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

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 GeneChipH 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 nigr

Normal Midbrain Dopaminergic Neuron Development and Function in miR-133b Mutant Mice

Midbrain dopaminergic (mDA) neurons control movement and emotion, and their degeneration leads to motor and cognitive defects in Parkinson's disease (PD). miR-133b is a conserved microRNA that is thought to regulate mDA neuron differentiation by targeting Pitx3, a transcription factor required for appropriate development of mDA substantia nigra neurons. Moreover, miR-133b has been found to be depleted in the midbrain of PD patients. However, the function of miR-133b in the intact midbrain has not been determined. Here we show that miR-133b null mice have normal numbers of mDA neurons during development and aging. Dopamine levels are unchanged in the striatum, while expression of dopaminergic genes, including Pitx3, is also unaffected. Finally, motor coordination and both spontaneous and psychostimulant-induced locomotion are unaltered in miR-133b null mice, suggesting that miR-133b does not play a significant role in mDA neuron development and maintenance in vivo.National Institute of Neurological Disorders and Stroke (U.S.)Poitras Center for Affective Disorders Researc

Neurochemical Measurement of Adenosine in Discrete Brain Regions of Five Strains of Inbred Mice

<div><p>Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models.</p></div

Total Adenosine, ATP, ADP, and AMP (pg/mg wet weight) in Brain Regions of 12 Month C7BL/6J mouse.

<p>Total Adenosine, ATP, ADP, and AMP (pg/mg wet weight) in Brain Regions of 12 Month C7BL/6J mouse.</p

Identification of optimal parameters for simultaneous detection of ADO, ATP, ADP and AMP.

<p>(A) To determine the optimum voltage necessary to fully oxidize ADO, ATP, ADP and AMP, we measured the number of oxidized molecules at voltages ranging from +700–+1700 mV. Construction of a hydrodynamic voltamogram (HDV), necessary to determine the optimal signal/noise (s/n) ratio, showed that +1300 mV was optimum for the simultaneous detection of the compounds. (B) To determine the accurate range and linearity of detection of ADO, ATP, ADP, and AMP, 0–100 ng of each standard was injected into the HPLC. Electrochemical detection of the oxidized molecules shows a linear slope for each of the compounds. (C) Representative chromatograms of simultaneous separation of ADO, ATP, ADP and AMP demonstrating linearity of the injected analytes.</p

Quantitation of ADO in 7 regions of the brain in 5 strains of mice.

<p>ANOVA demonstrated significant differences between mouse strains in each region examined. Post-hoc Bonferroni comparisons are shown for each brain region. N = 10 for each measurement.</p

Chronic exposure to high dose MPH results in microglial cell activation in the SNpc.

<p>Stereological estimates of Iba-1 positive microglia cells in the SNpc (A) the total number of morphologically-resting microglia and (B) the total number of morphologically-activated microglia following chronic administration of either saline (ctrl), 1 mg/kg MPH, 10 mg/kg MPH and 10 mg/kg MPH+MPTP. (n = 5). One-way ANOVA statistical test was performed to draw comparisons between the different groups followed by Bonferroni post-hoc tests.</p

mRNA expression of pro-inflammatory genes following acute administration of 10 mg/kg MPH in the striatum.

<p>Fold change values in mRNA expression presented are normalized against saline controls, in the striatum. The genes probed for include (A) <i>il-6</i>, (B) <i>tnf-á</i>, (C) <i>cox2</i> and (D) <i>il-1b</i>. *p≤0.02 compared to saline-controls (ctrl); **p≤0.02 compared to 10 mg/kg MPH-chronic dose, (n = 3). One-way ANOVA statistical test was performed to draw comparisons between the different groups followed by Bonferroni post-hoc tests.</p

Acute and chronic administration of MPH alters gene expression in the substantia nigra (SN).

<p>(A) Heat map representation of gene expression changes following chronic administration of either 1 mg/kg MPH or 10 mg/kg MPH in the SN (n = 3). qPCR analysis demonstrating normalized fold-change expression of (B) <i>bdnf</i>, (C) <i>gdnf</i>, (D) <i>dat1(slc6a3)</i>, (E) <i>vmat2(slc18a2)</i> and (F) <i>th</i> mRNA in SN (n = 3). *p≤0.02 vs saline-controls (ctrl); **p≤0.02 vs saline-controls and 10 mg/kg MPH-acute dose; #p≤0.02 10 mg/kg MPH acute-dose vs saline-controls (ctrl). One-way ANOVA statistical test was performed to draw comparisons between the different groups followed by Bonferroni post-hoc tests.</p