained there for 30 seconds. Rat performance was tracked using a video-tracking system (EthoVision XT16) [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eThe Probe Test\\u003c/b\\u003e (PD 28)\\u003c/p\\u003e \\u003cp\\u003eThis test was conducted on the 8th day of the experiment. The platform was removed before the test. All rats started from the north (N) position, closest to the platform's position. The rats were allowed to swim freely in the maze for 60 seconds. This test aimed to assess the quadrant preference and spatial strategy used by the rats and evaluate their memory of the spatial map [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eThe Memory Test\\u003c/b\\u003e (PD 32)\\u003c/p\\u003e \\u003cp\\u003eThis test took place on the 12th day of the experiment. The hidden platform was placed in the same position as during the learning phase. Each rat performed six trials. Each trial lasted 60 seconds. The memory test determined if the rats could remember the position of the hidden platform [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStrategies monitored\\u003c/h2\\u003e \\u003cp\\u003eWe monitored the same behavioral strategies, i.e., scanning and thigmotaxis, as in our previous studies [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eScanning\\u003c/h3\\u003e\\n\\u003cp\\u003eSuccessful animals eventually develop a scanning strategy where they explore different areas of the pool methodically. They swim in a way that covers different regions of the pool, efficiently locating the hidden platform. This is a sign of good spatial learning and memory.\\u003c/p\\u003e\\n\\u003ch3\\u003eThigmotaxis\\u003c/h3\\u003e\\n\\u003cp\\u003eIn the context of the MWM, thigmotaxis refers to a behavioral strategy where animals tend to swim close to the walls of the maze rather than exploring the open, central area of the pool. This behavior is often considered a natural response that reduces anxiety or stress in unfamiliar and potentially dangerous situations.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSample collection and processing\\u003c/h2\\u003e \\u003cp\\u003eTwenty-four hours after the end of the MWM testing period, animals were anesthetized with an overdose of chloralhydrate (400 mg/kg, Sigma-Aldrich) and given an intracardial perfusion of heparinized saline. The HP was removed, snap-frozen on dry ice, and stored at \\u0026minus;\\u0026thinsp;80\\u0026deg;C for further processing. Samples were divided for ELISA and Western Blott analysis. During processing, the samples for ELISA were homogenized in physiological saline containing 1 mM EDTA and 4 mM sodium metabisulfite (Sigma Aldrich) for a final concentration of 100 mg/ml. The homogenates were centrifuged for 10 minutes at 10,000 g in a cooled microcentrifuge; the supernatants were aliquoted and stored at \\u0026minus;\\u0026thinsp;80\\u0026deg;C until assayed. For the Western Blot analysis, HPs were homogenized in ice-cold T-PER\\u0026trade; Tissue Protein Extraction Reagent (78510, Thermofisher Scientific) mixed with cOmplete\\u0026trade; Protease Inhibitor Cocktail (Roche) using a Bead Raptor 12 (Omni) bead homogenizer. Homogenates were sonicated, using a Q700 Sonicator (QSonica LLC, Newtown, CT, USA), for 5 min (30 s on/off cycle) at 4\\u0026deg;C. Samples were centrifuged, and the supernatant was collected [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eELISA\\u003c/h2\\u003e \\u003cp\\u003eLevels of neurotransmitters and GFAP were measured with commercially available ELISA kits (LS-F11532-1 Rat GFAP (Sandwich ELISA) LS BIO; BA E-5500R, BA E-2400R BA E-5900R BA E-2500R, LDN Labor Diagnostika Nord GmbH \\u0026amp; Co.KG, Germany); analyses were performed according to the manufacturer's instructions. The absorbance was read at 450 nm on an 800\\u0026trade; TS microplate Absorbance Reader (BioTek) [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern Blot\\u003c/h2\\u003e \\u003cp\\u003eWe used a Western blot protocol previously described by Zloh and Fiserova [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Following the homogenization process, protein quantification was conducted using a BCA assay. The processed samples were diluted to the desired concentration and combined with dithiothreitol (DTT) and a 4x Laemmli Sample Buffer (#1610747, Bio-Rad laboratories). To denature proteins, samples were heated to 95\\u0026deg;C for 5 minutes. Subsequently, proteins were separated using SDS-PAGE on Mini-PROTEAN\\u0026reg; TGXTM Precast Protein Gels with 15 wells (#4561086, Bio-Rad Laboratories, Hercules, CA, USA), and then transferred to a polyvinylidene fluoride (PVDF) membranes (Immun-Blot PVDF Membrane, Bio-Rad Laboratories). The membranes were blocked with Every Blot Blocking Buffer (Bio-Rad Laboratories). To detect the proteins of interest, PVDF membranes were incubated overnight at 4\\u0026deg;C with the following rabbit monoclonal antibodies, all diluted 1:1000 in Every Blot Blocking buffer: α-Actinin (D6F6) XP\\u0026reg; Rabbit mAb #6487 and Doublecortin (E6O6A) Rabbit mAb #91954 (Abcam). The next day, the membranes were washed with Tris-buffered saline containing Tween-20 and subsequently incubated with Goat anti-rabbit IgG H\\u0026amp;L (HRP) (ab97051, Abcam) diluted 1:10,000 in Every Blot Blocking Buffer for one hour. Membrane images were captured using an Azure C300 Digital Imager and analyzed using AzureSpot software, version 14.2 (Azure Biosystems, Dublin, CA, USA). The amount of DCX in HP samples was then normalized against the levels of endogenous control α-Actinin [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analyses\\u003c/h2\\u003e \\u003cp\\u003eThe following statistical tests were used in the three experimental phases of the MWM: Learning, one-way ANOVA with multilevel repeated measure (Days \\u0026times; Trials/day); Probe Test, one-way ANOVA; Memory Test, one-way ANOVA with repeated measure (Trials). The Tukey post-hoc test was used to compare groups. A one-way ANOVA was used for Western Blot and ELISA data, and the Tukey post-hoc test was used to compare groups.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMorris Water Maze test\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eLearning\\u003c/h2\\u003e \\u003cp\\u003eIn terms of swimming distance and velocity, there were no significant differences relative to treatment. In terms of latencies in finding the hidden platform during the learning task, the treatment had a significant impact [F (2, 94)\\u0026thinsp;=\\u0026thinsp;6.009, p\\u0026thinsp;=\\u0026thinsp;0.0035]. On day two, group S had increased latency compared to group M (p\\u0026thinsp;=\\u0026thinsp;0.0026), as well as group C (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTreatment also had a significant impact on search errors during the learning task [F (2, 94)\\u0026thinsp;=\\u0026thinsp;4.185, p\\u0026thinsp;=\\u0026thinsp;0.0181]. On day two, there were significantly more search errors in Group M compared to Group S (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001) and Group C (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA shows the amount of time needed to find the hidden platform over six days of learning. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB shows the time spent in the quadrant where the platform was originally located (recall that in the Probe test, the platform was removed from the maze). Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC shows the time needed to find the hidden platform on the last day of testing, i.e., memory evaluation\\u0026mdash;differences between MA-treated and control animals: ***p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 (ANOVA and Tukey post-hoc test).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStrategies used during learning\\u003c/h2\\u003e \\u003cp\\u003eTreatment had no significant effect with regard to thigmotaxis; however, there was a significant impact on the time spent using the scanning strategy, which varied during learning days [F (2, 94)\\u0026thinsp;=\\u0026thinsp;4.409, p\\u0026thinsp;=\\u0026thinsp;0.0148] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e. On the first day, group M spent significantly less time using the scanning strategy compared to group S (p\\u0026thinsp;=\\u0026thinsp;0.0002), and on day two, group M spent significantly more time using the scanning strategy compared to group C (p\\u0026thinsp;=\\u0026thinsp;0.0001). On day four, group M spent significantly more time using the scanning strategy than group C (p\\u0026thinsp;=\\u0026thinsp;0.0072); on day five, group M spent significantly more time using the scanning strategy compared to group S (p\\u0026thinsp;=\\u0026thinsp;0.0001) and group C (p\\u0026thinsp;=\\u0026thinsp;0.0001), and on day six, group M spent significantly less time using the scanning strategy than group C (p\\u0026thinsp;=\\u0026thinsp;0.0001) and group S (p\\u0026thinsp;=\\u0026thinsp;0.0121).\\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA shows the time spent using the scanning strategy over six days of learning. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB shows the time spent using the thigmotaxic strategy over six days of learning. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC shows the time spent using the scanning strategy, and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD shows the time spent using the thigmotaxic strategy during the probe test. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE shows the time spent using the scanning strategy, and Figure F shows the time spent using the thigmotaxic strategy during the memory test. Difference between MA-treated and control animals: **p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 (ANOVA and Tukey post-hoc test).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eProbe\\u003c/h2\\u003e \\u003cp\\u003eWe saw no significant differences between groups during the Probe test on day eight (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC, D\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMemory\\u003c/h2\\u003e \\u003cp\\u003eTreatment had a significant effect on the distance traveled during the memory task [F (2, 141)\\u0026thinsp;=\\u0026thinsp;4.760, p\\u0026thinsp;=\\u0026thinsp;0.0100]; group S swam significantly further than group M (p\\u0026thinsp;=\\u0026thinsp;0.0274) and group C (p\\u0026thinsp;=\\u0026thinsp;0.0191). (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE, F\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTreatment also significantly affected swimming velocity during the memory task [F (2, 134)\\u0026thinsp;=\\u0026thinsp;6.372, p\\u0026thinsp;=\\u0026thinsp;0.0023]. Group M (p\\u0026thinsp;=\\u0026thinsp;0.0487) and S (p\\u0026thinsp;=\\u0026thinsp;0.0016) swam significantly faster than group C. With regard to latency, we saw no significant differences.\\u003c/p\\u003e \\u003cp\\u003eTreatment did not significantly affect the use of scanning vs. thigmotaxis strategies (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLevels of GFAP in the hippocampus\\u003c/h2\\u003e \\u003cp\\u003eTreatment significantly impacted GFAP levels in the HP [F (2, 26)\\u0026thinsp;=\\u0026thinsp;8.037, p\\u0026thinsp;=\\u0026thinsp;0.002]. The lowest levels of GFAP were measured in group C relative to group M (p\\u0026thinsp;=\\u0026thinsp;0.002) and group S (p\\u0026thinsp;=\\u0026thinsp;0.020) \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThis chart displays GFAP levels in the HP. Levels of GFAP are expressed per wet weight of the HP. The data are presented as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SEM (n\\u0026thinsp;=\\u0026thinsp;6 animals); *p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, **p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 (ANOVA and Tukey post-hoc test).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLevels of neurotransmitters in the hippocampus\\u003c/h2\\u003e \\u003cp\\u003eThere were no statistical differences in the levels of DA [F (2, 27)\\u0026thinsp;=\\u0026thinsp;0.332, p\\u0026thinsp;=\\u0026thinsp;0.720] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e, NA [F (2, 27)\\u0026thinsp;=\\u0026thinsp;1.040, p\\u0026thinsp;=\\u0026thinsp;0.3670] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e, GABA [F (2, 27)\\u0026thinsp;=\\u0026thinsp;0.607, p\\u0026thinsp;=\\u0026thinsp;0.552] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC\\u003cb\\u003e)\\u003c/b\\u003e, GLU [F (2, 27)\\u0026thinsp;=\\u0026thinsp;0.413, p\\u0026thinsp;=\\u0026thinsp;0.665] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD\\u003cb\\u003e)\\u003c/b\\u003e, or 5HT [F (2, 26)\\u0026thinsp;=\\u0026thinsp;0.135, p\\u0026thinsp;=\\u0026thinsp;0.873] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFigure three shows levels of neurotransmitters in the hippocampus. Levels of neurotransmitters are expressed per wet weight of the HP. The data are presented as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SEM (n\\u0026thinsp;=\\u0026thinsp;6).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eLevels of doublecortin in the hippocampus\\u003c/h2\\u003e \\u003cp\\u003eLevels of doublecortin were significantly affected by treatment [F (2, 9)\\u0026thinsp;=\\u0026thinsp;8.250, p\\u0026thinsp;=\\u0026thinsp;0.009]; levels were significantly lower in group M compared to group C (p\\u0026thinsp;=\\u0026thinsp;0.007 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e\\u003cb\\u003e).\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA shows the protein expression of DCX in the hippocampus of MA-treated, saline-treated, and control animals, and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB shows the representative Western Blot images. The data are presented as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SEM (n\\u0026thinsp;=\\u0026thinsp;5). The difference between MA-treated and control animals: **p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 (ANOVA and Tukey post-hoc test).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eThe present study is focused on the effect of MA exposure during the third postnatal week (PD 11\\u0026ndash;20), which contrasts with our previous studies that investigated the effect of MA administered during prenatal and early postnatal periods. The third postnatal week is the developmental period when the HP fully matures, making it vulnerable to external influences. Therefore, we hypothesized that MA exposure during this specific period could affect HP neurogenesis, neurotransmitter levels, activation of astrocytes, learning, and memory. Knowing the effect of MA exposure during this time will extend our knowledge of the long-term effects of MA on rat pups during development.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec25\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLearning and memory impairments\\u003c/h2\\u003e \\u003cp\\u003eSeveral studies deal with the effect of MA on memory and learning. Previous reports by Vorhees et al. and Daberkow et al. uggest that MA exposure during gestation and postnatal development in rats significantly alters learning and memory abilities [\\u003cspan additionalcitationids=\\\"CR39 CR40\\\" citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. MA-induced cognitive impairments found by Vorhees et al. and Ye et al. indicate that at the preclinical level, MA may lead to long-term impairments in a variety of cognitive domains [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. In our study, the MWM test did not uncover any MA-induced differences in memory and learning ability, except for smaller swimming distances in animals injected with MA and SA compared to the absolute controls (C). These results are partially in agreement with a study by Joca et al. (2014), which showed no significant impairment in cognitive functions in MWM and object recognition tests after exposure to MA during early adolescence in mice (4 \\u0026times; 7.5 mg/kg on PD 30 and 31)[\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. In a study by North et al. (2013), adolescent mice showed impairments in short-term memory and altered HP plasticity after repeated high exposures to MA (24 mg/kg/day for 14 days). North et al. also observed that after 7, 14, and 21 days of abstinence, MA-exposed mice exhibited a deficit in spatial memory and decreased HP plasticity [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. These varied and conflicting results indicate the importance of the timing of MA administration as well as the administered dose of the drug. Also of importance are the different results in several parameters between saline-treated and absolute control animals. One of our previous studies suggests that not only the drug but also the actual single injection can affect the behavior of laboratory animals [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. This fact is very important and points to the necessity of using absolute controls in these types of studies.\\u003c/p\\u003e \\u003cp\\u003eStudies of humans addicted to MA revealed that behavioral impairments are replicated in laboratory models, including repeated exposure to high doses of MA and self-administration of the drug over a prolonged period. These experimental paradigms, such as those described by Ito, Rogers ), and Recinto et al., result in cognitive dysfunction and memory impairments linked to hippocampus dysfunction[\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. Neurodegeneration and neurotoxicity in the HP are also seen in animal models of \\u003cem\\u003ebinge MA exposure\\u003c/em\\u003e and \\u003cem\\u003eMA self-administration\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. This suggests that there is a direct relationship between the toxic effects of MA and the resulting behavioral deficits caused by MA use [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e]. Further mechanistic investigations demonstrated that exposure to MA induced changes in the adaptability and physical composition of neurons in the hippocampus. As an illustration, the administration of MA in a concentrated and widespread manner decreases synaptic strength in CA1 pyramidal neurons associated with high-frequency stimulation between specific brain cells (known as long-term potentiation). This effect is achieved by activating D1 receptors and simultaneously increasing the initial level of communication between the neurons [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec26\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGFAP levels are elevated in MA-exposed animals\\u003c/h2\\u003e \\u003cp\\u003eSignificantly higher GFAP levels in the HP of MA-exposed animals in comparison to control animals are present in our study. GFAP is a protein produced by reactive astrocytes and can be a signaling mechanism for neural inflammation [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. Our results agree with previous studies, such as the study by McConnell et al. (2015), who reported microglial activation after MA exposure in mice [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. A study by Sheng et al. (1994) revealed that MA causes loss of DA neurons and an increase in reactive gliosis, which is indicated by increased numbers of cells stained with GFAP antibodies [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. Lau et al. (2000) showed that astrocytes cultured from different brain regions reacted differently to MA-induced oxidative stress formation.\\u003c/p\\u003e \\u003cp\\u003eThe most severe effect was seen in striatum astrocytes, which suggests that these astrocytes are more sensitive to oxidative stress induced by MA [\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. A comparative study by Thomas et al. (2004) showed that MA as well as MDMA (-3,4-methylenedioxymethamphetamine) administered 4 times at 2-hour intervals significantly increased activation of microglia in rodent brains [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. Microglia activation has been previously reported in several rodent and human studies. However, unlike our current study, these studies were performed on adult animals [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. A study by Peng et al. (2024) examined the role of TREM2 in MA-induced neuroinflammation using BV2 cells, wild-type C57BL/6J mice, CX3CR1GFP/+ transgenic mice, and TREM2 knockout mice [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003ePostmortem samples from the frontal cortex of individuals with a history of MA use were analyzed to assess TREM2, TLR4, IBA1, and IL-1β levels. TREM2, TLR4, IBA1, IL-1β, iNOS, and Arg-1 expression levels were assessed in BV2 cells and the frontal cortexes of both mice and human MA users. The findings indicated that expression levels of TREM2, TLR4, IBA1, and IL-1β were significantly increased in the BV2 cells of individuals using MA. Microglia exhibited clear activation in the frontal cortex of WT C57BL/6 mice and CX3CR1GFP/+ transgenic mice. Elevated protein levels of IBA1, TREM2, TLR4, and IL-1β were also observed in METH-induced mouse models. Further, TREM2-KO mice exhibited heightened microglial activation, neuroinflammation, and excitotoxicity resulting from MA exposure. These findings indicate that TREM2 could serve as a target for the regulation of MA-induced neuroinflammation [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec27\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMethamphetamine decreases DCX expression in the hippocampus\\u003c/h2\\u003e \\u003cp\\u003eIn our study, we saw significantly lower levels of expression of DCX in MA-exposed animals compared to group S and group C (i.e., controls). This phenomenon may be caused by decreased growth of new progenitor neural cells in the HP. This result agrees with previous studies, suggesting decreased HP neurogenesis after various MA administration protocols [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Mutations in this protein can cause defective neuronal migration; additionally, it has been reported that the absence of DCX causes imminent depletion of the progenitor pool during cortical development [\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eDCX is highly expressed on neural axons and may regulate microtubules in response to extracellular signals during development [\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e]. Studies have shown that DCX is expressed in various regions of the developing nervous system and is highly expressed in newly produced HP cells. This protein has been adopted as a marker for neuronal precursors and migrating neuroblasts during adult neurogenesis; it detects only newly generated neurons, hence its specificity [\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e]. Several studies also indicate that ablation of neurogenesis during adolescence is associated with impaired MWM performance in adulthood as well as with activation of microglia and neuroinflammation [\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e]. An extensive study by Galinato reported that cognitive impairments observed in individuals who abuse MA could be related to changes in the structure and function of HP neurons caused by MA use [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. Human imaging studies have demonstrated that chronic MA users have reduced hippocampal volume, specifically in the gray matter, and decreased responsiveness of the hippocampus. These findings, reported by Thompson et al. (2004), Schwartz et al. (2010), and many others, suggest that MA-exposed individuals have impaired hippocampal networking [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e]. Analysis of human brain tissue after death demonstrates that long-term MA use causes damage to the hippocampus, as shown in studies by Kitamura in 2009 and Kitamura et al. in 2010 [\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e]. This indicates a link between hippocampal dysfunction and toxicity in individuals addicted to MA. A study by Choi et al. (2018) aimed to examine the impact of MA on structural modifications and gene expression in the HP [\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e]. The HP demonstrated notable volumetric shrinkage in response to both acute and chronic MA compared to the HP of control subjects. The genes linked to cytoskeleton organization and phagocytosis were downregulated in the acute MA-treated cohort relative to the control group. Conversely, genes related to synaptic transmission, neuron differentiation regulation, and neurogenesis regulation were downregulated in the chronic MA-treated cohort. Our study verified that the expression patterns of ADM, BMP4, CHRD, PDYN, UBA1, profilin 2 (PFN2), ENO2, and NSE mRNAs were consistent with the findings from RNA-Seq, as determined by quantitative RT-PCR. Specifically, the expression levels of PFN2 mRNA and protein, which are crucial for actin cytoskeleton dynamics, were diminished by both acute and chronic MA treatment [\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec28\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eNeurotransmitter levels were not significantly affected by MA\\u003c/h2\\u003e \\u003cp\\u003eOur study found no significant differences in levels of DA between our test groups; however, it is well established that MA exposure profoundly alters DA transmission [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e], so this result was surprising. Then again, there are insufficient studies examining MA effects in adolescents or developing animals to draw conclusions. The relative resistance of the DA system to MA in the brains of adolescent rodents was previously reported by Kokoshka et al. (2000); however, the mechanism underlying this phenomenon is still unknown [\\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e]. Furthermore, concentrations of other neurotransmitters, such as 5HT, GABA, and NA, were also not significantly affected by MA. Levels of 5HT slightly decreased in MA-exposed animals, while NA levels slightly increased in MA-exposed animals. \\u003cem\\u003eIn vitro\\u003c/em\\u003e studies indicate that MA is twice as potent at releasing NA as DA, and its effect is 60-fold greater on NA than 5HT release [\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e]. Therefore, we speculate that our results are due to the MA dose being too low to evoke significant changes. It might also be that the period of MA exposure was too short to increase or decrease the function of these neurotransmitters. Additionally, in developing animals, there may be several processes, such as brain plasticity, which suppress the neurotoxic effects of MA on neurotransmitter systems. This is, however, our speculation, which needs to be confirmed with additional studies.\\u003c/p\\u003e \\u003cp\\u003eA tendency towards decreased GLU levels in MA-exposed animals compared to control animals was present in our study. These results correlated with performance in the probe and memory tests, where MA-exposed animals showed \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003esigns of decreasing tendency\\u003c/span\\u003e as well. Even though these differences were insignificant, the correlation between lower GLU levels and poorer memory in MA-exposed animals does not appear to be a coincidence. Something similar was seen in a study by Yamamoto et al. (1999) that found that GLU release from the HP was decreased in MA-sensitized rats compared to control; additionally, a study by Hori et al. (2010) showed that MA decreased GLU release from nerve terminals of the Shaeffer collateral [\\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e71\\u003c/span\\u003e]. However, some aforementioned studies also found increased GLU release after MA exposure [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"CONCLUSION\",\"content\":\"\\u003cp\\u003eOur study revealed that MA exposure during the third postnatal week in laboratory rats significantly induced levels of glial fibrillary acidic protein, which is a marker of reactive gliosis. The expression of doublecortin, a marker of neurogenesis inhibition, was also found to be reduced, although neither the behavioral (MWM) test nor neurotransmitter levels were affected. These results may be explained by the hypothesis that the molecular mechanisms of MA toxicity start much earlier, i.e., at the level of reduced HP neurogenesis and microglial activation. Nonetheless, the process is not strong enough to overcome the mechanisms of neuroplasticity and development and cause significant changes in cognitive functions or neurotransmitter levels. Changes in the markers of neurogenesis and microglial activation may also have multiple developmental causes.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe procedures used in this study were reviewed and approved by the Institutional Animal Care and Use Committee and met the requirements of the Czech Government put forth under the Policy of Humans Care of Laboratory Animals (No. 86/609/EEC) and the subsequent regulations of the Ministry of Agriculture of the Czech Republic.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflicts of interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no conflicts of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis research was supported by the research program Cooperatio Neurosciences from Charles University, grant project GA 21-30795S from the Grant Agency of the Czech Republic, and project PharmaBrain CZ.02.1.01/0.0/0.0/16_025/0007444 by OPVVV.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent to Publish declaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026rsquo; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBČ is a doctoral student responsible for all experimental procedures, statistical analysis, and the manuscript. R\\u0026Scaron; is a supervisor of BČ and the head of the department and laboratory where this study was conducted. KP, IF, and \\u0026Scaron;V were responsible for drug exposure, laboratory analyses, animal manipulations, and sample collections. All authors were involved in all parts of the present study. All authors contributed to the article and approved the submitted version.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors would like to express deep gratitude to Zuzana Ježd\\u0026iacute;kov\\u0026aacute; for her excellent technical assistance. Additionally, we want to thank Mgr. Miloslav Zloh for assistance with Western Blot analyses, Dr. Ivana Petr\\u0026iacute;kov\\u0026aacute;, Dr. Anna Mikuleck\\u0026aacute;, and Dr. Kateryna Nohejlov\\u0026aacute; for help with setting up and analyzing data from the Morris water maze. The authors also express their appreciation to Thomas Secrest for editing the manuscript.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eČechov\\u0026aacute; B, Šlamberov\\u0026aacute; R. Methamphetamine, neurotransmitters and neurodevelopment. Physiol Res. 2021;70(S3):S301\\u0026ndash;15.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDer-Ghazarian TS, Charmchi D, Noudali SN, Scott SN, Holter MC, Newbern JM, et al. Neural Circuits Associated with 5-HT(1B) Receptor Agonist Inhibition of Methamphetamine Seeking in the Conditioned Place Preference Model. ACS Chem Neurosci. 2019;10(7):3271\\u0026ndash;83.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJaehne EJ, Semaan H, Grosman A, Xu X, Schwarz Q, van den Buuse M. Enhanced methamphetamine sensitisation in a rat model of the brain-derived neurotrophic factor Val66Met variant: Sex differences and dopamine receptor gene expression. Neuropharmacology. 2023;240:109719.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBuck JM, Morris AS, Weber SJ, Raber J, Siegel JA. Effects of adolescent methamphetamine and nicotine exposure on behavioral performance and MAP-2 immunoreactivity in the nucleus accumbens of adolescent mice. Behav Brain Res. 2017;323:78\\u0026ndash;85.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCourtney KE, Ray LA. Methamphetamine: an update on epidemiology, pharmacology, clinical phenomenology, and treatment literature. Drug Alcohol Depend. 2014;143:11\\u0026ndash;21.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCleva RM, Gass JT, Widholm JJ, Olive MF. Glutamatergic targets for enhancing extinction learning in drug addiction. Curr Neuropharmacol. 2010;8(4):394\\u0026ndash;408.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHyman SE. Addiction: a disease of learning and memory. Am J Psychiatry. 2005;162(8):1414\\u0026ndash;22.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBarria A, Malinow R. Subunit-specific NMDA receptor trafficking to synapses. Neuron. 2002;35(2):345\\u0026ndash;53.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKoob GF, Volkow ND. Neurobiology of addiction: a neurocircuitry analysis. Lancet Psychiatry. 2016;3(8):760\\u0026ndash;73.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSartor GC, Aston-Jones G. Post-retrieval extinction attenuates cocaine memories. Neuropsychopharmacology. 2014;39(5):1059\\u0026ndash;65.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTorregrossa MM, Corlett PR, Taylor JR. Aberrant learning and memory in addiction. Neurobiol Learn Mem. 2011;96(4):609\\u0026ndash;23.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eThompson PM, Hayashi KM, Simon SL, Geaga JA, Hong MS, Sui Y, et al. Structural abnormalities in the brains of human subjects who use methamphetamine. J Neurosci. 2004;24(26):6028\\u0026ndash;36.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCossart R, Khazipov R. How development sculpts hippocampal circuits and function. Physiol Rev. 2022;102(1):343\\u0026ndash;78.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSemple BD, Blomgren K, Gimlin K, Ferriero DM, Noble-Haeusslein LJ. Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species. Prog Neurobiol. 2013;106\\u0026ndash;107:1\\u0026ndash;16.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKozareva DA, Cryan JF, Nolan YM. Born this way: Hippocampal neurogenesis across the lifespan. Aging Cell. 2019;18(5):e13007.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJun H, Mohammed Qasim Hussaini S, Rigby MJ, Jang MH. Functional role of adult hippocampal neurogenesis as a therapeutic strategy for mental disorders. Neural Plast. 2012;2012:854285.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKarila L, Petit A, Lowenstein W, Reynaud M. Diagnosis and consequences of cocaine addiction. Curr Med Chem. 2012;19(33):5612\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAlbani SH, McHail DG, Dumas TC. Developmental studies of the hippocampus and hippocampal-dependent behaviors: insights from interdisciplinary studies and tips for new investigators. Neurosci Biobehav Rev. 2014;43:183\\u0026ndash;90.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMandyam CD, Wee S, Crawford EF, Eisch AJ, Richardson HN, Koob GF. Varied access to intravenous methamphetamine self-administration differentially alters adult hippocampal neurogenesis. Biol Psychiatry. 2008;64(11):958\\u0026ndash;65.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKochman LJ, Fornal CA, Jacobs BL. Suppression of hippocampal cell proliferation by short-term stimulant drug administration in adult rats. Eur J Neurosci. 2009;29(11):2157\\u0026ndash;65.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJalayeri-Darbandi Z, Rajabzadeh A, Hosseini M, Beheshti F, Ebrahimzadeh-Bideskan A. The effect of methamphetamine exposure during pregnancy and lactation on hippocampal doublecortin expression, learning and memory of rat offspring. Anat Sci Int. 2018;93(3):351\\u0026ndash;63.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChe X, Bai Y, Cai J, Liu Y, Li Y, Yin M, et al. Hippocampal neurogenesis interferes with extinction and reinstatement of methamphetamine-associated reward memory in mice. Neuropharmacology. 2021;196:108717.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eShrestha P, Katila N, Lee S, Seo JH, Jeong JH, Yook S. Methamphetamine induced neurotoxic diseases, molecular mechanism, and current treatment strategies. Biomed Pharmacother. 2022;154:113591.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMcDonnell-Dowling K, Kelly JP. The Role of Oxidative Stress in Methamphetamine-induced Toxicity and Sources of Variation in the Design of Animal Studies. Curr Neuropharmacol. 2017;15(2):300\\u0026ndash;14.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 2009;32(12):638\\u0026ndash;47.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCornell J, Salinas S, Huang HY, Zhou M. Microglia regulation of synaptic plasticity and learning and memory. Neural Regen Res. 2022;17(4):705\\u0026ndash;16.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFriend DM, Keefe KA. Glial reactivity in resistance to methamphetamine-induced neurotoxicity. J Neurochem. 2013;125(4):566\\u0026ndash;74.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSengupta P. The Laboratory Rat: Relating Its Age With Human's. Int J Prev Med. 2013;4(6):624\\u0026ndash;30.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCrawford CA, Williams MT, Newman ER, McDougall SA, Vorhees CV. Methamphetamine exposure during the preweanling period causes prolonged changes in dorsal striatal protein kinase A activity, dopamine D2-like binding sites, and dopamine content. Synapse. 2003;48(3):131\\u0026ndash;7.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGraham DL, Amos-Kroohs RM, Braun AA, Grace CE, Schaefer TL, Skelton MR, et al. Neonatal +-methamphetamine exposure in rats alters adult locomotor responses to dopamine D1 and D2 agonists and to a glutamate NMDA receptor antagonist, but not to serotonin agonists. Int J Neuropsychopharmacol. 2013;16(2):377\\u0026ndash;91.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePetr\\u0026iacute;kov\\u0026aacute;-Hrebi\\u0026iacute;čkov\\u0026aacute; I, Ševčikov\\u0026aacute; M, Šlamberov\\u0026aacute; R. The Impact of Neonatal Methamphetamine on Spatial Learning and Memory in Adult Female Rats. Front Behav Neurosci. 2021;15:629585.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eŠlamberov\\u0026aacute; R, Mikuleck\\u0026aacute; A, Pometlov\\u0026aacute; M, Schutov\\u0026aacute; B, Hrub\\u0026aacute; L, Deykun K. Sex differences in social interaction of methamphetamine-treated rats. Behav Pharmacol. 2011;22(7):617\\u0026ndash;23.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHolubov\\u0026aacute; A, Luk\\u0026aacute;škov\\u0026aacute; I, Tom\\u0026aacute;šov\\u0026aacute; N, Šuhajdov\\u0026aacute; M, Šlamberov\\u0026aacute; R. Early Postnatal Stress Impairs Cognitive Functions of Male Rats Persisting Until Adulthood. Front Behav Neurosci. 2018;12:176.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMacuchova E, Nohejlova K, Sevcikova M, Hrebickova I, Slamberova R. Sex differences in the strategies of spatial learning in prenatally-exposed rats treated with various drugs in adulthood. Behav Brain Res. 2017;327:83\\u0026ndash;93.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eČechov\\u0026aacute; B, Mihalčikov\\u0026aacute; L, Vacul\\u0026iacute;n Š, Šandera Š, Šlamberov\\u0026aacute; R. Levels of BDNF and NGF in adolescent rat hippocampus neonatally exposed to methamphetamine along with environmental alterations. Physiol Res. 2023;72(S5):S559\\u0026ndash;71.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFišerov\\u0026aacute; I, Trinh MD, Elkalaf M, Vacek L, Heide M, Martinkov\\u0026aacute; S, et al. Isoprenaline modified the lipidomic profile and reduced β-oxidation in HL-1 cardiomyocytes: In vitro model of takotsubo syndrome. Front Cardiovasc Med. 2022;9:917989.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZloh M, Kutilek P, Štofkov\\u0026aacute; A. High-Contrast Stimulation Potentiates the Neurotrophic Properties of Muller Cells and Suppresses Their Pro-Inflammatory Phenotype. Int J Mol Sci. 2022;23(15).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDaberkow DP, Kesner RP, Keefe KA. Relation between methamphetamine-induced monoamine depletions in the striatum and sequential motor learning. Pharmacol Biochem Behav. 2005;81(1):198\\u0026ndash;204.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVorhees CV, Inman-Wood SL, Morford LL, Broening HW, Fukumura M, Moran MS. Adult learning deficits after neonatal exposure to D-methamphetamine: selective effects on spatial navigation and memory. J Neurosci. 2000;20(12):4732\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVorhees CV, Reed TM, Morford LL, Fukumura M, Wood SL, Brown CA, et al. Periadolescent rats (P41-50) exhibit increased susceptibility to D-methamphetamine-induced long-term spatial and sequential learning deficits compared to juvenile (P21-30 or P31-40) or adult rats (P51-60). Neurotoxicol Teratol. 2005;27(1):117\\u0026ndash;34.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVorhees CV, Skelton MR, Williams MT. Age-dependent effects of neonatal methamphetamine exposure on spatial learning. Behav Pharmacol. 2007;18(5\\u0026ndash;6):549\\u0026ndash;62.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYe T, Pozos H, Phillips TJ, Izquierdo A. Long-term effects of exposure to methamphetamine in adolescent rats. Drug Alcohol Depend. 2014;138:17\\u0026ndash;23.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJoca L, Zuloaga DG, Raber J, Siegel JA. Long-term effects of early adolescent methamphetamine exposure on depression-like behavior and the hypothalamic vasopressin system in mice. Dev Neurosci. 2014;36(2):108\\u0026ndash;18.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNorth A, Swant J, Salvatore MF, Gamble-George J, Prins P, Butler B, et al. Chronic methamphetamine exposure produces a delayed, long-lasting memory deficit. Synapse. 2013;67(5):245\\u0026ndash;57.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eŠlamberov\\u0026aacute; R, Nohejlov\\u0026aacute; K, Ochozkov\\u0026aacute; A, Mihalč\\u0026iacute;ikov\\u0026aacute; L. What is the role of subcutaneous single injections on the behavior of adult male rats exposed to drugs? Physiol Res. 2018;67(Suppl 4):S665\\u0026ndash;72.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRecinto P, Samant AR, Chavez G, Kim A, Yuan CJ, Soleiman M, et al. Levels of neural progenitors in the hippocampus predict memory impairment and relapse to drug seeking as a function of excessive methamphetamine self-administration. Neuropsychopharmacology. 2012;37(5):1275\\u0026ndash;87.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRogers JL, De Santis S, See RE. Extended methamphetamine self-administration enhances reinstatement of drug seeking and impairs novel object recognition in rats. Psychopharmacology. 2008;199(4):615\\u0026ndash;24.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGalinato MH, Takashima Y, Fannon MJ, Quach LW, Morales Silva RJ, Mysore KK, et al. Neurogenesis during Abstinence Is Necessary for Context-Driven Methamphetamine-Related Memory. J Neurosci. 2018;38(8):2029\\u0026ndash;42.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMandyam CD, Wee S, Eisch AJ, Richardson HN, Koob GF. Methamphetamine self-administration and voluntary exercise have opposing effects on medial prefrontal cortex gliogenesis. J Neurosci. 2007;27(42):11442\\u0026ndash;50.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSwant J, Chirwa S, Stanwood G, Khoshbouei H. Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus. PLoS ONE. 2010;5(6):e11382.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eClark KH, Wiley CA, Bradberry CW. Psychostimulant abuse and neuroinflammation: emerging evidence of their interconnection. Neurotox Res. 2013;23(2):174\\u0026ndash;88.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMcConnell SE, O'Banion MK, Cory-Slechta DA, Olschowka JA, Opanashuk LA. Characterization of binge-dosed methamphetamine-induced neurotoxicity and neuroinflammation. Neurotoxicology. 2015;50:131\\u0026ndash;41.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSheng P, Cerruti C, Cadet JL. Methamphetamine (METH) causes reactive gliosis in vitro: attenuation by the ADP-ribosylation (ADPR) inhibitor, benzamide. Life Sci. 1994;55(3):PL51\\u0026ndash;4.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLau JW, Senok S, Stadlin A. Methamphetamine-induced oxidative stress in cultured mouse astrocytes. Ann N Y Acad Sci. 2000;914:146\\u0026ndash;56.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eThomas DM, Dowgiert J, Geddes TJ, Francescutti-Verbeem D, Liu X, Kuhn DM. Microglial activation is a pharmacologically specific marker for the neurotoxic amphetamines. Neurosci Lett. 2004;367(3):349\\u0026ndash;54.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePeng Y, Yang G, Wang S, Lin W, Zhu L, Dong W, et al. Triggering Receptor Expressed on Myeloid Cells 2 Deficiency Exacerbates Methamphetamine-Induced Activation of Microglia and Neuroinflammation. Int J Toxicol. 2024;43(2):165\\u0026ndash;76.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGleeson JG, Lin PT, Flanagan LA, Walsh CA. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron. 1999;23(2):257\\u0026ndash;71.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAyanlaja AA, Xiong Y, Gao Y, Ji G, Tang C, Abdikani Abdullah Z, et al. Distinct Features of Doublecortin as a Marker of Neuronal Migration and Its Implications in Cancer Cell Mobility. Front Mol Neurosci. 2017;10:199.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTint I, Jean D, Baas PW, Black MM. Doublecortin associates with microtubules preferentially in regions of the axon displaying actin-rich protrusive structures. J Neurosci. 2009;29(35):10995\\u0026ndash;1010.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWeimer JM, Anton ES. Doubling up on microtubule stabilizers: synergistic functions of doublecortin-like kinase and doublecortin in the developing cerebral cortex. Neuron. 2006;49(1):3\\u0026ndash;4.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003evon Halbach B. Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus. Cell Tissue Res. 2011;345(1):1\\u0026ndash;19.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAchanta P, Fuss M, Martinez JL. Jr. Ionizing radiation impairs the formation of trace fear memories and reduces hippocampal neurogenesis. Behav Neurosci. 2009;123(5):1036\\u0026ndash;45.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRola R, Raber J, Rizk A, Otsuka S, VandenBerg SR, Morhardt DR, et al. Radiation-induced impairment of hippocampal neurogenesis is associated with cognitive deficits in young mice. Exp Neurol. 2004;188(2):316\\u0026ndash;30.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSchwartz DL, Mitchell AD, Lahna DL, Luber HS, Huckans MS, Mitchell SH, et al. Global and local morphometric differences in recently abstinent methamphetamine-dependent individuals. NeuroImage. 2010;50(4):1392\\u0026ndash;401.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKitamura O. Detection of methamphetamine neurotoxicity in forensic autopsy cases. Leg Med (Tokyo). 2009;11(Suppl 1):S63\\u0026ndash;5.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKitamura O, Takeichi T, Wang EL, Tokunaga I, Ishigami A, Kubo S. Microglial and astrocytic changes in the striatum of methamphetamine abusers. Leg Med (Tokyo). 2010;12(2):57\\u0026ndash;62.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChoi MR, Chun JW, Kwak SM, Bang SH, Jin YB, Lee Y, et al. Effects of acute and chronic methamphetamine administration on cynomolgus monkey hippocampus structure and cellular transcriptome. Toxicol Appl Pharmacol. 2018;355:68\\u0026ndash;79.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKokoshka JM, Fleckenstein AE, Wilkins DG, Hanson GR. Age-dependent differential responses of monoaminergic systems to high doses of methamphetamine. J Neurochem. 2000;75(5):2095\\u0026ndash;102.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI, et al. Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse. 2001;39(1):32\\u0026ndash;41.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHori N, Kadota MT, Watanabe M, Ito Y, Akaike N, Carpenter DO. Neurotoxic effects of methamphetamine on rat hippocampus pyramidal neurons. Cell Mol Neurobiol. 2010;30(6):849\\u0026ndash;56.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYamamoto H, Kitamura N, Lin XH, Ikeuchi Y, Hashimoto T, Shirakawa O, et al. Differential changes in glutamatergic transmission via N-methyl-D-aspartate receptors in the hippocampus and striatum of rats behaviourally sensitized to methamphetamine. Int J Neuropsychopharmacol. 1999;2(3):155\\u0026ndash;63.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"drug addiction, development, neurogenesis, neuroinflammation\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5742375/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5742375/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground:\\u003c/strong\\u003e Methamphetamine (MA) is one of the most abused illicit drugs in the world. Abuse of this drug among adolescents, given the still-developing physiological functions of the body, including the central nervous system (CNS), is problematic. The hippocampus (HP) is part of the limbic system and is associated with cognition, as well as the reward system, which is mediated by dopamine (DA). Disruption of this system is the primary mechanism underlying addiction and results in cognition deficiencies.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethods:\\u003c/strong\\u003e In this work, we focused on the influence of MA on the development of the rat HP during postnatal days 11–20. Subsequently, after MA application, we studied learning and memory processes using the Morris Water Maze (MWM). After the MWM, we measured the expression of the protein doublecortin (DCX), neurotransmitter levels of DA, GLU, 5HT, NA, and GABA, and glial fibrillary acidic protein (GFAP).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults:\\u003c/strong\\u003e MA significantly impairs HP neurogenesis when administered during the third postnatal week, as indicated by reduced expression of DCX, which correlates with increased levels of glial fibrillary acidic protein (GFAP), but these changes were not significantly reflected in learning abilities and memory formation nor the levels of neurotransmitters.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusion: \\u003c/strong\\u003eWe speculate that these mechanisms must be strongly expressed on multiple molecular levels to be able to cause cognitive changes. A significant role in this process is associated with the ability of young organisms to compensate and, to a certain extent, neurotoxic effects.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Methamphetamine exposure during the preweaning period alters hippocampal neurogenesis\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-01-08 11:39:19\",\"doi\":\"10.21203/rs.3.rs-5742375/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"619869ce-1ea8-4601-ba50-2ff3fb9add2f\",\"owner\":[],\"postedDate\":\"January 8th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-01-08T11:39:25+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-01-08 11:39:19\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5742375\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5742375\",\"identity\":\"rs-5742375\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}