Chronic methylphenidate induces increased quinone production and subsequent depletion of the antioxidant glutathione in the striatum.

BACKGROUND: Methylphenidate (Ritalin®) is a psychostimulant used chronically to treat attention deficit hyperactivity disorder. Methylphenidate acts by preventing the reuptake of dopamine and norepinephrine, resulting in an increase in these neurotransmitters in the synaptic cleft. Excess dopamine can be autoxidized to a quinone that may lead to oxidative stress. The antioxidant, glutathione helps to protect the cell against quinones via conjugation reactions; however, depletion of glutathione may result from excess quinone formation. Chronic exposure to methylphenidate appears to sensitize dopaminergic neurons to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We hypothesized that oxidative stress caused by the autooxidation of the excess dopamine renders dopaminergic neurons within the nigrostriatal pathway to be more sensitive to MPTP. METHODS: To test this hypothesis, male mice received chronic low or high doses of MPH and were exposed to saline or MPTP following a 1-week washout. Quinone formation in the striatum was examined via dot blot, and striatal GSH was quantified using a glutathione assay. RESULTS: Indeed, quinone formation increased with increasing doses of methylphenidate. Additionally, methylphenidate dose-dependently resulted in a depletion of glutathione, which was further depleted following MPTP treatment. CONCLUSIONS: Thus, the increased sensitivity of dopamine neurons to MPTP toxicity following chronic methylphenidate exposure may be due to quinone production and subsequent depletion of glutathione.

Neurogenesis within the hippocampus after chronic methylphenidate exposure.

Methylphenidate is a psychostimulant used to treat attention deficit hyperactivity disorder. Neurogenesis occurs throughout adulthood within the dentate gyrus of the hippocampus and can be altered by psychoactive medications; however, the impact of methylphenidate on neurogenesis is not fully understood. We investigated the effects of chronic low (1 mg/kg) and high (10 mg/kg) intraperitoneal doses of methylphenidate on neurogenesis in mouse hippocampus following 28 days and 56 days of treatment. Interestingly, methylphenidate, at both doses, increased neurogenesis. However, if methylphenidate treatment was not continued, the newly generated cells did not survive after 28 days. If treatment was continued, the newly generated neurons survived only in the mice receiving low-dose methylphenidate. To investigate the mechanism for this effect, we examined levels of proteins linked to cell proliferation 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), and beta-catenin. BDNF or GDNF levels were not significantly different between groups. However, hippocampal VEGF, TrkB, and beta-catenin were significantly increased in mice receiving low-dose methylphenidate for 28 days compared to controls. Interestingly, high-dose methylphenidate significantly decreased beta-catenin after 28 days and decreased VEGF, beta-catenin, and TrkB after 56 days compared to controls. Thus, low-dose methylphenidate appears to increase cell proliferation and cell survival in the hippocampus, and these effects may be mediated by increase in VEGF, TrkB, and beta-catenin. While high dose methylphenidate may initially increase neuronal proliferation, newly generated neurons are unable to survive long-term, possibly due to decrease in VEGF, TrkB and beta-catenin.

Methylphenidate exposure induces dopamine neuron loss and activation of microglia in the basal ganglia of mice.

BACKGROUND: Methylphenidate (MPH) is a psychostimulant that exerts its pharmacological effects via preferential blockade of the dopamine transporter (DAT) and the norepinephrine transporter (NET), resulting in increased monoamine levels in the synapse. Clinically, methylphenidate is prescribed for the symptomatic treatment of ADHD and narcolepsy; although lately, there has been an increased incidence of its use in individuals not meeting the criteria for these disorders. MPH has also been misused as a "cognitive enhancer" and as an alternative to other psychostimulants. Here, we investigate whether chronic or acute administration of MPH in mice at either 1 mg/kg or 10 mg/kg, affects cell number and gene expression in the basal ganglia. METHODOLOGY/PRINCIPAL FINDINGS: Through the use of stereological counting methods, we observed a significant reduction (∼20%) in dopamine neuron numbers in the substantia nigra pars compacta (SNpc) following chronic administration of 10 mg/kg MPH. This dosage of MPH also induced a significant increase in the number of activated microglia in the SNpc. Additionally, exposure to either 1 mg/kg or 10 mg/kg MPH increased the sensitivity of SNpc dopaminergic neurons to the parkinsonian agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Unbiased gene screening employing Affymetrix GeneChip® HT MG-430 PM revealed changes in 115 and 54 genes in the substantia nigra (SN) of mice exposed to 1 mg/kg and 10 mg/kg MPH doses, respectively. Decreases in the mRNA levels of gdnf, dat1, vmat2, and th in the substantia nigra (SN) were observed with both acute and chronic dosing of 10 mg/kg MPH. We also found an increase in mRNA levels of the pro-inflammatory genes il-6 and tnf-α in the striatum, although these were seen only at an acute dose of 10 mg/kg and not following chronic dosing. CONCLUSION: Collectively, our results suggest that chronic MPH usage in mice at doses spanning the therapeutic range in humans, especially at prolonged higher doses, has long-term neurodegenerative consequences.

Chloride transport inhibitors influence recovery from oxygen-glucose deprivation-induced cellular injury in adult hippocampus.

Cerebral ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro are characterized by major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na(+), Ca(2+), and Cl(-) and extracellular K(+). Recently, considerable attention has been focused on the cation-chloride cotransporters Na-K-Cl cotransporter isoform I (NKCC-1) and K-Cl cotransporter isoform II (KCC2), as they may play an important role in the disruption of ion gradients and subsequent ischemic damage. In this study, we examined the ability of cation-chloride transport inhibitors to influence the biochemical (i.e. ATP) and histological recovery of neurons in adult hippocampal slices exposed to OGD. In the hippocampus, 7 min of OGD caused a loss of ATP that recovered partially (approximately 50%) during 3 h of reoxygenation. Furosemide, which inhibits the NKCC-1 and KCC2 cotransporters, and bumetanide, a more specific NKCC-1 inhibitor, enhanced ATP recovery when measured 3 h after OGD. Furosemide and bumetanide also attenuated area CA1 neuronal injury after OGD. However, higher concentrations of these compounds appear to have additional non-specific toxic effects, limiting ATP recovery following OGD and promoting neuronal injury. The KCC2 cotransporter inhibitor DIOA and the Cl(-) ATPase inhibitor ethacrynic acid caused neuronal death even in the absence of OGD and promoted cytochrome c release from isolated mitochondria, indicating non-specific toxicities of these compounds.