Behavioral phenotype of maLPA1-null mice: increased anxiety-like behavior and
spatial memory deficits
L.J. Santin *,†, A. Bilbao ‡,a, C. Pedraza ‡, E. Matas-Rico ‡, D. López-Barroso †, E. Castilla-
Ortega †, J. Sánchez-López †, R. Riquelme §, I. Varela-Nieto §, P. de la Villa ††, M. Suardíaz
‡‡, J. Chun **, F. Rodriguez De Fonseca ‡ and G. Estivill-Torrús ‡.
† Departamento de Psicobiología y Metodología de las CC, Facultad de Psicología,
Universidad de Málaga. Campus de Teatinos, E-29071, Málaga, Spain.
‡ Laboratorio de Investigación, Grupo de Investigación en Neurofarmacología de
Transmisores Lipídicos, y Laboratorio de Medicina Regenerativa, Fundación IMABIS,
Hospital Carlos Haya, Avenida Carlos Haya 82, E-29010, Málaga, Spain.
§ Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de
Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid (UAM), Arturo
Duperier 4, E-28029, Madrid, Spain.
†† Departamento de Fisiología, Facultad de Medicina, Universidad de Alcalá, E-28871,
Alcalá de Henares, Madrid, Spain.
‡‡ Laboratorio de Función Sensitivomotora. Hospital Nacional de Parapléjicos, Finca de la
Peraleda s/n, E-45071, Toledo. Spain.
** Department of Molecular Biology, Helen L. Dorris Child and Adolescent Neuropsychiatric
Disorder Institute, The Scripps Research Institute, 10550 North Torrey Pines Road, ICND
118, La Jolla, California 92037, USA.
Behavior, but has yet to undergo copy-editing and proof correction. Please cite this article as an "Accepted Article"; doi:
This is an Accepted Article that has been peer-reviewed and approved for publication in the Genes, Brain and
a Present address: Department of Psychopharmacology, Central Institute of Mental Health,
Mannheim, 68159, Germany.
Correspondence to * L.J. Santin, Departamento de Psicobiología y Metodología de las CC,
Facultad de Psicología, Universidad de Málaga. Campus de Teatinos, 29071, Málaga,
Spain. E-mail: firstname.lastname@example.org
Received date: 11-Nov-2008
Revised date: 10-Jul-2009
Accepted date: 10-Jul-2009
Keywords: Lysophosphatidic acid, maLPA1-null mice, neurologic screening, water maze,
open field, elevated plus maze.
Running title: LPA1 receptor and behavior
Lysophosphatidic acid (LPA) has emerged as a new regulatory molecule in the
brain. Recently, some studies have demonstrated a role for this molecule and its LPA1
receptor in the regulation of plasticity and neurogenesis in the adult brain. However, no
systematic studies have been conducted to investigate whether the LPA1 receptor is
involved in behavior. Here we studied the phenotype of maLPA1–null mice, which bear a
targeted deletion at the lpa1 locus, in a battery of tests examining neurologic performance,
habituation in exploratory behavior in response to low and mild anxiety environments and
spatial memory. MaLPA1-null mutants showed deficits in both olfaction and somesthesis,
but not in retinal or auditory functions. Sensorimotor coordination was impaired only in the
equilibrium and grasping reflexes. The mice also showed impairments in neuromuscular
strength and analgesic response. No additional differences were observed in the rest of the
tests used to study sensoriomotor orientation, limb reflexes, and coordinated limb use. At
behavioral level, maLPA1-null mice showed an impaired exploration in the open field and
increased anxiety-like response when exposed to the elevated plus maze. Furthermore,
the mice exhibit impaired spatial memory retention and reduced use of spatial strategies in
the Morris water maze. We propose that the LPA1 receptor may play a major role in both
spatial memory and response to anxiety-like conditions.
Lysophosphatidic acid (LPA, 1-acyl-2-sn-glycerol-3-phosphate) is a phospholipid
that acts as an intercellular messenger and possesses growth factor-like activities. LPA
affects a variety of cell functions, including cell proliferation, differentiation, survival, and
migration (Moolenaar 2004; Ye et al. 2002; Anlinker & Chun 2004; Birgbauer & Chun 2006;
Chun 2005, 2007). The effects of LPA are mediated by a family of specific G protein-
coupled receptors (GPCRs) (Bandoh et al. 2000; Fukushima et al. 2001; Anliker & Chun
2004; Ishii et al. 2004). Among these receptors, LPA1 is a receptor coupled to Gi, Gq, and
G12/13 family heterotrimeric G proteins; it has high affinity for LPA, and its downstream
effectors are well characterized (Anlinker & Chun 2004).
To date, few studies have addressed a possible role of the LPA1 receptor in
behavior. Harrison et al. (2003) and Roberts et al. (2005) reported prepulse inhibition
impairment in LPA1-null mice. These studies suggest that LPA, acting through the LPA1
receptor, may mediate sensorimotor gating. LPA1-null mice display a reduced ability to filter
out irrelevant auditory stimulation, which may lead to the development of cognitive deficits.
Despite these findings, no studies testing the involvement of LPA1 in cognitive functions
such as learning and memory have been reported, although Dash et al. (2004) did
demonstrate enhancement of spatial memory in rats after post-training LPA microinjection
in the hippocampus. Hippocampal LPA receptor subtypes therefore seem likely to play a
role in adult cognitive function. However, the role of specific LPA receptors in adult animals
remains to be established.
In the present study, we assessed the role of the LPA1 receptor in sensorimotor,
emotional, and cognitive functions in adult mice. The study was performed in the maLPA1-
null mouse (Estivill-Torrús et al. 2008), a stable variant of the previously characterized
LPA1-null mutant (Contos et al. 2000). The maLPA1-null variant was obtained during the
propagation of LPA1-null mice. These mice carry a targeted disruption in the lpa1 gene.
They show normal survival but display defective hippocampal neurogenesis, decreased
levels of brain-derived neurotrophic factor (Matas-Rico et al., 2008), and altered cortical
development (Estivill-Torrús et al. 2008). The brain alterations seen in maLPA1-null mice
are accompanied in adult animals by behavioral defects that affect their performance in
neurological, emotional, and memory tasks. Neurological impairments were observed in
sensory functions (olfaction and somesthesis), limb reflexes, and coordinated limb use
(grasping reflex and equilibrium), as well as in neuromuscular strength. Though maLPA1-
null mice showed no impairment in either retinal or auditory functions, they exhibited
impaired exploration in the open field and increased anxiety-like responses in the elevated
plus maze test. Finally, maLPA1-null mice displayed impairments in spatial memory
retention and abnormal use of searching strategies. These findings strongly suggest that
the LPA1 receptor is involved in both spatial memory and emotional behavior.
Materials and Methods
The generation and characterization of maLPA1-null mice have been described
(Estivill-Torrús et al. 2008; Matas-Rico et al. 2008). The original null mice were obtained by
targeted gene disruption using homologous recombination and Cre-mediated deletion on a
129X1/SvJ background. These animals were then backcrossed with C57BL/6J mice.
Intercrosses of these mice, as well as with mice generated from one additional backcross
(Contos et al. 2000), were begun immediately. An LPA1-null mouse colony, termed
maLPA1-null from the Málaga variant of LPA1-null, was spontaneously derived during the
original colony expansion by crossing heterozygous foundation parents (maintained on the
original hybrid C57BL/6J × 129X1/SvJ background). Intercrosses were performed with
these mice and subsequently backcrossed for 15 generations with mice generated within
this mixed background. MaLPA1-null mice carrying the lpa1 deletion were born at the
expected Mendelian ratio, and they survived to adulthood. Targeted disruption of the lpa1
gene was confirmed by genotyping (according Contos et al. 2000); immunochemistry
confirmed the absence of LPA1 protein expression.
All experiments were conducted on age-matched male littermates from the following
genotypes: wild-type [malpa1(+/+)], maLPA1-null heterozygous [malpa1(+/–)] and homozygous
[malpa1(–/–)] mice. All mice were approximately three months old at the start of behavioral
testing. Mice were housed in groups of four on a 12-h light/dark cycle (lights on at 7:00
am). Water and food were provided ad libitum. Experiments were conducted between
10:00 am and 2:00 pm. The different types of experiments were carried out on different
groups of mice, such that no mouse participated in more than one phenotypic test. During
behavioral testing, the experimenters were blind to the genotypes of the mice. All
procedures were carried out in accordance with the European animal research laws
(European Communities Council Directive 86/609/EEC and 2003/65/CE, and Commission
Recommendation 2007/526/EC), as well as the Spanish National Guidelines for Animal
Experimentation and the Use of Genetically Modified Organisms (Real Decreto 1205/2005
and 178/2004, and Ley 32/2007 and 9/2003).
Neurologic screening and auditory and retinal function
Neurologic assessment was performed in a testing room where the animals were
previously habituated to the experimental conditions. All mice were taken from their home
cages to the testing room and were kept there for one hour before the neurological tests
were carried out. To test sensorimotor orientation and coordinated limb and neurological
function, the mice were subjected to a battery of tests taken from Marshall and Titelbaum
(1974), modified by Bjorklund et al. (1980), and extended to additional reflexes by Bures et
al. (1983). The following sensory reflexes were assessed: (a) somesthesis, in which a pin
prick was applied to six sites on the lateral surface of the animal body, combining dorsal
and ventral placements at rostral, middle, and caudal levels; (b) whisker touch, in which a
toothpick was brought close to the animal from the lower rear so as to avoid the visual field,
and then lightly brushed against the vibrissae; (c) snout probe, in which a toothpick was
gently rubbed against the snout of the mouse; (d) olfaction, where a small cotton swab
dipped in ammonia solution was slowly brought close to the mouse’s nose in a lateral-
medial direction; (e) corneal reflex, in which the animal was restrained with a hand while
the cornea was superficially stimulated with a fine, hair-tipped probe; (f) auditory startle, in
which an unexpected, loud acoustic stimulus was applied; and (g) head shaking, where the
mouse was placed on a small, elevated platform and tested for reaction to a puff of air
gently released through a narrow rubber tubing (internal diameter, 1 mm) to its pinna.
Limb reflexes and limb coordination were assessed using the following tests: (a)
surface righting reflexes, in which the animal was placed on its back onto a flat surface,
and the time for the animal to right itself was measured (b) forelimb suspension, where the
mouse was grasped by one forepaw and suspended, and the latency time for the animal to
grasp the hand with the free paw and use this to pull itself up onto the hand was recorded
(failure criterion, 10 s); (c) grasping test, in which the mouse was hung by its tail and the
forelimb palms were lightly touched with a stiff wire (diameter, 1 mm); (d) equilibrium tests,
in which the mouse was placed facing downwards on a wire mesh platform tilted 30º, after
which it was turned to face up the slope and then was finally placed on a horizontal
wooden bar (diameter, 2 cm; length, 30 cm) suspended 50 cm above the floor, and its
ability to stay on the bar was assessed; (e) placing reactions, where the mouse was
restrained at the edge of the table and one foreleg or hindleg was displaced so that it hung
over the edge.
The deficit in each orientation, limb use, and neurological test was rated on a three-
point scale: 0, absent; 1, weak; or 2, strong. Use of this battery of tests allowed us to
determine whether the maLPA1-null mutation affected a particular brain region, interfered
with a specific function, or affected the CNS as a whole (Bures et al. 1983). Ten malpa1(+/+)
mice, eight malpa1(+/–) mice, and eight malpa1(–/–) mice were used to assess neurologic
functions. Data were analyzed by a non-parametric Kruskal-Wallis test to assess the
variance of the neurological test between different groups. Subsequently, appropriate
paired comparisons were carried out using a Mann-Whitney U-test. A value of P < 0.05
was considered to be statistically significant. Additionally, the deficits in neurological test
were presented as a percent of incidences for each treatment.
The neurologic screening was completed using the hang wire and the tail flick tests
to test, respectively, neuromuscular strength and analgesic response. In the hang wire test,
the mouse was placed on a wire cage lid and the lid was gently moved back and forth,
enabling the mouse to grip the wire. The lid was then turned upside down at a height of 15
cm above the surface of the bedding material; mice can easily fall from this height and land
on their feet without injury. Latency to fall onto the bedding was recorded, with a cut-off
time of 60 seconds. Eight malpa1(+/+) mice, nine malpa1(+/–) mice, and 10 malpa1(–/–) mice
were used to assess neuromuscular strength. The tail flick test was performed using a
water tail flick test. The mouse was restrained for tip tail immersion into a 52 ± 0.5 ºC water
bath. The amount of time until the rodent flicked or moved its tail was recorded as the
latency time. Three trials (T1-T3), spaced 20 minutes apart, were conducted with each
animal. To avoid tissue damage, animals were never exposed to pain stimuli for more than
eight seconds. Thirteen malpa1(+/+) mice, eight malpa1(+/–) mice, and 14 malpa1(–/–) mice
were used to study the analgesic response. In both tests, data were analyzed by one-way
ANOVA followed by post-hoc comparisons (Fisher’s test).
To determine whether auditory or retinal function was altered in the absence of lpa1
expression, auditory brainstem responses (ABRs) and electroretinograms (ERGs) were
obtained from malpa1(+/+) and malpa1(–/–) mice. ABRs were measured in response to clicks
presented at a rate of 30 bursts/s. The mice were anesthetized with ketamine (100 mg ⁄ kg)
and xylazine (4 mg ⁄ kg) by intraperitoneal injection, and the ABR tests were performed in a
small sound-attenuating chamber. Analysis was performed on malpa1(+/+) and malpa1(–/–)
mice using 11 mice per genotype. ABRs were recorded with subcutaneous platinum needle
electrodes placed at the vertex (non-inverting input), right-side mastoid prominence
(inverted input), and tail. Electroencephalographic (EEG) activity was amplified and then
fed into an analog-to-digital converter [AD1, Tucker–Davis (TDT)]. Each averaged
response was based on 300–500 repetitions of the stimulus recorded over 10-ms epochs.
ABR waveforms were recorded in 5- to 10-dB steps decreasing incrementally from the
maximum amplitude of 90 dB SPL. The ABR threshold was defined as the stimulus level
that evoked a peak-to-peak voltage two SDs above mean background activity (Cediel et al.
2006; Ngan & May, 2001; http://www.eumorphia.org/EMPReSS/). ABR data were
expressed as mean ± S.E.M. and were statistically analyzed by t-test.
Electroretinographic recordings were made from four malpa1(+ /+ ) and four malpa1(–/–)
mice. Before recording, animals were adapted to the dark overnight; then they were
anesthetized and their pupils dilated with a topical drop of 1% tropicamide (Colircusí
Tropicamida; Alcon Cusí, SA, El Masnou, Barcelona, Spain). To optimize electrical
recording, 2% methocel (Ciba Vision AG, Hetlingen, Switzerland) was added to each eye
immediately before placing the corneal electrode. The non-registered eye was covered with
an opaque contact lens. Animals were placed in a Faraday cage, and experiments were
conducted in absolute darkness. Bipolar recording was performed between an Ag:AgCl
electrode fixed on a corneal lens and a reference electrode located on the head skin;
ground electrodes were located on the tail and nose. Scotopic flash ERGs were recorded
from each eye in response to light stimuli that consisted of light-emitting diodes (LED-white
light) centered on the visual axis and located 5 mm away from the cornea. Light stimuli
were presented for 5 ms at five increasing intensities ranging from 10-3 to 101 cd-s/m2. The
interval between light flashes was 10 seconds, and four to eight consecutive recordings
were averaged for each light presentation. The ERG signals were amplified, band-pass
filtered between 0.3 and 1000 Hz, and digitized at 10 kHz with a data acquisition board
(Power Laboratory 4ST; AD Instruments Pty. Ltd., Oxfordshire, UK). Recordings were
analyzed off-line by an investigator blinded to the experimental treatment of the animal
(Mayor-Torroglosa et al. 2005).
Activity and habituation in the open field and elevated plus maze
To identify differences in exploratory/motor activity, reactivity to novel or anxiety-
inducing environments, and habituation, we used the open field (OF) and the elevated plus
maze (EPM). In order to adapt the animals to the experimental conditions, each mouse
was manipulated by hand for 5 minutes/day for a week before testing. All mice were taken
from their home cages into the testing room and kept in the room for one hour before
The OF apparatus used in this experiment was a square, brightly illuminated (500
lux) wooden arena with dimensions of 50 × 50 × 38 cm. Each animal was placed in the
center of the apparatus, and its behavior was monitored for a total of 5 min using a real-
time video-tracking system (SMART 2.5, Panlab, Barcelona, Spain). Following the
recording of (novelty) behavior, each individual’s behavior was again recorded 24 h later
(familiarity) to evaluate the effects of reactivity to novelty and habituation mechanisms. For
data analysis, the OF was divided into two concentric rectangles: an outer zone, 8.3 cm in
from the walls, and an inner zone, 8.3 cm in from the outer zone. The distance moved and
the percentage of time spent in the center of the OF were taken as indices of exploratory
activity and anxiety-like behavior, respectively. Behavior in the open field was recorded for
nine malpa1(+/+) mice, 12 malpa1(+/–) mice, and 12 malpa1(–/–) mice. Significant differences in
the percentage of time and distance moved were determined by two-way analysis of
variance (ANOVA) with one repeated measure (novelty vs. familiarity). Simple main effects
were performed after significant interaction and Fisher's post-hoc comparisons were used
when appropriate. In order to control for possible differences in baseline activity in the three
genotypes (Bothe et al, 2004), we calculated the habituation activity change score [day 2
activity/day 1 + day2 activity)]. Comparisons among groups were performed using one-way
ANOVA followed by Fisher's post-hoc tests.
Unconditioned anxiety-like behaviors were assessed using an EPM consisting of two
open arms (30 x 5 cm), two enclosed arms (30 x 5 cm, with end and side walls 15 cm
high), and a connecting central platform (5 x 5 cm). The maze was raised to a height 38.5
cm above the floor and illuminated (100 lux) from the top. Each mouse was placed in the
intersection of the four arms of the maze and allowed to explore freely for 5 min (novelty).
After 24 h, the mouse was again placed into the maze for 5 min (familiarity). During this
test, mice were monitored using a real-time video-tracking system (SMART 2.5, Panlab).
An arm entry was defined as a mouse entering an arm of the maze with all four legs.
General activity/exploration was evaluated using the total number of entries into the arms.
Anxiety was assessed by comparing activity in the open vs. closed arms using the
following index: time spent in open arms/(time spent in open arms + time spent in closed
arms) (Malleret et al., 1999). Low values indicate high anxiety-like behavior levels, and high
values indicate low anxiety-like behavior levels. In this experiment, 8 malpa1(+/+) mice, 10
malpa1(+/–) mice, and 10 malpa1(–/–) mice were used. Data were analyzed by two-way
ANOVA with one repeated measure (novelty vs. familiarity), followed by post-hoc
comparisons using Fisher’s test.
Spatial memory in the water maze
To study spatial memory, we conducted place navigation in the Morris water maze
using 9 malpa1(+ / +) mice, 10 malpa1(+/ -) mice, and 10 malpa1(– / –) mice. Animals were
adapted to the experimental conditions for one week before behavioral testing. All mice
were taken from their home cages into the testing room and kept in the room for one hour
before the behavioral test. Mice were trained in a circular pool (diameter, 150 cm) filled with
water (24-26 °C) and made opaque with non-toxic white paint. The goal platform (diameter,
11 cm) could be placed anywhere in the pool at a distance of 30 cm from the pool edge.
The platform was submerged 1 cm beneath the surface of the water. The pool was placed
in an experimental room furnished with several place-fixed extra-maze cues. The pool
remained immobile in the room throughout the experimental period. A real-time video-
tracking system (SMART 2.5, Panlab) was used to record the animal’s movements in the
The experimental procedure was conducted over four days of spatial training,
followed by one day of reversal training. One day before training, all mice were habituated
to the experimental conditions, swimming in the pool without the escape platform for one
minute. This trial was used for checking whether the mice showed any preference or lack
of preference for any of the four quadrants that would be used later in the spatial learning
task (suppl. Fig. 1). In addition, the habituation trial was analyzed in order to study the
exploratory behavior of the mice. The pool was divided into three concentric circles (outer,
middle, and inner zones); the time spent and distance traveled by the mice in each zone,
as well as the distance traveled and the mean velocity in the pool, were obtained. Spatial
learning training was conducted on four consecutive days (days 1-4) with three trials per
day; the intertrial interval (ITI) was 15 minutes. For data analysis, the pool was divided into
four quadrants (A-D). The mice were able to escape from the water using a submerged
platform that was placed in the center of quadrant B, where it remained throughout the
experiment. The mice were introduced into the pool from one of the four release positions
in quadrant A, B, C, or D. The trial ended when the animal found the platform. When a
mouse did not find the platform within 60 s, the experimenter showed the animal the
platform location, where it remained for 10 s. After this period, the mouse was returned to
its cage for 15 minutes, after which it was introduced into the pool again. To test behavioral
flexibility, on day 5 the platform was moved to the opposite quadrant (quadrant A), where it
remained for three trials, with an ITI of 15 minutes (reversal learning task). The first 30
seconds of the first reversal trial were used to conduct a trial to probe spatial retention. This
period of time was used because none of the mice were able to find the novel platform
location during the first 30 seconds of the training. To analyze the spatial training and the
reversal task, escape latencies, distance swum, and velocity were recorded for each trial
and were collapsed into a block of three trials per training day. The percentage of time
spent swimming in the three concentric zones of the pool was calculated for the spatial
learning phase, in order to evaluate thigmotaxic behavior (i.e., peripheral pool time) and its
possible influence in spatial learning. Finally, the probe trial was analyzed by recording the
percentage of time spent in the trained (A) and non-trained quadrants (B, C, and D). Data
were analyzed using two-way ANOVA with repeated measures (habituation, spatial
learning and probe trial) and one-way ANOVA (reversal task). In the habituation trial, a
one-way ANOVA of both time spent and distance moved in each of the three zones, was
performed when genotype by zone interaction was reported. In this case, the Bonferroni
procedure was adopted to control the overall level of significance. In the probe trial, a
single ANOVA of time in the training quadrant was conducted when the genotype by
quadrant interaction was reported. Fisher's post-hoc comparisons were used when
To analyze the search strategies used by the mice in the pool, two independent
investigators blinded to mouse genotype determined a predominant search strategy for
each trial of the last day of the spatial training (day 4). The search paths of each mouse in
each trial were plotted using SMART 2.5 image software and were categorized into one of
the following mutually exclusive search strategies (Brody & Holtzman 2006): spatial
strategies, involving spatial direct, spatial indirect, and focal correct quadrant strategies;
systematic but non-spatial strategies, involving scanning, random, and focal incorrect
target strategies; and strategies involving repetitive looping paths, i.e., chaining, peripheral
looping, and circling strategies. The use of each search strategy was presented as a
percent of incidences in each trial performed during the last training day (day 4). Paired
comparisons were carried out using a Mann-Whitney U-test.
In order to study the possible influence of exploratory impairments (i.e., increased
thigmotaxic behavior) discovered during the habituation trial on spatial learning
performance and search strategies, Pearson correlations were calculated for each group
comparing the time spent in the outer zone during the habituation and time spent in the
target quadrant during the probe test. In addition, the degree of association between
thigmotaxic behavior during habituation and search strategy was also calculated for each
group, using the point biserial correlation coefficient (rpb).
Finally, in order to establish whether the water maze deficit reflects a non-specific,
sensorimotor or motivational performance deficit, various groups of mice were trained in a
visual-cued task. In this study, 10 malpa1(+ / +) mice, 6 malpa1(+/ -) mice, and 6 malpa1(– / –)
mice were used. Mice were trained in the water maze, adapted to the experimental
procedure, and received a habituation trial as described above. Twenty-four hours after
completing the habituation trial, the animals began training in the visual-cued task; this
training lasted for three days. Mice were trained to locate a visible, grey-colored platform
that rested 2 cm above the water surface. The platform was moved to a new location each
trial. The visual-cued task consisted of four trials, each starting from one of the four release
points, with an intertrial interval of 5 minutes. Mice were allowed to rest on the platform for
10 seconds. Data were analyzed using a two-way ANOVA with repeated measures
(genotype x training days), followed by post-hoc comparisons when appropriate (Fisher’s
Neurological abnormalities and preserved auditory and retinal function in maLPA1-
Kruskal-Wallis analysis showed that there was significant variance in somesthesis
(H = 7,5; df = 2; P < 0.05), olfaction (H = 8,699; df = 2; P < 0.05), grasping (H = 7,7; df = 2;
P < 0.05), and equilibrium (H = 7,82; df = 2; P < 0.05) tests between malpa1(+/+) and
maLPA1-null mice. Paired comparisons using Mann–Whitney U-tests revealed that the
absence of LPA1 receptor resulted in a significant impairment in somesthesis (U = 15; P <
0.05), olfaction (U = 49; P < 0.05), grasping (U = 56; P < 0.05), and equilibrium (U = 58.5;
P < 0.05) (Table 1). In contrast, the remaining sensory, limb reflex, and limb coordination
tests did not reveal any performance differences among the three groups (P > 0.05).
Neuromuscular strength analysis, assessed by the hang wire test, showed a
significant effect of genotype (F2,24 = 16.92; P < 0.001). The absence of LPA1 receptor was
associated with shorter latencies to fall compared with malpa1(+/+) and malpa1(+/–) mice (P <
0.05; Table 1). In the tail flick test, the three groups of mice exhibited different responses to
pain (F2,32 = 4.48; P < 0.001). The latency time of the pain response in malpa1(–/–) mice was
significantly longer than that of malpa1(+/+) and malpa1(+/–) mice (P < 0.05; Table 1); no
significant differences were found between malpa1(+/+) and malpa1(+/–) mice (P > 0.05).
ABR profiles showed similar responses in both wild-type and maLPA1-null animals
after stimulation. Animals of both genotypes showed a similar, five-peak wave pattern (Fig.
1a) and similar click-ABR thresholds, 59.1 ± 5.1 and 67.27 ± 3.32 dB SPL, respectively (t21
= 1.303; P > 0.05) (Fig. 1b). Thus, the two groups of mice did not differ in their inter-peak
latencies (I-II: t19 = -0.042; II-III: t19 = -1.44; III-IV: t19 = 0.50; IV-V: t18 = -0.6; I-III: t19 = -
1.325; III-V: t18 = -0.264; P > 0.05; Fig. 1c), corroborating the absence of defective auditory
response in maLPA1 null mice.
ERG analysis of visual function revealed no defective processing in mice lacking
LPA1 receptor. In 1-month-old maLPA1-null mice, average dark-adapted ERG waveforms
and amplitudes were similar to those seen in wild-type animals; no substantial differences
were observed for either a- or b-wave amplitudes over the stimulus intensity range used
(Fig. 1d). For example, at 0.3 cd-s/m2, the wild-type a-wave amplitude was 205.7 ± 28.5
µV, and the null value was 251.4 ± 17.1 µV; the wild-type b-wave amplitude was 477.1 ±
8.5 µV, and the null value was 480.0 ± 48.5 µV(n = 4). In addition, both a-wave and b-wave
dark-adapted thresholds were normal for both groups of mice. Thus, the absence of LPA1
receptor did not appear to affect the retinal pathway.
maLPA1-null mice show impaired activity in the open field and increased anxiety-like
behavior in the elevated plus maze under novelty conditions
In the OF test, the two-way ANOVA [genotype x trial (novelty vs. familiarity)]
revealed significant effects of genotype (F2,30 = 5.29, P < 0.01), trial (F1,30 = 38.08, P <
0.001), and interaction (F2,30 = 5.14, P < 0.01) in the total distance traveled (Fig. 2a).
Simple main effects analysis showed that the three genotypes traveled different distances
in the OF only during the first trial (F2,60 = 8.18, P < 0.01). Post hoc comparisons
demonstrated that both malpa1(+/+) and malpa1(+/–) mice traveled longer distances than
malpa1(–/–) mice P < 0.05). In addition, malpa1(+/+) and malpa1(+/–) mice traveled a shorter
distance during the second trial than they did during the first (F1,8 = 9.7, P < 0.01 and F1,8 =
58.34, P < 0.01 respectively). However, malpa1(–/–) mice showed the same exploration of
the OF in both conditions (F1,8 = 2.07, P > 0.05). Furthermore, malpa1(–/–) mice displayed
significantly lower intersession activity levels than did malpa1(+/+) mice when activity change
scores were analyzed [(F2,30 = 4.59, P < 0.05); (malpa1(+/+): 0.35 ± 0.032; malpa1(+/–): 0.41 ±
0.012; malpa1(–/–): 0.47 ± 0.035).
If the abnormal activity levels in the maLPA1-null mice are indicative of anxiety-like
behavior, one would expect clear differences in the percentage of distance traveled in the
center among the three genotypes. However, the two-way ANOVA [genotype x trial
(novelty vs. familiarity)] did not show any significant general effects of genotype (F2,30 =
0.13, P > 0.05) or interaction (F2,30 = 1.309, P > 0.05) (Fig. 2b). On the other hand, two-way
ANOVA revealed significant differences between the two trials (novel vs. familiar context;
F1,30 = 14.09, P < 0.001), suggesting that all the mice, regardless of genotype, spent a
smaller percentage of time in the center during the second trial (familiar context) than
during the first (novel context). Likewise, similar activity levels were evident for the activity
change scores in all the genotypes [(F2,30 = 1.77, P > 0.05); (malpa1(+/+): 0.41 ± 0.061;
malpa1(+/–): 0.30 ± 0.043; malpa1(–/–): 0.27 ± 0.056)]. These results do not support the
interpretation that enhanced anxiety-like behavior in maLPA1-null mice is the reason for
impaired exploration in this genotype.
In the EPM, two-way ANOVA conducted on the total number of entries in the arms
revealed a significant general effect of genotype (F2,25 = 3.98, P < 0.05) and trial (novelty
vs. familiarity) (F2,25 = 21.51, P < 0.001; Fig. 3a), indicating that, in EPM as in OF, mice of
all three genotypes showed less exploration during the second trial than during the first
trial. Post-hoc comparisons showed that malpa1(–/–) genotype mice exhibited less
exploration in both trials than the other two genotypes (P < 0.05).
The two-way ANOVA conducted on the anxiety index revealed a significant effect of
genotype (F2,25 = 7.12, P < 0.01) and trial (novelty vs. familiarity) (F2,25 = 5.38, P < 0.05; Fig.
3b). Post-hoc comparisons showed that the maLPA1-null mice exhibited more anxious-like
behavior than the other two genotypes (P < 0.05).
Impaired spatial memory retention in maLPA1-null mice
The Morris water maze was used to test spatial memory training. The habituation
trial analysis showed differences among the three genotypes in the velocity of swimming
(F2,26=4.44, P<0.05). In addition, malpa1(–/–) mice exhibited lower velocity than malpa1(+/+)
mice (P < 0.05), but not than malpa1(+/–) mice (P > 0.05) (Table 2). The two-way ANOVA
conducted on the time spent in the three zones of the water maze showed significant
differences among the three zones (F2,54 = 142.34, P < 0.001). Post hoc comparisons
revealed that the mice spent more time in the outer zone than in the other two zones (P <
0.05), and more in the middle zone than in the inner zone (P < 0.05). In addition,
interaction between genotype and zone was observed (F4,54 = 4.82, P < 0.05). The analysis
of the time spent in the three zones of the pool revealed genotype differences in the time
spent in the outer zone (F2,26 = 4.85, P < 0.0167) and in the middle zone (F2,26 = 5.042, P <
0.0167), but not in the inner zone (F2,26 = 2.694, P > 0.0167). Post-hoc comparisons
showed that malpa1(–/–) mice spent less time in both the outer and middle zones than did
malpa1(+/+) mice (P < 0.05), but not malpa1(+/–) mice (P > 0.05) (Table 2). With respect to the
distance moved in the three zones, the two-way ANOVA showed significant effects of
genotype (F2,26 = 4.04, P < 0.05), zone (F2,54 = 98.54, P < 0.001), and interaction (F4,54 =
2.607, P < 0.05). The analysis of the main effects, using post hoc comparisons, showed
that malpa1(–/–) mice swam shorter distances than malpa1(+/+) mice (P < 0.05), but not than
malpa1(+/–) mice (P > 0.05). In addition, the mice swam longer distances in the outer zone
than in the other two zones (P < 0.05), and longer in the middle zone than in the inner zone
(P < 0.05) (Table 2). The analysis of the distance moved in each of the three zones of the
pool revealed genotype differences in the middle (F2,26 = 5.666, P < 0.0167) but not in the
other two zones (inner zone: (F2,26 = 4.41, P > 0.0167); outer zone (F2,26 = 0.063, P >
0.0167)). Post-hoc comparisons revealed that malpa1(–/–) mice swam a shorter distance in
the middle zone than malpa1(+/+) mice (P < 0.05), but not than malpa1(+/–)
mice (P > 0.05;
The analysis of spatial learning revealed a spatial memory impairment in malpa1(–/–)
mice. Two-way ANOVA using repeated measures over the training days did not reveal a
significant main genotype effect in either escape latencies (F2,26 = 2.48, P > 0.05) or in the
distance moved (F2,26 = 0.77, P > 0.05; Fig. 4a and b respectively). However, in all groups,
day of training was found to affect escape latencies (F3,78 = 22.61, P < 0.001) and the
distance moved (F3,78 = 17.34, P < 0.001). Post-hoc comparisons showed that mice of all
genotypes were able to learn the location of the hidden platform, as revealed in the
reduction of escape latencies during the spatial training phase (first four days of testing) (P
< 0.05). The swimming velocity of the malpa1(–/–) mice did not increase during the training,
in contrast to the increase in swimming velocity observed in malpa1(+/+) and malpa1(+/–) mice
(Fig. 4c). The two-way ANOVA revealed an interaction effect (genotype x training days;
F6,78 = 2.27, P < 0.05). Simple main effects analysis demonstrated that the three genotypes
exhibited significantly different velocity during day 4 (F2,104 = 3.35, P < 0.05). Post hoc
comparisons revealed that malpa1(-/-) mice were slower than the other two genotypes (P <
0.05) (Fig. 4c).
During the reversal phase, no differences were observed among the three
genotypes on either escape latencies (F2,26 = 0.26, P > 0.05) or distances swum (F2,26 =
1.15, P > 0.05) (Fig. 4a and b). In contrast, the three genotypes exhibited different
swimming velocity during this phase (F2,26 = 3.46, P < 0.05). Post-hoc comparisons
revealed that malpa1(+/+) mice were faster than malpa1(-/-) mice (P < 0.05) (Fig. 4c).
The analysis of the percentage of time spent during the spatial learning in the outer,
middle and inner zones of the water maze showed that mice of all three genotypes
exhibited a strong overall preference for the middle zone of the water maze, where the
platform was located, spending less time in the outer and inner zones (F2,50 = 148.16, P <
0.001; LSD: outer vs. middle and inner; middle vs. inner (P < 0.05)) (Fig. 4d). Taken
together, these results indicate that the absence of LPA1 receptor is not associated with
enhanced thigmotaxis during spatial learning.
To test spatial memory retention (Fig. 4e), a probe trial was conducted during the
first 30 seconds of the first reversal trial. The two-way ANOVA (genotype x quadrant)
showed a strong effect of quadrant (F3,78 = 5.69, P < 0.01) and interaction (F6,78 = 3.74, P <
0.01; Fig. 4). Single ANOVA of time in the training quadrant showed a significant effect of
genotype (F2,26 = 4.31, P < 0.05). Post-hoc comparisons demonstrated that malpa1(+/+)
mice spent more time in the training quadrant than did malpa1(–/–) and malpa1(+/–) mice (P <
The alterations in behavior in the water maze of malpa1(-/-)) mice were not associated
with any sensorimotor or motivational deficits (suppl. Fig. 2). Mice of all three genotypes
correctly performed a visual-cued task, and there was no difference in the visual-cued task
performance (escape latencies: (F2, 19 = 3.42; P > 0.05) or in the distance swum (F2,19 =
2.11, P > 0.05)). Differences across the training days in both escape latencies (F2,38 =
28.81, P < 0.001) and distance swum (F2,38 = 14.6, P < 0.01) were found, showing that all
the mice were able to reduce their escape latencies and distance swum between day 1 and
the following days of the study (p < 0.05). No significant interaction effect was observed on
either escape latencies (F4,38 = 1.30, P > 0.05) or distance swum (F4,38 = 0.30, P > 0.05).
However, as was shown during the spatial learning, the mice belonging to the malpa1(–/–)
genotype exhibited a general reduction in their velocity (F2,19 = 5.06, P < 0.01; LSD:
malpa1(–/–) vs. malpa1(+/–) and malpa1(+/+) (p < 0.05)).
The analysis of strategy choice throughout the three trials during the last training day
(day 4) revealed that the groups employed different strategies in the water maze (Table 3).
The absence of LPA1 receptor affected the search strategy in the Morris water maze such
that malpa1(–/–) and malpa1(+/–) mice used fewer spatial strategies than malpa1(+/+) mice
(31%, 33%, and 56% respectively). Test monitoring demonstrated that deletion of the lpa1
gene changed the preferences of the mice in favor of non-spatial systematic strategies
(malpa1(+/+), 33%; malpa1(+/–), 56%; malpa1(–/–), 46%) and repetitive looping (malpa1(+/+),
11%; malpa1(+/–), 11%; malpa1(–/–), 23%). Paired comparisons showed that malpa1(+/+) mice
displayed significantly more spatial strategies than malpa1(+/–) and malpa1(–/–) mice (U =
310; P < 0.05; and U =285; P < 0.05 respectively).
Finally, correlational analysis showed that neither time spent in the target quadrant
during the probe test nor search strategy correlated with time spent in the outer zone
during habituation for any genotype. The results for time target quadrant/time outer zone
are as follows: malpa1(+/+), r = -0.59 (t7 = -1.237; P > 0.05); malpa1(+/–), r = -0.35 (t7 = -0.796;
P > 0.05); malpa1(–/–), r = 0.04 (t8 = 0.1154; P > 0.05). The results for search strategy/time
outer zone are as follows: malpa1(+/+), rpb = 0.16 (t7 = 0.46; P > 0.05); malpa1(+/–), rpb = -0.21
(t7 = -0.5089; P > 0.05); malpa1(–/–), rpb = 0.23 (t8 = 0.714; P > 0.05). These data suggest
that the increased thigmotaxis reported in the maLPA1-null mice during the habituation trial
is not associated with either impaired spatial memory retention or use of inappropriate
The neurological and behavioral phenotype of maLPA1-null mice documented in this
study strongly suggests that the LPA1 receptor is involved in several CNS-dependent
functions. However, it is unclear whether the effects in adult mice are directly mediated by
the receptor or are instead due to developmental abnormalities. Several reports have
indicated a critical role of LPA and LPA1 receptors on normal brain development (Anliker &
Chun 2004; Chun 2005; Estivill-Torrús et al. 2008; Choi et al. 2008); this may account for
some of the neurological and behavioral impairments observed in the maLPA1-null mice.
Nevertheless, previous observations suggest that the effects of LPA1 on many cerebral
processes may be context-dependent and may occur during both development and adult
life (Matas-Rico et al., 2008).
The observed neurological deficits in the maLPA1-null mice may affect behavioral
performance involving both motor and cognitive functions. However, the delection of LPA1
receptor does not appear to induce severe neurological deficits. Visual and auditory
functions are not impaired, and when sensory reflexes were assessed, only mild deficits in
somesthesis and olfaction were observed. Results from somatosensory tests, including the
tail-flick test, may be important when studies with painful stimulation are performed.
However, withdrawal from pain is probably not involved in the behaviors we evaluated in
our study. Weak olfaction deficits reported in the maLPA1-null mice may be involved in
exploratory and spatial tasks because olfactive cues are used when animals explore the
environment (Lavanex & Schenk 1997; Rossier & Schenk 2003). Nevertheless, no deficits
were observed in exploration by maLPA1-null mice during either NOR or EPM tasks,
suggesting that the impairments reported in those tasks are not due to impaired olfaction.
Our evaluation of limb reflexes and coordination demonstrated that knockout mice were
able to achieve a good level of coordination and placing. In addition, only a minor deficit
was observed in grasping and equilibrium tests; maLPA1-null mice displayed an adequate
response and only showed a reduced ability (time) to maintain equilibrium. The deficit
observed in the maLPA1-null mice that is most likely to have influenced the behavioral
tasks used in our study is related to muscular weakness. Tasks based on exploration, such
as OF and the water maze, may be influenced by muscular weakness; variables such as
distance traveled, escape latency, and velocity can be severely reduced. Although overall
speed was altered, distance and escape latencies were not significantly impaired in
maLPA1-null mice during spatial training in the water maze. Likely, hypolocomotion during
the first exposure to the OF may result from the muscular weakness of these animals. In
order to prevent or minimize bias in determining the cognitive and emotional deficits of
maLPA1-null mice in those tasks, we used some variables partially independent of these
alterations as indicative of emotional and cognitive impairments, such as the percentage of
time that the mice spent in a region of the maze.
The findings obtained in the open field showed a reduced exploratory reactivity of
maLPA1-null mice to a novel environment in comparison with the other two genotypes. This
overall decrease in activity might simply result from disturbances in motor functions,
leading to the observation of a floor effect in overall activity; it might also involve changes in
emotional variables. However, the exploration of the OF in the novelty condition is not
associated with anxiety-like behavior. In fact, locomotor activity in the center in response to
novelty was similar among the three genotypes, suggesting no significant genotype
differences in anxiety-like behavior in response to the novel environment. Thus, the
hypolocomotion of maLPA1-null mice in the OF is more likely to be due to their above-
mentioned motor impairments.
The role of the LPA1 receptor in the habituation of activity in the OF is suggested by
the activity levels during the second trial, especially when the activity change scores were
compared. It is also important to note that the abnormal intertrial habituation in maLPA1-
null mice probably cannot be explained by enhanced anxiety-like behavior, since no
significant differences exist among the genotypes in both the percentage of time spent in
the center of the OF. However, because the different activity levels on trial 1 of testing call
into question the interpretation of the results as habituation deficit, these data must be
interpreted cautiously. Further studies are required to clarify whether the low activity levels
in the maLPA1-null mice during the first trial may explain the impairment reported here.
In contrast to the OF results, on first exposure to the EPM, the three genotypes
exhibited a preference for the closed arms in the absence of significant differences in the
total arm entry score, a pattern previously seen in other genotypes (Holmes et al, 2000).
Notwithstanding, the low activity displayed by maLPA1-null mice in open arms indicates
enhanced anxious-like behavior under novelty conditions, which is specific to this
genotype. It is well known that prior exposure to the EPM alters baseline behavior on re-
exposure to the test (Dawson et al, 1994; Holmes et al, 2000), and that mice and rats with
increased reactivity to the novelty did not reduce open arm exploration during retest
(Holmes et al, 2000; Ballaz et al, 2007). In our experiment, when mice were re-exposed to
the EPM 24 hours after trial 1, all three genotypes exhibited a normal inter-trial reduction in
exploration of the EPM. The fact that the retest profile was not affected by the increased
anxiety-like behavior observed in the maLPA1-null mice suggests the involvement of
different mechanisms in the two behavioral processes. It is noteworthy that both impaired
exploration in the OF and enhanced anxiety-like behavior in the EPM when maLPA1-null
mice were tested cannot be attributed to a general locomotion impairment or to the motor
and sensory deficits reported in these knockout mice. Results reported in the EPM may on
occasion contradict those obtained in the OF; although these tasks are based on novelty
exploration and emotional response to environmental challenges, the results for each
depend strongly on the test conditions (Belzung & Griebel 2001). Therefore, different levels
of stress and emotion triggered by the tasks likely explain the differences in the results of
these tests in the two conditions studied.
Finally, our findings indicate that the absence of LPA1 receptor impairs spatial
memory. In the water maze, maLPA1-null mice exhibited impaired spatial retention during
the probe test and increased propensity to adopt inappropriate search strategies (i.e., non-
spatial strategies) during the last training day. Nevertheless, deletion of the lpa1 gene did
not cause a general spatial learning deficit because all genotypes showed similar learning
curves, improving their performance across the spatial training. Reduced overall speed
may result from the motor alterations observed during neurological tests. The impairment in
speed in the maLPA1-null mice coincides with the longer latency scores observed on days
3 and 4, suggesting the influence of speed on this variable. However, no significant
differences in escape latencies were observed among the genotypes during spatial training
on these same days. It is therefore likely that the absence of differences among the
genotypes in their escape latencies is due to the mild impairment in speed of the mice used
in our study. Escape latency impairment has been reported in mice with stronger reduction
of overall speed than we have seen in our study (Stein et al, 2006). In contrast, poor
performance during the probe trial and the use of inappropriate search strategies have also
been observed in malpa1(+/-) mice, but without speed impairments, arguing against the
influence of speed deficits in the behavioral impairments reported here.
The cognitive nature of the impairment in maLPA1-null mice can also be questioned
on the grounds that anxiety enhancement and abnormal motor behavior increase
thigmotaxic behavior in the water maze (Petrosini et al. 1996; Rodgers 1997). In fact,
during habituation of maLPA1-null mice, increased peripheral exploration is consistent with
the anxious-like behavior enhancement previously demonstrated in these animals.
Thigmotaxis has been used as an index of anxiety-like behavior that may interfere with the
normal acquisition of a spatial learning task (Whishaw 1995; Champagne et al. 2002).
However, enhanced thigmotaxis in maLPA1-null mice does not seem to be the reason for
the deficits reported here. During the habituation trial, animals of all three genotypes
displayed a tendency to swim in the peripheral zone. Thigmotaxis is the normal behavior
when rodents are exposed for the first time to the water maze; it is replaced by more
accurate strategies when animals are repeatedly trained in this task (Whishaw 1995). The
maLPA1-null mice used in this study exhibited the normal pattern of significant thigmotaxic
behavior during the habituation trial. During spatial training in the water maze, this stronger
preference was replaced in maLPA1-null mice as well as in the other two genotypes by
search strategies more centered in the middle zone of the water maze. Thus, taken
together, our data suggest that enhanced thigmotaxis in maLPA1-null mice during the
habituation trial may be due to anxiety-like behavior enhancement or motor impairment, but
the fact that reduction of this behavior across the spatial training occurred in the way
expected for normal animals argues against its influence on the impairments that we
observed in the water maze. In relation to this finding, thigmotaxic behavior in the maLPA1-
null mice during the habituation trial does not correlate with either searching strategies or
performance during the probe test.
The absence of deficits in the visual-cued task argues against the involvement of
sensorimotor or motivational alterations in the performance of the malpa1-null mice in the
water maze. Our data support the notion that the cognitive deficits observed in these mice
are not due to increased emotionality, sensorimotor or motivational deficits.
In summary, our data show that the LPA1 receptor has a role in generating or
controlling anxiety-like behavior, as well as in cognitive processes such as spatial memory.
These results support a role for LPA signaling via LPA1 receptors in major neuropsychiatric
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This work was supported by grants from the Human Frontier Science Programme, (JC,
FRDF), MEC SEJ2007-61187 (LS), FIS 01/3032 (GE), FIS 02/1643, FIS PI07/0629 (GE),
Red CIEN (G03/06) (FRDF) (Instituto de Salud Carlos III, Ministerio de Sanidad) and the
National Institutes of Health (USA) MH51699 and MH01723 (JC). The author E. Castilla-
Ortega and J. Sánchez-López were supported by a FPU Grant of the Spanish Ministerio de
Educacion y Ciencia (AP-2006-02582 and AP-2007-03719 respectively). We are grateful to
animal housing facilities of University of Málaga for maintenance of mice and technical
Figure 1: Auditory and retinal function in mice lacking LPA1 receptor. (a)
Representative auditory brainstem responses (ABR) recordings for two wild-type
(malpa1(+/+)) and two maLPA1-null (malpa1(–/–)) mice. Typical waveforms comprising four or
five peaks are distinguishable in a time period of about 8 ms following stimulation and
similar for both genotypes. Test for mice used 10-dB steps down from the maximum
amplitude of 90 dB SPL. (b) Average ABR thresholds for click stimulus of malpa1(+/+) and
malpa1(–/–) mice. Data presented as mean ± SEM. No significant differences were observed
between the two groups (n = 11; P < 0.01). (c) Graph showing the determination of 30 pps
80dB SPL click-ABR inter-peak latencies, in ms, for one wild-type (malpa1(+/+)) and one
maLPA1-null (malpa1(–/–)) mice. Statistical analysis (T-test; n = 11) demonstrated no
differences in latencies attributable to absence of LPA1 receptor. (d) Representative dark-
adapted ERG tracings for one wild-type (malpa1(+/+)) and maLPA1-null (malpa1(–/–)) mice at
0.3 cd-s/m2 stimulus intensity and corresponding mean (±SEM) amplitudes of the a- and b-
Figure 2: Open field exploration in mice lacking LPA1 receptor. (a) Data represent
mean (± SEM) distance moved in the OF. malpa1(+/+) and malpa1(+/–) showed decreased
motor activity (distance traveled) during the second trial (familiarity) compared to the first
trial. However, malpa1(–/–) genotype showed the same activity in both trials. In addition,
malpa1(–/–) mice traveled less distance than the other two genotypes only during the first
trial (novelty). (* P < 0.01 (novelty vs. familiarity); + P < 0.05 (malpa1(+/+) vs. malpa1(–/–) ; & P <
0.05 (malpa1(+/–) vs. malpa1(–/–)). (b) Data represent mean percentage of time (±SEM) spent
in the center of the OF. The three genotypes spent less percentage of time in the center
zone during the second trial (familiarity) than during the first trial (novelty) (P < 0.05).
Figure 3: Increased anxiety-like behavior in mice lacking LPA1 receptor in the
elevated plus maze. (a) Data represent mean (± SEM) number of total transitions in the
EPM. All the genotypes showed decreased motor activity during the second trial
(familiarity) compared to the first trial (novelty) (P < 0.05 ). In addition, malpa1(–/–) genotype
exhibited less exploratory activity than the other two genotypes (P < 0.05). (b) Data
represent anxiety index calculated in the EPM. malpa1(+/+) and malpa1(+/–) exhibited less
anxiety-like behavior during both first and second trials (novelty vs. familiarity) than
Figure 4: Spatial learning in mice lacking LPA1 receptor. Mice of all genotypes learned
to locate the hidden platform position, as shown by decreasing escape latencies (a) and
distance moved (b) in the acquisition phase (days 1-4). The reversal phase analysis (day
5) showed no differences among the three genotypes either in escape latency (a) or in
distance moved (b) in the Morris water maze (P > 0.05). Analysis of velocity (c) revealed
that malpa1(+/+) and malpa1(+/–) mice increased velocity over the training days (P < 0.05)
while LPA1-null mice failed to increase velocity through the spatial training (P > 0.05). (d)
The percentage of searching time in three different zones in the water maze (inner, middle
and outer zone) showed that the three genotypes spent more time in the middle than in the
other two zones during the spatial training (p < 0.05). No differences were seen among the
genotypes (p > 0.05). (e) Data represent mean (± SEM) of percentage of total time spent in
each quadrant during the probe test in the water maze. malpa1(–/–) and malpa1(+/–) mice
genotypes were not able to remember the location of the platform in the target quadrant
(B). * P < 0.05; malpa1(+/+) vs. malpa1(+/–) and malpa1(–/–).
Supplementary Figure 1: Quadrant preference during the habituation trial in the
water maze. No differences in preference (or lack of preference) during the habituation
trial were observed among the three genotypes.
Supplementary Figure 2: Visual-cued task in the water maze. All genotypes were able
to learn a visual-cued task as shown by decreasing escape latencies (a) and distance
moved (b) during the training (days 1-3). No differences either in the escape latencies (a)
or distance moved (b) in the water maze, were observed among the three genotypes (P >
0.05). However, the analysis of velocity (c) revealed that malpa1(-/-) mice were slower than
the other two genotypes in the visual-cued task (P < 0.05).
Neurological screening of malpa1
(+ / +), malpa1
(+ / –) and malpa1
(– / –) mice
Genotype Absent deficit (0) Weak deficit (1) Strong deficit (2)
(+ / +)
(+ / –)
(– / –)
Genotype Absent deficit (0) Weak deficit (1) Strong deficit (2)
(+ / +)
(+ / –)
(– / –)
Genotype Absent deficit (0) Weak deficit (1) Strong deficit (2)
(+ / +)
(+ / –)
(– / –)
Genotype Absent deficit (0) Weak deficit (1) Strong deficit (2)
(+ / +)
(+ / –)
(– / –)
(E) Tail flick and hangwire tests
Genotype Tail flick test Hangwire test
(+ / +)
(+ / –)
(– / –)
2.0 ± 0.15 s
1.9 ± 0.32 s
2.6 ± 0.17 s*
51.1 ± 4.5 s
46.8 ± 4.3 s
18.3 ± 4.7 s*
(A,B,C,D) Data are expressed as percentage of mice
(E) Data are expressed as mean ± SEM escape latencies
* P < 0.05, malpa1
(– / –) vs. malpa1
(+ / +) and malpa1
(+ / –)
Habituation of malpa1
(+ / +), malpa1
(+ / –) and malpa1
(– / –) mice in the Morris water maze
(+ / +)
(+ / –)
(– / –)
Data are expressed as mean ± SEM
* P < 0.05, malpa1
(+ / +)
(– / –)
Search strategies in the Morris water maze of malpa1
(+ / +), malpa1
(+ / –)
(– / –) mice
(+ / +)
(+ / –)
(– / –)
Data are expressed as the percentage of mice.
Time in center (%)
Mean (+/- SEM) distance (cm)
Mean (+/- SEM) number of total entries
DAY 2 DAY 3 DAY 4 Download full-text
Mean (+/- SEM) distance (cm)
DAY 2 DAY 3 DAY 4
Mean (+/- SEM) escape latencies (s)
DAY 2 DAY 3 DAY 4
Mean (+/- SEM) velocity (cm/s)
Time in quadrant (%)
Quadrant A Quadrant B Quadrant C Quadrant D