Dysregulation of intracellular dopamine stores revealed in the R6/2 mouse striatum

Abstract

Huntington's disease (HD) is a fatal, neurodegenerative movement disorder characterized by preferential and extensive striatal degeneration. Here, we used fast-scan cyclic voltammetry to study the mobilization and efflux of reserve pool dopamine (DA) in striatal brain slices from HD model R6/2 mice. When applying stimulus trains of 120 pulses, evoked DA release in wild-type (WT) slices was greater than that in R6/2 slices at the higher frequencies (50 and 60 Hz). To quantify cytosolic and reserve pool DA levels, amphetamine-induced DA efflux was measured after pre-treatment with either tetrabenazine or alpha-methyl-p-tyrosine. Slices from 12-week-old R6/2 mice released less DA than slices from WT mice, while no difference was noted in slices from 6-week old mice. The vesicular release of reserve pool DA, mobilized by treatment with cocaine, was shorter lived in R6/2 slices compared with WT slices even though peak DA release was the same. Moreover, the number of DA reserve pool vesicles in R6/2 mice was less than half of that in WT. Therefore, our data suggest that the same number of DA molecules are present in each reserve pool vesicle in WT and R6/2 mice and that these vesicles are readily mobilized in both genotypes; however, R6/2 mice have fewer DA reserve pool vesicles available for mobilization.

Figure 1

Striatal DA release, evoked by the application of 120-pulse stimulus pulse trains, is impaired in R6/2 brain slices compared to WT brain slices. (A) Representative plots and cyclic voltammograms obtained by FSCV at carbon-fiber microelectrodes for R6/2 and WT mice are shown. The representative plots where taken using a 60 Hz stimulation frequency. The duration of the stimulation is represented by the bar under each plot. The cyclic voltammograms confirm the presence of DA. (B) DA release was evoked by 20, 30, 40, 50, and 60 Hz 120-pulse stimulus trains. [DA]_stim was significantly greater in WT brain slices compared to R6/2 brain slices when evoked using 50Hz and 60 Hz stimulus frequencies (*p < 0.05 at 50 and 60 Hz; n = 5 R6/2 mice and 6 WT mice). Although striatal DA release appears to increase with increasing frequency in WT slices, differences in release between application frequencies are not significant within either genotype (one-way ANOVA).

Figure 2

AMPH induced DA efflux after TBZ treatment is impaired in 12-week old R6/2 mice compared to WT mice. (A) Representative data from a 6-week old WT mouse. The brain slice was exposed to 20 µM TBZ while applying single, 4 ms duration stimulus pulses (one pulse every 5 minutes) until DA release disappeared. The slice was then exposed to 20 µM AMPH for 25 minutes, and [DA]_AMPH was measured. A CV is provided at peak release and confirms the presence of DA. Stimulated release plots were collected every 5 minutes, but are shown every 10 minutes for clarity. (B) In 12-week old R6/2 mice, [DA]_AMPH is significantly less than in WT mice (*p < 0.05; n = 7 R6/2 mice and 8 WT mice), but at 6-weeks of age there is not a significant difference between the two sets of mice (p > 0.05; n = 6 R6/2 and 7 WT mice).

Figure 3

AMPH induced DA efflux after αMPT treatment is diminished in older R6/2 mice. (A) Representative data from a 6-week old WT mouse. Slices were treated with 50 µM αMPT until the stimulated DA release peak disappeared. The slice was then exposed to 20 µM AMPH for 25 minutes and [DA]_AMPH was measured. The last 20 minutes of AMPH treatment are shown in this particular plot. A CV is provided at peak release and confirms the presence of DA. Stimulated release plots collected every 10 minutes are shown for clarity. (B) AMPH induced DA efflux was not significantly different between 6-week old R6/2 and WT brain slices (p > 0.05; n = 5 R6/2 and 3 WT mice). [DA]_AMPH was significantly decreased in slices from 12-week old R6/2 mice compared to those from age-matched WT control mice (*p < 0.05; n = 5 R6/2 mice and 4 WT mice).

Figure 4

Reserve pool vesicles are depleted in 12-week old R6/2 mice. Slices were treated with 50 µM αMPT while applying a 4 ms duration single-pulse electrical stimulus every 5 minutes. Once the stimulated DA release peak disappeared, 20 µM COC was added to the brain slice and the application of stimulus pulses continued. Stimulated DA release was measured by FSCV. (A) Representative data from R6/2 and WT slices. Reserve pool mobilization by COC resulted in measurable stimulated DA release. Stimulated release plots collected immediately after the addition of αMPT and the disappearance of the DA release peak have been omitted in this figure for clarity. (B) Pooled data from R6/2 and WT mice. Each data point represents the average value (± SEM) of [DA]_stim obtained from 5 R6/2 mice and 6 WT mice. The maximum peak amplitudes obtained in R6/2 and WT slices were not significantly different.