Repeated Oral Methylphenidate Administration Evokes Changes in Brain Plasticity Proteins in Juvenile Wistar Kyoto Rats: Evidence for Sex-Related Differences
Patrícia Soares-Couto, Susana Isabel Sá, Vera Marisa Costa, Ana Dias-Carvalho, Mariana Ferreira, Félix Dias Carvalho, Andreas Meisel, João Paulo Capela
TL;DR
This study shows that methylphenidate affects brain plasticity proteins differently in male and female juvenile rats.
Contribution
The study reveals sex-related differences in brain protein responses to methylphenidate in juvenile rats.
Findings
MPH reduced GAP43 and synaptophysin in male rats' cerebellum and CA1 region.
MPH increased PSD-95 and GAPDH in the striatum and diencephalon of males.
MPH-treated females showed a significant decrease in PSD-95 levels in the PFC.
Abstract
Methylphenidate (MPH) is first-line pharmacotherapy for Attention Deficit Hyperactivity Disorder (ADHD). Misdiagnosis and misuse raise concerns about exposing children and adolescents to MPH. This study aimed to assess how clinically relevant oral doses of MPH influence the expression of brain proteins involved in synaptic plasticity and neuronal growth in both sexes. Thirty-seven Wistar-Kyoto (WKY) rats (18 males and 19 females) were divided into an MPH group (daily oral dose of 5 mg/kg MPH in a 5% sucrose solution) and a control group (equivalent volume of 5% sucrose solution). Daily gavage administration started on postnatal day (PND) 15 and lasted for 15 days, with sacrifice at PND 30. In five brain regions [prefrontal cortex (PFC), striatum, hippocampus, cerebellum, and diencephalon], GAP43, GAPDH and PSD-95 levels were measured by Western blot. Additionally, MAP2 and synaptophysin…
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Taxonomy
TopicsAttention Deficit Hyperactivity Disorder · Neurotransmitter Receptor Influence on Behavior · Genetic Neurodegenerative Diseases
Introduction
Attention Deficit Hyperactivity Disorder (ADHD) is one of the most prevalent neuropsychiatric disorders in childhood and adolescence [1], affecting approximately 7.6% of children and 5.6% of adolescents worldwide [2]. According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, ADHD is characterized by a triad of symptoms of hyperactivity, inattention, and impulsivity [3, 4]. These symptoms can have a significant impact on the lives of affected individuals, influencing their academic and professional performance, as well as their family and social relationships [5, 6].
Despite being a highly prevalent condition, the etiology and pathophysiology of ADHD remain poorly understood. A complex interaction among genetic, environmental, and neurobiological factors contributes to the development of the condition [7]. Previous studies have reported a diverse profile of structural, functional, connectivity, and neurochemical alterations in individuals with ADHD, which are responsible for the heterogeneous symptomatology characteristic of the disorder [8]. These alterations include structural reductions in various cortical [9, 10] and subcortical regions [9, 11], changes in neuronal networks [12, 13] and their connectivity [14], as well as inadequate regulation of neurotransmitters such as dopamine and noradrenaline [7].
Unfortunately, to date, there is no disease-modifying therapy that cures or reverses the underlying pathology of ADHD. The management of ADHD essentially focuses on relieving symptoms and minimizing their impact on daily life through pharmacological and non-pharmacological strategies [7]. Methylphenidate (MPH) is considered the first-line pharmacological intervention due to its proven efficacy in reducing ADHD symptoms [15–17]. Its therapeutic effect is associated with stimulation of the hypofunctional catecholaminergic system. MPH inhibits the reuptake of dopamine and noradrenaline by binding to their respective transporters, thereby increasing the concentration of these neurotransmitters in the synaptic cleft [16, 18]. This effect contributes to the restoration of neurochemical balance and consequently to the normalization of brain activity, particularly in the hypothalamus, striatum, and dorsolateral and medial prefrontal cortex (PFC) [16, 19].
Although MPH is considered safe and effective in the treatment of ADHD, its use in non-clinical contexts has generated some debate [20]. The diagnosis of ADHD is challenging, as it is essentially based on the identification of clinical signs [17, 21]. The lack of specific biomarkers or tests to identify or confirm the presence of the condition [17, 21] makes this process highly subjective [22, 23]. As a result, concerns have been raised regarding the increased prescription of MPH associated with misdiagnoses, particularly in children [20, 22]. At the same time, MPH consumption among young people seeking to improve academic performance has increased [20, 24]. This scenario becomes even more worrying considering that childhood and adolescence are critical periods of brain development and maturation [25], during which pharmacological influences can significantly impact neuronal structure and function [26]. Therefore, non-medical consumption of MPH is considered a significant public health concern [20], which can have harmful effects. In fact, stimulant misuse, including MPH, by athletes or by students as smart drugs, has been linked to psychosis and other neurological or psychiatric complications in non-ADHD populations [27].
Previous studies have shown that MPH can induce changes in synaptic plasticity in the hippocampus and PFC [28–31]. Additionally, Schmitz and coworkers found that chronic treatment with MPH (2.0 mg/kg, intraperitoneal (i.p.), for 30 days) in juvenile male Wistar rats postnatal day (PND) 15 impaired neuronal survival and growth, evidenced by a reduction in the number of neurons and astrocytes in the hippocampus, an increase in inflammatory [Tumor Necrosis Factor alpha (TNF-α) and Interleukin 6 (IL-6)] and apoptotic markers (cleaved caspase 3), as well as a decrease in essential neurotrophins (brain-derived neurotrophic factor and nerve growth factor) [24]. Most of these studies have used non-clinical doses, parenteral administration, focused solely on males, or evaluated later developmental stages, leaving the effects of oral MPH on synaptic plasticity during early development largely unknown.
In this context, this study aimed to evaluate the effects of repeated clinically relevant oral doses of MPH on the levels of pre- and post-synaptic protein markers involved in synaptic plasticity, neuronal development, and neurite outgrowth, with a special focus on identifying possible sex-related differences. In fact, females are largely overlooked in studies with MPH conducted in laboratory animals. For this purpose, healthy juvenile male and female Wistar-Kyoto (WKY) rats were used as an in vivo model that mimics the non-medical use of MPH or misdiagnosis during childhood and early adolescence in humans.
Materials and Methods
Drugs and Chemicals
For clear referencing of materials, all major chemicals and kits used in experimental procedures, ranging from drug treatment, western blot, and immunohistochemistry analysis, are listed in Table 1. Additionally, primary and secondary antibodies used in Western blot and immunohistochemistry analysis are listed in Table 2.Table 1. List of major chemicals used in drug treatment, western blot, and immunohistochemistry analysisChemicalsSupplierThreo-methylphenidate hydrochloride (MPH)Tocris Bioscience (Bristol, United Kingdom)SucroseLaborspirit, Lda (Loures, Portugal)DC™ protein assay kitBIO-RAD Laboratories, Inc. (Hercules, CA, USA)Mini-Protean TGX stain-free gelsBIO-RAD Laboratories, Inc. (Hercules, CA, USA)Trans-blot turbo transfer packsBIO-RAD Laboratories, Inc. (Hercules, CA, USA)4 × Laemmli Sample bufferBIO-RAD Laboratories, Inc. (Hercules, CA, USA)Precision plus protein dual color standardsBIO-RAD Laboratories, Inc. (Hercules, CA, USA)10 × Tris/Glycine/SDS BufferBIO-RAD Laboratories, Inc. (Hercules, CA, USA)Clarity™ Western enhanced chemiluminescence (ECL) substrateBIO-RAD Laboratories, Inc. (Hercules, CA, USA)Triton X-100 and FluorSave™Merck (Darmstadt, Germany)4′,6-diamidino-2-phenylindole (DAPI)Fisher Scientific (Loughborough, UK)Ponceau S solutionSigma-Aldrich (Algés, Portugal)Sodium deoxycholateSigma-Aldrich (Algés, Portugal)Trizma® hydrochlorideSigma-Aldrich (Algés, Portugal)β-mercaptoethanolSigma-Aldrich (Algés, Portugal)TWEEN® 20Sigma-Aldrich (Algés, Portugal)Sodium chlorideJosé Manuel Gomes dos Santos, Lda (Odivelas, Portugal)Sodium dodecyl sulfate (SDS)Panreac Applichem (Barcelona, Spain)Table 2. List of primary and secondary antibodies used in Western blot and immunohistochemistry analysisSupplierCat. No.DilutionPrimary AntibodyMouse anti-microtubule-associated protein 2 (MAP2)Merck (Darmstadt, Germany)ab948561:300Rabbit monoclonal anti-synaptophysinAbcam (Cambridge, UK)ab321271:300Rabbit monoclonal anti-growth-associated protein 43 (GAP43)Abcam (Cambridge, UK)ab758101:5000Rabbit monoclonal anti-postsynaptic density protein 95 (PSD-95)Abcam (Cambridge, UK)ab2381351:1000Rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)Abcam (Cambridge, UK)ab94851:200Secondary AntibodyPolyclonal anti-rabbit, DyLight 488Vector Laboratories (Oxford, UK)green, DI-10881:1000Texas Red horse anti-mouseVector Laboratories (Oxford, UK)red, TI-20001:1000Goat anti-rabbit IgG H&L (HRP)Abcam (Cambridge, UK)ab970511:10,000
Experimental Protocol
WKY rats were used as animal model. WKY rats are frequently used as a control group in studies involving spontaneously hypertensive rats (SHR), one of the most researched animal models of ADHD. The fact that the WKY did not show neuropathological alterations associated with ADHD made it possible to mimic a non-pathological context of exposure to MPH [32, 33].
Initially, four pairs of 4-week-old WKY rats were acquired from Charles River Laboratories (Saint Germain Nuelles, France) and allowed to grow until 3 months of age in the animals’ facility of the Faculty of Medicine of the University of Porto. Subsequently, couples were mated. This breeding resulted in four litters, comprising a total of 37 WKY rats (18 males and 19 females). Rats were housed in acrylic cages with their mothers during the experimental period. During the entire protocol, animals were maintained under strictly controlled environmental conditions, including a temperature of 22 ± 2 °C, 40% relative humidity, and a 12-h light–dark cycle. Throughout the study period, the animals were provided with food and water ad libitum. To identify the animals, non-permanent tattoos were applied to their paws and marks were made on their tails, three to four days prior to the protocol.
To ensure animal welfare and minimize suffering, all animal handling procedures were carried out under continuous veterinary supervision in accordance with the guidelines defined by the European Council Directive (2010/63/EU), transposed into Portuguese legislation (Decreto-Lei no. 113/2013). This study was approved by the Local Committee Responsible for Animal Welfare (Reference ORBEA_127_2023/0607) and by the competent national authority, represented by the Portuguese National Authority for Animal Health (Ref. 65,106/25-S).
Drug Treatment
The animal protocol began with an acclimation phase in which, starting at ten days of age, the animals received oral administration of a 5% sucrose solution. This approach aimed to mitigate procedure-induced stress and facilitate habituation to the handler, thereby ensuring smoother subsequent drug administration by promoting a positive association with the handling experience. Adding sucrose also helps ensure the easy, complete, and efficient ingestion of the drug throughout treatment, thereby avoiding the possible unfavorable taste of MPH.
Considering that the oral route is the primary route of MPH administration in children, to mimic oral administration in humans, rats were administered MPH by oral gavage. To avoid any animal harm, a flexible plastic cannula appropriate for oral gavage in animals of this age was used (Instech, USA). On PND 15 (analogous to human childhood) [34], animals were randomly assigned to two groups. The treated group (n = 17, 8 males and 9 females) received a daily dose of MPH (5 mg/kg in a 5% sucrose solution), while the control group (n = 20, 10 males and 10 females) received an equivalent volume of the 5% sucrose solution [35]. Administration continued for 15 consecutive days, from PND 15 to PND 29, with doses individually adjusted based on the animal’s weight (100 µL of drug or vehicle per 25 g animal weight). Twenty-four hours after the last administration at PND 30, the animals were sacrificed. All administrations were carried out in the morning, between 9 and 11 a.m., and the weight of each animal was monitored throughout the treatment, as well as any clinical signs that could indicate changes in welfare. During the entire experimental period, animals were kept with their respective mothers until the day of sacrifice, to avoid any stress that could arise from separating them at such a young age [36].
This protocol was designed to mimic daily oral treatment with MPH, using a clinically relevant dose equivalent to that administered to children in pediatric ADHD treatment protocols**.** The dose was selected based on pharmacokinetic modeling to achieve peak plasma levels within the clinical range, and it has been previously used by other groups [35, 37, 38]. The treatment period studied (PND 15 to PND 29) corresponds approximately to childhood and early adolescence in humans [39], which are critical phases in brain development and maturation [25]. This timeframe is particularly significant, as it coincides with the typical onset of ADHD symptoms and with the period when most misdiagnoses occur [17, 40].
Sacrifice and Tissue Collection
On PND 30, animals were deeply anesthetized in a closed chamber containing isoflurane, an inhalation anesthetic, until they were completely sedated. Once no response to stimuli was confirmed, exsanguination was immediately performed via the inferior vena cava, using a hypodermic heparinized needle. Animals were then decapitated, and their brains removed and weighed. A mid-sagittal cut was made to separate the hemispheres, which were then alternately assigned for western blot or histological analysis. The hemispheres used for the western blot analysis were grossly dissected to isolate major brain regions of interest, including the diencephalon, frontal cortex (mainly prefrontal cortex, PFC), cerebellum, striatum, and hippocampal formation (hippocampus) as a whole. No further subdivision of the cortex, the striatum, or the hippocampal formation into specific subregions was performed at this stage. These samples were collected in RIPA buffer (Radio Immuno Precipitation Assay buffer, containing 0.1% sodium dodecyl sulfate, 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM sodium chloride, 50 mM Tris) supplemented with protease inhibitors (10 mM sodium fluoride, 1 mM sodium metavanadate, 0.25 mM phenylmethylsulfonylfluoride, and Sigma protease inhibitor cocktail, pH 8.0) and 1 mM dithiothreitol, and frozen at -80 ºC until they were analyzed. The hemispheres used for histological analysis were immersed in 4% paraformaldehyde in phosphate buffer saline (PBS), pH 7.4, for 24 h at 4 °C with gentle agitation for histological processing. After the fixation period, the samples were washed in water for 20 min and then transferred to Olmos solution [30% sucrose, 30% ethylene glycol, and 1% polyvinylpyrrolidone-40 in PBS], where they were stored at -20 °C until further processing.
Immunohistochemistry
After processing the brain samples, as previously described, the tissue blocks obtained were mounted in agarose on a Leica VT1000S vibratome with the rostral surface oriented upwards. These blocks were then sectioned into 40 μm-thick coronal sections from the olfactory bulb to the caudal limit of the hippocampal formation, and the sections were collected in PBS and subsequently washed in Olmos solution to remove the agarose. Until immunofluorescence analysis, the sections were preserved at -20 °C in the Olmos cryoprotectant solution.
These coronal sections were used for the immunofluorescence detection of MAP2 and synaptophysin of hippocampal formation (CA1, CA3, dentate gyrus, and hilus), striatum (ventral and dorsal striatum), and frontal cortex (PFC and motor cortex) subregions, according to previously published procedures [41, 42]. These subregions were identified and delineated using optical and histological criteria in accordance with the Paxinos and Watson rat brain atlas [43] and are detailed in Supplementary Data File 3. A systematic random sampling procedure was used, with sections sampled at a ratio of 1:12. The previously sliced sections were washed three times in PBS for 10 min to completely remove the Olmos solution. Subsequently, the sections were treated with 30% hydrogen peroxide (H₂O₂) in PBS for 10 min to quench erythrocyte autofluorescence, followed by three additional PBS washes (10 min each). After the final wash, sections were blocked with 5% normal horse serum in PBS containing 0.5% Triton X-100 for 1 h at room temperature to prevent nonspecific binding. Following this blocking step, primary antibodies, anti-MAP2, and anti-synaptophysin, diluted 1:300 in PBS 0.1% Triton X-100, were added to the tissue sections and incubated for 72 h at 4 °C with gentle agitation. Then, the sections were mounted on gelatin-coated slides and incubated with the respective fluorescent secondary antibodies for 1 h at room temperature, protected from light. The secondary antibodies used were polyclonal anti-rabbit, DyLight 488 (green; DI-1088), and polyclonal anti-mouse Texas Red (red; TI-2000), both diluted 1:1000 in PBS containing 0.25% Triton X-100. After three final PBS washes (10 min each) to remove any unbound antibodies, the sections were air-dried and cover-slipped with Fluorsafe TM mixed with DAPI (1:100 dilution). Antibody performance was validated prior to analysis, and only markers showing robust detection in immunohistochemical conditions were included in the respective assays. The antibodies used for MAP2 and synaptophysin were validated for immunohistochemistry detection in fixed tissue.
Finally, brain sections immunolabeled with MAP2 and synaptophysin antibodies were photographed using a Carl Zeiss Axio Imager 2.0 microscope equipped with a color camera and Carl Zeiss AxioVision software Rel. 4.8 (New York, USA). Images were manually scanned from regions including the PFC, motor cortex, ventral striatum, dorsal striatum, and hippocampal formation (dentate gyrus, CA1, and CA3) at 200 × total magnification, using the following exposure times: 40 ms for DAPI, 60 ms for DyLight 488, and 75 ms for Texas Red. Image intensity per area was measured using ImageJ 1.52a software; whenever necessary, the area of interest was outlined using the polygon selection tool, and all areas of interest were analyzed.
Tissue Preparation for Western Blotting
To process the samples for western blot analysis, the brain regions were first homogenized on ice using a sonicator for 20 s to ensure complete tissue disintegration. Following homogenization, the samples were centrifuged (16,060 g, 15 min, 4 °C), and the supernatants were recovered for protein quantification.
The protein content of the brain samples was determined using the commercial DC Protein Assay kit, according to the manufacturer’s instructions. Standards of bovine serum albumin were prepared in a range of concentrations between 0 and 2000 µg/mL. Absorbance was measured on a plate reader (BioTek PowerWaxe X, Vermont, USA) at 750 nm.
Western Blot
Sample volumes were adjusted to ensure that 50 μg of protein was loaded onto the electrophoresis gel. Next, following the procedure described by Dias-Carvalho et al. [42], brain homogenates were reduced with Laemmli buffer [0.5 M Tris–HCl, pH 6.8, 4% (w/v) SDS, 15% (v/v) glycerol, 0.04% (w/v) bromophenol blue, and 20% (v/v) β-mercaptoethanol] in a 1/2 (v/v) ratio. This mixture was then heated at 100 °C for 5 min. For electrophoresis, pre-made Mini-PROTEAN® TGX Stain-Free™ gels were used. The gels were run under a constant voltage of 170 V in running buffer [25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS], allowing proteins to migrate through the gel according to their molecular weights.
After electrophoresis, the gels were transferred to a nitrocellulose membrane using the Trans-Blot® Turbo™ Transfer System (Bio-Rad®, CA, USA) at a constant current of 2.5 A for 7 min. Transfer efficiency and sample integrity were verified by staining the membranes with Ponceau S. Once the proteins were successfully transferred, the membranes were blocked to avoid non-specific binding for 1 h at room temperature with agitation in a 5% (w/v) nonfat dry milk solution in TBS-T [100 mM Tris (pH 8.0), 1.5 M NaCl, and 0.5% Tween 20]. After this step, the membranes were incubated overnight at 4 °C with constant agitation with primary antibodies anti-GAP43 (1:5000), anti-PSD-95 (1:1000) or anti-GAPDH (1:200). To optimize antibody incubation and reduce background noise, each membrane was cut between the molecular weight markers of 50 and 75 kDa. This allowed the simultaneous incubation of antibodies against GAP43 and PSD-95 in different sections of the membrane under optimal conditions. The membrane section used for the GAP43 antibody was later reused for incubation with the anti-GAPDH antibody. After incubation with the primary antibodies, the membranes were washed three times (10 min each) with TBS-T to remove any unbound antibodies or contaminants. Following the washing step, the membranes were incubated with secondary antibodies, anti-rabbit (1:10,000) or anti-mouse (1:5000) horseradish peroxidase, for 25 min at room temperature under agitation. All antibodies were diluted in 5% (w/v) nonfat dry milk in TBS-T. After a final washing step, immunoreactive bands were detected using ECL reagents, following the manufacturer’s instructions. Images were captured and scanned using the Gel Doc XR imaging system (Bio-Rad®, CA, USA) and analyzed with Image Lab software (Bio-Rad®, CA, USA, version 6.0.1) for arbitrary units of optical density (OD). The average of the control bands of each membrane was calculated. Then, these average values were divided by the lowest value obtained among the control bands of two membranes, creating a normalization factor by which the remaining samples were divided. These OD values were divided by 1000, and the data were presented in graphs as arbitrary units of OD.
Moreover, before incubation with the antibodies, the membranes were stained with Ponceau S to assess equal sample loading.
Statistical Analysis
Results were expressed as mean ± standard deviation (SD). Outliers were identified using the ROUT’s test. Statistical analysis of the animals’ weight was carried out by the two-way analysis of variance (ANOVA) with repeated measurements test. For other data comparisons, a t-test was used to compare two groups: control versus treated. Differences were considered significant at p values lower than 0.05. Statistical analysis was conducted using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).
Results
Treatment with MPH did not Impact Body Weight Gain in WKY Rats
To evaluate the influence of the drug on body growth, the animals were weighed daily throughout the entire study until the day of sacrifice. The results were analyzed separately for males and females, with values expressed as a percentage of initial weight in grams per day (gr/day). Figure SF1 in the supplementary data file 1 presents the percentage of body weight gain per day in males (A) and females (B). Data reveal no statistically significant changes in body weight gain among control and MPH-treated rats at any treatment period. Also, no notable sex differences were observed in terms of body weight gain (data not shown).
MPH-Treated Female WKY Rats Showed Decreased PSD-95 Levels in PFC
Figure 1 shows the effects of MPH (5 mg/kg) on GAP43, PSD-95, and GAPDH protein levels in the PFC of male and female WKY rats. All corresponding Ponceau S images and western blots from all analyzed datasets are provided in Supplementary Data File 2.Fig. 1. Western blotting analysis of GAP43 (A, B), PSD-95 (C, D) and GAPDH (E, F) levels in prefrontal cortex (PFC) of male [control n = 8; MPH n = 7] and female [control n = 8; MPH n = 7] Wistar Kyoto (WKY) rats, in the control and MPH-treated groups. The control and treated group samples were processed after daily oral administration of sucrose solution or 5 mg/kg MPH, respectively, for 15 consecutive days. The values obtained were presented as optical density (OD), expressed in arbitrary units (a.u.). Statistical comparisons were conducted using the t-test: *p < 0.05, control versus MPH 5 mg/kg treated animal. Protein loading was confirmed by the Ponceau S staining (Figures in Supplementary Data File 2)
For GAP43, the levels of this synaptic protein were not significantly altered in either males or females following MPH administration compared with their respective controls (Fig. 1A and B). In contrast, analysis of PSD-95 revealed a significant reduction in protein levels in female rats treated with MPH relative to female controls (Fig. 1D). However, no significant differences in PSD-95 levels were detected between control and MPH-treated male rats (Fig. 1C). Finally, GAPDH levels remained unchanged in both sexes following exposure to MPH (Fig. 1E, F).
MPH-Treated WKY Males Showed Increased PSD-95 Levels in the Striatum
Figure 2 presents the levels of the proteins GAP43, PSD-95, and GAPDH in the striatum of WKY rats, daily exposed to a clinically relevant dose of 5 mg/kg MPH.Fig. 2. Western blot analysis of GAP43 (A, B), PSD-95 (C, D), and GAPDH (E, F) levels in the striatum of juvenile male [control n = 8; MPH n = 6] and female [control n = 8; MPH n = 7] Wistar Kyoto (WKY) rats, in the control and MPH-treated groups. The control and treated group had daily oral administration of sucrose solution or 5 mg/kg MPH, respectively, for 15 consecutive days. The values obtained were presented as optical density (OD), expressed in arbitrary units (a.u.). Statistical comparisons were conducted using the t-test: **p < 0.01, control versus MPH 5 mg/kg treated animals. Protein loading was confirmed by the Ponceau S staining (Figures in Supplementary Data File 2)
No changes were observed in GAP43 levels in males (Fig. 2A). In contrast, levels of PSD-95 were significantly higher in MPH-treated male WKY rats compared to their respective controls (Fig. 2C). In females, no differences were observed in either of the two neuronal proteins analyzed (Fig. 2B, D). GAPDH levels remained unchanged following exposure to a clinically relevant dose of MPH in both male (Fig. 2E) and female (Fig. 2F) WKY rats.
MPH Decreased the Levels of GAP43 Protein in the Cerebellum of Male WKY Rats
Figure 3 illustrates the effects of oral administration of 5 mg/kg MPH on the levels of GAP43, PSD-95, and GAPDH proteins in the cerebellum of juvenile male and female WKY rats.Fig. 3. Western blot analysis of GAP43 (A, B), PSD-95 (C, D), and GAPDH (E, F) levels in the cerebellum of juvenile male [control n = 8; MPH n = 6] and female [control n = 8; MPH n = 7] Wistar Kyoto (WKY) rats, in the control and MPH-treated groups. The control and treated group had daily oral administration of sucrose solution or 5 mg/kg MPH, respectively, for 15 consecutive days. The values obtained were presented as optical density (OD), expressed in arbitrary units (a.u.). Statistical comparisons were conducted using the t-test: *p < 0.05, control versus MPH 5 mg/kg treated animals. Protein loading was confirmed by the Ponceau S staining (Figures in Supplementary Data File 2)
The determination of neuronal proteins GAP43 and PSD-95 in cerebellar homogenates revealed that MPH administration affected GAP43 levels only in male WKY rats (Fig. 3A), resulting in a decrease in GAP43 levels, without changes in females (Fig. 3B). PSD-95 levels remained unchanged in both male and female WKY MPH-treated rats (Fig. 3C, D). Additionally, MPH did not induce any significant alterations in GAPDH levels in either males or females compared to their respective control groups (Fig. 3E, F).
MPH Alters GAPDH Levels in the Diencephalon of WKY Rats
Figure 4 shows the levels of GAP43, PSD-95, and GAPDH in the diencephalon of juvenile WKY rats, differentiated by sex, following repeated exposure to clinically relevant doses of MPH.Fig. 4. Western blot analysis of GAP43 (A, B), PSD-95 (C, D), and GAPDH (E, F) levels in the diencephalon of juvenile male [control n = 8; MPH n = 7] and female [control n = 7; MPH n = 7] Wistar Kyoto (WKY) rats, in the control and MPH-treated groups. The control and treated groups had daily oral administration of sucrose solution or 5 mg/kg MPH, respectively, for 15 consecutive days. The values obtained were presented as optical density (OD), expressed in arbitrary units (a.u.). Statistical comparisons were conducted using the t-test: *p < 0.05, control versus MPH 5 mg/kg treated animals. Protein loading was confirmed by the Ponceau S staining (Figures in Supplementary Data File 2)
The western blot analysis (Fig. 4) revealed that the groups treated with 5 mg/kg MPH exhibited similar levels of GAP43 and PSD-95 in the diencephalon compared to the control groups, in both males (Fig. 4A, C) and females (Fig. 4B, D). However, MPH-treated males revealed a significant increase in GAPDH levels (Fig. 4E). This trend was not observed in female WKY rats (Fig. 4F).
MPH Administration did not Affect GAP43, PSD-95, nor GAPDH Levels in the Hippocampus of WKY rats
Data presented in Fig. 5 illustrate the impact of orally administered clinically relevant doses of 5 mg/kg MPH on the levels of GAP43, PSD-95, and GAPDH in the hippocampus of male and female WKY rats, during the developmental period between PND 15 and PND 29.Fig. 5. Western blot analysis of GAP43 (A, B), PSD-95 (C, D), and GAPDH (E, F) levels in the hippocampus of juvenile male [control n = 8; MPH n = 7] and female [control n = 8; MPH n = 7] Wistar Kyoto (WKY) rats, in the control and MPH-treated groups. The control and treated group had daily oral administration of sucrose solution or 5 mg/kg MPH, respectively, for 15 consecutive days. The values obtained were presented as optical density (OD), expressed in arbitrary units (a.u). Statistical comparisons were conducted using the t-test: control versus MPH 5 mg/kg treated animals. Protein loading was confirmed by the Ponceau S staining (Figures in Supplementary Data File 2)
As observed for the diencephalon, treatment with MPH (5 mg/kg) did not result in significant differences in the levels of GAP43 (Fig. 5A, B) or PSD-95 (Fig. 5C, D) in the hippocampus of juvenile WKY rats, compared to the control groups (5% sucrose solution). This lack of effect was observed in both male and female subjects. GAPDH levels in the hippocampus also remained unchanged following MPH treatment (Fig. 5E, F).
No sex-Specific Differences Were Found in GAP43, PSD-95, or GAPDH Basal Levels Across Multiple Brain Regions of Juvenile WKY Rats
The graphic plots presented in Figure SF2 in the supplementary data file 1 show the comparison of GAP43, PSD-95, and GAPDH basal control protein levels between male and female WKY rats at PND 30, across five brain regions: PFC (Fig. SF2A, B, C), striatum (Fig. SF2D, E, F), hippocampus (Fig. SF2G, H, I), cerebellum (Fig. SF2J, K, L), and diencephalon (Fig. SF2M, N, O).
The western blot results indicated that at PND 30, no significant differences were detected in the basal levels of GAP43, PSD-95, or GAPDH proteins in any of the brain regions analyzed when comparing control male and control female WKY rats.
MPH did not Alter Synaptophysin and MAP2 Immunoreactivity Levels in the PFC of WKY Rats Exposed to Therapeutic Dosing of MPH
Figure 6 shows the graphic plots for synaptophysin (Fig. 6A, B) and MAP2 (Fig. 6C, D) immunoreactivity in the PFC of juvenile WKY rats orally treated with MPH during the developmental period from PND 15 to PND 29. As illustrated, MPH did not induce any significant changes in MAP2 and synaptophysin levels in the PFC of male (Fig. 6A, C) and female (Fig. 6B, D) WKY rats, when compared to the control group.Fig. 6. Immunohistochemistry analysis of protein levels of synaptophysin (A, B, E, F), and MAP2 (C, D, G, H) in juvenile male [control n = 4; MPH n = 4] and female [control n = 5; MPH n = 4] Wistar Kyoto (WKY) rats, across cortex regions namely the PFC (A, B, C, D), and motor cortex (E, F, G, H). The control and treated group had daily oral administration of sucrose solution or 5 mg/kg MPH, respectively, for 15 consecutive days. The values obtained are presented as optical density (OD) per area, expressed in arbitrary units (a.u)/µm2. Statistical comparisons were conducted using the t-test: control versus MPH 5 mg/kg treated animals. Representative immunohistochemistry images can be found in Supplementary Data File 3
Representative immunohistochemistry images of the analyzed area and its iconography are available in Supplementary Data File 3.
Synaptophysin and MAP2 Immunoreactivity Levels Remained Unaffected in the Motor Cortex of Juvenile WKY Rats After MPH Administration
The graphic plots presented in Fig. 6 show the immunoreactivity of synaptophysin (Fig. 6E, F) and MAP2 (Fig. 6G, H) in the motor cortex of juvenile WKY rats. The analysis revealed that synaptophysin and MAP2 levels did not differ significantly between the treated and control groups in the motor cortex. These findings were consistent across both male (Fig. 6E, G) and female (Fig. 6F, H) WKY rats.
Striatal Synaptophysin and MAP2 Immunoreactivity Levels in WKY Rats were Unchanged After Repeated MPH Exposure
The results presented in Fig. 7 indicate that administration of a clinically relevant dose of MPH did not alter the overall immunoreactivity of synaptophysin (Fig. 7A, B) and MAP2 (Fig. 7C, D) in the striatum of juvenile males and females WKY rats. Specific analysis of the dorsal (Fig. 7E, F, G, H) and ventral (Fig. 7I, J, K, L) regions confirmed that synaptophysin (Fig. 7E, F, I, J) and MAP2 (Fig. 7G, H, K, L) levels remained unchanged compared to controls, in both sexes.Fig. 7. Immunohistochemistry analysis of protein levels of synaptophysin (A, B, E, F, I, J), and MAP2 (C, D, G, H, K, L) in juvenile male [control n = 4; MPH n = 4] and female [control n = 5; MPH n = 4] Wistar Kyoto (WKY) rats, across striatum regions (A, B, C, D), and in the dorsal striatum (E, F, G, H), and ventral striatum (I, J, K, L). The control and treated group had daily oral administration of sucrose solution or 5 mg/kg MPH, respectively, for 15 consecutive days. The values obtained are presented as optical density (OD) per area, expressed in arbitrary units (a.u)/µm^2^. Statistical comparisons were conducted using the t-test: control versus MPH 5 mg/kg treated animals. Representative immunohistochemistry images can be found in Supplementary Data File 3
Synaptophysin Immunoreactivity Levels were Reduced in the CA1 Region of the Hippocampal Formation of Male Rats Following Therapeutic-Relevant MPH Administration
The data presented in Fig. 8 illustrate the impact of orally administering clinically relevant doses (5 mg/kg MPH) during the developmental phase, between PND 15 and PND 29, on synaptophysin and MAP2 immunoreactivity levels in the hippocampal formation of male and female WKY rats.Fig. 8. Immunohistochemistry analysis of protein levels of synaptophysin (A, B, E, F, I, J, M, N, Q, R), and MAP2 (C, D, G, H, K, L, O, P, S, T) in juvenile male [control n = 5; MPH n = 4] and female [control n = 5; MPH n = 4] Wistar Kyoto (WKY) rats, across hippocampus regions (A, B, C, D) and in the CA1 (E, F, G, H), CA3 (I, J, K, L), dentate gyrus (M, N, O, P), and hilus (Q, R, S, T). The control and treated group had daily oral administration of sucrose solution or 5 mg/kg MPH, respectively, for 15 consecutive days. The values obtained are presented as optical density (OD) per area, expressed in arbitrary units (a.u)/µm^2^. Statistical comparisons were conducted using the t-test **p < 0.01, control versus MPH 5 mg/kg treated animals. Representative immunohistochemistry images can be found in Supplementary Data File 3
These results indicate that the experimental groups (treated with MPH at 5 mg/kg) exhibited average levels of synaptophysin (Fig. 8A, B) and MAP2 (Fig. 8C, D) similar to those of the control groups (treated with 5% sucrose solution).
The separate histological analysis in the hippocampal subregions CA1, CA3, dentate gyrus, and hilus enabled the assessment of any potential changes in synaptophysin and MAP2 levels in these specific subregions in the experimental model due to exposure to MPH.
The levels of synaptophysin and MAP2 showed no significant variations between the groups treated with 5 mg/kg of MPH and the control groups, across most of the analyzed hippocampal regions, including the CA3 (Fig. 8I, J, K, L), dentate gyrus (Fig. 8M, N, O, P), and hilus (Fig. 8Q, R, S, T). However, in the CA1 region of male WKY rats, treatment with MPH resulted in a significant reduction in synaptophysin levels (Fig. 8E), a trend not observed in female WKY rats (Fig. 8F). Furthermore, the levels of MAP2 in the CA1 region remained unchanged in both male (Fig. 8G) and female (Fig. 8H) rats.
Male and Female WKY Juvenile Rats Revealed Different Basal Levels of Synaptophysin and MAP2 Immunoreactivity
Sex-related differences in the basal immunoreactivity levels of synaptophysin and MAP2 were identified in the whole striatum of WKY rats at PND 30, with males displaying higher levels of both proteins, as presented in Figs. 9A and 9B. Interestingly, when the histological data from each striatal subregion were analyzed separately, it was observed that synaptophysin levels were significantly higher in the dorsal striatum (Fig. 9C) of male WKY rats compared to females. However, in the ventral striatum (Fig. 9E), no statistically significant differences in synaptophysin levels were detected between the sexes. These findings suggest that the overall differences in synaptophysin levels observed in the upper graphs are primarily attributable to variations in the dorsal striatum. Although an overall increase in MAP2 levels was observed in the whole striatum of male WKY rats compared to females (Fig. 9B), this difference was not confirmed when the dorsal (Fig. 9D) and ventral striatum (Fig. 9F) were analyzed separately.Fig. 9. Comparative analysis of protein levels of synaptophysin, and MAP2, obtained by immunochemistry, between juvenile male [control n = 4] and female [control n = 5] Wistar Kyoto rats (WKY) rats, across striatum regions, and in the dorsal striatum (C, D), and ventral striatum (E, F). The values obtained are presented as optical density (OD) per area, expressed in arbitrary units (a.u.)/µm^2^. Statistical comparisons were conducted using the t-test: *p < 0.05 male versus female animals. Representative immunohistochemistry images can be found in Supplementary Data File 3
In the other brain regions (graphic plots presented in the supplementary data file 1), namely the PFC (Fig. SF3A, B), the motor cortex (Fig. SF3C, D), and all hippocampal subregions analyzed (Fig. SF4), there were no significant differences in the basal levels of synaptophysin and MAP2 immunoreactivity between male and female WKY rats.
Discussion
Our paper reports a protocol using both female and male WKY rats that was designed to mimic daily oral MPH treatment, using a clinically relevant dose equivalent to that administered to children with ADHD, thereby achieving plasma levels in animals within the clinical range. Table 3 summarizes the main protein alterations observed in the brains of WKY rats following exposure to MPH.Table 3. Summary of sex-specific changes in the expression levels of PSD-95, GAPDH, and GAP43 obtained by Western blot, and in the immunoreactivity levels following immunohistochemistry analyses of synaptophysin, observed in some of the analyzed brain regions (prefrontal cortex [PFC]; striatum, cerebellum, hippocampus CA1, and diencephalon) of Wistar Kyoto (WKY) rats exposed to 5 mg/kg MPH. Arrows indicate relative changes compared to controls: ↑ increased, ↓ decreasedPFCStriatumDiencephalonCerebellumCA1Male↑ PSD-95↑ GAPDH↓ GAP43↓SynaptophysinFemale↓ PSD-95
One of the known adverse effects of MPH is weight loss [44], which raises the possibility that the drug could negatively impact growth in WKY rats. However, in this study, rats treated with therapeutic doses of MPH from PND 15 to PND 29 showed similar weight gains to controls, which aligns with findings from a previous study conducted in our laboratory [35]. Other studies have evaluated the impact of MPH under different experimental conditions. A study published in 2015, showed a tendency towards weight loss in Wistar rats (180-200 g) following oral administration of MPH (2, 5, 8 mg/kg) twice daily for 4 weeks [45]. Similarly, Gray and colleagues reported a significant reduction in body weight after administering rats a 5 mg/kg dose of MPH, administered i.p. twice daily, from PND 7 to PND 35 [46]. In another study, prolonged exposure to MPH also affected the growth of Sprague–Dawley rats: four-week-old rats treated with 4, 10, 30, or 60 mg/kg of MPH for 3 months revealed a dose-dependent decrease in body weight [47]. Thus, the absence of significant differences in weight gain in our study may be explained by the relatively low clinical dosage, the route of administration, and the specific treatment window selected—after the neonatal phase but before the periadolescent period. On the other hand, some authors suggest that changes in body weight may be a natural symptom of ADHD, and not necessarily a direct effect of treatment [48, 49]. In this context, it would be important to compare the data obtained from WKY rats with models of ADHD, such as the spontaneously hypertensive rat.
GAP43 is a protein highly expressed in axon terminals and growth cones during embryonic and perinatal development, where it regulates processes such as neuron growth and the formation of synaptic connections [50]. Western blot analysis revealed that MPH treatment reduced GAP43 levels only in the cerebellum of male WKY rats; meanwhile, no changes were observed in other brain areas or in female rats. Decreased levels of GAP-43 have been linked to disruptions in synaptic connectivity and neurogenesis [51]. However, our findings should be interpreted considering GAP43’s role in cerebellar development, where its levels are essential for granule cell differentiation and migration, as well as for axonal growth and synaptogenesis. GAP43 mRNA levels are typically high in immature neurons (up to PND 7) and gradually decrease as the brain matures [15]. When GAP43 is not expressed, the centrosome and mitotic spindles become misaligned during brain growth, resulting in a significant decrease in cerebellum size [50]. Thus, the reduction in GAP43 levels observed in juvenile male WKY rats induced by MPH suggests changes in structural integrity and plasticity of the cerebellum. This effect is concerning, given the crucial role of the cerebellum in motor coordination, balance, and cognitive function [52]. In another study, oral administration of the same dose of MPH (5 mg/kg/day on weekdays orally) to WKY rats for 30 days, from PND 28 to PND 55 (period analogous to late childhood through late adolescence in humans) resulted in decreased levels of GAP43 in the hippocampus [20]. The differences in response to MPH compared to our findings may be primarily attributed to the distinct developmental periods of the rats.
Our study also revealed changes in PSD-95 levels in the PFC and striatum of juvenile WKY rats exposed to MPH. PSD-95, localized at the excitatory synapses, is responsible for the organization of excitatory synapses, regulating the location and stabilization of glutamate receptors, which, when stimulated, promote long-term potentiation and long-term depression processes [53, 54]. In addition, PSD-95 plays an important role in the morphogenesis of dendritic spines, which also contributes to synaptic strength [55]. The reduction of PSD-95 levels observed in the PFC of WKY females may indicate alterations in dendritic spine morphology due to MPH exposure. Also, the decrease in dendritic spines impairs long-term potentiation, weakens synaptic strength, and excitatory signal transmission [56]. These findings suggest that exposure to clinical doses of MPH alters synaptic plasticity during PFC development in WKY females, which is relevant, as the PFC during early childhood exhibits a dendritic spine density 2 to 3 times higher than that observed in the adult PFC [57]. Moreover, in rodents, synaptic density in the PFC peaks around the 4th PND, a period of high synaptic plasticity [57]. Considering that the study evaluated the effect of MPH on the PFC of rats between PND 15 and PND 29, the alterations in PSD-95 levels may compromise synaptic maturation and potentially lead to long-term changes in processes regulated by the PFC. A study conducted on WKY rats (PND 28 to PND 55) found that oral exposure to a similar dose of MPH (5 mg/kg/day on weekdays) resulted in decreased levels of PSD-95 in the hippocampus. This decrease was also associated with impaired memory in MPH-treated rats, highlighting the link between neuronal changes and cognitive deficits [20]. It is also important to emphasize the roles of dopamine and adrenergic receptors in the context of MPH actions in the PFC, which appear to involve indirect stimulation of α_2_ adrenoceptors and D_1_ receptors [58]. It is worth noting that greater expression of D_1_, but less of D_2_, in female cortex compared with males at PND 30 has been previously reported [59]. Therefore, the differential response of PSD-95 expression among sexes following MPH can also be related to dimorphic expression in dopaminergic markers in the PFC. On the other hand, α_2A_ were colocalized in dendritic spines, known for the presence of PSD-95, within the PFC [60]. In cultured cortical neurons, α_2A_ stimulation promoted dendritic growth and enhanced PSD-95 expression [61]. These in vitro results do not align with our animal results, which showed no increase but instead a decrease in PSD-95 in females treated with MPH.
In contrast to what was observed in the PFC of female WKY rats, repeated treatment with MPH resulted in a significant increase in PSD-95 levels in the striatum of male WKY rats. Since MPH primarily acts on the dopaminergic system [62] and the striatum is densely innervated by dopaminergic projections [58], the synaptic plasticity associated with increased PSD-95 levels may represent an adaptive response to the drug-induced enhancement of dopaminergic neurotransmission. This interpretation aligns with the existing literature, which suggests that in striatal neurons, PSD-95 may negatively regulate synaptic activity mediated by D_1_ receptors, thereby preventing excessive dopaminergic activity [63, 64]. Although we did not find basal differences in PSD-95 levels between males and females across the analyzed brain areas, subtle subregion-specific differences have been reported. In adult Sprague–Dawley rats, females have, on average, larger and more intensely labeled PSD-95 puncta in the nucleus accumbens than males, which explains the differential response to psychostimulants between the sexes [65].
GAPDH is a glycolytic enzyme known for its role in cellular metabolism and other processes, including RNA transport, DNA replication and repair, membrane trafficking (endocytosis and exocytosis), cytoskeleton organization, apoptosis, and necrosis [66, 67]. Given GAPDH’s role in glycolysis, the increased GAPDH levels observed in the diencephalon of treated males may reflect a compensatory response to the drug, indicating higher energy demands due to the drug’s enhancement of dopaminergic neurotransmission [68]. However, no changes were observed in other brain areas, and it remains unclear why changes were only seen in this area. This study also highlights the erroneous idea that GAPDH can be a reliable brain housekeeping protein, given that several studies found significant variations in GAPDH levels in the brain, both at the mRNA and protein levels [69].
Synaptophysin is an integral membrane glycoprotein widely expressed in neuronal synaptic vesicles [70], and in the context of neurodevelopment, synaptophysin represents an important marker of synaptic formation and plasticity in the brain [71–73]. In the CA1 region of male WKY rats, exposure to MPH led to a notable reduction in synaptophysin immunoreactivity levels. A decrease in synaptophysin might indicate that MPH acts on either the number or effectiveness of synaptic vesicles available for neurotransmitter release. On the other hand, reduced levels of this protein can be detrimental to synaptic plasticity and neuronal development, as evidenced in mice lacking synaptophysin [73], which is relevant since synaptic plasticity in the hippocampus is necessary for learning and for memory encoding, a process for which the CA1 region is responsible [74].
Our findings differ from those of Coelho-Santos and colleagues, who reported that oral administration of MPH (5 mg/kg/day on weekdays) to WKY rats from PND 28 to PND 55 did not alter synaptophysin levels in the hippocampus as assessed by western blotting [20]. This difference between the two studies may be attributed to the different developmental periods studied and to the possibility of region-specific analysis. In fact, in rodents, the critical period of synaptogenesis occurs during the first three postnatal weeks, during which the number of synapses in the dentate gyrus is less than 1% of an adult [75]. In parallel, in the developing rat hippocampus, there is a significant increase in the spine density between PND 7 and PND 28, and in long-term potentiation intensity, from PND 10 to PND 20 [76]. Given that the period evaluated in this study (PND 15 to PND 29) coincides precisely with this phase of intense plasticity, it seems that the hippocampus might be particularly susceptible to the changes induced by MPH compared to later stages, when synapses have already matured and are more stable. On the other hand, analysis of brain sections allowed us to detect alterations specifically in the CA1 region of the hippocampus, which might have been missed by the whole hippocampus analysis performed by Coelho-Santos.
MAP2 is a neuron-specific cytoskeletal protein associated with microtubule stabilization and dendritic arborization during neuronal development [77]. Contrary to what was observed for other proteins tested, the administration of MPH during PND 15 to PND 29 did not alter MAP2 immunoreactivity levels in any of the brain regions analyzed. To date, no studies have been found in the literature to corroborate our results, making this the first study to evaluate MAP2 in models exposed to clinically relevant doses of MPH.
In healthy WKY rats, sex differences in behavioral responses to MPH have been reported [78]. It would be expected that MPH could also manifest differently in males and females at the neurobiological level, namely in the levels of synaptic proteins. We observed a tendency towards greater changes in synaptic proteins in male WKY rats compared to females when exposed to the same clinically relevant oral dose of MPH. Male rats showed reduced levels of GAP43 and synaptophysin immunoreactivity levels in the cerebellum, and an increased expression of GAPDH and PSD-95 was noted in the diencephalon and striatum. Meanwhile, reduced synaptophysin immunoreactivity levels were observed in the CA1 subregion of treated males. In females, only the PFC was affected, with reduced PSD-95 protein levels. These results indicate a sex-specific response, with male rats appearing more susceptible to the effects of MPH. Our study highlights the need to consider sex as a critical variable in animal studies involving MPH. There is a conspicuous lack of data on females, making it difficult to fully understand how MPH affects each sex. In fact, several clinical trials have highlighted that girls with ADHD receive less medication, and the efficacy/effectiveness of MPH differs between the sexes in several measures [79].
Considering the greater impact of MPH on males, it became important to assess whether, at the baseline level, there were sex differences in the proteins analyzed in control animals. Overall, at 30 days of age, we found only differences in the basal levels of MAP2 and synaptophysin within the striatum. A recent study conducted on male and female adult Wistar rats reported that soma size in the NAc shell, a subarea of the striatum, was larger in males than in females across multiple estrous cycle phases [80], which is consistent with our data showing higher MAP2 levels in the striatum of males when compared to female WKY rats. Differences in synaptophysin levels may reflect more pronounced synaptic plasticity in males at this age. Further studies focusing on the dorsal striatum are needed to confirm this hypothesis.
This work has several limitations. We evaluated the levels of proteins involved in synaptic plasticity, but we did not evaluate synaptic plasticity through direct measurements, which could reveal objective brain area-specific differences. We complemented our Western blotting with neuronal counts via immunocytochemistry to circumvent potential inherent flaws, such as uneven bands and potential artifacts associated with the first technique. The use of a single strain of animals, particularly WKY rats, may reveal limitations in extrapolating the herein obtained results to other rat strains or even humans.
Conclusion
Our results suggest that therapeutic doses of MPH in healthy models can induce significant changes in neuronal development and synaptic plasticity. Considering the widespread use of MPH in childhood and the limited literature on the effects of exposure to clinical doses of MPH, especially during the early stages of development, this study addresses critical gaps, offering new insights into how MPH may influence synaptic plasticity mechanisms. Importantly, we identified significant sex-related differences in the brain’s response to MPH, which should be carefully considered in future animal studies investigating the neurobiological effects of this drug. Finally, our findings may open new avenues for research in ADHD models, as the ability of MPH to influence neuroplasticity could, in theory, have important therapeutic implications.
Supplementary Information
Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 701 KB)Supplementary file2 (DOCX 3595 KB)Supplementary file3 (DOCX 6278 KB)