Effect of long‐term methylphenidate exposure on neurogenesis in the hippocampus

Abstract

Attention Deficit Hyperactivity Disorder (ADHD) is a brain disorder marked by a pattern of inattention and hyperactivity that disrupts everyday functions. This disorder is commonly treated with the drug methylphenidate (MPH), which inhibits reuptake of the neurotransmitters norepinephrine and dopamine, thereby increasing the levels of these catecholamines in the synaptic cleft. In addition to its therapeutic use, MPH is commonly abused by students to increase focus and wakefulness while studying for exams. In order to better understand the long‐term effects of MPH and its pharmacologic activities, we examined the impact of 4 weeks of MPH treatment on neurogenesis in the hippocampus. Interestingly, both low (1 mg/kg) and high (10 mg/kg) doses of MPH delivered twice daily, increased the rate of neurogenesis after 4 weeks. Neurogenesis was measured using 5′‐ethinyldeoxyuridine (EdU), and mice were subjected to a 2 day methylphenidate‐free washout, injected with EdU, then brains were collected 24 hours later. Additionally, we examined the survival of the new cells 4 weeks after EdU injection; here, half of the mice received injections (saline, 1 mg/kg, and 10 mg/kg MPH) for an additional 4 weeks and half were maintained in standard housing with no injections. Mice were sacrificed via transcardial perfusion with 4% paraformaldehyde, and newly generated cells were stained for EdU using Click‐IT chemistry and were double‐labeled using immunohistochemistry for the neuronal marker, NeuN. Cell counts were performed and ratios of EdU+/NeuN+ cells were compared. Although both 1 mg/kg and 10 mg/kg MPH increased the ratio of EdU+/NeuN+ cells, the EdU+/NeuN+ ratios were no different from control (saline) if drug was not continued. Interestingly, if 1 mg/kg MPH was continued for an additional 4 weeks, survival of newly generated cells was enhanced; however, this was not the case for the high dose (10 mg/kg) of MPH. In an effort to investigate the mechanism for MPH‐induced changes in hippocampal neurogenesis, we investigated the levels of several proteins linked to cell growth and survival in the hippocampus, including brain derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), tropomyosin receptor kinase B (TrkB, the receptor for BDNF), and beta‐catenin. For these studies, mice were divided into 6 cohorts: 2 groups receiving 10 mg/kg of MPH for 4 or 8 weeks, 2 groups receiving 1mg/kg for 4 or 8 weeks, and 2 control groups injected with saline for 4 or 8 weeks. All 6 groups were injected twice a day, 5 days a week. After either 4 weeks or 8 weeks, mice were decapitated, brains dissected, and tissues were flash‐frozen in liquid nitrogen. Then, levels of BDNF or GDNF were examined using enzyme‐linked immunosorbent assays (ELISAs), and VEGF, TrkB, and beta‐catenin expression was investigated using simple western. Interestingly, 1 mg/kg MPH appears to increase VEGF, TrkB, and beta catenin after 4 weeks. In the animals treated with 10 mg/kg MPH, despite the observed increases in neurogenesis after 4 weeks of treatment, beta catenin levels were decreased compared to control at 4 weeks, and VEGF, TrkB, and beta catenin levels were decreased at 8 weeks. In conclusion, long‐term exposure to MPH increases neurogenesis rate in the hippocampus; this effect with low doses of MPH may be related to the increased expression of VEGF, TrkB, and beta catenin.Support or Funding InformationThis research was supported by the ETSU Research Development Committee and the Department of Pharmaceutical Sciences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.