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

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

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

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

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

Chronic MPH dosing alters dopamine turnover in the striatum.

<p>Striata were microdissected from the brains of mice administered 90 days of saline (Ctrl), 1 mg/kg MPH or 10 mg/kg MPH and were processed for HPLC analyses. Total striatal levels of (A) dopamine and (B) the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) are presented as percentage of saline-treated controls (Ctrl). (C) Dopamine turnover is presented as the ratio of DOPAC/DA. *p≤0.01 compared to saline-controls (Ctrl) (n = 8). 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