Non-Motor and Motor Features in LRRK2 Transgenic Mice
Zoe ¨ Bichler1,2*, Han Chi Lim3, Li Zeng2,3, Eng King Tan1,2,4*
1Behavioral Neuroscience Laboratory, National Neuroscience Institute, Singapore, Singapore, 2Duke-NUS Graduate Medical School, Program in Neuroscience and
Behavioral Disorders, Singapore, Singapore, 3Neural Stem Cell Laboratory, National Neuroscience Institute, Singapore, Singapore, 4Department of Neurology, National
Neuroscience Institute, Singapore General Hospital, Singapore, Singapore
Background: Non-motor symptoms are increasingly recognized as important features of Parkinson’s disease (PD). LRRK2
mutations are common causes of familial and sporadic PD. Non-motor features have not been yet comprehensively
evaluated in LRRK2 transgenic mouse models.
Objective: Using a transgenic mouse model overexpressing the R1441G mutation of the human LRRK2 gene, we have
investigated the longitudinal correlation between motor and non-motor symptoms and determined if specific non-motor
phenotypes precede motor symptoms.
Methodology: We investigated the onset of motor and non-motor phenotypes on the LRRK2R1441GBAC transgenic mice and
their littermate controls from 4 to 21 month-old using a battery of behavioral tests. The transgenic mutant mice displayed
mild hypokinesia in the open field from 16 months old, with gastrointestinal dysfunctions beginning at 6 months old. Non-
motor features such as depression and anxiety-like behaviors, sensorial functions (pain sensitivity and olfaction), and
learning and memory abilities in the passive avoidance test were similar in the transgenic animals compared to littermate
Conclusions: LRRK2R1441GBAC transgenic mice displayed gastrointestinal dysfunction at an early stage but did not have
abnormalities in fine behaviors, olfaction, pain sensitivity, mood disorders and learning and memory compared to non-
transgenic littermate controls. The observations on olfaction and gastrointestinal dysfunction in this model validate findings
in human carriers. These mice did recapitulate mild Parkinsonian motor features at late stages but compensatory
mechanisms modulating the progression of PD in these models should be further evaluated.
Citation: Bichler Z, Lim HC, Zeng L, Tan EK (2013) Non-Motor and Motor Features in LRRK2 Transgenic Mice. PLoS ONE 8(7): e70249. doi:10.1371/
Editor: Huaibin Cai, National Institute of Health, United States of America
Received February 26, 2013; Accepted June 18, 2013; Published July 30, 2013
Copyright: ? 2013 Bichler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors thank Singapore Millennium Foundation, National Medical Research Council and Duke NUS Graduate Medical School for their support. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (ZB); firstname.lastname@example.org (EKT)
Parkinson’s Disease (PD), a common neurodegenerative disease,
has a complex etiology where both genetic and environmental
factors play a role. The diagnosis of PD is essentially based on the
motor features that appear relatively late in the time course of the
disease, such as resting tremor, rigidity and bradykinesia, by which
time more than 70% of the dopaminergic neurons have
degenerated . Numerous non-motor symptoms contribute to
significant disability in PD [2,3,4]. Olfactory loss, rapid eye
movements sleep behavior disorder, mood disorders and consti-
pation are commonly described in patients [4,5]. Increased co-
morbidity of non-motor symptoms was associated with greater PD
severity and some have even been suggested to be a risk factor for
PD as they might precede for years the clinical diagnosis [6,7,8].
Understanding the etiopathology of the disease may thus help
unraveling specific early biomarkers and would certainly improve
the design of future therapies [9,10,11].
Mutations of the Leucine-Rich Repeat Kinase 2 (LRRK2) gene
are the most common causes of sporadic and autosomal dominant
PD [12,13,14,15,16]. LRRK2 is a large protein of 2527 amino
acids and multiple protein domains, with pathogenic mutations
distributed throughout its length: G2019S is the most common
mutation identified, with a prevalence of 13 to 40% depending on
the ethnic races. R1441G is the second most common site of
pathogenic LRRK2 substitutions. We have previously identified
other common genetic variants such as G2385R that are unique to
Most clinical studies on non-motor symptoms are cross sectional
in design, as it is difficult to conduct short-term longitudinal studies
on the progression of such phenotypes in humans. Several animal
models for PD have been developed, but most of them focus on
motor and neuropathological features and they do not recapitulate
[18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]. Recently, Li and
collaborators have created a mouse line that sums up the main
features of human PD . These mice overexpress the R1441G
mutation of the human LRRK2 gene by means of a Bacterial
Artificial Chromosome (BAC). The LRRK2R1441GBAC transgenic
(Tg) mice displayed an age-dependent slowness of movements that
begun at 6 months old, and, at 10–12 months of age, the motor
deficit was associated with diminished dopamine release and
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axonal pathology of nigrostriatal dopaminergic projections. These
mice had also an increased level of neuroinflammation inducing
neurotoxicity . However, the authors did not conduct a
thorough evaluation of non-motor features. In addition, motor
features were not reported on mice above the age of 12 months.
Unpublished data from the Jackson’s laboratory (http://jaxmice.
jax.org/strain/009604.html) suggest that the motor features could
not be replicated.
To address limitations of current literature, we conducted a
detailed longitudinal study of both motor and non-motor features
in the LRRK2R1441Gmutant mouse line. Our main hypothesis was
that motor symptoms correlate with non-motor symptoms over the
course of the disease.
Decreased Locomotor Activity in LRRK2R1441GTg Aged
As motor symptoms are considered the hallmark of PD in
human patients, we first identified the onset of motor disabilities in
LRRK2R1441Gmice. Tg and NTg (non-transgenic) littermates
coming from several litters were exposed to different tasks
measuring locomotion, vertical activity, balance and coordination.
Compared to their age- and gender-matched controls, Tg mice
displayed subtle motor deficits only after the age of 16 months,
though no gross dysfunction could be observed. Indeed, in the
standard cylinder test, both groups of mice reared the same
number of time within the 5 min session from 4 to 16 months old,
but at twenty months old, Tg mice reared much less than their
controls NTg (Welch’s t test, t(6.036)=2.783, p,0.05, Figure 1A).
Tg mice were still well coordinated in their movements as they
performed as good as their controls in the accelerated rotarod
(Figure 1B). In order to check any decrease of limb muscular
tonus that could be responsible for the decreased vertical ability
observed in the cylinder test, we measured the ability of the mice
to remain clinging to an inverted cage lid (grip strength test,
Figure 1C). No difference between Tg and NTg could be found
concerning the mean latency until they fall from the elevated cage
lid, showing that both groups had similar muscular tonus capacity
and grip strength at this age.
To measure the spontaneous activity, mice were put in the open
field apparatus during 15 min (Photobeam Activity System, PAS-
Open Field, San Diego Instrument, USA). Horizontal and vertical
activity was automatically recorded as well as the fine activity,
which combined actions like grooming, exploration on four limbs
and sniffing. We first considered the total amount of activity within
the 15 min test (Figure 1D). In general, mice increased their
activity at 9 months old, but decreased it by half at 16 months old
(Age effect with MANOVA with ‘‘age’’ and ‘‘genotype’’ as in-
p,0.001; Horizontal activity: F(6,131)=6.123, p,0.001). These
changes were statistically significant only for Tg animals (Geno-
type effect with MANOVA with ‘‘age’’ and ‘‘genotype’’ as in-
between subject factors, Vertical activity: n.s.; Horizontal activity:
F(1,131)=4.622, p,0.05; followed by post-hoc comparisons,
Figure 1Da and 1Db). From 16 months old, Tg mice were
constantly less active than NTg controls, with a significant
t(8.975)=8.646, p,0.05). A similar profile of activity was observed
over time concerning the amount of photobeam activity in the
center (MANOVA with ‘‘age’’ and ‘‘genotype’’ as in-between
subject factors, Age effect: F(6,131)=5.239, p,0.001, Genotype
effect: n.s., Figure 1Dc), and from 16 months old, the Tg mice
spent significantly less time than the controls in the center (Welch’s
old (Welch’st test,
t test p,0.05 at 16 and 20 months old, with t(11.805)=5.418 and
In summary, Tg animals decreased their activity in the open
field compared to NTg littermate controls after the age of 16
months old. This was not compensated by an augmentation of fine
behaviors. Indeed, although 16 months old Tg mice increased
their fine activities compared to NTg (p,0.05, Figure 1Dd), the
photobeam activity reflecting the number of fine behaviors were
not changed otherwise, suggesting that Tg mice displayed an
overall diminution of locomotor activity only.
We further compared the activity of both groups according to
intervals of 3 min time per 15 min session (Figure 2). While the
overall activity curves of the mice were similar for both NTg and
Tg before one year old, Tg animals showed constantly less
horizontal ambulation and rearing behaviors than NTg mice from
16 months old (MANOVA with ‘‘genotype’’ and ‘‘3-min-
intervals’’ as in-between subject factors, main effect of the
F(1,74)=14.233, p,0.001, 20 months old F(1,54)=24.032,
p,0.001; andin Vertical
F(1,74)=7.811, p,0.01 and 20 months old F(1,54)=8.565,
p,0.01). As previously shown, Tg mice increased their fine
movements at 16 months old compared to their NTg littermates
(MANOVA with ‘‘genotype’’ and ‘‘3-min-intervals’’ as in-between
subject factors, main effect of the genotype: F(1,74)=10.118,
p,0.01). This resting behavior was however not different at older
ages, and thus cannot compensate the general decreased activity
observed. Also, this analysis confirmed that 16 months old and
older Tg mice behaved less in the center of the arena compared to
controls (MANOVA with ‘‘genotype’’ and ‘‘3-min-intervals’’ as in-
between subject factors, Genotype effect: 16 months old:
F(1,74)=14.617, p,0.001, 20 months old: F(1,54)=17.874,
p,0.001). The results were similar if we considered the percentage
of photobeam activity over the total photobeam activity (data not
shown, MANOVA with ‘‘genotype’’ and ‘‘3-min-intervals’’ as in-
between subject factors, Genotype effect: 16 months old:
p,0.001), suggesting that this might not reflect only a reduced
locomotor capacity but rather a centrophobic response due to a
possible fear of open areas. Since this can be an indicator of
anxiety level, we analyzed the mice in other tasks which are
sensitive to mood disorders-like behaviors.
activityat 16months old
months old: F(1,54)=9.375,
LRRK2R1441GMice did not Display Anxiety or Depression-
like Behaviors with Age
Tg and NTg littermates were tested at 6, 14 and 19 months old
in the elevated plus maze. This task measures the willingness of the
mice to explore opened and closed areas and informs on the level
of anxiety-like behavior of the animals in a new environment. We
did not observe any tendency of Tg mice to be more anxious than
the NTg (Figure 3A). The distance run was similar across ages
and genotypes in this 5 min task and the time spent in the opened
arms augmented regularly from 6 to 19 months old suggesting an
increased exploratory behavior of all mice with age.
Mice were then tested in the tail suspension and forced
swimming tests. These tests evaluate the motivation of the animals
to escape from a despaired situation, and reflect the depressed
state of the animal as compared to the depression-like behavior
displayed by humans. Different batches of NTg and Tg mice were
tested at different ages from 6 to 19 months old in both tests
(Figure 3B–C). They all behaved the same, though the Tg mice
were slightly more active (i.e. less immobile) than their controls
from 16 months old onwards in the tail suspension test (mean
immobility time at16 months oldNTg=118.01629.99,
Behavioral Phenotype of LRRK2 Mice
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Tg=85.91618.69; at 19 months old NTg=98.68616.88,
Tg=60.42614.35, ANOVA test with ‘‘age ‘‘ and ‘‘ genotype’’
as in-between factors, Age effect: F(4,76)=2.539, p,0.05,
Genotype effect: F(1,76)=0.101, n.s.). This was in accordance
with the increased exploratory activity found in the elevated plus
maze. Similar results were obtained in the forced swimming test
with a constant slight decrease of the immobility time with age, but
no difference between the two genotypes (ANOVA test with ‘‘age’’
and ‘‘genotype’’ as in-between factors, Age effect: F(4,73)=1,695,
n.s., Genotype effect: F(1,73)=0.337, n.s.).
LRRK2R1441GMice Displayed Normal Sensory Responses at
To determine whether the overexpression of the R1441G
mutant form of human LRRK2 gene would affect sensory functions
in mice, we checked olfaction abilities with the buried and the
block tests, and sensitivity to pain with the formalin test. The two
olfaction tests did not reveal any impairment in the ability to smell
and recognize either social odors (Figure 4Aa-d) or food odor
following deprivation (Figure 4Ae-f). In the block test, where
mice should recognize a familiar odor in scented wooden cubes,
Tg and NTg mice tested at 6 and 14 months showed the same
pattern independently of their genotype. Interestingly, mice were
more interested in sniffing the familiar blocks at 6 months old,
whereas 14 months old mice had a preference for the unknown
congener’s odors (Figure 4Ab). No difference between NTg and
Tg was detected in the habituation period of the 6 first trials in
contact with the blocks scented with the familiar odor of the home
cage. In the buried test, 6 months old mice smelled very well the
odor of the food following food deprivation, independently of the
genotype. Tg mice were not impaired, and were even faster in
eating the piece of food in the control trial than the NTg
Figure 1. Motor abilities of the BAC LRRK2R1441GTg and NTg mice with age. A. Number of rearing in the cylinder test. B. Latency to fall in the
accelerated rotarod paradigm. C. Mean latency of staying gripped to the grid of a reverted cage lid. D. General activity in the open field test in a
15 min test: rearing (a), horizontal activity (b), activity in the center of the apparatus (c), and total number of fine activities (d). Data are means 6 SEM,
number of animals are indicated in the corresponding bar of the histogram. *p,0.05, **p,0.01, ***p,0.001 with MANOVA followed by post-hoc
comparison (Bonferroni), and Student t test for single comparisons or Welch’s t test when variances were unequal.
Behavioral Phenotype of LRRK2 Mice
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littermates (surface trial, mean latency to find and eat the piece of
food NTg=42.9869.20; Tg=23.21610.00; Figure 4Af).
To determine if there was any pain-dependent-sensory
dysfunction, we performed the formalin test. Mice were injected
with 20 microl of 2.5% formalin in the right hind paw and
observed for 30 min. The pain response followed a two-phase
curve. The first phase occurred immediately after injection and
lasted no more than 2 to 4 min; it is considered as an acute
response of local pain. The second phase occurred about 10 to
15 min later corresponding to the response of the central nervous
system . Different batches of NTg and Tg mice were tested at
6, 9 and 21 months old. All mice showed similar pain sensitivity
without any difference in both phases (Figure 4B).
LRRK2R1441GAged Mice had Good Learning Abilities
We then asked whether the LRRK2R1441GBAC Tg mice
displayed learning and memory deficits. Since the sensory function
in the paw was not impaired in the formalin test, we tested mice of
both genotypes in the passive avoidance task, where the mouse
should learn to avoid a mild electric shock given through the grid
floor by inhibiting its behavior to enter in the preferred dark
compartment. Twenty-one-months old NTg and Tg mice learned
similarly well the task as they increased their latency to step
through the dark compartment within trials (ANOVA test with
‘‘genotype’’ and ‘‘trials’’ as in-between subjects factors, main effect
of trials F(1,110)=3.929, p,0.001). No difference could be seen
between the genotypes (genotype as factor F(1,110)=1.218, n.s.,
Figure 5A). The mice were retested 24 h after the last trial to
check the retention memory. All the mice had a very poor
performance in remembering to stay in the light box in this trial.
This result might reflect impairment in long term memory abilities
linked to ageing, as it was not dependent on the genotype.
LRRK2R1441GMice Displayed Gastrointestinal Dysfunctions
from 6 Months Old
Constipation is one of the major problems encountered by
PD patients. We thus investigated whether the R1441G
mutation in the LRRK2 gene would have an influence in the
stool consistency and compared the production and water
content of droppings of NTg and Tg mice at different ages.
Figure 2. Activity with age in 3 min intervals in the 15 min Open Field test. Horizontal, vertical and fine activity as well as total activity in the
center of the arena, of mice at 6, 12, 16 and 20 months old. These results come from the experiments shown in Figure 1 but expressed in 3 min
intervals. Data are means 6 SEM. *p,0.05, **p,0.01, ***p,0.001, with Welch’s t test within two values of the graph, MANOVA test analysis with
‘‘genotype’’ and ‘‘intervals’’ as factors when considering the genotype effect.
Behavioral Phenotype of LRRK2 Mice
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Although no statistical difference between NTg and Tg animals
could be observed at any ages, the overall dropping analysis
indicated that Tg mice displayed gastrointestinal problems.
While the NTg mice had a constant stool consistency over their
life time (ANOVA test comparison with ‘‘genotype’’ and ‘‘age’’
as factors, main Age effect for Water content: F(7,78)=0.649,
n.s.; for Dry Stool Weight: F(7,78)=0.324, n.s.), Tg animals
displayed differences in the water content and weight of dry
stool collection from 6 months old, indicating gastrointestinal
dysfunctions that oscillate between constipation and diarrhea
(ANOVA test comparison with ‘‘genotype’’ and ‘‘age’’ as
factors, Age effect for Water content F(7,67)=2.928, p,0.05;
for Dry Stool Weight F(7,67)=3.776, p,0.01). At 6 months
old, Tg mice were clearly constipated. The amount of water in
their stool and the total dry stool weight was statistically lower
compared to the baseline described at 2 months old. In
comparison, the droppings of NTg animals contained a stable
amount of water and dry stool from 2 to 9 months old (baseline
Tg=73.5066.26, Figure 5B–C). As Tg mice were getting
older, the amount of water in the stool increased, coming back
to ‘‘normal’’ (at 9 and 12 months old) then higher compared to
the initial baseline, with a maximum at 14 months old (Welch’s
t test analysis not detailed here but shown in the graphs with
*p,0.05, **p,0.01, ***p,0.001, Figure 5B–C).
In a longitudinal study, we have demonstrated that the
overexpression of the R1441G mutated form of the human
LRRK2 gene induced mild motor deficits as well as gastrointestinal
dysfunctions in mice aged from 4 to 21 months. However, apart
from gastrointestinal dysfunctions, it did not trigger major changes
in a wide range of non-motor phenotypes, including smell and
pain sensitivity, mood disorders-like behaviors, and cognition
The motor abilities of the LRRK2R1441GTg mice were assessed
in a battery of tasks measuring horizontal and vertical ability, grip
strength, balance and coordination. Compared to age and gender-
matched control littermates, the horizontal and vertical activity of
Tg mice was decreased from 16 months old onwards in the open
field (Figure 2), and at 20 months old in the cylinder test
(Figure 1). However, the Tg mice had a good coordination in the
rotarod test, and had a grip strength similar to the NTg
littermates. The motor impairment was thus restricted to a
decrease in horizontal and vertical activity, appearing at an
advanced age comparable with the late onset of motor dysfunc-
tions observed in human PD patients.
Figure 3. Anxiety and depression-like behaviors in BAC LRRK2R1441GTg and NTg mice with age. A. Distance, percentage of time spent in
the opened and closed arms, and percentage of entries done in the opened and closed arms in a 5 min test on the elevated plus maze. B. Immobility
time recorded in a 6 min tail suspension test. C. Immobility time recorded in a 6 min forced swimming test. Data are means 6 SEM, number of
animals are indicated in the corresponding bar of the histogram. The brackets indicate data given only as indication; these data were not included in
the statistics due to the small size of the group considered.
Behavioral Phenotype of LRRK2 Mice
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Though very similar to the knock-out mouse model for the
LRRK2 gene published by Hinkle and coll., , these results are
not in accordance with those reported by Li and coll. . They
showed that the LRRK2R1441GTg mice decreased their rearing
activity in the cylinder test from the age of 6 months old. At 10 to
12 months old, the Tg mice were heavily impaired and showed a
complete akinesia. Although these phenotypes were very convinc-
ing (supporting documents with videos in ), we were unable to
reproduce their results. We believe that animal housing, breeding
or other experimental differences are not alone able to explain
such discrepancies. We verified that the mice belonged to the same
mouse line, as confirmed by the systematic mice genotyping
performed by PCR with specific primers amplifying the transgene,
and the verification of the transgene expression by Western Blot
analysis (Figure S1).
According to the literature, other LRRK2 mouse models have
also failed to show specific PD motor deficits. Indeed, mouse lines
overexpressing the wild type form or the G2019S mutation of the
LRRK2 gene displayed hyperactivity and increased motor function
[24,27], while other lines overexpressing the same G2019S
mutation or knock-in for the R1441C mutation did not exhibit
particular motor disturbances [24,25,31], apart from increased
thigmotaxis (preference for the area close to the walls) .
Similarly, knock-out mice for the LRRK2 gene displayed only
increased thigmotaxic behavior in the open field and increased
abilities in the rotarod . No strong impairment has been
reported to date, which is in favor with the idea that LRRK2 may
not be entirely responsible for the motor dysfunction observed in
PD patients. Moreover, these lines did not present dopaminergic
cell loss, but only impaired dopaminergic neurotransmission,
which might explain why the motor functions were only mildly
Although the R1441G mutation of the LRRK2 human gene may
have little effect on the development of motor features, it might
regulate other physiological systems, as shown by the numerous
non-motor symptoms associated to PD in humans [36,37]. Indeed,
Tg mice displayed a centrophobic behavior in the open field (or
increased thigmotaxis), shown by the decrease in time spent in the
center of the open field arena compared to control littermates
(Figure 2). This could be due to an augmentation of fear or
anxiety-like behavior. However, no difference was observed
between naive NTg and Tg animals at 6, 14 and 19 months in
the elevated plus maze, a test assessing anxiety-like behaviors. At
this latter age, Tg mice were even more exploratory than the NTg
(% time in open arms at 19 months NTg=58.4165.37 and
Tg=73.7467.53, n.s., Figure 3A), though not more active
Tg=10.3161.77 m). Although the centrophobic response ob-
served in the open field is typically associated to an increase in
anxiety-like behavior, it might also be a lack of motivation to
explore the new environment, which could be linked to a
depressive-like state. However, by testing the animals in despair
tests, we couldn’t find any tendency of the Tg mice to adopt the
typical depressive-like behavior (to become immobile in the tail
suspension or forced swimming test) compared to their controls. In
contrast, Tg mice aged 16 months and more were even more
active in the tail suspension test compared to NTg littermates
(immobility time of Tg was slightly decreased compared to the
immobility time of NTg mice, Figure 3B).
The motor dysfunctions of the LRRK2R1441GBAC Tg mice
might be attenuated by the high level of activity observed in this
mouse line in several tasks. This hyperactivity might be due to the
genetic background as FVB mice are known to have a spontaneous
high activity compared to other strains [38,39]. FVB/NJ mice are
also homozygous for the retinal degeneration allele Pde6brd1,
resulting in blindness by wean age (http://jaxmice.jax.org/strain/
001800.html). Due to this visual defect, FVB mice might develop
stronger senses such as smell or touch, and any sensorial
dysfunction similar to PD patients might be easily detectable. In
our experiments, Tg and NTg had similar abilities to recognize
familiar mouse odors in the block test or find hidden food in the
buried test, in accordance with the literature . Six-months old
Tg mice were even faster to grab and eat the food than their
littermate NTg controls in the probe trial of the buried test
(Figure 4Af). This could be due to a higher reactivity (faster in
performing the test), as there were no evidence that the olfactory
Figure 4. Sensory functions of LRRK2R1441GTg and NTg mice
with age. A. Olfaction tests at 6 and 14 months old. a-d. Wooden
block test average time sniffing the familiar block in trial 1 to 6 (a), block
preferences in trial 7 (b), and time sniffing the blocks within trials at 6
months (c) and 14 months (d). e–f. Latency to find and begin to eat the
hidden food within trials in 8 NTg and 6 Tg mice aged 6 months old,
and (e) average of latencies to find and eat the hidden food in the same
mice (f). ‘‘D1’’ to ‘‘D5’’ represent ‘‘Day 1’’ to ‘‘Day 5’’ and ‘‘Surf’’ or
‘‘Surface’’ means ‘‘Surface test’’ occurring on the 6thday of testing. B.
Time licking and biting the injured paw in the early and late phases of
the 30 min formalin test in mice at 6, 9 and 21 months old. Data are
means 6 SEM, number of animals are indicated in the corresponding
bar of the histogram.
Behavioral Phenotype of LRRK2 Mice
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function was better. Interestingly, at the same age, all the mice
preferred to explore a wooden block scented with familiar home-
cage odor rather than a block impregnated with the odors of
unknown congeners. The preference was reversed at 14 months
old, where mice were more interested in exploring the block
scented with different animal beddings, which is the result
expected from a normal adult mouse . In summary, the
R1441G mutation had no particular effect on the olfactory
response of the mice. In fact, this correlates with clinical studies
since patients carrying the R1441G mutation do not display main
olfactory deficits, in contrast to the majority of PD patients
Similarly, Tg mice did not show any difference in pain
sensitivity. The formalin test assesses the way an animal responds
to moderate continuous pain generated by injured tissue. It is
characterized by two different neurological responses towards the
pain stimuli: (i) a first phase, starting immediately after injection of
formalin, and responding to the local stimulation of nociceptors,
and (ii) a second phase occurring later in time and reflecting the
response of the central nervous system towards the stimulus,
especially the spinal cord. PD patients display chronic unexplained
pain and a decrease in pain tolerance [37,44]. We therefore
expected the Tg mice to show an increase of the pain sensitivity
following the injection of the formalin in the paw, and in particular
in the second phase, which is considered as a chronic response
. However, Tg animals tested at 6, 9 and 21 months of age
were not impaired in both phases, suggesting that R1441G
mutation do not act on pain dysfunctions. Like many phenotypes,
the responses observed in the formalin test are regulated by the
genetic background [45,46,47]. Since FVB/NJ mice have a
greater sensitivity to injuries in general , any susceptibility to
pain displayed by the Tg mice should thus be seeable. A short
study measuring the tactile reaction of the mice when a piece of
adhesive tape was put between their ears did not detect any
difference between NTg and Tg mice in the latency to remove the
tape (data not shown). This corroborated the results observed in
the olfaction and pain test and confirmed the absence of any
sensory deficits in these mice, even at older ages.
Cognitive impairments in PD range from mild forms of
cognitive dysfunction to overt dementia. Although not all patients
develop these symptoms, they may arise at late stages of the PD
course and are usually associated with more rapid progression of
PD disability [37,48]. From our study, LRRK2 might not play a
role in cognitive dysfunctions, in particular working memory.
LRRK2R1441Gmice showed indeed similar learning abilities than
the NTg littermates in the passive avoidance test at 21 months old.
Both groups of mice have learnt well to avoid the electric chock
within 10 trials at an old age, strongly suggesting that these mice
will not develop any working memory impairment due to the
transgene. Their impairment in visual acuity did not disturb their
performance in this test since they could learn well. It has been
indeed shown that they have enough visual sensitivity to
distinguish a dark area from a bright lighted one . Yet, the
good performances of the mice in the passive avoidance test do not
allow us to conclude about the possible role of the R1441G
mutation on cognitive abilities. Further tests should be conducted
Figure 5. Cognition abilities in the passive avoidance test and gastrointestinal dysfunction in BAC LRRK2R1441GTg and NTg mice. A.
Latency to step through the white compartment with trials in the passive avoidance test. 5 NTg and 6 Tg aged 21 months old were tested up to 10
consecutive times the first day and 24 h after the last trial. B–C. Water content in the stool collected in one hour and dry stool weight after
evaporation of the water content of Tg and NTg mice aged from 2 to 21 months old. Data are means 6 SEM, number of animals are indicated in the
corresponding bar of the histogram, *p,0.05, **p,0.01, ***p,0.001 with Welch’s t test for unequal variances.
Behavioral Phenotype of LRRK2 Mice
PLOS ONE | www.plosone.org7 July 2013 | Volume 8 | Issue 7 | e70249
to see whether the LRRK2R1441Gmice are impaired in other kind of
tasks that are more related to Parkinsonian phenotypes, i.e.
procedural- and associative-learning abilities characteristic for
striatal impairments. Furthermore, as mentioned before, a large
number of tests could not be conducted in these mice due to their
genetic background. By breeding the line with another strain to
remove the homozygosity of the allele Pde6brd1, other tests like the
two-object recognition tasks, mazes involving spatial recognition,
or the five-choice serial reaction time task for attention deficits
could be assessed, for a better cognitive characterization.
One of the clearer phenotypes displayed by the LRRK2R1441G
Tg mice was gastrointestinal dysfunctions. The consistency of the
droppings observed in the one-hour stool collection test was
deregulated over ages, beginning with a constipation state at 6
months old. This follows the symptoms described in clinical
studies, as one of the most common problems observed in PD
patients (including patients carrying the R1441G mutation) are
gastrointestinal and urinary symptoms [36,50]. Constipation
appears at an early age and other dysfunctions of the digestive
system occur frequently at all stages of the disease, such as bowel
movements, or even colon or rectal cancers .
In conclusion, we have shown that overexpression of the
R1441G mutation of the human LRRK2 gene in mouse did not
cause significant behavioral changes (fine behaviors, olfaction,
pain sensitivity, mood disorders or cognitive impairments) apart
from gastrointestinal dysfunction from 6 months old followed by
mild motor deficits at older ages. The observations on olfaction
and gastrointestinal function in this model validate findings in
human carriers. Compensatory mechanisms modulating the
progression of PD at late stages in these animal models should
be further evaluated.
Materials and Methods
BAC LRRK2R1441Gmutant Tg and NTg mice were provided by
The Jackson Laboratory
Tg(LRRK2*R1441G)135Cjli/J) and further bred under the same
FVB/NJ background. The transgene segregated heterozygously
and the genotype of the animals was determined by PCR on tail
samples as advised by The Jackson Laboratory. Food and water
were given ad libitum. Mice were marked randomly at the ears and
were tested in all experiments in the same order according to their
identification number (number/cage), following standard blocked
randomization of testing and data processing . The experi-
ments and analysis were always performed blind of the treatment
This study was carried out at the Animal Research Laboratory/
Surgical Science and Research Laboratory located in Tan Tock
Seng Hospital and co-managed by Tan Tock Seng Hospital and
the National Neuroscience Institute, in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
protocol was approved by the Tan Tock Seng Hospital - National
Neuroscience Institute (TTSH-NNI) Institutional Animal Care
and Use Committee (IACUC) of Singapore (Ref TNI-11/2/003),
and all efforts were made to minimize suffering.
Tg and NTg male mice were tested at 4, 6, 9, 12, 14, 16, 18, 19,
20 and 21 months old. At each age, different tests were done so
that mice were naı ¨ve as much as possible, except for the open field
and the one-hour stool collection tests that were conducted at all
ages. Only one test per day was performed, in 3 h time from
9:00 AM to 3:00 PM. No more than 5 tests were conducted on the
same group of age, from the less to the more stressful one,
conducting one test every other day. The formalin test, forced
swimming test, the buried test and the passive avoidance test were
considered the most stressful tests. Only one of these tests was
performed in one mouse at a particular age, and always as the last
test of the series, letting 1 to 3 months rest before the next set of
experiments. On the testing day, animals were put in the test room
at least 20 min before testing in order to acclimatize, and were
systematically weighted before the task. The experimenter was
always blind of the genotype when performing the tests. The
details of the behavioral tests are described below, respecting as
possible the order of the assessment schedule.
Elevated plus maze.
The grey colored high-tech metal alloy
maze consisted of a 60 cm elevated plus-shaped apparatus with
four arms (35 cm length65 cm width), two of them being
surrounded with walls of 15 cm height (Ugo Basile, Italy). Mice
were gently placed on the central platform facing a closed arm and
were allowed to freely explore the maze for 5 min. The apparatus
was cleaned immediately after each session with cotton pads
wetted with 60% ethanol. The test was automatically analyzed
with the ANY-mazeTMvideotracking system (Stoelting, USA).
Mice were allowed to explore freely a cleaned
photobeam activity transparent chamber for 15 min (43 x 43 x
43 cm, PAS Open Field, San Diego Instrument, USA). Each
mouse was gently placed in the middle of the arena at the start of
the test session. Horizontal and vertical activity was recorded
automatically and data were analyzed in intervals of 60 to 180 sec
over the 15 min session. A central area (X:4, Y:4 in the PAS Open
Field system) was designed in order to detect thigmotaxis
(preference to move close to the walls) and anxiety-like behavior
related to the time spent in the center of the arena. The number of
droppings was recorded at the end of the test. Immediately after
each session, the apparatus was cleaned with cotton pads wetted
with 60% ethanol.
Mice were put into a transparent Plexiglas
cylinder (12 cm diameter, 20 cm high) surrounded by two mirrors
allowing the observer to see the animal in all directions. The
behaviors were video recorded and the number of rearing in the 5
first min was then analyzed manually on the video with a counter.
Immediately after each session, the apparatus was cleaned with
cotton pads wetted with 60% ethanol.
One hour stool collection.
between 11:00 AM and 14:00 PM as described by . Each
mouse was placed in a separate clean cage and observed
throughout a 60 min collection period. Fecal pellets were collected
immediately after expulsion and placed in sealed tubes to avoid
evaporation. Tubes were weighted to obtain the wet weight of the
stool, then dried overnight at 65uC and weighted again to obtain
the dry weight. Stool frequency, dry stool weight and total water in
the stool were calculated.
Mice were put on a cage lid placed 10 cm
below a table covered by towels. The lid was softly shacked to
allow the mouse to grip more intensively the mesh, and slowly
inverted. The latency to fall was recorded with a stopwatch
(Marble, accuracy of 0.01 sec). Mice underwent 4 trials of a
maximum of 2 min with 10 to 15 min intertrial. Results were
means of all trials.
The protocol used was modified from . Mice
were put on a rod of an accelerating rotarod for a maximum of
5 min (3 to 30 rpm, 5 min ramp, Ugo Basile, Italy). The latency to
fall from the rotating rod was taken on 2 days, with 4 trials per day
and an intertrial period of 15–20 min. The 3 last trials of the
second day served as a mean value for locomotor abilities.
Tests were conducted at all ages
Behavioral Phenotype of LRRK2 Mice
PLOS ONE | www.plosone.org8 July 2013 | Volume 8 | Issue 7 | e70249
Immediately after each session, the apparatus was cleaned with
Tail suspension test.
Mice were individually suspended by
the tail to a horizontal wooden bar 40 cm from the bench top
using an adhesive tape placed approximately 1 cm from the tip of
the tail. Typically, mice demonstrated several escape-oriented
behaviors interspersed with temporally increasing bouts of
immobility. The behaviors were videotaped throughout a 6 min
test and the immobility time defined as lack of all movements
except for whisker movement and respiration was analyzed with a
Forced swimming test.
Mice were placed individually in a
cylinder (height 25.5 cm, diameter 12 cm), containing 14 cm
water maintained at 23–25uC. As for the tail suspension test, mice
showed typically escape-oriented behaviors by swimming along
the cylinder walls, interspersed with immobility, floating in an
upright position and making only small movements to keep its
head above the water. Behaviors were videotaped and the 6-min
test videos were analyzed by a well-trained experimenter with a
Tests were performed as described in .
Individually housed animals were exposed to five wooden cubes
(2.5 cm3) placed inside the cage for one week, during which the
bedding was not changed. On the 8thday, the blocks were
removed and placed in a plastic bag with the cage and animal
number. The first 6 trials consisted of exposing the animal in a
clean cage with four of his own blocks, (changing the blocks in
each trial, i.e. ABCD, then BCDE, then ABCE, etc). The 7thtrial,
test trial, consisted of exposing the animal with 3 blocks from his
own cage plus one from the cage of another animal. Each
exposure lasted 30 sec with 5 min intertrial. The overall exper-
iment was videotaped, and the time spent sniffing the blocks was
recorded. Preference in the last trial was expressed with the
following formula: ((time sniffing only the block from unknown
cage)-(time sniffing only the blocks from his cage))/(total sniffing
Tests were performed as described in .
Animals were food restricted up to 90% of their body weight. The
tests begun only when mice had reached a stable weight (after 3 to
4 days). The buried test was performed once a day during 5 days.
The mouse was put in a new cage where a piece of honey cereal
bar (about 250 mg of Oats’n Honey from Nature ValleyH
Crunchy Granola bar) was hidden at 0.5 cm of the top of a
3 cm high layer of clean bedding. The latency to dig up and begin
to eat was taken. Mice were given a maximum of 5 min to find the
food. On the 6thday, the piece of food was put on the surface of
the clean bedding and the latency to begin to eat was taken.
Mice were shortly placed in a restraint
cylinder (type 50 ml Falcon tube which extremity was cut) and
injected in the right hind paw plantar surface (i.pl.) with 20 microl
of 2.5% formalin (in NaCl 0.9%, equiv. 1.84% of formaldehyde,
injection done with a 50 microl Hamilton syringe). They were then
immediately placed into a glass cylinder (20 cm diameter),
surrounded by mirrors to avoid any obstructed view of the
animal. The amount of time spent licking and biting the injected
paw and leg was considered as indicator of pain and was recorded
in 5 min intervals over 30 min. Each animal was tested only once.
Passive avoidance test.
The test was performed according
to . The passive avoidance apparatus (GEMINI Avoidance
System, San Diego Instrument, USA) consisted of a test station
divided into two compartment enclosures and grid floor (22.8 x
20.3 x 20.3 cm each), one brightly illuminated with a ‘‘cue’’ light
and the other dark. In the acquisition session, the mouse was
placed in the start compartment and after a 2-sec orientation
phase the door was opened allowing the mouse to freely explore all
the apparatus. Once the mouse entered the dark compartment
(detected by photobeams) the door was closed and the mouse was
punished by a mild foot-shock (3 sec, 0.3 mA). At the end of each
trial (lasting max 120 sec), the subject returned to the home cage
for a 60 sec inter-trial interval. Mice not entering the escape
compartment within 120 sec were arbitrarily assigned a latency
time of 120 sec. The session terminated either after two
consecutive trials without a stepping-through response within
120 sec, or after 10 trials without completion of this passive
avoidance acquisition criterion. When the experimental subject
reached the criterion before the 10thtrial, a 120 sec latency score
was assigned to each trial omitted. The Retention session took
place 24 h later, and consisted of one trial not punished by foot
shock. The Retention trial ended when the animal either gave the
step-through response or remained in the start compartment for
The behaviors of the mice were analyzed according to the
testing possibilities: the activity was automatically recorded either
with the photobeam activity system in the open field (PAS-Open
field, San Diego Instrument, USA), or displayed automatically on
the rotarod apparatus (Ugo Basile, Italy) and passive avoidance
Gemini system (San Diego Instrument, USA), or by means of the
ANY-mazeH videotracking system (Stoelting, USA) in the elevated
plus maze apparatus. Data in the cylinder, tail suspension test,
forced swimming test, olfactory tests, and pain test were collected
manually with a counter or a stopwatch (0.01 sec accuracy).
Statistics were done using SPSS Software, v18.0. Two kinds of
analysis were usually done: (i) Analysis of variances by ANOVA
test for univariate analysis or MANOVA test for multivariate
analysis followed by post-hoc comparisons (Bonferroni), in order to
compare the scores/variables with multiple factors, i.e. genotype,
age, intervals, and/or trials, and (ii) if adequate, a second analysis
comparing the scores between genotypes at a specific age or for the
same group at different ages with the unpaired Student’s T-Test or
Welch’s t test for unequal variance. Equality of variances was
assessed by Levene’s Test. Statistical significance was considered
only when p,0.05.
Protein extraction and western blotting.
were washed with PBS and lysed in RIPA buffer containing
phosphatase and protease inhibitors. Protein concentration was
determined by Bio-Rad protein assay. Equal amounts of protein
(20 microg) were separated by electrophoresis on SDS-PAGE gel,
and transferred onto nitrocellulose membrane (Millipore). The
membranes were blocked with 5% milk in TBST for 1 h at room
temperature, followed by probing with specific primary antibodies
in 5% milk in TBST for overnight at 4uC (anti-LRRK2, 1:1000,
69879 from Santa Cruz Biotechnology). Secondary antibody was
diluted in 5% milk in TBST, and the incubation was done for 2 h
at room temperature. Protein bands were visualized using ECL
detection kit (GE Healthcare).
brain areas. Representative Western Blots of proteins extracted
from the brain of Tg and NTg LRRK2*R1441G BAC mice at 12
months old. Actin is represented as loading control.
Expression of the LRRK2 protein in different
Behavioral Phenotype of LRRK2 Mice
PLOS ONE | www.plosone.org9 July 2013 | Volume 8 | Issue 7 | e70249
Irene Soon, He Bing Guan Yuan, and Nurul Dini Binte Abdul Rahim are
warmly acknowledged for breeding and genotyping the mice.
Conceived and designed the experiments: ZB EKT. Performed the
experiments: ZB HCL. Analyzed the data: ZB. Contributed reagents/
materials/analysis tools: ZB LZ EKT. Wrote the paper: ZB EKT. Revised
the manuscript: HCL LZ.
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