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Keywords:

  • Anxiety;
  • cognition;
  • GPR92;
  • LPA5;
  • lysophosphatidic acid;
  • mouse;
  • nociception

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Lysophosphatidic acid (LPA) is a bioactive lipid acting on the nervous system through at least 6 different G protein-coupled receptors. In this study, we examined mice lacking the LPA5 receptor using an extensive battery of behavioral tests. LPA5-deficient mice showed decreased pain sensitivity in tail withdrawal, faster recovery in one inflammatory pain procedure (complete Freund's adjuvant-induced inflammation) and attenuated responses under specific neuropathic pain conditions. Notably, deletion of LPA5 also induced nocturnal hyperactivity and reduced anxiety in the mutant mice. Several exploratory tasks revealed signs of reduced anxiety in LPA5 knockout mice including increased visits to the arena center and reduced thigmotaxis in the open field, and more open arm entries in the elevated plus maze. Finally, LPA5 knockout mice also displayed marked reduction in social exploration, although several other tests indicated that these mice were able to respond normally to environmental stimuli. While learning and memory performance was not impaired in LPA5-deficient mice, we found differences, e.g., targeted swim strategy and reversal learning, as well as scheduled appetitive conditioning that might indicate differential motivational behavior. These results imply that LPA5 might be involved in both nociception and mechanisms of pain hypersensitivity, as well as in anxiety-related and motivational behaviors. These observations further support the proposed involvement of LPA signaling in psychopathology.

Lysophosphatidic acid (LPA, 1-acyl-2-sn-glycerol-3-phosphate) is a bioactive membrane lipid that acts on at least six distinct G protein-coupled receptors (LPA1–6). Arguably one of the least known of these still enigmatic receptors, LPA5 (previously known as GPR92), is widely expressed in various parts of the body including brain and peripheral nervous system (Kotarsky et al. 2006; Lee et al. 2006; Oh et al. 2008). Endogenous LPA release following tissue damage stimulates peripheral nociceptors (Inoue et al. 2004; Leo et al. 2008; Ueda 2006) and controls immune responses (Fukushima et al. 2001; Graler & Goetzl 2002; Kotarsky et al. 2006; Oh et al. 2008). The high expression of LPA5 in some cells of the mouse dorsal root ganglion suggests a role for this LPA receptor in acute and neuropathic pain (Kotarsky et al. 2006; Oh et al. 2008; Sheardown et al. 2004).

In addition, LPA signaling has also been implicated in development and adult functioning of the mammalian brain (Dubin et al. 2010). For example, LPA has been shown to initiate neurite reorganization by activation of Rho/ROCK pathway (Lee et al. 2006; Tigyi 2010) and is a principal activator of the ERK pathway that plays a central role in synaptic and developmental plasticity (Oh et al. 2008; Thomas & Huganir 2004). Furthermore, LPA has been shown to play a role in neuroplasticity-dependent brain functions like pain and memory (Ji et al. 1999, 2003; Ma & Quirion 2005). Inhibitors of this pathway do alleviate pain in several experimental models (Ji et al. 1999; Kawasaki et al. 2004; Ma & Eisenach, 2003; Yu & Yezierski 2005), but also interfere with various hippocampus- and neocortex-dependent behaviors. Some authors also indicated that the role of LPA receptors in brain development could underlie their involvement in neurodevelopmental disorders (Harrison et al. 2003). In particular, using murine knockout models, LPA1 has been shown to be involved in hippocampus-dependent learning, emotional behavior and in normal development of inborn, neonatal behavior (Ahn et al. 2009; Blanco et al. 2012; Castilla-Ortega et al. 2010; Contos et al. 2000; Matas-Rico et al. 2008; Santin et al. 2009). It should be noted, however, that there is presently little information available about the involvement of other LPA receptors, especially LPA5, in any central function or dysfunction.

Therefore, the present study investigates the behavioral significance of LPA5-mediated signaling using LPA5-deficient mice. We compared LPA5-deficient to wild-types mice using a comprehensive and multifaceted test battery (Callaerts-Vegh et al. 2006; Leo et al. 2008). Various clinically relevant pain responses were evaluated as well as neuromotor abilities, social and emotional behaviors, and cognitive performance that was assessed in different learning and memory tasks. The involvement of LPA5 in nociception might identify this receptor as a potential pain target, whereas various other observations reported here might relate to the putative involvement of LPA signaling in some neurodevelopmental psychopathologies.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Generation of LPA5 gene knockout mice

LPA5 gene knockout mice [inbred 129S9/SvEvH background (Festing et al. 2009)] were developed in collaboration with Takeda Cambridge Ltd (Cambridge, United Kingdom). Homologous recombination resulted in the deletion of the seven transmembrane spanning regions of lpa5, which was replaced by a frame independent fusion with LacZ upstream of a selection cassette consisting of the neo gene in the same orientation as the lpa5 gene (see Fig. 1a for schematic presentation). EcoRI digests generated specific fragments detectable by Southern blotting (Fig. 1b). For a detailed description of the generation of LPA5 gene knockout mice see Appendix S1, Supporting Information.

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Figure 1. Generation of LPA5 gene knockout mice. (a) Targeted disruption of lpar5. Illustration shows the lpa5r coding exon structure (open boxes, UTR; solid boxes open reading frame; hatched region, 7tm domain); the targeting construct containing an IRES lacZ reporter cassette (stippled box labeled IRES lacZ) followed by a promoted neomycin phosphotransferase selectable marker (stippled box labeled with neo); the resultant genomic structure following homologous recombination; and the location of the external probe used in the Southern blot shown in (b). BspHI sites and EcoRI sites are depicted as B and E. (b) Genomic analysis of LPA5+/+, +/− and −/− mice using external hybridization probe. The BspHI hybridizing fragment is 9.4 kb in an undisrupted allele and 13.6 kb after targeting. The EcoRI hybridizing fragment is 8.4 kb undisrupted and 5.4 kb after targeting. (c) Successful recombination in LPA5−/− mice was demonstrated by cell-specific expression of lacZ in tissues shown to be rich in lpa5, such as ganglia and small intestine.

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Behavioral testing

LPA5 gene knockout mice were bred by homozygous crossings and F3 offspring were used for behavioral testing. For pain-related behavior, male mice (age 8–15 weeks) were used and all animals were subjected to only one test [with the exception of testing acute thermal sensitivity such as tail withdrawal, hot plate (HP) and Hargreaves' test (HT)]. Pain assessment was performed at Janssen Pharmaceutica. Male animals were housed individually under controlled housing conditions (12 h light–dark cycle, 21°C room temperature) with ad libitum access to food and water. The remaining behavioral tests were performed in the Laboratory of Biological Psychology (KULeuven) in a cohort of female mice (age 8–15 weeks). Animals were group housed (2–4 animals per cage) on a 12 h dark–light cycle (light on at 8:00 AM, testing during light period) with free access to food and water, except during some of the behavioral testing. In all the experiments, LPA5−/− mice were directly compared with same gender and same generation controls (LPA5+/+) and subjected to the following test order: cage activity, rotarod, grip strength, open field, social exploration, elevated plus maze, defensive burying, passive avoidance, Morris water maze, acoustic startle, contextual fear conditioning and appetitive conditioning (see Appendix S1 for time table). All experiments were carried out in accordance with the European Communities Council Directives (86/609/EEC) and were approved by the local ethical committee.

Assessment of pain behavior

Nociceptive withdrawal responses were evaluated following previously described methods in experimentally naïve animals (Leo et al. 2008). Noxious stimuli included heat (HP, tail withdrawal, HT) and punctate stimuli (von Frey), abdominal stretch responses (writhing test) to acetic acid (1%, 0.1 ml/10 g), flinching in the formalin test (1%, 25 µl), heat sensitivity after carrageenan subplantar injection (2%, 25 µl) and neuropathic pain-related behavior after spared nerve injury (SNI). Additionally, complete Freund's adjuvant (CFA)-induced inflammation was evaluated by assessing paw volume using a plethysmometer, and thermal sensitivity in the HT. Peripheral inflammation was produced in the mice by a 25-µl subcutaneous (s.c.) injection of 1 mg/ml CFA solution into the subplantar side of the left hind paw using a syringe with a 30-gauge needle (controls received an s.c. injection of 25 µl saline). Mice were tested for thermal sensitivity in Hargreaves' heat test. A cut-off time of 20 seconds was adopted to prevent tissue damage. Before injection, baseline latencies were measured twice with an interval of 15 min. Post-injection latencies were measured after 4, 24 and 48 h. To quantify the magnitude of the inflammatory response produced by CFA, paw edema was measured using a plethysmometer (IITC Inc. Life Science, Woodland Hills, CA, USA). The paw is inserted in water, contained in a special water cell of which the resistance is changed because of the immersion of the animals paw. This resistance change is calibrated in milliliter (ml) and shown on an electronic monitor. Paw volumes were measured once prior to injection and 4, 24 and 48 h after CFA challenge.

The chronic constriction injury (CCI) model was used as a second neuropathic pain model. A procedure originally described by Bennett and Xie 1988 was adapted for use in mice. Surgical procedures were performed under gas anesthesia with a mixture of isoflurane (5% for induction and 3% for maintenance) and oxygen. After skin and muscle incision of the left hind leg, the sciatic nerve was exposed and three ligatures constricting the nerve diameter by approximately 1/3 to 1/2 with chromic catgut (6-0, Ethicon Inc, Somerville, NJ, USA) were placed around the nerve, proximal to the trifurcation. A sham operation was performed by exposing, but not ligating the sciatic nerve. The muscle and skin layer were closed separately by using 6-0 Vicryl® and 6-0 Mersilk® sutures (Ethicon Inc), respectively. Sham mice received the same surgical procedure without touching any nerve. Similar to the SNI mice, responses to mechanical (von Frey) or cold (acetone) stimulation as well as weight bearing were measured before and after the injury at regular time points (2, 6, 9, 14 and 21 days post-surgery).

General activity and neuromotor and sensorimotor performance

General activity and neuromotor coordination were assessed by ambulatory activity monitoring in the home cage during a 23-h period, motor coordination/balance on accelerating rotarod and grip strength tasks (D'Hooge et al. 2005). Briefly, cage activity was recorded using a lab-built activity logger connected to 3 IR photo beams. Mice were placed individually in 20 × 30 cm2 transparent cages located between the photo beams. Over a period of 24 h, activity was measured and expressed as number of beam crossings. For motor balance test, mice were first trained at constant speed (4 rpm, 2 min) before starting with four test trials (intertrial interval, 10 min). During these test trials, the animals had to balance on a rotating rod that accelerated from 4 to 40 rpm in 5 min, and time until they dropped from the rod was recorded (up to the 5-min cut-off). Grip strength was measured using a device consisting of a T-shaped bar connected to a digital dynamometer (Ugo Basile, Comerio, Italy). Mice were placed before the bar, which they usually grabbed spontaneously and gently pulled backward until they released the bar (maximal readouts were recorded). Ten such measurements were obtained for each animal. Acoustic startle was assessed using a protocol adapted from a startle/freezing cage system (Panlab©, Barcelona, Spain). The mouse was placed inside a small animal holder restricting free movements and, after 5 min habituation (with 65 dB white noise as background), presented 10 ×10 startle stimuli (sound pressure level ranging from 75–120 dB, 4 kHz, 40 milliseconds, inter stimulus interval on average 15 seconds) in a random order. Startle responses were recorded for 100 milliseconds from the onset of the startle stimuli and averaged by sound pressure level. Startle responses were recorded on an arbitrary scale from 0 to 100, and maximal response as well as latency to stimuli were determined. Startle threshold was defined as the lowest sound level pressure (dB) that evoked a significant startle compared to baseline levels (65 dB).

Exploration and anxiety assessment

Several exploration and anxiety-related tests were carried out according to previously described protocols. Open field and social exploration was examined using a 50 × 50 cm2 square arena of transparent plexiglass (Callaerts-Vegh et al. 2006; D'Hooge et al. 2005). Animals were dark adapted for 30 min and placed individually in the brightly illuminated arena (465 lux) for 11 min (1 min habituation and 10 min recording). Movements of the mice in the arena were recorded using EthoVision video tracking equipment and software (Noldus bv, Wageningen, The Netherlands). Total path length and corner crossings were included as measures of locomotor activity. These measures are highly correlated and mainly indicate thigmotactic walking near the walls of the arena. Entries into the center of the field were recorded as a measure of conflict resolution or anxiolysis. For social exploration assessment, the arena contained a 15-cm round cage with two unfamiliar female mice. Exploratory activity was tracked for 11 min (1 min habituation and 10 min recording) using EthoVision software (Noldus) using predefined center and corner areas of the open field.

The elevated plus maze was used to assess anxiety-related exploration (Callaerts-Vegh et al. 2006). The arena consisted of a plus-shaped maze with two arms (5 cm wide) closed by side walls and two arms without walls. Mice were placed at the center of the maze and were allowed to explore freely for 11 min (1 min habituation and 10 min recording). Exploratory activity was recorded by five IR beams (four for arm entries and one for open arm dwell) connected to a computerized activity logger.

To evaluate defensive burying mice were placed individually in transparent plastic cages (15 × 26 × 42 cm) containing 5 cm of sawdust and 24 identical glass marbles (∼1.5 cm diameter) evenly spaced 2 cm from the cage wall (Moechars et al. 2006; Njung'e & Handley, 1991). The cages were placed on a platform 80 cm above the floor and under bright illumination. After 30 min, the mice were returned to their home cage and the number of marbles buried two thirds by saw dust was counted.

Spatial memory in the water maze

Spatial memory abilities were examined in a standard hidden-platform Morris water maze using acquisition, retention (i.e., long-term memory) and reversal learning protocols. A 150-cm circular pool was filled with water, opacified with non-toxic white paint and kept at 26°C as previously described (Callaerts-Vegh et al. 2006; D'Hooge et al. 2005). A 15-cm round platform was hidden 1 cm beneath the surface of the water at a fixed position. Mice were trained for 10 days (Mondays through Fridays, weekends at rest). Each daily trial session consisted of four swimming trials (15–30 min intertrial interval) starting randomly from each of four starting positions. Mice that failed to find the platform within 2 min were guided to the platform, where they remained for 15 seconds before being returned to their cages. The probe trial was conducted after 5 and 10 trial sessions (Monday mornings before continuation of acquisition or reversal trials). During a probe trial, the platform was removed from the pool and the search pattern of the mice was recorded for 100 seconds. After 2 weeks of acquisition and the second probe trial, the platform was moved to the opposite position to measure cognitive flexibility in spatial navigation during reversal learning. For reversal learning, the animals were tested on one single day in five consecutive reversal trials (15–30 min intertrial interval), starting randomly from all four starting positions (reversal trial 1 and 5 were from the same position). Several parameters were analyzed: cumulative distance to the platform position (5 Hz sampling frequency), latency to find the platform, swim speed and platform crossings. Swimming paths of the animals were recorded using EthoVision video tracking equipment and software (Noldus bv). We also analyzed in detail search strategies by categorizing individual swim paths as either spatial (spatial direct, spatial indirect, focal correct), non-spatial systematic (random, scanning, focal incorrect) or repetitive looping (circling, chaining, peripheral looping) categories (Brody & Holtzman 2006) by applying a multi-class support vector machine (SVM; (Cortes & Vapnik 1995) model that was using 809 tracks scored by one human observer (based on static track images) (see Appendix S1 for detailed description). When different strategies were used during one acquisition trial, the strategy used predominantly was defined as the category. In addition, we visualized swimming paths during probe trials creating heat plots indicating preferential location with a custom-made software written in MATLAB (Balschun et al. 2010; Van der Jeugd et al. 2011). Intense colors (from blue to red) indicated a higher presence in those specific areas.

Fear conditioning and appetitive conditioning learning

Passive avoidance learning was examined as previously described in a two compartment box with a shock grid (Callaerts-Vegh et al. 2006; D'Hooge et al. 2005). Dark-adapted mice were placed in the light compartment and latency to enter the dark compartment was measured. Upon entry into the dark compartment, the door was closed and a 2-second foot shock (0.2 mA) was applied. The mouse was then removed from the box and placed in its home cage. Twenty-four hours later, the dark-adapted mouse was again placed in the light box and latency to enter the dark compartment was measured.

Context- and cue-dependent fear conditioning was studied by measuring freezing responses, a reliable measure of innate and conditioned fear in rodents (Moechars et al. 2006). Mice were fear conditioned by presenting 2 CS (30 seconds, 4 kHz, 80 dB, inter-stimulus interval 1 min) which co-terminated with a 2 seconds, 0.3 mA footshock. Twenty-four hours later, memory retention to conditioning context and to the auditory cue were evaluated.

Scheduled appetitive conditioning was tested in automated operant chambers (Coulbourn Instruments, Allentown, USA), essentially as described previously (Callaerts-Vegh et al. 2006; Goddyn et al. 2008). Mice were trained in daily 30-min sessions on a conditioning protocol with increasing difficulties in rewarding schedule to obtain food, starting with continuous reinforcement (CRF), switched to fixed ratio (FR3, 5 and 10) and variable ratio (VR) reinforcement, and finally ended with a variable 30-second interval schedule (VI30). Transition to a following schedule depended on predefined criteria, such as minimal number of nosepokes or minimal number of sessions. Responses in each schedule were averaged over sessions.

Statistical analysis

Data are presented as mean ± SEM. Differences between mean values were determined using t-test or two- or three-way repeated measures analysis of variance (RM anova) procedures with Tukey tests for post-hoc comparison when applicable (IBM SPSS Statistics v19). When appropriate (violation of D'Agostino-Pearson normality assumption), Mann–Whitney Rank Sum test was used to compare two groups. All statistical tests were performed at the α level of significance at 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Generation of LPA5 gene knockout mice

The replacement of the coding region in exon 3 of lpa5r by LacZ resulted in cell-specific staining in dorsal root ganglia, trigeminal ganglia (Fig. 1c) and in the small intestine in adult mice. No LacZ staining was detected in other central nervous system structures (data not shown).

Assessment of pain behavior

Acute nociception

Of the three assays performed to assess acute thermal sensitivity, we observed genotype-dependent thermal nociception in LPA5−/− mice only in the tail withdrawal test (TWR; Fig. 2a), not in the HP (Fig. S1a, Supporting Information) nor in HT(Fig. S1b). TWR was performed at multiple stimulus intensities ranging from 47 to 53°C. A clear and stable temperature effect was seen, i.e., at higher temperatures, response latency was reduced (RM anova F3,126 = 107.77, P < 0.001). Tail flick response, which is considered to be a spinal reflex, was significantly reduced in LPA5-/- mice (F1,42 = 4.66, P < 0.05), while statistically the interaction was not significant (factor genotype × temperature F3,126 = 1.54). This indicates that LPA5−/− mice withdrew their tail later compared to LPA5+/+ at 53°C (q = 4.16, P < 0.005). In contrast, HP and HT tests did not show a significant genotype effect (see Appendix S1). Also, mechanical thresholds in the automated von Frey test were indistinguishable between LPA5+/+ (left paw: 6.7 ± 0.5 g; right: 6.8 ± 0.3 g) and LPA5−/− mice (left paw: 6.4 ± 0.3 g; right: 6.4 ± 0.3 g).

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Figure 2. Differences in nociception between LPA5+/+ and LPA5−/− mice. (a) In the tail withdrawal test (n = 22 per genotype), LPA5−/− mice (open bars) withdrew their tail later than LPA5+/+ mice (black bars). (b) Thermal hyperalgesia caused by inflammation after complete Freud's adjuvant (CFA, circle symbols) injections lasted shorter LPA5−/− mice (white circles) than LPA5+/+ mice (black circles) (n = 10–13, per genotype). Twenty-four hours after injection, thermal thresholds were back to saline levels (square symbols) in LPA5−/− mice. (c) Increased response times to acetone were seen after chronic constriction injury (CCI, circle symbols) in LPA5+/+ and LPA5−/− mice when compared to sham-operated animals (square symbols). However, LPA5−/− mice (white circles) developed cold allodynia to a lesser extent than LPA5+/+ (black circles) after injury. Data are presented as mean ± SEMs. Symbol # indicates significant differences between LPA5+/+ and LPA5−/− mice (#P < 0.05, ##P < 0.01). Symbol * indicates significant differences between conditions, i.e., saline vs. CFA and CCI vs. sham, respectively (*P < 0.05, **P < 0.01).

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Inflammatory pain conditions

In all but one chemically induced inflammation models, we observed no differences between the genotypes (see Fig. S2).

The effect of the CFA-induced inflammatory pain was investigated using a 3-way RM anova with categorical factors condition (NaCl, CFA), genotype (LPA5−/− and LPA5+/+), over four repeated measures time (pre, 4 h, 24 h and 48 h) (Fig. 2b). We observed a significant effect of condition (F1,42 = 5.18, P < 0.03) and a significant interaction of condition × genotype (F1,42 = 4.5, P < 0.05). Thermal hyperalgesia was displayed 4 h after CFA injection by both LPA5−/− and LPA5+/+ mice (Fig. 2b), however, at 24 h the thermal sensitivity of LPA5−/− mice was back at baseline levels, whereas LPA5+/+ mice still displayed increased sensitivity to thermal stimulation. Forty-eight hour post-CFA, thermal sensitivity had normalized in all groups to baseline.

Neuropathic pain conditions

The spared (tibial) nerve injury (SNI) and the CCI models were employed to evaluate neuropathy-related behavioral responses. Three different tests measured the degree of neuropathic pain using mechanical allodynia (von Frey test), cold allodynia (acetone test) and spontaneous display of discomfort (weight bearing in the balance box).

In the SNI model, both genotypes displayed similar decreases in mechanical threshold from post-surgery day 2 through 21 compared to their sham-operated littermates (Fig. S3). Similarly, we observed no genotype differences in CCI-induced neuropathy in von Frey and balance box procedures (Fig. S4). In contrast, in the acetone challenge test, LPA5−/− mice developed a lesser degree of cold allodynia than LPA5+/+ (Fig. 2c: 3-way RM anova for genotype F1,42 = 4.63, P < 0.04; for condition F1,42 = 93.54, P < 0.001, no significant interaction effect)..

General activity, neuromotor and sensorimotor assessment

Cage activity recording revealed significantly increased nocturnal activity in LPA5−/− mice compared to LPA5+/+ (F1,38 = 4.76, P < 0.05; Fig. S5a). LPA5−/− mice performed better on the accelerating rotarod and had on average a later fall-off latency than LPA5+/+ (Fig. S5b; F1,38 = 4.77, P < 0.05). No difference between genotypes was observed in the grip strength test (462 ± 16 mN and 490 ± 18 mN for LPA5+/+ and LPA5−/− mice, respectively).

Both groups show similar startle behavior to loud sound bursts. Increasing levels of startle sounds increased significantly the startle response with a threshold of 105 dB in both groups (Fig. S5c, F9,324 = 42.8; P < 0.001). No significant effect of genotype or interaction genotype × intensity was found in the maximum startle response. Similarly, a significant effect for stimulus intensity in latency to startle was found (Fig. S5d, F9,324=57.99; P < 0.001), irrespective of genotype.

Exploratory activity and anxiety measures

In the open field task (Fig. 3a–d), LPA5−/− mice displayed signs of increased locomotion and reduced anxiety. In accordance with their increased nocturnal cage activity, LPA5−/− mice covered more distance during their stay in the arena (t38 = 2.09, P < 0.05; Fig. 3a). During the tracking period, LPA5−/− mice stayed significantly longer in the center of the arena (t38 = 2.45, P < 0.02, Fig. 3b) and with a shorter initial delay compared to LPA5+/+ (t38 = 3.27, P < 0.005, Fig. 3c). They spent significantly less time along the walls of the arena (thigmotaxis; 598 ± 1.1 seconds and 563 ± 12 seconds for LPA5+/+ and LPA5−/−, respectively, t38 = 2.95, P < 0.01) but showed more exploratory rearing bouts (Fig. 3d, t38 = 2.95, P < 0.01).

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Figure 3. Inactivation of LPA5 reduces anxiety in open field exploration. LPA5−/− mice (open bars, n = 20) were significantly more active (a and b), spent more time in the center (c) and ventured quicker toward the center (d) than LPA5+/+ mice (black bars, n = 20). Data are presented as mean ± SEM. *P < 0.05, ** P < 0.01 student's t-test comparison.

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In the social exploration task, LPA5−/− mice displayed signs of decreased social exploration with similar locomotor activity (path length 16.1 ± 2.3 m and 13.56 ± 1.9 m for LPA5+/+ and LPA5−/−, respectively, t38 = 0.85). Indeed, they spend marginally less time in the center of the arena that now contained a cage with two unknown mice (Mann–Whitney U = 134.5, P = 0.057, Fig. 4a), and remained close to the walls; however, the contrast was not significant compared to LPA5+/+ (U = 164, P = 0.33, Fig. 4b). In LPA5+/+ mice, with similar general locomotion compared to LPA5−/−, several social exploration measures were increased in comparison to the open field test illustrating their inquisitive response to the mice in the arena center. In contrast, LPA5−/− mice still moved around, but displayed little social exploration.

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Figure 4. Inactivation of LPA5 reduces social exploration. LPA5−/− mice (open bars, n = 20) spent less time close to the cage (a) and remained closer to the walls (b) than LPA5+/+ mice (black bars, n = 20). Data are presented as mean ± SEM.

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The anxiolytic phenotype of the LPA5−/− mice was further confirmed in the elevated plus maze. No differences were found in general exploratory activity measures such as total arm entries (105 ± 9 and 105 ± 7 in LPA5+/+ and LPA5−/− mice, respectively). However, anxiety-related measures such as time spend in open arms (Fig. 5a, t37 = 2.66, P < 0.02) or the ratio of closed/open arm entries (Fig. 5b, t37 = 2.57, P < 0.02) were significantly altered in LPA5−/− mice.

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Figure 5. Inactivation of LPA5 reduces anxiety in the elevated plus maze. LPA5−/− mice (open bars, n = 20) ventured more frequently into open arms (a) and showed increased ratio of open/closed arm visits (b) than LPA5+/+ mice (black bars, n = 20). Data are presented as mean ± SEM. *P < 0.05 student's t-test comparison.

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In contrast, no difference in defensive burying behavior was observed between the two genotypes (8 ± 1.4 and 11 ± 1.4 marbles buried for LPA5+/+ and LPA5−/−, respectively).

Learning and memory tasks

In passive avoidance learning, step-through latency was not significantly different between genotypes, neither during the initial training phase (22.6 ± 7 seconds and 8.4 ± 1.3 seconds for LPA5+/+ and LPA5−/− mice, respectively), nor 24 h later during the memory-related test phase (273 ± 18 seconds and 231 ± 28 seconds for LPA5+/+ and LPA5−/− mice, respectively). In the fear conditioning protocol (Fig. S6a), LPA5−/− mice displayed significantly less freezing than controls during the baseline exposure to the training cage prior to fear conditioning (F1,38 = 7.93; P < 0.01), which might further relate to their hyperactivity/anxiolytic phenotype. Exposure to tone-shock stimuli induced freezing behavior to the same extent in both groups. Twenty-four hours later, both groups showed robust fear memory-related freezing to both the context (i.e., cage environment) and the tone (CS).

In the spatial navigation test of the Morris water maze, we found some subtle spatial learning and memory impairments between the two genotypes (two LPA5+/+ mice were removed from analysis because of persistent floating). During acquisition training (Fig. 6a), both groups learned to locate the hidden platform (indicated as reduction in cumulative distance to the platform location). RM anova indicated a significant effect for session (F9,306 = 32.61, P < 0.0001) and significant interaction genotype × session (F9,306 = 1.98, P < 0.05) without a significant effect for genotype. However, the comparison of the learning curve using an exponential curve fit [Y = Y0 × exp(−k × X)] indicated that LPA5−/− reached a better performance level than LPA5+/+ (comparison of curve fitting parameters: F3,354 = 6.064, P < 0.001). When we analyzed the individual swim paths according to three main search strategies (Fig. 6b, spatial, non-spatial and repetitive looping), we found a significant increase in spatial based strategies in LPA5−/− during acquisition compared to LPA+/+ mice (Fig. 6c, RM anova for factor genotype and repeated factor trial: genotype F1,36 = 4.35, P < 0.05, trial F9,28 = 15.73, P < 0.001, genotype × trial F9,28 = 2.57, P < 0.03). Interspersed probe trials after 5 and 10 acquisition sessions indicated that both genotypes show similar significant preference for the target quadrant (Fig. 6e). When a mouse is placed inside the pool, it will, after a brief orientation, swim straight toward the position the platform should be. So we compared the swim behavior in the first 25 seconds of the second probe trial using heat plot analysis where dwell frequency is indicated by coloration from red through blue (Fig. 6d). While the swim trajectory from the release point to the target position is similar in both genotypes, LPA5−/− were swimming directly toward the target position and searched close by for the platform (dark red areas), while the distribution for LPA5+/+− shows a more wide spread pattern and reduced accuracy for the target. Similarly, we compared the frequency of platform crossings during the first 25 seconds between the genotypes (Fig. 6f) using a two-way RM anova (factor genotype as between measure and probe as within measure). We found no effect for factor probe and a marginal effect for genotype (F1,36 = 3.38, P = 0.07), without interaction.

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Figure 6. Spatial acquisition in the Morris water maze. (a) During spatial acquisition, LPA5−/− mice (open symbols, n = 20) reached a better performance level (cumulative distance to platform) when compared to LPA5+/+ (black symbols, n = 18). (b) Path analysis according to search strategies showed that LPA5−/− mice employ increasingly spatial (blue) strategies, while non-spatial (green) and repetitive looping (white) strategies were used to a lesser extent. (c) The comparison of spatial strategies use was significantly increased in LPA5−/− mice (white symbols) compared to LPA5+/+ (black symbols). (d) Heat plot analyses of the first 25 seconds of the second probe trial reflect the increase accuracy in search strategy for LPA5−/− mice compared to LPA5+/+. (e) However, probe trials to test for quadrant preference did not show a genotype difference. (f) Similarly, frequency of target location crossings during the first 25 seconds of each probe trial was marginal increased in LPA5−/− mice compared to LPA5+/+ (two-way RM anova F1,36 = 3.38, P = 0.07). Results for spatial acquisition are averaged over four daily trials and plotted as sessions. Results for probe trials are averages over 100 seconds (e) or the first 25 seconds (f). Data are presented as mean ± SEM.

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During reversal training, the platform was placed in the opposite quadrant and animals had to readjust their spatial memory to locate the novel platform position. Cumulative distance to the platform decreased over trials, but LPA5−/− mice were significantly more efficient to navigate to the novel positions (Fig. 7a). RM anova indicated a significant effect for genotype (F1,144 = 17.78, P < 0.0005), for trial (F4,144 = 15, P < 0.0001) as well as a significant interaction (F4,144 = 2.56, P < 0.05). Analysis of swim speed (not shown) or thigmotaxis (Fig. 7b) revealed no difference between the genotypes during reversal trials.

image

Figure 7. Reversal learning in the Morris water maze. During reversal learning, LPA5−/− were faster adjusting to the new platform location and reducing the cumulative distance (a), while thigmotaxis (expressed as % of total duration) was not affected by genotype (b). For reversal learning, individual swim trials were averaged by genotype. Data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001 indicates significant differences between LPA5−/− and LPA5+/+ mice (RM anova, post-hoc comparison with Bonferroni correction).

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Scheduled appetitive conditioning

During appetitive conditioning, all mice learned to poke for food rewards and increased responding over consecutive schedules (Fig. S6b, RM anova F5,185 = 166.9, P < 0.0001). However, LPA5−/− mice reached a higher response rate than LPA5+/+ mice (genotype effect: F1,37 = 8.58, P < 0.01; and genotype × schedule interaction: F39,185 = 3.13, P < 0.01). While during CRF schedule both genotypes responded equally, LPA5−/− mice showed increased responding with FR, VR and VI schedules. During the final VI30 trials (seven sessions in total), nose poke rates still increased until they reached a plateau in both groups.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

In the present study, we examined the possible involvement of LPA5 in various behavioral functions using mice with targeted deletion of this receptor. The significance of LPA5 for nociceptive signaling was examined by using a test battery that models various clinically relevant pain conditions. This included thermally and mechanically induced acute nociceptive responses and pain responses after inflammation or neuropathy. Furthermore, we investigated the role of LPA5 in a wide-range of tests focused on social and emotional behaviors as well as various aspects of cognitive performances.

LPA5-deficient mice showed decreased sensitivity in the TWR, which indicates a possible role of LPA5 in thermal nociception based on spinal reflexes. Similarly, LPA1-deficient mice displayed increased tail flick latencies compared to controls (Santin et al. 2009). In contrast, responses to radiant heat in HT that rely on reflexive (withdrawal) and organized (licking) behavior, as well as the HP test that is mainly based on organized responding (licking/shaking), were not significantly different between genotypes.

The possible contribution of LPA5 to inflammation-evoked responses was assessed by different tests. Formalin-induced biphasic flinching and carrageenan-induced thermal hyperalgesia were unaltered, whereas CFA-induced thermal hyperalgesia was reduced in LPA5-deficient mice. Immediately following local tissue damage, peripheral nerve endings are exposed to mediators that induce hypersensitivity to subsequent stimulation (Jones 2001). If the painful stimulus or inflammatory state is prolonged, several adaptive changes may occur along the pain pathway. The alterations in CFA-induced thermal sensitivity in LPA5-deficient mice could be attributed to changes in LPA5-expressing C fibers or elsewhere in the peripheral or central nervous system (Kotarsky et al. 2006; Oh et al. 2008). However, the inconsistencies between the different test models indicates that LPA5 might be involved in some, but not all sensitization processes. Also, Kinloch & Cox (2005) predicted a strong role of the LPA5 receptor in various forms of neuropathic pain, which is confirmed by changes in cold allodynia in LPA5-deficient mice reported here. However, there were again inconsistencies between different measures, which indicates that LPA5 may play a rather subtle or modulatory role in these forms of pain. Furthermore, it is noteworthy, that the 129 genetic background in the LPA5 might have blunted possible subtle effects in pain-related behavior (Leo et al. 2008).

More consistently across testing procedures, LPA5-deficient mice displayed an anxiolytic phenotype. This was shown by several observations including increased exploration of the central area and reduced thigmotaxis in the open field and increased open arm exploration in the elevated plus maze. It could also account for the marginally reduced step-through latency during passive avoidance training and reduced baseline freezing in the fear conditioning procedure. These changes could be attributed to reduced vigilance or general emotional blunting in LPA5-deficient mice, but results of neuromotor tests, startle response recording and defensive marble burying illustrate that these mice are still able to respond efficiently to some environmental stimuli. Interestingly, the observed anxiolytic phenotype is the opposite of the observed anxiety-like phenotype in LPA1-deficient mice (Castilla-Ortega et al. 2010; Santin et al. 2009). LPA1-deficient mice reportedly were more anxious in open field exploration and elevated plus maze. This anxiolytic phenotype in LPA5 deficient mice did not generalize to social exploration, because our task could be interpreted as conflict resolution in encountering novel conspecifics that might include elements of anxiety. Indeed, presentation of two novel mice increased center directed exploration in LPA5+/+ mice, but failed to have an effect in LPA5−/− mice. While social approach behavior can be influenced by a variety of non-anxiety related variables, such as differences in olfactory processes and differences in visual acuity, it also includes an aspect of social interaction motivation. Interestingly, this finding seems to relate specifically to social behavior because in other motivational tests LPA5-deficient mice rather displayed higher responses (e.g., appetitive motivated operant conditioning). Furthermore, in the Morris water maze, LPA5-deficient mice displayed improved spatial accuracy and search strategies, which was apparent in the initial and in the reversal learning. In contrast, LPA5-deficient mice failed to show changes in passive avoidance learning and fear conditioning.

Overall, these observations indicate that LPA5-deficient mice, which are otherwise functioning quite normally, specifically display changes in emotional and motivational behaviors. Little is known about the behavioral significance of LPA receptors in general, except for LPA1, which is still the most extensively studied LPA receptor. Mice lacking LPA1 displayed defects in rotarod performance, open field and elevated plus maze exploration, and in water maze and hole board learning (Castilla-Ortega et al. 2010; Santin et al. 2009). Interestingly, LPA1-deficient mice displayed a behavioral phenotype that is somewhat a mirror image of that described here in LPA5-deficient mice. For example, LPA1-deficient mice display balance defects, hypoactive open field exploration and increased anxiety-related behavior in the elevated plus maze test, whereas LPA5-deficient animals had improved balance on the rotarod, were clearly less anxious, and showed reduced fear responses compared to controls. Also, LPA1-deficient mice failed to acquire a spatial strategy to solve the hidden-platform water maze, while LPA5-deficient animals showed a more efficient search strategy than controls. Some of these observed differences between LPA1 and LPA5 might be because of their differential expression pattern during neurodevelopment (Ohuchi et al. 2008). In particular, in the developing mouse brain (E9.5−12.5) LPA5 expression appeared to be diffuse, with higher expression in areas of the prospective forebrain and rostral midbrain (Ohuchi et al. 2008), while LPA1 expression was observed predominantly in the rostral forebrain. Rostral midbrain areas and in particular mesolimbic dopamine neurotransmission have been linked to motivational behavior (Li et al. 2010).

In summary, mice lacking the LPA5 receptor showed decreased sensitivity to acute pain stimuli in tail withdrawal latency, faster recovery in CFA-induced inflammatory pain procedure, and attenuated responses under some neuropathic pain conditions. LPA5 deletion also induced nocturnal hyperactivity, anxiolytic effects, increased appetitive responding and reduced social exploration. While learning and memory performance was unaffected, we found an increase in behavioral flexibility and motivational behavior, such as improved targeted swim strategy and scheduled appetitive conditioning. These results imply that LPA5 might be involved in nociception, anxiety-related and motivation driven behaviors, which further supports the proposed involvement of LPA signaling in psychopathology.

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  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

The authors wish to thank Leen van Aerschot and Adrian Lo for technical assistance. Z.C.V. and S.L. were funded by a research agreement between Janssen Pharmaceutica and Leuven Research & Development (LRD), and an R&D grant of the Flemish science and technology fund IWT. B.V. was funded by a project grant of the Flemish research organization (FWO, grant G.0562.10). Additional funding was provided by F.W.O. (grant G.0327.08) and K.U.Leuven Research Council (IDO 06/004). The authors declare no conflict of interest or financial interest.

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information
FilenameFormatSizeDescription
gbb840-sup-0001-appendixS1.docWord document69K Appendix S1: Generation of LPA5 gene knockout mice.
gbb840-sup-0002-FigureS1.tifTIFF image2224K Figure S1: Thermal nociception between LPA5+/+ and LPA5−/− mice. (a) In the hot plate test (n = 22), there were no differences between LPA5+/+ (black symbols) and LPA5−/− (white symbols). (b) The test of Hargreaves (n = 12) displayed similar sensitivity for radiant heat in both genotypes. Bars represent mean response latencies (± SEM) following thermal stimulation.
gbb840-sup-0003-FigureS2.tifTIFF image2049K Figure S2: Pain response after chemical-induced inflammation between LPA5+/+ and LPA5−/− mice. Reaction to drugs (circle symbols) was compared to vehicle injections (square symbols). (a) After formalin challenge (n = 12), LPA5+/+ (closed bars) and LPA5−/− mice (open bars) displayed similar flinching behavior. (b) In the carrageenan test (n = 18), both LPA5+/+ (closed symbols) and LPA5−/− mice (open symbols) developed profound thermal hyperalgesia after carrageenan-injection, whereas NaCl-injected mice did not. (c) Complete Freud adjuvants injection induced paw swelling in both LPA5+/+ and LPA5−/− mice. At 4-h post-injection, paw edema was more pronounced in LPA5−/− mice compared to LPA5+/+ mice. Data are presented as means ± SEM. *Indicates significant differences between conditions , i.e., carrageenan or CFA injection vs. NaCl injection (*P < 0.05, **P < 0.01). #Indicates significant differences between LPA5+/+ and LPA5−/− mice (#P < 0.05, ##P < 0.01).
gbb840-sup-0004-FigureS3.tifTIFF image3173K Figure S3: Neuropathic pain behavior in LPA5+/+ and LPA5−/− mice after spared (tibial) nerve injury (SNI, a–c). After SNI (n = 3–9), the extent of mechanical and cold allodynia as well as changes in weight bearing were similar between genotypes. (a) SNI (circle symbols) induced robust mechanical allodynia in both LPA5+/+ (black symbols) and LPA5−/− mice (white symbols) compared to sham operated animals (squares symbols). (b) The acetone test showed mild increased response times after injury (circle symbols) in both LPA5+/+ and LPA5−/− mice. (c) Both SNI-operated genotypes showed a clear weight shift to the right non-injured side.
gbb840-sup-0005-FigureS4.tifTIFF image1670K Figure S4: Neuropathic pain behavior in LPA5+/+ and LPA5−/− mice after chronic constriction injury (CCI) After CCI (circle symbols, n = 10–14), both LPA5+/+ (black symbols) and LPA5−/− mice (white symbols) developed mechanical allodynia and showed similar changes in weight bearing. (a) Both LPA5+/+ and LPA5−/− CCI-operated mice showed similar decreased mechanical thresholds from day 2 until day 21 post-surgery, whereas sham-operated mice (square symbols) stayed stable over time. (b) No significant genotype effects were found in the weight distribution after CCI. Again, both genotypes showed a clear weight shift to the right non-injured side. Data are presented as means ± SEM. *Indicates significant differences between conditions, i.e., SNI or CCI-operated vs. sham-operated animals (*P < 0.05, **P < 0.01).
gbb840-sup-0006-FigureS5.tifTIFF image498K Figure S5: General activity, neuromotor and sensorimotor assessment in LPA5 mice. LPA5−/− animals (white symbols) had increased nocturnal activity (a), and improved performance in 4 consecutive trials on the accelerating rotarod (b) compared to LPA5+/+ animals (black symbols). When presented with startle stimuli, similar startle amplitude (c) and latency (d) were observed between LPA5+/+ and LPA5−/− mice. Data are presented as means ± SEM.
gbb840-sup-0007-FigureS6.tifTIFF image2077K Figure S6: Contextual and cued fear conditioning and scheduled appetitive conditioning. (a) During fear conditioning, LPA5−/− mice (white symbols) were more active during baseline recording before the tone-shock presentations than LPA5+/+ mice (black symbols). Both groups displayed stable freezing response to shocks, context, unrelated context (PreCS) and cue (CS). No differences between groups were found in any other condition. (b) LPA5−/− mice reached during appetitive conditioning consistently higher nose poke levels over increasing rewarding schedules compared to LPA5+/+ mice. Data are presented as means ± SEM (n = 19–20 per genotype). *P < 0.05, **P < 0.01, student's t-test comparison.

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