Differential degradation of motor deficits during gradual dopamine depletion with 6-hydroxydopamine in mice

Abstract

Parkinson's disease (PD) is a movement disorder whose cardinal motor symptoms arise due to the progressive loss of dopamine. Although this dopamine loss typically progresses slowly over time, currently there are very few animal models that enable incremental dopamine depletion over time within the same animal. This type of gradual dopamine depletion model would be useful in studies aimed at the prodromal phase of PD, when dopamine levels are pathologically low but motor symptoms have not yet presented. Utilizing the highly characterized neurotoxin 6-hydroxydopamine (6-OHDA), we have developed a paradigm to gradually deplete dopamine levels in the striatum over a user-defined time course - spanning weeks to months - in C57BL/6 mice. Dopamine depletions were achieved by administration of five low-dose injections (0.75μg) of 6-OHDA through an implanted intracranial bilateral cannula targeting the medial forebrain bundle. Levels of dopamine within the striatum declined linearly with successive injections, quantified using tyrosine hydroxylase immunostaining and high-performance liquid chromatography. Behavioral testing was carried out at each time point to study the onset and progression of motor impairments as a function of dopamine loss over time. We found that spontaneous locomotion, measured in an open field, was robust until ∼70% of striatal dopamine was lost. Beyond this point, additional dopamine loss caused a sharp decline in motor performance, reaching a final level comparable to that of acutely depleted mice. Similarly, although rearing behavior was more sensitive to dopamine loss and declined linearly as a function of dopamine levels, it eventually declined to levels similar to those seen in acutely depleted mice. In contrast, motor coordination, measured on a vertical pole task, was only moderately impaired in gradually depleted mice, despite severe impairments observed in acutely depleted mice. These results demonstrate the importance of the temporal profile of dopamine loss on the magnitude and progression of behavioral impairments. Our gradual depletion model thus establishes a new paradigm with which to study how circuits respond and adapt to dopamine loss over time, information which could uncover important cellular events during the prodromal phase of PD that ultimately impact the presentation or treatability of behavioral symptoms.

Keywords: Parkinson’s disease; basal ganglia; compensation; gradual depletion.

Figure 1

Gradual depletion paradigm linearly reduces dopamine over time. A, Schematic of sagittal section of mouse brain showing the position of the infusion cannula in the MFB. Str = striatum, Thal = thalamus, MFB = medial forebrain bundle, SNc = substantia nigra pars compacta. B, Cannulae placements in the MFB, confirmed postmortem. Left and right MFBs from coronal sections spanning the cannulae placements are shown, and Bregma coordinates are notated. Representative bilateral cannulae placements from three representative mice are indicated with triangles, squares, and inverted triangles. C, Representative images of TH immunofluorescence (TH-IR), taken from the dorsal striatum of mice treated with saline, two doses of 0.75 µg 6-OHDA, or five doses of 0.75 µg 6-OHDA. D, Quantification of TH-IR in the dorsal striatum, normalized to saline controls, following repeated injections of 0.75 µg or a single injection of 5 µg 6-OHDA. Values are expressed as percentage of dopamine remaining. Throughout figure, error bars are SEM. KW, χ²(6) = 106.218, p < 0.0001, pairwise, **p < 0.005 from saline. E, Quantification of TH-IR in the ventral striatum, normalized to saline controls, following repeated injections of 0.75 µg or a single injection of 5 µg 6-OHDA. Values are expressed as percentage of dopamine remaining. KW, χ²(6) = 51.744, p < 0.0001, pairwise, **p < 0.005 from saline. F, Quantification of TH-IR in the dorsal and ventral striatum following the 5th injection of 0.75 µg of 6-OHDA. Data from each mouse, as well as population averages are shown. Student’s t-test, t = −3.896, **p = 0.002. G, Quantification of HPLC detected levels of dopamine in the dorsal straitum, normalized to saline controls, following repeated injections of 0.75 µg or a single injection of 5 µg 6-OHDA. Values are expressed as percentage of dopamine remaining. Fit line from the graph of TH-IR levels is overlaid for comparison. KW, χ²(6) = 69.554, p < 0.0001, pairwise, **p < 0.005 from saline.

Figure 2

Levels of dopamine metabolites and other monoamines measured with HPLC during gradual dopamine depletion paradigm. A, Quantification of HPLC detected levels of the dopamine metabolites homovanillic acid (HVA), 3,4-Dihydroxyphenylacetic acid (DOPAC), and 3-methoxytyramine (3-MT). Tissue samples were taken from the dorsal striatum of mice who received repeated injections of 0.75 µg 6-OHDA or a single injection of 5 µg of 6-OHDA. Values are normalized to saline controls for each time point. Throughout figure, error bars are SEM. Fit line from the graph of HPLC dopamine levels is overlaid for comparison. ANOVA, HVA: F[6,73] = 51.085; DOPAC: F[6,73] = 55.560; 3-MT: F[6,73] = 34.572, all p < 0.0001, Dunnett T3, *p < 0.05 from saline; **p < 0.005 from saline. B, Quantification of HPLC detected levels of norepinephrine in the dorsal striatum at all time points of the gradual depletion paradigm and in acutely depleted mice. Values are normalized to saline controls. ANOVA, F[6,73] = 9.709, p < 0.0001, Dunnett t, *p < 0.05 from saline; **p < 0.005 from saline. C, Quantification of HPLC detected levels of serotonin in the dorsal striatum at all time points of the gradual depletion paradigm and in acutely depleted mice. Values are normalized to saline controls. ANOVA, F[6,73] = 4.083, p = 0.01, Dunnett t, *p < 0.05 from saline; **p < 0.005 from saline. Serotonin levels in the dorsal striatum from each mouse, normalized to saline controls, plotted as a function of striatal dopamine levels.

Figure 3

Effects of gradual dopamine depletion on spontaneous locomotion in an open field. A, Example plot tracks from a saline control mouse, a gradually depleted mouse that has received 5 injections of 0.75 µg 6-OHDA, and an acutely depleted mouse that has received 1 injection of 5 µg 6-OHDA. B, Total distance traveled over 10 minutes in saline control animals, gradually depleted animals, and acutely depleted animals. Throughout the figure, error bars are SEM. KW, χ²(6) = 49.674, p < 0.0001, pairwise, *p = 0.012 from saline; **p < 0.005 from saline. C, Total distance traveled by saline control animals compared to gradually and acutely depleted mice with 0–20% dorsal striatal dopamine remaining. KW, χ²(2) = 31.091, p < 0.0001, pairwise, **p < 0.005. D, Average velocity of saline control animals, gradually depleted animals, and acutely depleted animals. KW, χ²(6) = 49.642, p < 0.0001, pairwise, *p = 0.012 from saline; **p < 0.005 from saline. E, Average velocity of saline control animals compared to gradually and acutely depleted mice with 0–20% striatal dopamine remaining. KW, χ²(2) = 31.141, p < 0.0001, pairwise, **p < 0.005. F, Percentage of time spent immobile by saline control animals, gradually depleted animals, and acutely depleted animals. KW, χ²(6) = 40.429, p < 0.0001, pairwise, *p < 0.05 from saline; **p < 0.005 from saline. G, Percentage of time spent immobile in saline control animals compared to gradually and acutely depleted animals with 0–20% striatal dopamine remaining. KW, χ²(2) = 31.679, p < 0.0001, pairwise, **p < 0.005.

Figure 4

Effects of gradual dopamine depletion on rearing behavior. A, Example of a full extension rear. B, Average number of rears of saline control animals, gradually depleted animals across all dopamine levels, and acutely depleted animals during 10 minutes in beaker. Throughout the figure, error bars are SEM. KW, χ²(6) = 42.623, p < 0.0001, pairwise, **p < 0.005 from saline. C, Average number of rears of saline control animals, gradually depleted animals with 0–20% dorsal striatal dopamine, and acutely depleted animals with 0–20% dorsal striatal dopamine. KW, χ²(2) = 23.977, p < 0.0001, pairwise, **p < 0.005. Figure 5. Effects of gradual dopamine depletion on motor coordination assessed with a vertical pole task. A, Image of mouse descending pole and schematic of timed parameters of pole task. B, Average time needed to complete the entire task for saline control animals, gradually depleted animals, and acutely depleted animals. Throughout the figure, error bars are SEM. KW, χ²(6) = 52.592, p < 0.0001, pairwise, **p < 0.005 from saline. C, Average total time of saline control animals compared to gradually and acutely depleted animals with 0–20% striatal dopamine remaining. KW, χ²(2) = 39.537, p < 0.0001, pairwise, *p < 0.05. D, Average turn down latency (TDL) of saline control animals, gradually depleted animals, and acutely depleted animals. KW, χ²(6) = 37.667, p < 0.0001, pairwise, **p < 0.005 from saline. E, Average TDL of saline control animals compared to gradually and acutely depleted animals with 0–20% dorsal striatal dopamine, and acutely depleted animals with 0–20% striatal dopamine remaining. KW, χ² (2) = 29.908, p < 0.0001, pairwise, **p < 0.005. F, Average traversal time of saline control animals, gradually depleted animals, and acutely depleted animals. KW, χ²(6) = 58.834, p < 0.0001, pairwise, *p < 0.05 from saline; **p < 0.005 from saline. G, Average traversal time of saline control animals compared to gradually and acutely depleted animals with 0–20% striatal dopamine remaining. KW, χ²(2)= 43.068, p < 0.0001, pairwise, *p < 0.05.

Figure 5

Effects of gradual dopamine depletion on motor coordination assessed with a vertical pole task. A, Image of mouse descending pole and schematic of timed parameters of pole task. B, Average time needed to complete the entire task for saline control animals, gradually depleted animals, and acutely depleted animals. Throughout the figure, error bars are SEM. KW, χ²(6) = 52.592, p < 0.0001, pairwise, **p < 0.005 from saline. C, Average total time of saline control animals compared to gradually and acutely depleted animals with 0–20% striatal dopamine remaining. KW, χ²(2) = 39.537, p < 0.0001, pairwise, *p < 0.05. D, Average turn down latency (TDL) of saline control animals, gradually depleted animals, and acutely depleted animals. KW, χ²(6) = 37.667, p < 0.0001, pairwise, **p < 0.005 from saline. E, Average TDL of saline control animals compared to gradually and acutely depleted animals with 0–20% dorsal striatal dopamine, and acutely depleted animals with 0–20% striatal dopamine remaining. KW, χ² (2) = 29.908, p < 0.0001, pairwise, **p < 0.005. F, Average traversal time of saline control animals, gradually depleted animals, and acutely depleted animals. KW, χ²(6) = 58.834, p < 0.0001, pairwise, *p < 0.05 from saline; **p < 0.005 from saline. G, Average traversal time of saline control animals compared to gradually and acutely depleted animals with 0–20% striatal dopamine remaining. KW, χ²(2)= 43.068, p < 0.0001, pairwise, *p < 0.05.

Figure 6

Time course of gradual depletions can be varied and extended over a month. A, Quantification of TH-IR in the dorsal striatum normalized to saline controls following repeated injections of 0.75 µg 6-OHDA, administered every seven days, every three days, or a single injection of 5 µg of 6-OHDA. Values are normalized to saline controls and expressed as percentage of dopamine remaining. Throughout the figure, error bars are SEM. B, Quantification of TH-IR in the dorsal striatum, normalized to saline controls, following the last injection of the gradual seven-day, gradual three-day, and acute paradigms. KW, χ²(3) = 110.653, p < 0.0001, pairwise, **p < 0.005. C, Example plot tracks from a gradually depleted mouse that has received seven injections of 0.75 µg 6-OHDA every seven days, a gradually depleted mouse that has received five injections of 0.75 µg every three days, and an acutely depleted mouse that has received one injection of 5 µg. D, Total distance traveled during 10 minutes in the open field arena by saline control animals compared to mice with 0-20% dopamine remaining that were depleted with the seven-day gradual, three-day gradual, or acute dopamine depletion paradigms. KW, χ²(3) = 36.831, p < 0.0001, pairwise, *p < 0.05. E, Average velocity of saline control animals compared to animals with 0-20% dopamine that were depleted with the seven-day gradual, three-day gradual, or acute dopamine depletion paradigms. KW, χ²(3) = 36.881, p < 0.0001, pairwise, *p < 0.05. F, Percentage of time spent mobile in saline control animals and animals with 0-20% dopamine that received 0.75 µg every 7 days, 0.75 µg every 3 days, or a single injection of 5 µg of 6-OHDA during open field. KW, χ²(3)= 40.548, p < 0.0001, pairwise, **p < 0.005. G, Percentage of time spent immobile in saline control animals and animals with 0–20% dopamine that received 0.75 µg every 7 days, 0.75 µg every 3 days, or a single injection of 5 µg of 6-OHDA during open field. KW, χ²(3)= 34.129, p < 0.0001, pairwise, **p < 0.005.

Figure 7

Differential progression motor deficits in gradually depleted mice. A, Normalized behavior of gradually depleted animals plotted as a function of striatal dopamine levels. Percentage of time spent mobile normalized to saline controls is plotted as a representative of open field activity, total number of rears normalized to saline controls is plotted for rearing, and turn down latency normalized to saline controls is plotted as a representative of pole task performance. B, Schematic showing the differential degradation of behavior of animals whose dopamine was gradually depleted.