Neurological Disorders – Huntington’s Disease
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Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder. It is characterized by motor disturbance, cognitive decline and psychiatric manifestations. The disease typically starts in midlife and symptoms progress over the course of 15–20 years until death. About 6% of cases start before the age of 21, which present a faster progression. The mutation responsible for HD is an unstable expansion of CAG repeats in the gene encoding Huntington. Numerous genetic mouse models of the disease have been generated and PsychoGenics has phenotypically characterized extensively both transgenic and knock-in models of the disease (Menalled et al., 2010). The R6/2 transgenic mouse model was the first model developed (Mangiarini et al., 1996) and it is widely used to understand the pathogenesis of the disease as well as for drug evaluation. These mice present severe weight loss, progressive motor, cognitive and psychiatric deficits, all of which can be assessed by the tests enumerated below. In addition, the life expectancy of R6/2 mice carrying around 120 CAG repeats is reduced (median survival of 18-20 weeks). In the present time, PsychoGenics houses several animal models of HD, two different lines of R6/2 and various knock in lines which have been fully characterized.
R6/2 HD mutant mice
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- Arousability
- Body Weight
- Contextual Fear Conditioning
- Grip Strength
- Open Field
- Rotarod
- Rear and Climbing
- Survival
- Two Choice Swim Tank Test – Cued
BAC HD mutant mice
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Huntington’s Disease – Arousability
State of arousal, operationally defined in this assay as reactivity to removal of the top of the home cage, is reduced in R6/2 compared to Wild Type (WT) mice. This measure has been suggested as a behavioral model of the apathy characteristic of Huntington’s disease.
Huntington’s Disease – Body Weight
Body weights are markedly reduced in R6/2 mice, with the onset of reduced growth becoming apparent early in life.
Huntington’s Disease – Contextual Fear Conditioning
The formation of contextual memories is dependent upon the integrity of medial temporal lobe structures including the hippocampus and amygdala. In this assay mice are trained to remember that a particular salient context (conditioned stimulus; CS) is associated with an aversive event, in this case a mild foot shock (the unconditioned stimulus, US). Animals that show good learning freeze upon re-presentation of the context (Contextual Fear Conditioning). For some lines, we also quantify freezing to a tone stimulus paired with a foot shock US (Cued Fear Conditioning).
The R6/2 mice show a deficit in contextual fear conditioning compared to Wild Type littermates (WT), when studied at 11 weeks of age.
Huntington’s Disease – Grip Strength
Grip strength is used to assess muscular strength in limb muscles. The animal is lowered toward the platform and gently pulled backwards with consistent force by the experimenter until it releases its grip. The grip force is recorded on the strain gauge.
R6/2 mice exhibit weaker Grip Strength compared to wild type mice at around 9 - 10 weeks of age.
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Huntington’s Disease – Open Field
The open field test (OF) is used to assess motor activity. The enclosure is configured to split the open field into a center and periphery zone. Animals that have higher levels of anxiety or lower levels of activity tend to stay in the corners of the OF enclosures. Mice that have high levels of activity and low levels of anxiety tend to spend more time in the center of the enclosure.
Reduced locomotion is observed in R6/2 mice starting at 4-6 weeks of age compared to Wild Type (WT) littermates, and this difference becomes more pronounced with age.
Huntington’s Disease – Rotarod
Motor coordination and exercise capacity are assessed by rotarod. Tests are performed on separate days, with multiple trials per day. Mice are placed on the rotarod and the speed is gradually and uniformly increased over a maximum period of time. The time that each mouse remains on the rotating rod before falling is recorded.
Impaired Rotarod performance observed in R6/2 mice compared with wild type littermates with significant differences observed between the mutant R6/2 mice and their Wild Type (WT) littermates as early as 6 weeks of age.
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Huntington’s Disease – Rear and Climbing
Rearing-climbing behavior is used to assess motor movement and coordination. The mouse is placed on a flat surface and a closed-top wire mesh cylinder 15 cm x 20 cm tall is placed over the mouse. The animal's behavior is videotaped and the proportion of mice that climb and the latency to climb are measured over a 5 minute period.
Beginning at 4 weeks of age, R6/2 mice are observed to climb less with age compared to Wild Type (WT) controls. No climbing is observed after 14 weeks of age.
Huntington’s Disease – Survival
Whereas the life expectancy of a normal mouse is approximately 2 years, R6/2 mice with 120 CAG repeats exhibit a significantly reduced median survival of 18-20 weeks.
Huntington’s Disease – Two Choice Swim Tank test - Cued
The swim tank is a simple learning protocol where mice learn to escape an aversive water environment by climbing onto a platform. The location of the platform is signaled by a cue (light) at one end of the aquarium, so mice need to learn to associate the cue with the position of the platform. Mice are placed in the center of a water-filled aquarium and the choice of direction towards the platform is recorded. Once an acquisition criterion has been reached, the light is placed on the other end of the tank and mice enter a reversal learning phase of the task where they must now learn that the escape platform is located on the dark side of the tank.
Reversal: Mutant R6/2 mice show pronounced cognitive impairment compared to WT mice during reversal in the cued two choice swim test.
Huntington’s Disease – Circadian function
Neurodegenerative diseases are marked by psychiatric and motor problems are often accompanied by deficits in sleep and circadian function. As expected, deficits in sleep-wake patterns have profound consequences in many physiological functions. In Huntington’s Disease patients (HD), for examples, abnormal sleep patterns correlate with symptom severity, specifically in depression and cognitive dysfunction, and in caudate atrophy [4,5,6]. We studied the behavior of the BAC HD model [7,8] using an 24/7 automated home-cage system and custom-built computer vision assessment of behavior, PhenoCube™. Using this system we could, in just 6 days, measure a significant lengthening of the circadian period in the mutants. The results reinforce previous findings in the R6/2 mouse and in a transgenic rat model of HD [2,3], indicating that circadian dysfunction is a fundamental feature of HD. These results also provide the validation of PhenoCube™ as a system for the fast assessment of circadian function.
Mutant mice carried the full-length human mutant huntingtin, with 97 glutamine repeats under the control of endogenous htt regulatory machinery on a bacterial artificial chromosome (BAC). All mice tested here (32 BAC female mice and 32 female WT controls) were generated on an FVB/n x C57Bl6 F1, created by crossing BAC hemizygous FVB/n male animals with WT C57Bl6 female animals. The congenic line used for these breedings, originally sourced from the laboratory of X. William Yang at the UCLA David Geffen School of Medicine, Los Angeles, is maintained by crossing hemizygous male BAC mice with FVB/n female animals. Mice were implanted with RFID electronic chips (DataMars, OH) for identification. The animals housed in OptiRAT® cages (Animal Care Systems, CO – groups of 8 animals of the same genotype) for several months prior to the experiment, with free access to water in 4 gated corners and to standard 5001 lab chow except as described. Experiments were conducted using eight modified IntelliCage units (IC, New Behavior AG), each with a camera mounted on top of the cage for computer vision analysis. The cages were maintained with constant dim red light (7 lux using a photographic bandpass filter (LDP LLC, NJ) that eliminates long wavelength light frequencies not visible to mice). Visits to the corners are monitored through the RFID chips.
Huntington’s Disease – Motivation
Apathy, which can be defined as a lack of motivation characterized by diminished goal-oriented behavior, is a known symptom of Huntington’s disease and other neurodegenerative disorders. A typical and well controlled assay to probe this domain in rodents in the progressive ratio task, a procedure where food-restricted animals are required to expend progressively increasing effort to obtain successive food reinforcements.
BAC HD mice, a transgenic model of Huntington’s disease, show reduced motivation to work for food reinforcement in our progressive ratio task. The figure illustrates the mean response rate recorded per reinforcer earned for a group of 80 week BAC HD mice vs. littermate control animals. These data show comparable response levels in all animals where fewer than 20 responses are required per reinforcer, but then show a clear reduction in responding in the BAC HD mice relative to controls as the ratio becomes more demanding.
The total number of errors (including reference memory and working memory errors) decreases over 4 days of training.
