Quantification of reserve pool dopamine in methionine sulfoxide reductase A null mice.

The abnormal regulation of dopamine (DA) has been associated with multiple neurodegenerative disease states (Bird and Iversen, 1974, Morgan et al., 1987), yet the role of DA reserve pool storage and mobilization in the pathophysiology of these conditions is now just being revealed. Many of these conditions, such as Parkinson’s disease, Huntington’s disease, and Lou Gehrig’s disease, have been associated with the increased production or reactive oxygen species, thereby enhancing the degree of cell oxidative stress in the brain (Patten et al., 2010, Cohen, 1983, Perez-Severiano et al., 2004). Interestingly, elevated DA levels have also been associated with enhanced oxidative stress (Oien et al., 2008b). Indeed, one model of oxidative stress, methionine sulfoxide reductase A knockout (MsrA−/−) mice, have been reported to have chronically high brain DA levels (Oien et al., 2008b). These mice lack the antioxidant enzyme MsrA, which is part of the Msr system. Methionine sulfoxide posttranslational modifications can be reversed by the Msr system, which consists of MsrA (reduces S methionine sulfoxide enantiomer) and MsrB (reduces R methionine sulfoxide enantiomer) (Moskovitz, 2005). The MsrA−/− mouse is hypersensitive to oxidative stress, accumulates higher levels of carbonylated protein, and expresses brain pathologies associated with neurodegenerative diseases (Moskovitz et al., 2001, Pal et al., 2007). Recent studies have shown that these mice have abnormally high DA levels in the brain at the ages of 6 and 12 months, compared to wild type (WT) control mice. Additionally, these high levels parallel an increased presynaptic DA release when stimulated in vitro without drug treatments. A possible mechanism for an increase in stimulated DA release in MsrA−\− mice involves the mobilization of reserve pool DA. In general, DA-containing vesicles are believed to be separated into three pools: the readily releasable pool (RRP), the recycling pool, and the reserve pool (Neves and Lagnado, 1999, Rizzoli and Betz, 2005). The RRP undergoes exocytosis upon mild stimulation and is replenished by the mobilization of the recycling pool vesicles. The reserve pool, mobilized upon prolonged periods of synaptic activity (Neves and Lagnado, 1999), is the largest pool consisting of 80–90% of the total vesicles (Rizzoli and Betz, 2005). Pharmacological manipulations, using a combination of alpha-methyl-p-tyrosine (aMPT) and either cocaine (COC) or amphetamine (AMPH) (Venton et al., 2006, Ortiz et al., 2010), have been used to quantitatively measure reserve pool dopamine. Other factors, such as calcium transport, may also influence the amplitude of stimulated dopamine release plots. Transient increases in intracellular calcium concentration trigger vesicular exocytosis (Nachshen and Sanchez-Armass, 1987, Kume-Kick and Rice, 1998) as well as the movement of RRP and reserve pool vesicles (Rose et al., 2002). Moreover, the increase in oxidative stress may result in calcium dysregulation. For example, the activity of calmodulin, a calcium regulatory protein that activates the plasma membrane calcium ATPase (PMCA), diminishes due to oxidative post-translational modifications as tissues age (Michaelis et al., 1996). The oxidation of specific methionines in calmodulin results in about a 50% reduction of PMCA activation (Bartlett et al., 2003), thereby impairing the ability of cells to clear calcium from the cell (Palacios et al., 2004). Oxidized calmodulin can accumulate in brain tissues as a result of low antioxidant levels and it is speculated that oxidation of methionines on calmodulin may be acting as a molecular switch in calcium regulation, oxidative stress, and DA release (Chen et al., 2001, Bigelow and Squier, 2005). To investigate possible mechanisms underlying elevated DA content and release found in MsrA−/− mice, fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to measure the mobilization and efflux of reserve pool DA in striatal brain slices from MsrA−/− mice and WT control mice (Oien et al., 2008b). We hypothesized that the DA reserve pool is enhanced in MsrA−/− mice compared to WT control mice. In order to measure reserve pool DA, slices were pre-treated with αMPT and then treated with either AMPH, to measure the efflux of reserve pool DA, or with COC, to measure the stimulated release of mobilized DA reserve pool vesicles. Collectively, our results suggest that reserve pool DA is more abundant in the MsrA−/− striatum and that the number of vesicles is greater compared to WT controls.