Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation

Abstract

Transgenic mice expressing exon 1 of the human Huntington's disease (HD) gene carrying a 141-157 CAG repeat (line R6/2) develop a progressive neurological phenotype with motor symptoms resembling those seen in HD. We have characterized the motor deficits in R6/2 mice using a battery of behavioral tests selected to measure motor aspects of swimming, fore- and hindlimb coordination, balance, and sensorimotor gating [swimming tank, rotarod, raised beam, fore- and hindpaw footprinting, and acoustic startle/prepulse inhibition (PPI)]. Behavioral testing was performed on female hemizygotic R6/2 transgenic mice (n = 9) and female wild-type littermates (n = 22) between 5 and 14 weeks of age. Transgenic mice did not show an overt behavioral phenotype until around 8 weeks of age. However, as early as 5-6 weeks of age they had significant difficulty swimming, traversing the narrowest square (5 mm) raised beam, and maintaining balance on the rotarod at rotation speeds of 33-44 rpm. Furthermore, they showed significant impairment in prepulse inhibition (an impairment also seen in patients with HD). Between 8 and 15 weeks, R6/2 transgenic mice showed a progressive deterioration in performance on all of the motor tests. Thus R6/2 mice show measurable deficits in motor behavior that begin subtly and increase progressively until death. Our data support the use of R6/2 mice as a model of HD and indicate that they may be useful for evaluating therapeutic strategies for HD, particularly those aimed at reducing the severity of motor symptoms or slowing the course of the disease.

Fig. 1.

Body weights of wild-type and R6/2 transgenic mice. Symbols indicate the means ± SEM body weight (gram) of wild-type (n = 22) and R6/2 transgenic (n = 9) mice. The body weight of R6/2 transgenic mice gradually declines from 12 weeks of age. Asterisksindicate significant differences between wild-type control R6/2 transgenic mice (**p < 0.01).

Fig. 2.

Swimming performance of wild-type and R6/2 transgenic mice in the swimming tank. Wild-type (n= 22) and R6/2 transgenic (n = 9) mice were tested in the swimming tank for two trials per day for 3 consecutive days at 5–6 weeks of age, retested at 8–9 weeks of age, and then weekly thereafter. Separate measurements were made of the latency (A), the number of hindlimb kicks (B), and the number of forelimb kicks (C) to swim 60 cm to a visible escape platform.Vertical bars indicate means ± SEM of each measure across all trials at each age. R6/2 transgenic mice display swimming impairments at the earliest age tested, 5–6 weeks, which progressively worsen over subsequent weeks. Asterisks indicate significant differences between wild-type control and R6/2 transgenic mice (*p < 0.05, **p < 0.01).

Fig. 3.

Balance and motor coordination on the raised beams. Wild-type (n = 22) and R6/2 transgenic (n = 9) mice were trained to walk across six progressively more difficult beams of square and round cross-section to reach an enclosed safety platform. The latency to cross (A–F) and the number of footslips made (G–L) were recorded on each trial. All animals were given two trials on each of the graded square and round beams, in each case progressing from the widest to the narrowest, for 3 consecutive days at 5–6 weeks of age, retested at 8–9 weeks of age, and then weekly thereafter. Individual symbols represent means ± SEM of the two groups on each test and age. R6/2 transgenic mice exhibit a progressive decline in beam-walking ability with age and beam difficulty. Asterisks indicate significant differences between wild-type control and R6/2 transgenic mice (*p < 0.05, **p < 0.01).

Fig. 4.

Balance and motor coordination on the rotarod. Wild-type (n = 22) and R6/2 transgenic (n = 9) mice were subjected to eight different speeds on an accelerating rotarod (5–44 rpm), receiving two trials per speed. The means ± SEM of the duration of balance or latency to fall (maximum trial length = 60 sec) for the two trials at each speed level was recorded. Animals 5–6 weeks old were tested to establish baseline performance, and animals were retested at 8–9 weeks of age and then weekly thereafter. R6/2 transgenic mice 5–6 weeks old exhibit difficulty maintaining balance on the rotarod at the highest rotation speeds (G, H) and display a progressive decline in performance on the rotarod with increasing rotation speed and age. Asterisks indicate significant differences between wild-type control and R6/2 transgenic mice (*p < 0.05, **p < 0.01).

Fig. 5.

Representative walking footprint patterns of 13- to 14-week-old wild-type (A) and R6/2 transgenic (B) mice. Qualitatively, the generated patterns clearly differ showing that R6/2 transgenic mice display irregularly spaced shorter strides and an uneven left–right step pattern as compared with the evenly spaced and accurately positioned footprints of the wild-type control mice.

Fig. 6.

Quantitative analysis of the walking footprint patterns produced by wild-type and R6/2 transgenic mice, based on measurements of stride length (A), hindbase width (B), frontbase width (C), and distance between front and hind footprint placement or overlap (D). At 5–6 weeks of age the footprint patterns of R6/2 transgenic mice are indistinguishable from those produced by wild-type control mice, but by 8–9 weeks of age, R6/2 transgenic mice exhibit a significantly shorter stride length, a broader frontbase width, and a greater distance between front footprint/hind footprint overlap, as compared with wild-type control mice.Symbols indicate means ± SEM by mice of each group at each age on each measure. Asterisks indicate significant differences in the performance of wild-type control and R6/2 transgenic mice (*p < 0.05, **p < 0.01).

Fig. 7.

Prepulse inhibition of the acoustic startle response in wild-type (n = 22) and R6/2 transgenic (n = 9) mice. Symbols indicate mean ± SEM of the response of the two groups at different ages to white noise stimuli of 120 dB (A–E) and 105 dB (F–J) with either the baseline startle response recorded to the primary acoustic startle stimulus alone (A, F, respectively) or with 100 msec prepulse warning stimuli of rising intensities (2, 4, 8, and 16 in B–Eand G–J, respectively). R6/2 transgenic mice exhibit impaired PPI by 8–9 weeks of age, and this impairment progressively declines with age. Asterisks indicate significant differences in performance between the two genotypes (*p < 0.05, **p < 0.01).