Dose Dependent Effects of Methylphenidate on Hippocampal Long-Term Potentiation and Gene Regulation in an Amisulpride-Based Mouse Model of ADHD

Abstract

The attention deficit hyperactivity disorder (ADHD) is a complex neuropsychiatric condition with high prevalence, particularly in children. Alterations in dopaminergic neurotransmission are a well established feature of ADHD and also affect the hippocampus, a key structure for learning and memory. This study investigates dose dependent effects of the ADHD medication methylphenidate (MPH) on hippocampal synaptic plasticity using <em>in vitro</em> electrophysiology and transcriptomics. Acute hippocampal slices from 28–34 day old male C57BL/6J mice received extracellular long term potentiation (LTP) stimulation using microelectrode array in the presence of MPH (0.2 µM, 20 µM, or 200 µM) and/or the D2 receptor antagonist amisulpride (Ami; 5 µM or 20 µM). Field EPSPs (fEPSPs) were recorded and analyzed for slope and amplitude. Transcriptome analysis was performed using microarrays and included volcano plots, principal component analysis, fold change analysis, gene ontology enrichment, and Venn analysis. Statistical evaluation was conducted using one way ANOVA and Tukey's HSD test. <br/> Electrophysiological recordings revealed that 5 µM Ami increased the fEPSP slope, whereas co-application of 200 µM MPH abolished this effect. The suppressive action of high-dose MPH may involve inhibition of NMDAR, reduced activation of the CaMKII/PKA/PKC/PI3K/ERK/Ca²⁺ signaling cascade, diminished AMPAR insertion, and decreased eIF4E/4E BP/S6/CREB activity. Transcriptome analysis after one-way ANOVA identified several intersectional genes for 0.2 µM and 20 µM MPH that were oppositely regulated at 200 µM MPH. Subsequent Tukey's HSD post-hoc testing revealed that the differentially expressed genes (DEGs) <em>Nudt21</em> and <em>Nin</em> were significantly downregulated, whereas <em>Capn2</em> was significantly upregulated for 0.2 µM and 20 µM MPH compared with ACSF controls. All three DEGs interact with GSK3β and the Wnt/β catenin and mTOR/axin pathways, which regulate hippocampal LTP and contribute to ADHD risk and pathology. <br/> Overall, this thesis elucidates potential mechanisms underlying dose dependent MPH effects on hippocampal LTP in the healthy brain and in ADHD patients and delivers important implications for chronic MPH misuse.