Selective discrimination learning impairments in mice expressing the human Huntington's disease mutation.
ABSTRACT Cognitive decline is apparent in the early stages of Huntington's disease and progressively worsens throughout the course of the disease. Expression of the human Huntington's disease mutation in mice (R6/2 line) causes a progressive neurological phenotype with motor symptoms resembling those seen in Huntington's disease. Here we describe the cognitive performance of R6/2 mice using four different tests (Morris water maze, visual cliff avoidance, two-choice swim tank, and T-maze). Behavioral testing was performed on R6/2 transgenic mice and their wild-type littermates between 3 and 14.5 weeks of age, using separate groups of mice for each test. R6/2 mice did not show an overt motor phenotype until approximately 8 weeks of age. However, between 3.5 and 8 weeks of age, R6/2 mice displayed progressive deterioration in specific aspects of learning in the Morris water maze, visual cliff, two-choice swim tank, and T-maze tasks. The age of onset and progression of the deficits in the individual tasks differed depending on the particular task demands. Thus, as seen in humans with Huntington's disease, R6/2 mice develop progressive learning impairments on cognitive tasks sensitive to frontostriatal and hippocampal function. We suggest that R6/2 mice provide not only a model for studying cognitive and motor changes in trinucleotide repeat disorders, but also a framework within which the functional efficacy of therapeutic strategies aimed at treating such diseases can be tested.
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ABSTRACT: Huntington's disease (HD) is an inherited neurodegenerative disorder that primarily affects the medium-size GABAergic neurons of striatum. The R6/2 mouse line is one of the most widely used animal models of HD. Previously the hallmarks of HD-related pathology have been detected in photoreceptors and interneurons of R6/2 mouse retina. Here we aimed to explore the survival of retinal ganglion cells (RGCs) and functional integrity of distinct retinal cell populations in R6/2 mice. The pattern electroretinography (PERG) signal was lost at the age of 8 weeks in R6/2 mice in contrast to the situation in wild-type (WT) littermates. This defect may be attributable to a major reduction in photopic ERG responses in R6/2 mice which was more evident in b- than a-wave amplitudes. At the age of 4 weeks R6/2 mice had predominantly the soluble form of mutant huntingtin protein (mHtt) in the RGC layer cells, whereas the aggregated form of mHtt was found in the majority of those cells from the 12-week-old R6/2 mice and onwards. Retinal astrocytes did not contain mHtt deposits. The total numbers of RGC layer cells, retinal astrocytes as well as optic nerve axons did not differ between 18-week-old R6/2 mice and their WT controls. Our data indicate that mHtt deposition does not cause RGC degeneration or retinal astrocyte loss in R6/2 mice even at a late stage of HD-related pathology. However, due to functional deficits in the rod- and cone-pathways, the R6/2 mice suffer progressive deficits in visual capabilities starting as early as 4 weeks; at 8 weeks there is severe impairment. This should be taken into account in any behavioral testing conducted in R6/2 mice.PLoS ONE 12/2014; 9(12):e113317. · 3.53 Impact Factor
- 46th AIAA/ASME/ASCE/AHS/ASC Structures, Structural Dynamics and Materials Conference; 04/2005
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ABSTRACT: Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by the expansion of a polyglutamine stretch within the huntingtin protein (HTT). The neurological symptoms, that involve motor, cognitive and psychiatric disturbances, are caused by neurodegeneration that is particularly widespread in the basal ganglia and cereberal cortex. HTT is ubiquitously expressed and in recent years it has become apparent that HD patients experience a wide array of peripheral organ dysfunction including severe metabolic phenotype, weight loss, HD-related cardiomyopathy and skeletal muscle wasting. Although skeletal muscles pathology became a hallmark of HD, the mechanisms underlying muscular atrophy in this disorder are unknown. Skeletal muscles account for approximately 40% of body mass and are highly adaptive to physiological and pathological conditions that may result in muscle hypertrophy (due to increased mechanical load) or atrophy (inactivity, chronic disease states). The atrophy is caused by degeneration of myofibers and their replacement by fibrotic tissue is the major pathological feature in many genetic muscle disorders. Under normal physiological conditions the muscle function is orchestrated by a network of intrinsic hypertrophic and atrophic signals linked to the functional properties of the motor units that are likely to be imbalanced in HD. In this article, we highlight the emerging field of research with particular focus on the recent studies of the skeletal muscle pathology and the identification of new disease-modifying treatments.Frontiers in Physiology 10/2014; 5:380.
Selective Discrimination Learning Impairments in Mice Expressing
the Human Huntington’s Disease Mutation
Lisa A. Lione,1,3,4Rebecca J. Carter,1Mark J. Hunt,1Gillian P. Bates,5A. Jennifer Morton,1and
Stephen B. Dunnett2,3
Departments of1Pharmacology and2Experimental Psychology, and3Medical Research Council, Cambridge Centre for
Brain Repair, University of Cambridge, United Kingdom,4Parke-Davis Neuroscience Research Centre, Cambridge, CB2
2QB United Kingdom, and5Division of Medical and Molecular Genetics, Guy’s Hospital, London, SE1 9RT United
Cognitive decline is apparent in the early stages of Huntington’s
disease and progressively worsens throughout the course of
the disease. Expression of the human Huntington’s disease
mutation in mice (R6/2 line) causes a progressive neurological
phenotype with motor symptoms resembling those seen in
Huntington’s disease. Here we describe the cognitive perfor-
mance of R6/2 mice using four different tests (Morris water
maze, visual cliff avoidance, two-choice swim tank, and
T-maze). Behavioral testing was performed on R6/2 transgenic
mice and their wild-type littermates between 3 and 14.5 weeks
of age, using separate groups of mice for each test. R6/2 mice
did not show an overt motor phenotype until ?8 weeks of age.
However, between 3.5 and 8 weeks of age, R6/2 mice dis-
played progressive deterioration in specific aspects of learning
in the Morris water maze, visual cliff, two-choice swim tank, and
T-maze tasks. The age of onset and progression of the deficits
in the individual tasks differed depending on the particular task
demands. Thus, as seen in humans with Huntington’s disease,
R6/2 mice develop progressive learning impairments on cog-
nitive tasks sensitive to frontostriatal and hippocampal func-
tion. We suggest that R6/2 mice provide not only a model for
studying cognitive and motor changes in trinucleotide repeat
disorders, but also a framework within which the functional
efficacy of therapeutic strategies aimed at treating such dis-
eases can be tested.
Key words: transgenic mice; Huntington’s disease; cognition;
behavior; Morris water maze; T-maze
Expansions of unstable CAG trinucleotide repeats within the
coding regions of target genes cause at least eight different genetic
neurodegenerative diseases, the most prevalent being Hunting-
ton’s disease (HD) (for review, see Paulson, 1999). HD is char-
acterized by motor, cognitive, and psychological disturbances
(Vonsattel and DiFiglia, 1998). Although HD is traditionally
considered to be a “motor” disorder, cognitive decline is an early
and pivotal feature (Mohr et al., 1991; Foroud et al., 1995; Lange
et al., 1995; Lawrence et al., 1996, 1998).
The primary neuropathology in HD is a selective neuronal loss
in the striatum and cortex followed by more widespread atrophy
and neuronal loss in other brain regions in the later stages of the
disease (Vonsattel and DiFiglia, 1998). Several animal models for
HD have been described, including lesions of the striatum in-
duced by excitotoxins (e.g., quinolinic acid) and metabolic poi-
sons (e.g., 3-nitropropionic acid). These models show striatal
pathology similar to that seen in HD (Coyle and Schwarcz, 1976;
McGreer and McGreer, 1976; Beal et al., 1986, 1993; Bossi et al.,
1993; Brouillet et al., 1993, 1995), and also replicate some of the
motor and cognitive symptoms of the disease (Borlongan et al.,
1995; Brouillet et al., 1995; Furtado and Mazurek, 1996; Palfi et
al., 1996; Emerich et al., 1997; Kodsi and Swerdlow, 1997; Shear
et al., 1998a,b). However, a major disadvantage of these neuro-
toxic models is that they lack the genetic pathogenesis and the
progressive nature of HD.
The recent development of transgenic mouse models of HD
(Mangiarini et al., 1996; Reddy et al., 1998; Hodgson et al., 1999;
Schilling et al., 1999; Shelbourne et al., 1999) provide new ways of
examining the mechanisms underlying the progression of HD as
well as the genetic basis of the disease. The R6/2 transgenic
mouse, which expresses the first exon of the human HD gene
carrying 141–157 CAG repeat expansions, develops a number of
the key features of HD, including the progressive motoric dete-
rioration (Mangiarini et al., 1996; Dunnett et al., 1998; Carter et
al., 1999) and the appearance of neuronal intranuclear inclusions
(NIIs) (Davies et al., 1997).
Cognitive function in R6/2 mice has not been well studied. We
reported recently that 8-week-old R6/2 transgenic mice are im-
paired in a spatial navigation cognitive task using the Morris
water maze (Murphy et al., 1998). However, although R6/2 mice
showed a clear deficit in performance of this task, the design of
the Morris water maze task was such that the relative contribu-
tion of motor, sensory, and cognitive factors could not easily be
resolved. Hence, the slower learning of R6/2 mice may have
reflected, in part, a motor and/or visual deficit, rather than a
purely cognitive impairment.
In the present study, a series of tests were chosen in which the
cognitive tasks could be manipulated separately from the sensory,
motor, and motivational conditions within each test. We have
Received June 23, 1999; revised Aug. 27, 1999; accepted Sept. 9, 1999.
This work was supported by grants from the Hereditary Disease Foundation,
Medical Research Council, Parke-Davis Neuroscience Research Centre (UK), and
the Wellcome Trust (UK). L.A.L. is supported by Parke-Davis Neuroscience Re-
search Centre. R.J.C. is supported by the Medical Research Council. We thank Mrs.
Chris Riches and Mr. Trevor Humby for valuable technical assistance.
L.A.L. and R.J.C. contributed equally to this work.
Correspondence should be addressed to Dr. Lisa Lione, Parke-Davis Neuro-
science Research Centre, Cambridge University Forvie Site, Robinson Way, Cam-
bridge, CB2 2QB, UK. E-mail: Lisa.Lione@wl.com.
Copyright © 1999 Society for Neuroscience 0270-6474/99/1910428-10$05.00/0
The Journal of Neuroscience, December 1, 1999, 19(23):10428–10437
used four cognitive tests (the Morris water maze, visual cliff
avoidance, two-choice swim tank, and T-maze) to examine spa-
tial, visual, reversal, and alternation discrimination learning and
MATERIALS AND METHODS
Animals (R6/2 transgenic mice). The R6/2 line of transgenic mice was
generated as previously described (Mangiarini et al., 1996). A colony of
R6/2 transgenic mice was established in the Department of Pharmacol-
ogy, University of Cambridge, and the line was maintained by back-
crossing onto CBA ? C57BL/6 F1 animals. All mice used in the study
were taken from the 14–19th generations of back-crossing.
Mice were housed together in numerical birth order in groups of mixed
genotype, and data were recorded for analysis by mouse number. All
mice were tested during the light phase of a 12 hr light/dark cycle. Until
the appearance of the hindlimb grooming behavior, transgenic mice
could not be distinguished by observation from littermates in their home
cage. Therefore, until the grooming behavior appeared (between 8 and 9
weeks of age; see Mangiarini et al., 1996), the experimenters were blind
to the genotype of the mice. Although data collected after the onset of
an overt phenotype was not collected blind, it should be noted that the
home cage observation for overt phenotype was performed and recorded
separately from the behavioral tests. Because the abnormal grooming in
its early stage is difficult to distinguish from normal grooming behaviors,
this meant that until the abnormal grooming movements occurred reg-
ularly, the experimenters did not routinely know the genotype of a
particular mouse (see below). Once the grooming behavior and other
phenotypic changes became pronounced (usually between 10 and 14
weeks), the experiments could no longer be conducted blindly.
R6/2 mice suffer from diabetes (Hurlbert et al., 1999). In our colony,
diabetes is common in R6/2 mice ?12–14 weeks of age. However, we
have never seen elevated blood glucose levels in mice ?8 weeks of age
(our unpublished observations). Therefore, during the critical testing
period of our study (3–8 weeks of age), any deleterious neurological
effects reported to be associated with diabetes are unlikely to contribute
to the behavioral phenotype that we describe in the present study.
Genotyping. Genotyping was confirmed by PCR based on a modifica-
tion of the method of Mangiarini et al. (1996). Tail tips were removed
from each mouse at 3 weeks of age, and DNA extraction and PCR were
subsequently performed as described (Mangiarini et al., 1996), with the
exception that the following primers were used in the place of those
previously published: 31329 HD (5? to 3?: ATG AAG GCC TTC GAG
TCC CTC AAG TCC TTC) and 33934 HD (5? to 3?: GGC GGC TGA
GGA AGC TGA GGA). In all cases, mice that exhibited a progressive
impairment in the behavioral tests (see below) were genotyped to be
transgenic (and vice versa).
Behavioral testing. Behavioral testing for spatial, visual, reversal, and
alternation discrimination learning and memory was assessed in R6/2
transgenic mice using the Morris water maze, visual cliff avoidance,
two-choice swim tank, and T-maze tests. Testing started at 3 weeks of
age, at which time transgenic mice displayed no overt detectable motor
Separate groups of mice were tested for each of the spatial, visual,
reversal, and alternation discrimination learning and memory tasks.
During the acquisition phase of the Morris water maze and two-choice
swim tank tests, five transgenic mice consistently failed to show motiva-
tion for swimming and made no attempt to escape from the water tank.
These mice (designated “floaters” on the two tests) consistently exhibited
latencies of ?60 or ?120 sec, respectively. Data from these mice were
excluded from the analysis. During habituation in the T-maze, two
transgenic mice did not eat the banana-flavored pellets each day, hence
data from these mice were also excluded from the analysis.
Morris water maze. Spatial and nonspatial learning was assessed in a
Morris water maze modified for use in mice (Stewart and Morris, 1994).
A circular water tank, made from black polypropylene (diameter, 100 cm;
height, 40 cm) was filled to a depth of 25 cm with water (23°C) and
rendered opaque by the addition of a small amount of nontoxic white
paint powder. Four positions around the edge of the tank were arbitrarily
designated north (N), south (S), east (E), and west (W); this provided
four alternative start positions and also defined the division of the tank
into four quadrants: NE, SE, SW, and NW. A square clear Perspex
escape platform (10 ? 10 ? 2 cm) was submerged 0.5 cm below the water
surface and placed at the midpoint of one of the four quadrants. A video
camera was fixed 1.6 m above the center of the swim tank, and all
swimming trials were recorded. Mice were tested daily, from 3 weeks of
age, over 19 d.
On the first 3 d, mice were trained on a visible platform task. The
platform was made visible by the attachment of a high-contrast striped
flag. Mice were trained for 4 trials per day (with an intertrial interval of
?10 min). The start position (N, S, E, or W) and the location of the
platform (NE, SE, SW, or NW) were pseudorandomized across trials.
Mice were allowed up to 60 sec to locate the escape platform, and their
escape latency and pathlengths were recorded. Mice that failed to locate
the platform within the time limit were ascribed an escape latency of 60
sec and were placed on the platform by hand. All mice were then allowed
to stay on the platform for 15 sec, before being removed and returned to
the home cage during the intertrial interval.
From day 4, mice were trained for four trials per day to swim to the
submerged platform, which was now “hidden” by the removal of the flag.
The platform remained in the midpoint of the SW quadrant. Training
continued for 11 d. On day 14 the mice received a single probe trial,
during which the escape platform was removed from the tank, and the
swimming path of each mouse was recorded over 60 sec while it searched
for the missing platform. The mice then received a further four trials
with the hidden escape platform returned to the SW quadrant.
From day 15, reversal training commenced. The escape platform was
moved to the midpoint of the NE quadrant, and the mice were trained to
swim to this new position for four trials per day, over days 15–19. All
trials were videotaped and subsequently analyzed manually using
purpose-designed image analysis software (HVS, Hampton, UK).
Visual cliff avoidance. To assess visual acuity, several groups of age-
matched mice were tested for their ability to avoid a visual cliff. Visual
cliff avoidance was tested in an open-topped box (60 ? 60 cm square ?
30 cm high; Dunnett et al., 1998). The four walls of the box were made
from white plywood, and the base was made from clear Perspex. The box
was positioned on the edge of a laboratory bench so that half of the base
was placed on the bench (“bench side”), and the other half over the edge
of the bench, suspended 1 m above the floor (“open side”). Anglepoise
lamps (60 W) were positioned 60 cm above and 60 cm below the base of
the box. The lamps were positioned in such a way to highlight the edge of
the bench (the “visual cliff”) and to illuminate both the bench and open
side of the box. Mice were placed in a central 7 ? 5 cm “start area”, in
the middle of the base at the edge of the cliff, and their activity was
recorded for 5 min using a video camera fixed 1 m above the center of the
box. Three separate groups of mice (aged 3, 4, and 6 weeks of age at the
start of testing) were used, with each group of mice thereafter being
retested on a weekly basis until 10 weeks of age. The videos were
subsequently analyzed for the percentage of time each mouse spent in
the start area, bench side and open side of the box, and in which direction
the first step outside of the start area was taken.
Two-choice swim tank. Acquisition of a simple left–right visual dis-
crimination task was performed in a modified version of the swim tank
test as described previously (Carter et al., 1999). Briefly, the two-choice
swim tank is a water-filled corridor adapted from a glass aquarium,
100-cm-long and 6-cm-wide, and filled to a depth of 20 cm with water
maintained at a temperature of 23°C. The swim tank was completely
surrounded by 1-m-high gray boards, thus totally obscuring surrounding
spatial cues in the experimental room. Two vertical black lines on the
side of the swim tank marked a horizontal distance 40 cm from either end
of the tank and provided a 20 cm start area in the middle. A visible
escape platform made from black Perspex (6-cm-square, 20.5-cm-high,
with the top surface 0.5 cm above the water level) was placed in a
pseudorandom order at either the left or right end of the swim tank for
each trial. At the beginning of each trial, mice were placed in the start
area facing one side wall so that no directional bias for swimming was
In acquisition training, a 60 W Anglepoise light was positioned over
the escape platform; in reversal learning, the light was positioned over the
end of the swim tank opposite to the platform. The main light source in
the experimental room was dimmed to provide a greater contrast be-
tween the lit and unlit ends of the swim tank. Thus, mice were trained to
swim toward or away from the light stimulus to reach the escape platform
in the acquisition and reversal training, respectively.
During acquisition, mice were given 10–20 trials per day (with an
intertrial interval of 5–30 min) for 7 d. On each trial, a mouse was
considered to have made a correct choice if, and only if, it swam directly
to the platform. An incorrect choice was recorded if (1) the mouse swam
out of the start area in the opposite direction, (2) the mouse swam out of
the start area in the correct direction but returned across the start area,
Lione et al. • Progressive Learning Deficits in R6/2 MiceJ. Neurosci., December 1, 1999, 19(23):10428–10437 10429
or (3) the mouse failed to reach the platform within 120 sec. Analysis was
based on the percentage of correct choices of the first 10 trials performed
each day. Additional training trials were given each day until the mice
made 10 correct choices to a maximum of 20 trials in total.
A first study was performed in which 3-week-old mice were trained for
7 d in this manner, within which time they reached criterion level (?90%
correct choices of their first 10 trials). These mice were then retested
twice weekly to assess retention of learning and ability to discriminate a
light stimulus over time. On their final day of testing, at 9 weeks of age,
the light stimulus over the escape platform was removed to investigate
the importance of the light cue in this task.
In the second experiment, four additional groups of mice were sub-
jected to 7 d of acquisition training. These mice were aged 3, 5, 7, and 10
weeks at the start of testing. After acquisition training, each group was
allowed to rest for 3 d before undergoing a single day of reversal training.
During reversal training all mice were given a total of 30 trials, with an
intertrial interval of 5–30 min. Data were analyzed in blocks of five trials,
and the mean number of correct choices within each trial block was used
for analysis. The performance criterion was set at achieving four correct
trials in a five trial block within the 30 trials. On each trial, mice were
considered to have made a correct choice when they swam directly
toward the escape platform and an incorrect choice when they swam in
the opposite direction to the escape platform.
T-maze. Alternation, spatial, and nonspatial learning was assessed in a
T-maze adapted from a radial eight-arm maze for mice (Molinari et al.,
1996). A T-maze was formed by using the central enclosure and three of
the arms; two at 180° to each other, and one at 90° to these (the stem of
the T). The maze was constructed from clear Perspex with access to the
remaining five arms prevented by clear Perspex guillotine doors that
remained lowered throughout the study. The stem and arms of the T were
each 45-cm-long and 8-cm-wide and surrounded by a 20-cm-high clear
Perspex enclosure. Clear Perspex guillotine doors provided entry to each
arm, and these doors could be raised and lowered remotely. A 10-cm-
long area at the end of the stem of the T provided the start area. The
maze was elevated 50 cm above the floor. A number of distal cues were
placed around the maze. Reinforcement was provided by a 20 mg
banana-flavored pellet (Bio-serve) placed in a white cup at the end of
each arm. Mice were maintained on a 16 hr schedule of food deprivation
throughout the period of testing in the T-maze; water was available ad
libitum. Body weights were monitored daily and maintained at 85%
Before training, all mice were given 3 d of 6 min/d access to the baited
maze with the three doors open to habituate them to the environment.
The number of arms entered and the number of pellets consumed during
this phase did not differ between R6/2 and control mice, providing
evidence against any form of nonspecific motor and motivational impair-
ment. Mice were then given 3 d of 10 “forced” alternation trials before
reinforced alternation training began.
At the start of each alternation training session, both arms were baited,
and all guillotine doors were closed. The mouse was then placed in the
start area and left for 10 sec before all three doors were raised simulta-
neously. After the mouse’s entry into an arm (defined as placing all four
feet in the arm), all doors were reclosed, confining the mouse to its
chosen arm. The mouse was allowed to receive its food reward and then
returned to the start area. The first trial of any session each day was a
“free choice” trial in which a food pellet was placed in both left and right
food cups, and the result was taken as the mouse’s initial choice, after
which the mouse was required to alternate 10 times to receive its food
reward. On each subsequent trial, a mouse was considered to have made
a correct choice if it entered the opposite arm to that last visited. An
error was recorded if the mouse entered the same arm to that last visited.
The process was repeated with as little intertrial delay as possible, until
the mouse had completed the required number of trials (criterion being
10 correct choices). Analysis was based on the number of correct trials of
the first 10 trials and the total number of errors made. The latency
between leaving the start area and entering the chosen arm was also
recorded. Mice were given one training session a day, and additional
trials were given until the mice made 10 correct choices. Training
continued until mice reached a performance level of eight or nine correct
choices of the first 10 trials.
Black–white discrimination. Acquisition of the black–white discrimina-
tion task was also performed in the T-maze. One of the arms was entirely
covered with black cardboard (black), and the other was covered with
thick translucent tracing paper with a 60 W Anglepoise lamp positioned
directly above (white). Although these coverings totally obscured sur-
rounding spatial cues in the room, they provided a dark arm and a
contrasting light arm. One half of each group of mice received reinforce-
ment in the dark arm; the other half received reinforcement in the light
arm. The position of the arms in the apparatus remained on the same side
from trial to trial with the constraint that for half of each group the
reinforced arm was on the left, and for other half on the right. This
allowed mice to solve the discrimination from spatial, visual, or posi-
tional information. Each mouse was placed in the start area and re-
mained there for 10 sec until all doors were raised simultaneously. After
the mouse’s entry into one of the arms, all doors were reclosed, confining
the mouse to its chosen arm. The mouse was allowed to receive its food
reward and then returned to the start area. On each trial, a mouse was
considered to have made a correct choice if it entered into the same
reinforced arm to that last visited. An error was recorded if the mouse
entered into the opposite arm to that last visited; hence, the mice had to
learn which arm was correct. Mice received up to 20 trials each day until
the criterion of five consecutive correct trials had been achieved. The
choice of arm and latency between leaving the start area and entering the
chosen arm was recorded on each trial. After acquisition, the dark and
light arm positions were reversed, whereby the respective reinforced arm
was positioned on the opposite side of the maze to the previous session.
Training continued again to a criterion of five consecutive correct trials.
This reversal procedure was repeated five times. Analysis was based on
the number of trials to criterion of five consecutive correct choices and
the number of errors made.
After black–white discrimination, mice were trained on the same
procedure without the black–white cues. Distal cues were reintroduced
around the maze as for alternation training. On the sixth and eleventh
reversal the maze remained in the same position, but a fourth arm, 180°
to the stem of the T, was introduced as an alternative start arm. Mice
were placed in each start arm on alternate trials. On each trial, a mouse
was considered to have made a correct choice if it entered into the same
arm to that last visited. An error was recorded if the mouse entered into
the opposite arm to that last visited. Hence, on each trial the mice now
had to make a different turn to make a correct choice, relying entirely on
spatial information and removing any possible contribution to solving the
task from positional information. In this way, the proportion with which
each group of mice used spatial versus positional cues to solve the task
Statistical analysis. Behavioral data were subjected to ANOVA (Gen-
stat software package, version 3.2) with two between-subject factors
(genotype and gender) and with repeated measures on one within-
subject factor (age/day/block of trials) as appropriate to the particular
test. In cases of a significant interaction (genotype ? age/day/block of
trials) Sidak’s test was used for multiple independent post hoc pairwise
comparisons between transgenic and wild-type mice at each relevant age,
day, or block of trials (Rohlf and Sokal, 1995). Within each group,
changes in performance with training were evaluated using Dunnett’s
test. A critical value for significance of p ? 0.05 was used throughout the
In no case was there any significant genotype ? gender interactions.
Consequently, although the data from males and females were separated
in all analyses, data has been pooled for clarity of presentation of the
Morris water maze
The visible platform version of the Morris water maze test was
used to test nonspatial learning and to assess whether spatial
learning deficits detected in the R6/2 line are a result of deficient
escape motivation or impairment of visual and/or motor perfor-
mance. As illustrated in Figure 1, both groups showed marked
improvements in escape latencies over the 3 d of training (day,
F(2,82)? 54.21; p ? 0.001). However, the two groups did not differ
either in overall level of performance or change across days
[genotype, F(1,41)? 0.00; genotype ? day, F(2,82)? 2.24, both not
significant (NS)]. An identical pattern of results was seen in the
pathlength measure (Fig. 1B; day, F(2,82)? 69.74, p ? 0.001;
10430 J. Neurosci., December 1, 1999, 19(23):10428–10437Lione et al. • Progressive Learning Deficits in R6/2 Mice
genotype, F(1,41)? 0.21; genotype ? day, F(2,82)? 3.48, both NS).
The swim speed of R6/2 mice did not differ from that of controls
on any day (Fig. 1C; genotype, F(1,41)? 0.95; genotype ? day,
F(2,82)? 0.66, both NS).
Spatial learning: acquisition
The results of the spatial learning task in the Morris water maze
are also shown in Figure 1. In learning to find the fixed hidden
platform, control and R6/2 mice exhibited comparable latency
and pathlength measures on the first 2 d of spatial learning.
However, the R6/2 mice failed to improve on subsequent days as
rapidly as the control mice and, from the third day of spatial
training (day 6 onward), R6/2 mice showed significantly longer
latency and pathlength measures than controls (genotype ? day:
latency, F(10,410)? 2.97, p ? 0.001; pathlength, F(10,410)? 2.85, p
? 0.01). The swim speed of R6/2 mice did not differ from that of
controls on any day (Fig. 1C; genotype ? day: F(10,410)? 1.46, NS).
the probe trials, R6/2 and control groups spent significantly ?25% of their
time in the platform quadrant, indicating that all mice had learned the
platform location (A). The implications of A are refuted by the observa-
tion that R6/2 mice crossed what had been the exact location of the
platform significantly less frequently than did controls (B) and further-
more that R6/2 mice did not show a preference for platform crossings
over equivalent sites in the other quadrants. R6/2 mice spent signifi-
cantly more time in the peripheral outer pool zone and significantly
less time in the middle pool zone relative to control mice (C). Vertical
bars indicate the SEM. Asterisks indicate significant preferences be-
tween measures (**p ? 0.01).
Impairment of probe trial performance in R6/2 mice. During
Escape latency (A), pathlength (B), and swimming speed (C) of control
(n ? 20) and R6/2 (n ? 23) mice during acquisition of visible (days 1–3),
hidden (days 4–14), and reversal (days 15–19) learning. R6/2 mice were
unimpaired in swimming to a visible platform compared with controls
(A–C). The latency (A) and pathlength (B) to escape to the hidden
platform was impaired in R6/2 mice relative to control mice. Note that
control and R6/2 mice did not differ in initial escape latency (A), path-
length (B), or swimming speed (C), indicating that R6/2 mice did not
show nonspecific sensory impairment. R6/2 mice were impaired relative
to controls in ability to learn reversal place learning of a hidden platform.
There was no significant difference in swimming speed between R6/2 and
control mice during the visible and hidden trials, but during reversal trials
swimming speeds were significantly different between the two groups.
Symbols indicate means ? SEM by mice of each group on each measure.
Asterisks indicate significant differences between control and R6/2 mice
(*p ? 0.05; **p ? 0.01).
Impairment of Morris water maze learning in R6/2 mice.
Lione et al. • Progressive Learning Deficits in R6/2 MiceJ. Neurosci., December 1, 1999, 19(23):10428–10437 10431
On the probe trial, both control and R6/2 mice exhibited
evidence for spatial learning, spending above chance time swim-
ming in the platform quadrant (Fig. 2A), and the two groups did
not differ significantly (genotype, F(1,41)? 0.05; genotype ?
quadrant, F(3,123)? 0.71, both NS). However on a more stringent
measure of spatial navigation, measured by counting the “cross-
ings” (the number of times the mice cross the correct location of
the training platform), control mice made significantly more
crossings than R6/2 mice (Fig. 2B; genotype, F(1,41)? 22.39, p ?
0.001; genotype ? platform, F(3,123)? 4.50, p ? 0.01). Further-
more, R6/2 mice spent significantly less time swimming in the
exact location of platform compared with controls (control,
10.4 ? 0.8 sec; R6/2 transgenic, 6.0 ? 1.0 sec; p ? 0.01). An
alternative measure of navigation accuracy is the proportion of
time spent in the inner, middle, and outer zones of the maze.
R6/2 mice spent significantly more time in the outer annuli and
significantly less time in the middle annuli than control mice (Fig.
2C; genotype ? zone, F(2,82)? 25.54, p ? 0.001), indicating that
transgenic and control mice used different platform-searching
strategies. During the probe trial, R6/2 mice swam more slowly
over the full 60 sec test than their wild-type littermates (control,
22.8 ? 0.5 cm/sec; R6/2 transgenic, 19.0 ? 0.6 cm/sec; t ? 5.05;
df ? 41; p ? 0.001).
Spatial learning: reversal
On the first day of reversal training (day 15), when the escape
platform was moved from the center of the SW quadrant to the
center of the NE quadrant, escape latency and pathlength in-
creased transiently in both control and R6/2 transgenic groups, as
the mice initially continued to search for the platform in the
previous location. Thereafter, both control and R6/2 mice rapidly
learned to swim to the “new” NE location. However, as before,
R6/2 mice showed significantly longer latencies and pathlengths
compared with the wild-type mice (Fig. 2A,B; genotype, F(1,41)?
35.07 and 32.34, both p values ? 0.001, for latency and pathlength,
Whereas control mice maintained stable swimming speeds
throughout the study, R6/2 mice swam significantly more slowly
from day 16, and their swim speeds continued to decline (Fig. 1C;
genotype ? day, F(4,164)? 3.40, p ? 0.01). This early deficiency
in motor performance is consistent with our previous findings
(Carter et al., 1999).
Visual cliff avoidance
To assess whether the impaired performance of 3.5- to 5.5-week-
old R6/2 transgenic mice in the Morris water maze could be
attributed to impaired vision using more complex stimuli than
simple light brightness discrimination, we studied mouse behavior
in the visual cliff avoidance test. In this test, the amount of time
spent in the open side of the box is used as a measure of
impairment in visual acuity, although motivational changes in
fear or an inability to learn to avoid the cliff edge may also lead to
failure in this test. Here, control and R6/2 mice were tested in the
visual cliff between 3 and 10 weeks of age. Our previous data
show that only the performances of R6/2 mice at the final time
point were likely to be affected by motor deficits. Between 3 and
7 weeks of age, there were no significant differences between
control and R6/2 mice in the proportion of time spent on the
bench versus open side of the visual cliff box (Fig. 3A). Addition-
ally, the amount of time spent on the bench side of the box
increased significantly in both groups of mice from week 4 (effect
of age, F(7,284)? 20.32, p ? 0.01). However, whereas the wild-
type mice continued to spend the majority of their time on the
bench side during all subsequent trials, reflecting habituation, the
R6/2 mice regularly ventured into the center of the visual cliff box
and frequently crossed the cliff edge without hesitation from 8
weeks of age (Fig. 3; genotype, F(1,62)? 24.74; genotype ? age,
F(7,284)? 17.45, both p values ? 0.01).
A similar pattern of behavior was shown when the direction of
the first step outside the start area was analyzed. There were no
significant differences between control and R6/2 mice from 3 and
7 weeks of age (Fig. 3B), with mice in both groups stepping onto
the bench side, in preference to the open side, of the visual cliff
box. However, after 7 weeks of age, whereas control mice con-
tinued to step onto the bench side first, the direction of first step
taken by R6/2 mice reverted to chance (genotype, F(1,62)? 11.25;
genotype ? day, F(7,284)? 4.43, both p values ? 0.01).
Two-choice swim tank
In the first experiment, 3-week-old control and R6/2 mice learned
to discriminate bright and dimmed light and respond accordingly
within 7 d of training. The mean percentage of correct choices
of mice were tested between 3 and 10 weeks of age. Visual cliff avoidance
of mice was measured using the percentage of time spent on the bench
side of the visual cliff open field, as well as the direction of the first step
outside of the start area (0 equates to open side step and 1 equates to
bench side step). Control (n ? 34) and R6/2 (n ? 30) mice spent
significantly more time on the bench side of the visual cliff box from 3–7
weeks of age (A). From 8 weeks of age, R6/2 mice spent significantly less
time on the bench side relative to controls. A similar profile was seen for
control and R6/2 mice for the direction of first step measure (B). Symbols
indicate means ? SEM at each age. Asterisks indicate significant differ-
ences between control and R6/2 mice (**p ? 0.01).
Deficient visual cliff avoidance in R6/2 mice. Separate groups
10432 J. Neurosci., December 1, 1999, 19(23):10428–10437Lione et al. • Progressive Learning Deficits in R6/2 Mice
made of the first 10 trials for R6/2 mice did not differ significantly
from controls on any day (Fig. 4A; genotype, F(1,29)? 0.99;
genotype ? day, F(16,464)? 0.64, both NS). Subsequent retesting
revealed that control and R6/2 mice retained their learning,
performing at criterion until 8.5 weeks of age. On the final retest
at 9 weeks of age, the light stimulus over the visible escape
platform was removed. Performance declined to chance levels for
both groups (Fig. 4A), suggesting that all mice were using the
light stimulus as their visual cue, and that R6/2 mice are able to
detect a distant bright light until at least 8.5 weeks of age. It is
noteworthy that R6/2 mice swam significantly more slowly than
controls from 5 weeks of age, as shown by their increased latency
to swim the 40 cm distance (Fig. 4B; genotype, F(1,29)? 28.94,
p ? 0.001; genotype ? day, F(16,464)? 11.02, p ? 0.01), although
this did not affect choice accuracy.
In the second experiment, the two-choice swim tank task was
used to examine visual and reversal discrimination learning in
more detail. Separate groups of mice were used, aged 3, 5, 7, and
10 weeks old at the start of testing. R6/2 mice were not impaired
in task acquisition at 3–6 weeks of age (Fig. 5A,B; genotype ?
day, 3–4 weeks: F(6,168)? 1.54; 5–6 weeks: F(6,174)? 2.07, both
NS). R6/2 mice exhibited significant deficits at 7–8 weeks of age,
although they could still learn the task (Fig. 5C; genotype ? day,
7–8 weeks: F(6,174)? 4.82, p ? 0.001). By 10–11 weeks of age, the
ability of R6/2 mice to acquire this task was severely impaired
compared with their age-matched littermate controls (Fig. 5D;
genotype ? day, F(6,168)? 7.13, p ? 0.001).
When the task was reversed, both control and R6/2 mice
initially exhibited a performance deficit at all ages tested, as they
continued to swim toward the previously reinforced visual cue,
the light stimulus. However, for control mice, this performance
deficit was transient, and at all ages tested they learned to swim
away from the light stimulus within 15–20 trials and performed to
criterion within 30 trials (Fig. 5E–H). In contrast, whereas 4.5-
week-old R6/2 mice learned to dissociate the original visual cue
from reinforcement and learned to swim away from the light
stimulus as rapidly as age-matched controls (Fig. 5E; genotype,
F(1,28)? 0.24; genotype ? trial bin, F(5,140)? 1.01, both NS),
older R6/2 mice showed performance deficits. Although 5- to
6-week-old R6/2 mice could learn the simple visual discrimina-
tion during acquisition as well as their littermate controls, they
were impaired when they were subsequently forced to reverse the
discrimination (Fig. 5F; genotype, F(1,29)? 26.69; genotype ?
trial bin, F(5,145)? 16.83, both p values ? 0.01). These R6/2 mice
failed to learn the reversal task after 30 trials, persistently swim-
ming in the direction of the light. This deficit in reversal discrim-
ination learning was also seen in 8.5- and 11.5-week-old R6/2 mice
(Fig. 5G,H; 8.5 week: genotype, F(1,29)? 19.28; genotype ? trial
bin, F(5,145)? 9.72, both p values ? 0.01; 11.5 week: genotype,
F(1,28)? 36.19; genotype ? trial bin, F(5,140)? 7.06, both p
values ? 0.01).
The T-maze test was used to test alternation, spatial, and non-
spatial learning in R6/2 mice. R6/2 mice made significantly fewer
alternations in the T-maze and more errors relative to controls
from day 1 onwards (Fig. 6A; genotype, F(1,26)? 55.54 and 59.10,
both p values ? 0.001; genotype ? block of days, F(5,130)? 55.54
and 59.10, both p values ? 0.001, for the number of alternations
and errors, respectively). Initial and final levels of performance
significantly differed between R6/2 mice and controls, indicating
that the observed distinction may be attributable to nonspecific
sensory, motor, or motivational deficits of the R6/2 mice. Neither
group differed in their mean latency to enter an arm on any day
(data not shown). This indicated that R6/2 mice did not have
impairments in locomotor activity and that motivation was
In the second phase of the T-maze study, whereby mice were
rewarded for discriminating a black or white arm, there was no
evidence of any nonspecific deficit in R6/2 mice compared with
controls (Fig. 6B). In fact, R6/2 mice learned the black–white
discrimination more rapidly than controls, requiring significantly
fewer trials to reach criterion and producing fewer errors (t ?
4.14 and 3.51; both df ? 25 and p ? 0.01 for the number of trials
to criterion and errors, respectively). On the subsequent reversals
of this task there was no significant difference on the number of
trials to criterion and errors made between each group (Fig. 6B;
genotype, F(1,25)? 3.59 and 0.54, both NS, for the number of
trials to criterion and errors, respectively).
dent of swimming dysfunction in R6/2 mice. Control (n ? 17) and R6/2
(n ? 14) mice were tested between 3 and 9 weeks of age. Control and R6/2
mice displayed a comparable degree of acquisition of a two-choice swim
tank visual discrimination task at 3–4 weeks of age, and both groups
maintained performance criterion until 8.5 weeks of age (A). On removal
of the light stimulus at 9 weeks of age, performance declined to chance
levels for both groups. R6/2 mice displayed swimming impairments from
5 weeks of age, which progressively worsened, as compared with controls
(B). Motoric dysfunction failed to impair performance of R6/2 mice in
the two-choice swim tank task. Vertical bars indicate means ? SEM of
each measure across all trials at each age. Asterisks indicate significant
differences between control and R6/2 mice (*p ? 0.05; **p ? 0.01).
Normal visual discrimination learning and memory indepen-
Lione et al. • Progressive Learning Deficits in R6/2 MiceJ. Neurosci., December 1, 1999, 19(23):10428–10437 10433
On removal of the black/white cues, the number of trials to
criterion and errors made by R6/2 mice did not differ from
controls on acquisition (Fig. 6C; t ? 0.76 and 0.44, both df ? 24,
and NS, for the number of trials to criterion and errors, respec-
tively) and any reversal, except the “opposite” reversals six and
eleven (in which all mice were placed in the opposite start arm on
alternate trials) (Fig. 6C; number of trials to criterion: genotype,
F(1,24)? 28.80; genotype ? reversal, F(11,264)? 4.11, both p
values ? 0.001; number of errors: genotype, F(1,24)? 24.71;
genotype ? reversal, F(11,264)? 3.10, both p values ? 0.001).
Hence, whereas control mice spatially learned when placed in the
opposite start arm to make a turn in the opposite direction to
enter the same arm as on the previous trial, R6/2 mice persistently
turned in the same direction irrespective of their start position.
Furthermore, the deficit displayed by R6/2 mice on this task was
significantly worse on reversal 11 compared with that on reversal
6 ( p ? 0.01).
This study provides the first evidence for progressive learning and
memory deficits in R6/2 mice. We describe deficits in a number of
tasks assessing spatial, visual, reversal, and alternation discrimi-
nation learning and memory. These deficits cannot be attributed
to a loss of motor or visual function because impairments in
spatial, reversal, and alternation learning were observed as early
as 3.5–6.5 weeks of age, when R6/2 mice were not impaired in the
motor and visual components of these tasks. Furthermore, differ-
ent patterns of deficit were seen in tests with similar sensory and
motor demands, with the extent of the deficit depending on the
cognitive demands of the task (Fig. 7).
Cognitive deficits in R6/2 mice
Behavioral testing of R6/2 mice started at 3 weeks of age, several
weeks before subtle (5–6 weeks), or overt (8–9 weeks) motor
symptoms appeared (Carter et al., 1999). At the earliest age
tested (3.5 weeks), it was apparent that R6/2 mice have a spatial
learning deficit in the Morris water maze test. This is consistent
with our previous study, which found that older R6/2 mice (7–8
weeks) exhibit severe impairments in spatial learning of the
hidden platform task (Murphy et al., 1998). The slower learning
of older R6/2 mice may reflect a motor deficit, rather than a
purely cognitive impairment. However, the impaired perfor-
mance of young R6/2 mice reported here cannot be secondary to
motor deficits or lack of motivation to escape from the water,
because control and R6/2 mice initially showed identical path-
lengths and swim speeds. Moreover, although R6/2 mice swim
crimination learning in R6/2 mice. Separate groups of
control (n ? 10–18) and R6/2 (n ? 12–21) mice were
tested between 3 and 10 weeks of age. Control and R6/2
mice displayed a comparable degree of acquisition of a
two-choice swim tank visual discrimination task at 3–4
(A) and 5–6 (B) weeks of age, however R6/2 mice dis-
played significantly slower learning than controls at 7–8
weeks of age (C), and by 10–11 weeks of age (D) acqui-
sition was severely impaired in R6/2 mice. In contrast,
acquisition of reversal discrimination was impaired in
R6/2 mice from 6.5 weeks of age (E–H). Symbols indicate
means ? SEM by mice of each group at each age on both
measures. Asterisks indicate significant differences be-
tween control and R6/2 mice (*p ? 0.05; **p ? 0.01).
Selective deficits in visual and reversal dis-
10434 J. Neurosci., December 1, 1999, 19(23):10428–10437Lione et al. • Progressive Learning Deficits in R6/2 Mice
more slowly with increasing age, impaired water maze learning
was associated with increased pathlengths, indicating that older
R6/2 mice swam further than controls without apparent difficulty.
It was also notable during the probe trial that R6/2 mice exhibited
a different platform search strategy from controls, swimming
preferentially in the periphery of the water tank. “Thigmotaxis”
(seen commonly in normal animals in the early phase of learning
Morris water maze tasks) is exacerbated in rodents with striatal
lesions (Devan et al., 1999). This suggests there is a striatal
component to the dysfunction in the R6/2 mice.
The possibility that visual impairments in R6/2 mice contribute
to impairments in water maze performance is an important
confound. Thus, we tested R6/2 mice on two visual discrimination
tasks: visual cliff avoidance and two-choice swim tank. The visual
cliff task showed that R6/2 mice avoid a visual cliff until at least 7
weeks of age but display a progressive reduction in avoidance
thereafter. The increased time spent over the edge of the cliff is
unlikely to reflect increased exploratory activity, because control
and R6/2 mice have comparable levels of locomotor activity in an
open field until 10 weeks of age (Dunnett et al., 1998). The
performance of older (?8 weeks) R6/2 mice in the visual cliff may
be confounded by visual dysfunction, although it is possible that
an impaired ability to learn to avoid the cliff edge is responsible
for their deteriorating performance. Reduced anxiety in R6/2
mice (File et al., 1998) may also contribute to these deficits.
The performance of R6/2 mice on the two-choice swim tank
task showed that mice trained at 3–4 weeks can perceive a visual
light cue until at least 8.5 weeks of age. Subsequently removing
the light cue causes the performance of control and R6/2 mice to
decline to chance levels, demonstrating that all mice use the light
as their definitive visual cue. Data from both cliff and swim tank
tests support the idea that young R6/2 mice have sufficiently
normal vision to perform the tasks and support the suggestion
that poor performance of R6/2 mice in the Morris water maze
and swim tank is attributable to cognitive rather than nonspecific
sensory or motor deficits.
Impairments in cognitive performance in the two-choice swim
tank were particularly revealing. The ability of young R6/2 mice
to learn the task (acquisition) was not affected; however, by 7–8
weeks, learning was mildly impaired, and by 10 weeks learning
was severely compromised. Impairments on the reversal trials
were also seen, but were much more severe, with maximum
impairment seen by 6.5 weeks. The reversal learning deficits in
the swim tank at least, cannot be attributed to a loss of motor or
visual function, because pretrained R6/2 mice continue to per-
form accurately in this task at that same age. It is more likely that
(n ? 15) were impaired in T-maze alternation (A) but unimpaired in a
simple T-maze black–white visual discrimination test (B), compared with
controls (n ? 13). Whereas control mice adopted spatial strategies, R6/2
mice used nonspatial strategies (C). Symbols indicate means ? SEM by
mice of each group on each measure. Asterisks indicate significant differ-
ences between control and R6/2 mice (*p ? 0.05; **p ? 0.01).
Alternation learning impairments in R6/2 mice. R6/2 mice
pairment in spatial, visual, reversal, and alternation learning in R6/2 mice.
R6/2 mice showed spatial learning impairments in the Morris water maze
from 3.5 weeks (A), alternation learning impairments in the T-maze from
5 weeks (B), and reversal learning impairments in the two-choice swim
tank from 6.5 weeks (C). Visual discriminative learning impairments
were first observed from 7–8 weeks (D, E), and deterioration in retention
of a previously learned visual task was not seen until 8.5 weeks (F). Filled
bars indicate onset of impairment.
Schematic representation of the earliest age of onset of im-
Lione et al. • Progressive Learning Deficits in R6/2 MiceJ. Neurosci., December 1, 1999, 19(23):10428–10437 10435
R6/2 mice have an age-dependent deficit in cognitive flexibility,
because they appear to be incapable of making this strategy
Between 5 and 6.5 weeks R6/2 mice are deficient in their ability
to alternate in a T-maze. This may reflect either a disturbance in
the expression of innate motivational program or a tendency of
R6/2 mice to perseverate. When the same mice were trained
subsequently in a specific visual discrimination paradigm, R6/2
mice reached criterion more rapidly than controls, although this
may simply reflect the fact that they were starting from a lower
baseline of alternation performance. Certainly, once trained at
7–9 weeks, R6/2 mice and controls showed comparable ability to
learn the reversal, again indicating that the acquisition deficit is
not attributable to a visual deficit. Spontaneous alternation rep-
resents an innate species-specific pattern of motivated behavior
that may be related to optimal foraging strategies in the wild
(Dember and Fowler, 1958). However, to alternate, animals must
have a functionally intact short-term memory. Significantly, spon-
taneous alternation is disrupted by striatal, prefrontal, and hip-
pocampal lesions (Divac et al., 1975; Johnson et al., 1977).
To determine whether a spatial and/or a nonspatial strategy is
adopted by mice in this task, they were placed in the opposite
start arm on alternate trials. R6/2 mice appear to be deficient in
spatial learning, because they continued to adopt nonspatial in-
tramaze (and/or positional) cues, eventually solving the task to a
very low level of accuracy. In contrast, control mice adopt normal
spatial learning and reach criterion within 10 trials.
Similarities between cognitive deficits in R6/2 mice
and HD patients
The cognitive impairment in HD is believed to be a consequence
of the profound frontostriatal pathology associated with the dis-
ease (Vonsattel and DiFiglia, 1998). Striatal dysfunction is impli-
cated in the specific cognitive deficits seen in HD patients (Lange
et al., 1995; Lawrence et al., 1996, 1998). The nature of cognitive
decline seen in patients with HD includes a decline in visuospa-
tial skills, cognitive flexibility, and recall memory. Notably, Law-
rence et al. (1996, 1998) reported impairments involving changing
a previously learned response, with HD patients showing deficits
in a visual discrimination/set-shifting task by exhibiting perse-
verative responding to the original stimulus. This response may
be analogous to the impaired performance of R6/2 mice in the
reversal phase of the swim tank and their inability to alternate in
the T-maze. An inability to switch from one set of learned
responses to another therefore appears to be a fundamental
feature of patients with HD as well as R6/2 mice. HD patients
(Swerdlow et al., 1995) and R6/2 mice (Carter et al., 1999) also
exhibit deficient inhibitory control in a number of neuropsycho-
logical paradigms, including prepulse inhibition. However the
neural basis for these deficits remains unresolved.
Possible mechanisms underlying cognitive dysfunction
The behavioral tests used here were designed to test frontostriatal
function in R6/2 mice. Although R6/2 mice show no striatal
neuronal loss (Mangiarini et al., 1996; Davies et al., 1997), inclu-
sions (Davies et al., 1997) and neurotransmitter abnormalities
(Cha et al., 1998) are found in the frontostriatal region of R6/2
mice, suggesting that the cognitive changes reported here are
attributable to neuronal dysfunction (rather than cell death). The
cognitive deficits in HD are associated with frontostriatal pathol-
ogy, however striatal dysfunction and cognitive deficits in the
absence of cell death are seen in early stage HD (Myers et al.,
1988). Interestingly, animal models of HD with frontostriatal
lesions display cognitive deficits, attributed to the selective stria-
tal damage, which are similar to those reported here (Divac et al.,
1967; Furtado and Mazurek, 1996; Palfi et al., 1996; Emerich et
al., 1997; Shear et al., 1998a,b). The present data are consistent
with a frontostriatal basis for the deficit.
NII formation in R6/2 mice is not restricted to the striatum,
and an alternative explanation for the observed learning deficits is
the existence of hippocampal impairments in R6/2 mice. Lesions
of the hippocampus can cause maze-learning deficits in rodents
(Morris et al., 1982; Logue et al., 1997), and R6/2 mice show NII
pathology in this area (Davies et al., 1997). In addition, we have
shown that R6/2 mice exhibit a reduced long-term potentiation
and a lasting long-term depression in the CA1 area of the hip-
pocampus (Murphy et al., 1998), electrophysiological phenomena
that have been implicated in learning and memory (Tsien et al.,
1996; Stevens and Sullivan, 1998). Similar changes in plasticity
have been seen in other transgenic models of HD, although these
groups did not report on the cognitive phenotype (Hodgson et al.,
1999; Usdin et al., 1999). Thus, it seems possible that changes in
synaptic plasticity, or other aspects of hippocampal dysfunction,
contribute to the deficits in learning and memory that we describe
Our study describes specific deficits in several forms of learning in
R6/2 mice. We provide evidence for a specific and progressive
pattern of discriminative learning impairments in R6/2 mice that,
as in humans, have no clear “age of onset”, because the age at
which an impairment becomes apparent is dependent on the
sensitivity of the test and the particular demands of each task.
These results indicate that transgenic mice, such as the R6/2
strain, may not only be useful for studying the relationship be-
tween cognitive and motor components of neurological dysfunc-
tion, but also for providing models of specific diseases for assess-
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