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The protective/neurotoxic role of fractalkine (CX3CL1) and its receptor CX3C chemokine receptor 1 (CX3CR1) signaling in neurodegenerative disease is an intricate and highly debated research topic and it is becoming even more complicated as new studies reveal discordant results. It appears that the CX3CL1/CX3CR1 axis plays a direct role in neurodegeneration and/or neuroprotection depending upon the CNS insult. However, all the above studies focused on the role of CX3CL1/CX3CR1 signaling in pathological conditions, ignoring the relevance of CX3CL1/CX3CR1 signaling under physiological conditions. No approach to date has been taken to decipher the significance of defects in CX3CL1/CX3CR1 signaling in physiological condition. In the present study we used CX3CR1−/−, CX3CR1+/− and wild-type mice to investigate the physiological role of CX3CR1 receptor in cognition and synaptic plasticity. Our results demonstrated for the first time that mice lacking CX3CR1 receptor show contextual fear conditioning and Morris water maze deficits. CX3CR1 deficiency also affects motor learning. Importantly, mice lacking the receptor have a significant impairment in long term potentiation (LTP). Infusion with IL-1β receptor antagonist significantly reversed the deficit in cognitive function and impairment in LTP. Our results reveal that under physiological conditions, disruption in CX3CL1 signaling will lead to impairment in cognitive function and synaptic plasticity via increased action of IL-1β.
Microglia are the resident immune cells in the central nervous system (CNS) that act as macrophages (Aloisi, 2001;Harrison et al., 1998). Microglia can rapidly respond to the detection of homeostatic disturbances by inducing an immune response, which consists of a transient, up-regulation of inflammatory molecules and neurotrophic factors (Batchelor et al., 1999;Miwa et al., 1997;O’Donnell et al., 2002;Nakajima and Kohsaka, 2004). Through this immune response, microglia protect proper brain function and remove cells damaged from an acute injury. When chronic inflammation occurs, prolonged activation of microglia trigger the release of several neurotoxic products and proinflammatory cytokines including IL-1β, IL-6, and Tumor necrosis factor (TNFα) (Colton and Gilbert, 1987).
Microglia are restrained by numerous micro-environmental influences, many of which are produced by neurons (Hanisch and Kettenmann, 2007;Cardona et al., 2006;Lyons et al., 2007). Fractalkine (CX3CL1) is a chemokine that is constitutively expressed by healthy neurons and identified as a novel neuroimmune regulatory protein. CX3CL1 signals to microglia, which inhibits microglial activity under inflammatory conditions (Harrison et al., 1998;Ransohoff et al., 2007). In contrast to other chemokines, CX3CL1 binds to only one receptor, CX3CR1 (Harrison et al., 1998). In the brain CX3CR1 is exclusively expressed by microglia (Harrison et al., 1998). CX3CL1 can bind to CX3CR1 either as a membrane bound protein or a soluble ligand following constitutive cleavage by ADAM10 and ADAM17 metalloproteases (Bazan et al., 1997). Interactions between CX3CL1 and CX3CR1 contribute to microglial ability to maintain a resting phenotype. However, when neurons are injured, CX3CL1 levels decrease, which results in microglia recruitment and activation.
Through interactions with microglia, CX3CL1 serves as an endogenous neuronal modulator and controls the over-production of iNOS, IL-1β, TNFα and IL-6. Interestingly, CX3CL1/CX3CR1 signaling has been linked to human neurodegeneration, highlighted by the identification of the V249I and T280M polymorphisms in CX3CR1. These polymorphisms are associated with the neuroinflammatory disorder, human age-related macular degeneration (Tuo et al., 2004;Chan et al., 2005). Furthermore, mice that are deficient in CX3CR1 have increased microglial cell expression of IL-1β in response to lipopolysacharide (LPS) stimulation. This increased IL-1β expression is associated with increased neuronal cell death in the hippocampus (Ransohoff et al., 2007) and CX3CR1-deficent mice have increased susceptibility to neurotoxins such as MPTP (Cardona et al., 2006). However, all of the studies mentioned have focused on the role of CX3CL1/CX3CR1 signaling in pathological conditions, ignoring the relevance of CX3CL1/CX3CR1 signaling under physiological conditions. Our lab has previously established the impact of functional disruption of CX3CR1 in a non-pathological condition. We have demonstrated that CX3CR1−/− mice have decreased hippocampal neurogenesis (Bachstetter et al., 2009). Furthermore, antagonism of CX3CR1 in young rats leads to increased hippocampal protein levels of IL-1β and decreased neurogenesis. We hypothesize that disruption of CX3CL1/CX3CR1 signaling will result in alterations of the physiological activities of the brain. Here, we demonstrate under physiological conditions, disruption of CX3CL1/CX3CR1 signaling leads to impairments in motor learning, cognitive function and synaptic plasticity through increased inflammation in the CNS.
All experiments were conducted in accordance with the National Institute of Health Guide and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of the University of South Florida, College of Medicine or the University of Florida, as appropriate. CX3CR1−/− mice were backcrossed to the C57BL/6J background for greater than 10 generations and were obtained from Jackson Laboratories (Bar Harbor, Maine). Colonies of the CX3CR1+/− and CX3CR1−/− mice were maintained at the University of South Florida and genotyping performed as previously described (Bachstetter et al., 2009). Three month-old male CX3CR1+/− and CX3CR1−/− littermates and C57BL/6J (wild-type) were used in the experiments. Mice were pair-housed in environmentally controlled conditions (12:12 h light:dark cycle at 21 ± 1°C) and provided food and water ad libitum. Animals that developed skin lesions were excluded from the Morris water maze test to avoid the development of infection.
Three month-old male (n = 5/genotype) CX3CR1+/−, CX3CR1−/− littermates and C57BL/6 (wild-type; WT) received two intraperitoneal (i.p.) injections within 12 hours interval of bromodeoxyuridine (BrdU) (5-bromo-2-deoxyuridine; Sigma, St. Louis, MO) at dose of 50 mg/kg. Animals were euthanatized 24 hours following the last injection.
Three month-old male CX3CR1−/− and C57BL/6 (wild-type; WT) mice were implanted with a brain infusion cannula connected with an osmotic mini-pump. Before implantation, the pumps were incubated in sterile saline for at least 48 h at 37 °C to prime the pumps. For implantation, mice were anaesthetized with isofluorane and placed in a stereotaxic frame. A guide cannula was implanted in the left ventricle AP, 1.0; ML, −0.5; DV, 2.5 mm) and connected to the osmotic minipump (Alzet Model, 1004: pumping rate, 0.11 μl/h; total volume, 100 μl), which was inserted subcutaneously. Pumps were weighed before implantation and at the end of the experiment to ensure complete delivery of their content. r-metHu IL-1ra, 100 μg/ml/day (kind gift from Amgen,Thousand Oaks CA), was infused through the cannula which was connected to the filled mini-pump. The infusion started on the day of the surgery and continued for 4 weeks. Control animals received the same volume of heat-inactivated IL-1ra (heat-inactivated for 45 minutes in a water bath at 90°C) or artificial cerebrospinal fluid (ACSF). Behavioral analysis started 15 days following the beginning of the infusion. Mice that during the course of behavioral testing showed signs of distress or infections during to surgical procedure were not included in the following behavioral studies.
For immunohistochemistry studies, animals were anaesthetized with pentobarbital (50 mg/kg, i.p.). The mice were transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in PBS. The brains were postfixed in 4% paraformaldehyde for 12 h, after which they were transferred into 30% sucrose in PBS for at least 16 h at 4°C. Exhaustive sagittal sections of the left hemisphere were made at 40μm using a Microm cryostat (Richard-Allan Scientific, Kalamazoo Michigan) and stored in cryoprotectant at 4°C. Animals used for biochemical studies were euthanatized by rapid decapitation. Brain tissues were separately dissected and rapidly frozen in liquid nitrogen before storage at −80°C. Both hippocampi were homogenized using an electric tissue homogenizer in 1:10 weight to volume ratio of ice-cold RIPA buffer (Millipore; Billerica MA) containing protease inhibitors and EDTA (Pierce; Rockford IL). Following homogenization, sample lysates were centrifuged at 10,000 x g at 4°C for 15 minutes, the cleared supernatant was collected.
Total protein was measured using BCA assay (Pierce). IL-1β and TNFα concentrations were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kits (eBioscience; San Diego CA) following manufacturer’s protocol.
50 μg of total protein per lane was loaded onto a 10% SDS-polyacrylamide gel (BioRad; Hercules CA) for electrophoresis. Proteins were transferred onto a nitrocellulose membrane for immunodetection. The membrane was blocked for 1-hour in 5% non-fat dry milk (NFDM) in PBS-tween (PBS-T, 0.1%). Antibodies for rabbit phospho-p38 (Cell Signaling; Boston MA, 1:1000) and mouse beta-actin (Sigma; 1:3000) were incubated overnight at 4°C in 1% NFDM in PBS-T. Following washes, anti-rabbit and anti-mouse secondary antibodies (LiCor; Lincoln NE) were incubated for 1-hour at room temperature in 1% NFDM in PBS-T. Membranes were scanned using LiCor Odyssey Imager and raw intensity for each band was measured using LiCor Odyssey image analysis software.
Except where specifically indicated, standard staining procedures were conducted on free-floating sections using every sixth section for the entire hippocampus beginning with a random start and including sections before and after the hippocampus to ensure that the entire structure was sampled. Sections were blocked in 10% normal serum from the species that secondary antibody was raised in, with the addition of 0.1% Triton X-100. Sections were incubated, with primary antibody (Abcam anti Iba1 1:500) diluted in 3% normal serum with 0.1% Triton X-100, overnight at 4°C. For immunohistochemistry biotinylated secondary antibodies (horse anti-goat 1:2000) were diluted in 3% normal serum with 0.1% Triton X-100 and were incubated for 2 hours at room temperature. Enzyme detection was done using avidin-biotin substrate (ABC kit, Vector Laboratories, Burlingame, CA) followed by color development in diaminobenzidine solution (Sigma, St. Louis, MO). For immuno-detection of BrdU labeling, protocol was followed as previously described (Bachstetter et al., 2009;Gemma et al., 2007). Briefly, sections were pretreated with 50% formamide/2× SSC (0.3 M NaCl, 0.03 M sodium citrate) at 65°C for 2 hours, rinsed in 2 × SSC. BrdU was detected using a mouse anti-BrdU (1:100; Roche; Indianapolis IN, clone B44). Doublecortin (DCX) is a marker of migrating neurons that is expressed for approximately three weeks after the cell is born and has been shown to be a reliable indicator of neurogenesis (M. S. Rao and A. K. Shetty, 2004; S. Couillard-Despres et al., 2005). For DCX immuno-detection, incubation in primary antibody was done for 36 hours at 4°C using a polyclonal goat antibody C-terminus of human DCX (1:200; Santa Cruz biotechnology, Santa Cruz, CA, USA).
To determine cell numbers, the optical fractionator method of unbiased stereological cell counting techniques (M. J. West et al., 1991) was used with a Nikon Eclipse 600 microscope and quantified using Stereo Investigator software (MicroBrightField, Colchester, VT). The virtual grid (175 × 175) and counting frame (100 × 100) were optimized to count at least 200 cells per animal with error coefficients less than 0.07. Outlines of the anatomical structures were done using a 10x/0.45 objective and cell quantification was conducted using a 60x/1.40 objective.
Slides with Iba1 stained sections were scanned using a Zeiss Mirax slide scanner. Following scanning, digital images were analyzed for Iba1 positive staining using Zeiss Neuroquant IAE analysis software. Briefly, an investigator blind to the study randomly placed a measurement grid within the DG of each HPC section. Each measurement grid had the same dimensions and therefore same measurement area. Iba1 positive cells were defined in the analysis software based upon threshold to include both the cell body and processes, while eliminating background values. Data were obtained solely from the randomly placed measurement grid on an average of five sites per animal within the DG. Raw data were normalized to account for variations in the number of sampling sites. Results are given as the average ratio of Iba1 positive area relative to total measurement area.
Aged-matched 3 month old wild-type, CX3CR1−/−, and CX3CR1+/− mice were first tested for overall balance, motor coordination and motor learning. This test was performed on an accelerating rotarod apparatus (Ugo Basile, Italy) with a 3 cm diameter rod starting at an initial rotation of 4 RPM accelerating to 40 RPM over 5 minutes. Mice were tested for the time spent on the rod during each of four trials with a 30 min inter-trial interval. Each trial was completed when the mouse fell off the rod (distance of 12 cm) onto a spring-cushioned lever.
The open field was used as a standard test of general activity. Animals were monitored for 15 minutes in a 40 cm square open field with a video tracking software (ANY-Maze, Stoelting, Illinois), under moderate lighting. General activity levels were evaluated by determining the total amount of distance traveled. Anxiety levels were assessed by the pattern of exploration of in the open-field (center versus periphery).
Anxiety was also assessed through the elevated plus maze (EPM). The EPM consisted of two well-lit open arms (35 cm) facing each other and two enclosed arms (30.5 cm) also facing each other. Each arm is attached to a common center platform (4.5 cm square) and elevated 40 cm off the floor. The mouse was placed in the center platform and allowed to explore for 5 min. Video tracking software measured movement in each section (ANY-Maze, Stoelting, Illinois).
Fear conditioning was used to assess memory formation that is especially suited to test for proper hippocampus function. Animals were placed in the fear conditioning apparatus (Panlab, Spain) for 2 minutes, then a 30 second acoustic conditioned stimulus (CS; 80 dB tone) was delivered and a 0.5 mA shock (unconditioned stimulus-US) was applied to the floor grid during the last 2 seconds of the CS. Training consisted of two CS-US pairings with a 1.5 minute interval between each. The mice were placed in the chamber and monitored for freezing to the context 24 hours after training (no shocks or conditioned stimulus given). Immediately after the contextual test, mice were placed into a novel context and exposed to the CS for 3 minutes (cued fear conditioning). Learning was assessed by measuring freezing behavior (i.e. motionless position) for 2 consecutive seconds. Shock threshold was assessed by placing an animal in the same conditioning chamber as used for associative fear conditioning. Foot shock intensity started at 0.075 mA and was increased by 0.05 mA every 30 s. The shock intensity to induce flinching, jumping, and vocalization were determined and the shock intensity was recorded. The experiment was terminated at the shock intensity sufficient to induce vocalization.
The Morris hidden platform water maze (MWM) consisted of a circular pool (1.38 m diameter) filled with non-toxic opaque water at room temperature with an escape platform (10 cm diameter) hidden beneath the water (3 cm). Each mouse was placed in the pool in a pseudorandom order and given 60 seconds to locate the escape platform. When the mouse found the platform or if the mouse failed to find the platform within 60 seconds, it was placed on the platform where it remained for 30 seconds. Each mouse was given four trials per day with an inter-trial interval of 1 hour. The time to find the platform (escape latency), the total distance traveled, and the swim speed of the animals were recorded by video tracking software (ANY-Maze, Stoelting, Illinois). The mice were then towel-dried and placed in a cage with a heating pad underneath until dry and returned to their home cage. On day 7, all mice were subjected to one probe trial in which the platform was removed and each animal had 60 seconds to search the pool for the platform. Since wild-type mice did not show improvement in learning ability to find the platform on day 7, a second probe trial was performed on day 11, following 3 additional continuous days of training.
Mice were euthanatized by rapid decapitation and hippocampi were dissected out for electrophysiological experimental paradigms as previously described (Beffert et al., 2005;Peters et al., 2006;Weeber et al., 2002). Field excitatory post-synaptic potentials (fEPSPs) were obtained from area CA1 stratum radiatum with the use of a glass microelectrode filled with ACSF (2–4 mΩ). fEPSPs were evoked through stimulation of the Schaffer collaterals using a 0.1 ms biphasic pulse delivered every 20 s. After a consistent response to a voltage stimulus was established, threshold voltage for evoking fEPSPs was determined and the voltage was increased incrementally every 0.5 mV until the maximum amplitude of the fEPSP was reached (I/O curve). All other stimulation paradigms were induced at the same voltage, defined as 50% of the stimulus voltage used to produce the maximum fEPSP amplitude, for each individual slice. Paired-pulse facilitation (PPF) was induced with two paired-pulses given with an initial delay of 20 ms and the time to the second pulse incrementally increased 20 ms until a final delay of 300 ms was reached.
Slices that received IL-1ra (100 μg/ml kind gift from Amgen, Thousand Oaks CA) were bathed in ACSF containing IL-1ra for at least 15 min prior to the start of recordings. A fEPSP LTP baseline response was recorded every 20 sec for 20 min. The tetanus used to evoke LTP was high-frequency stimulation (HFS), which consisted of 2 trains of 1 sec, 100 Hz pulses, with an inter-train interval of 20 s. Following HFS, fEPSPs were recorded for 60 min. The slopes of fEPSPs were averaged into 1 min bins and graphed. Potentiation was measured as the increase of the mean fEPSP descending slope following TBS normalized to the mean fEPSP descending slope of baseline recordings.
All hippocampal slices were bathed in ACSF containing 50 uM APV for at least 15 min prior to recordings. Slices that received IL-1a were bathed in ACSF containing IL-1ra for at least 15 min prior to the start of recordings. A fEPSP PTP baseline response were recorded every 3 sec for 1 min. The tetanus used to induce PTP was high-frequency stimulation (HFS), which consisted of a 1 sec, 100 Hz pulses. Following HFS, fEPSPs were recorded every 3 sec for 5 min. The slopes of fEPSPs were averaged into 12 sec bins and graphed. Potentiation was measured as the increase of the mean fEPSP descending slope following PTP normalized to the mean fEPSP descending slope of baseline recordings.
Mice were sacrificed and hippocampi were dissected out for DAPI staining in the exact same manner as electrophysiological experimental paradigms. Hippocampal slices were placed in a submerged holding chamber containing oxygenated ACSF for no less than two hours before being transferred to 4% PFA for fixation for approximately 16 hours. Following fixation, 40 μm sections were made through entire dissected region and mounted on superfrost slides. Visualization of cell nuclei was achieved using DAPI VectaShield hardest mounting media (Vector Labs, Burlingame, CA). Fluorescent images were taken using Olympus FluoView FV1000 confocal microscope. Hippocampal DAPI positive cells were quantified from 16-bit grayscale images using ImageJ software for each of the three genotypes (n=5 per group).
Statistical analysis comparing the 3 genotypes was done using a one-way analysis of variance (ANOVA) or two-way ANOVA with the Tukey-Kramer or Bonferroni post-hoc test,. Repeated Measure ANOVA was used to analyze Rotarod data, followed Bonferroni post-hoc. Statistical analysis was done in the GraphPad Prism software.
Previously, we demonstrated that CX3CR1−/− young mice have a significant decrease in hippocampal neurogenesis when compared to heterozygous littermates. To determine whether both alleles are required for normal receptor function, we counted the number of doublecortin (DCX) positive cells in the subgranular zone (SGZ) of the dentate gyrus in CX3CR1−/−, CX3CR1+/−, and wild-type mice. Using the optical fractionator method of design-based stereology, we observed a significant decrease in the number of DCX+ cells in the SGZ of the dentate gyrus in CX3CR1−/− mice compared to CX3CR1+/− and wild-type mice (Figure 1a; one-way ANOVA, p< 0.0001). Interestingly, loss of one allele is sufficient to impair receptor function (Tukey’s post-hoc; wt vs +/− , p< 0.001; wt vs −/−, p< 0.001; +/− vs −/−, p< 0.05). To determine if this decrease is due to reduced cell proliferation, mice were injected twice (8 hours interval) with BrdU (50mg/kg). Mice were euthanized the following day. Quantification of the number of BrdU+ cells revealed a significant decrease in the number of BrdU+ cells in the CX3CR1−/− mice compared to the CX3CR1+/− and wild-type mice, indicating reduced cell proliferation (Figure 1b; Tukey’s post-hoc; wt vs +/−, p< 0.05; wt vs −/−, p< 0.001; +/− vs −/−, p< 0.05).
Coordination and motor skill acquisition were tested using an accelerating rotorod. The amount of time an animal stays on the rotorod is an indicator of its general level of balance and coordination. In general, mice improve their performance over time with training, which is an indicator of motor learning. In the first study, aged-matched littermate wild-type (n = 10), CX3CR1−/− (n = 12) and CX3CR1+/− (n = 12) mice were tested for the time spent on the rod during each of four trials per day, for two consecutive days. No differences were observed between genotypes during the first day of training (Fig 2a). On the second day of training (trials 5-8), wild-type mice remained on the rod for longer periods of time. However, neither the CX3CR1−/− or the CX3CR1+/− mice significantly improvement motor coordination on the second day of training (Figure 2a; repeated measures ANOVA, p<0.0001.). Wild-type mice performed significantly better on trial 8 compared to trial 1 than did CX3CR1−/− and CX3CR1+/− mice (Figure 2b), suggesting motor learning deficits in CX3CR1−/− and CX3CR1+/− mice. There were no significant differences on the first trial of testing between all experimental groups, which suggests that there is no difference in baseline motor skills. Taken together, these data suggest that CX3CR1−/− and CX3CR1+/− mice have deficits in motor learning, indicating that cerebellar or striatal-dependent learning may be compromised in these mice.
To examine spontaneous locomotor activity in response to a novel environment, CX3CR1−/−, CX3CR1+/− and wild-type mice were tested in the open field behavioral task. The open field task monitors activity in a brightly lit, novel environment and is a necessary control for interpretation of associative fear conditioning. Spontaneous locomotor activity was assessed as the total amount of distance traveled in the chamber. Also, anxiety levels can be measured in the open field task through assessment of the distances traveled in the center versus perimeter of the chamber. This task exploits the natural tendency of mice to avoid open areas. There were no significant differences in total distance traveled (Figure 2c). In addition, no differences were observed between distance traveled in the center zone compared to the perimeter zone and the time spent in each zone (data not shown). Anxiety levels were also measured using elevated-plus maze task. Mice generally prefer to be in the closed arms of the maze, but their natural curiosity compels them to explore their environment. Their level of anxiety was determined by the amount of time spent exploring the open arms of the maze versus the time spent in the closed arms. CX3CR1−/− and CX3CR1+/− spent similar amounts of time in the open arms and in the center zone compared to wild-type mice (data not shown). Taken together, the absence of the CX3CR1 receptor does not impair spontaneous locomotor activity nor cause excessive anxiety-like behavior.
Hippocampal-dependent associative learning and memory was assessed in CX3CR1−/−, CX3CR1+/− and wild-type mice using contextual fear conditioning (CFC) and cued fear conditioning. Animals were trained with a standard two-shock protocol as previously described (Weeber et al., 2002). Freezing to the context (hippocampal-dependent) or to the conditioned stimulus in a novel context (hippocampal- and amygdala-dependent) was used as an index of memory formation. During training, CX3CR1−/− and CX3CR1+/− displayed similar freezing behavior compared to wild-type mice (Figure 3a). When placed in the context twenty-four hours following training, CX3CR1−/− and CX3CR1+/− show a decrease in freezing to the context compared to wild-type mice (Figure 3b; One-way ANOVA, Bonferroni post-hoc = p< 0.001). Interestingly, CX3CR1+/− mice were significantly different from the CX3CR1−/− mice, further suggesting that the loss of one allele is sufficient to affect receptor function. When placed in the novel environment and presented with the conditioned stimulus, there were no significant differences between CX3CR1−/−, CX3CR1+/− and wild-type mice, suggesting that the behavioral deficits may be limited to hippocampal-dependent behaviors (Figure 3c).
To further assess hippocampal-dependent learning and memory, the same group of mice was tested in the hidden platform water maze (HPWM) task. There were no significant differences in escape latencies between experimental groups during training (Figure 3d). However, both CX3CR1 −/− and CX3CR1+/− mice had a significant decrease in the number of target platform crossings during the probe trial compared to wild-type mice (Figure 3e; One-way ANOVA followed by Bonferroni post-hoc: p<0.05). No differences were observed between experimental groups on the visible platform test (data not shown). These data further support the role of the CX3CR1 receptor to modulate hippocampal-dependent learning and memory.
LTP is the most widely studied cellular model of memory. The observation that CX3CR1 −/− and CX3CR1 +/− mice have deficiencies in both associative and spatial memory led us to determine whether the hippocampal-dependent memory deficits observed in these mice correlate with a reduction in synaptic plasticity. Both CX3CR1 −/− and CX3CR1 +/− mice showed significantly reduced hippocampal-dependent LTP compared to wild-type controls (Figure 4a-c). Interestingly, examination of basal synaptic transmission revealed no differences between experimental groups (data not shown), suggesting that the decrease in LTP may be due to post-high frequency stimulation-dependent cellular mechanisms and may not represent differences in overall synaptic transmission. Additionally, short-term plasticity evaluated by paired-pulse facilitation (PPF) is found to be normal in these mice, further suggesting that presynaptic function is not affected through decreased or absent levels of CX3CR1 (Figure 4c; one-way ANOVA followed by Tukey’s post hoc: *, p<0.01).
It has been shown that hippocampal slice preparations enhance microglial activation (Haynes et al., 2006). Given the well-characterized effects of CX3CR1 signaling in modulating microglial activation, it was important to examine the hippocampal slice preparations for baseline alterations in microglial activation and/or neurotoxicity between WT, Cx3cr1+/−, and Cx3cr1−/− genotypes. DAPI staining showed no difference between genotype in microglia alteration and or neurotoxicity (data not shown)
The IL-1 receptor antagonist, IL-1ra, is the naturally occurring receptor antagonist of IL-1β (Rothwell et al., 1997;Dinarello, 1997). Recently, we demonstrated in young rats that infusion of IL-1ra completely reverses the decrease in hippocampal neurogenesis induced by the co-administration of CX3CR1 blocking antibody (Bachstetter et al., 2009). Next, we wanted to determine if basal levels of hippocampal IL-1β were altered in CX3CR1−/− mice. We analyzed the hippocampus of these mice for protein expression of IL-1β. Hippocampi from CX3CR1−/− and CX3CR1 +/− mice have increased IL-1β expression compared to wild-type mice (Figure 5a; One-way ANOVAs, p<0.01).
The MAP kinase p38 is a key signal transduction factor involved in the production of IL-1β and TNFα (Raingeaud et al., 1995;Rouse et al., 1994;Guesdon et al., 1993). Due to the importance of p38 in IL-1β signaling, we wanted to determine whether hippocampal levels of phospho-p38 were altered in CX3CR1 deficient mice. Not surprisingly, phospho-p38 protein levels were significantly increased in the hippocampus of CX3CR1−/− and CX3CR1+/− mice when compared to wild-type mice (Figure 5b). CX3CR1−/− mice have impaired motor learning that may represent cerebellar dysfunction. We next determined whether deficiencies in CX3CR1 affected cerebellar TNFα protein levels. TNFα expression was significantly increased in CX3CR1−/− mice compared to wild-type (Fig 5c). Taken together, CX3CR1 deficiency leads to increased expression of IL-1β, p38 and TNFα, suggesting increased inflammation of the CNS.
Loss of CX3CR1 leads to over-activation of microglia following an insult. To determine under basal conditions whether lack of CX3CR1 receptor leads to increased activation of microglial cells, we immunostained hippocampal sections from CX3CR1 −/−, CX3CR1+/− and wild-type mice for Iba-1, a marker for both resting and activated microglia. The area of Iba-1 staining is significant higher in CX3CR1−/− when compared to wild-type and CX3CR1+/− mice (Figure 5d, p < 0.05, CX3CR1−/− vs wild-type ), reflecting an increased volume of microglial cells reflective of morphology associated with activated microglia.
Increased levels of IL-1β negatively modulate induction and maintenance of LTP (Cunningham et al., 1996;Kelly et al., 2003). Because mice lacking the CX3CR1 receptor have increased levels of IL-1β, we determined if the IL-1 receptor antagonist, IL-1ra, reverses the impairment in synaptic plasticity observed in the CX3CR1−/− mice. To determine if IL-1ra can improve synaptic plasticity in CX3CR1 deficient mice, hippocampal slices were perfused with varying concentrations of IL-1ra and post-tetanic potentiation was induced. In agreement with the LTP results, hippocampal slices from CX3CR1−/− mice had deficits in PTP compared to wild-type mice (Figure 6a). However, when hippocampal slices from CX3CR1−/− mice were perfused with 100 μg/ml of IL-1ra, the deficit in PTP was rescued (Figure 6a). Blocking the function of IL-1β completely reversed the impairment in PTP. Our observation that PTP in hippocampal slices from CX3CR1−/− was rescued with perfusion of IL-1ra prompted us to determine if the long-lasting LTP deficit in CX3CR1−/− mice could be rescued as well. Perfusion of 100 μg/ml of IL-1ra to hippocampal slices from CX3CR1−/− completely rescued the deficit in LTP induction and maintenance (Figure 6b). Taken together, these results suggest that the deficits seen in CX3CR1−/− are due to increased levels of IL-1β, further implicating a detrimental increase of inflammation in the CNS.
To determine whether the impairment in motor learning and cognitive function was also dependent on IL-1β , we performed a second study in which wild-type and CX3CR1−/− mice were infused with either IL-1ra, heat-inactivated IL-1ra, or ACSF. In agreement with the first study, mice were tested starting from the least invasive task (open field) to the most invasive task (Morris water maze(McIlwain et al., 2001).
Although in the first study there were no significant impairments in general locomotor activity, explorative behavior and anxiety levels of the CX3CR1 deficient mice and wild-type, it was crucial to retest the performance of new groups of mice, to exclude the possibility that the surgery procedure could impair general motor activity or enhance anxiety levels . Wild-type mice infused with either ACSF (n = 11), heat-inactivated IL-1ra (n = 12),IL-1ra (n = 11), and CX3CR1−/− infused with either ACSF (n = 14) or IL-1ra (n = 13) were first tested in the open field behavioral task. Spontaneous locomotor activity was assessed as the total amount of distance traveled in the chamber. As observed in the first study there were no significant differences between genotypes in total distance traveled. Furthermore, no significant differences were observed between treatments (data not shown). In addition, the distance traveled in the center zone compared to the perimeter zone and the time spent in each zone was similar between groups (data not shown). Anxiety levels were also measured using elevated-plus maze task. Their level of anxiety was determined by the amount of time spent exploring the open arms of the maze versus the time spent in the closed arms. As previously shown, all groups spent similar amounts of time in the open arms and in the center zone compared to wild-type mice and no effect of IL-1ra was observed (data not shown).
Wild-type mice (ACSF, n = 11; heat-inactivated IL-1ra,n = 12; IL-1ra, n = 11) and CX3CR1−/− (ACSF, n = 14; IL-1ra, n = 13) were then tested on rotarod as described above. No differences were observed between genotypes during the first day of training (Fig 7A). On the second day of training (trials 5-8), wild-type mice remained on the rod for longer periods of time compared to CX3CR1−/− mice. However, neither CX3CR1−/− treated with IL-1ra or CX3CR1−/− control mice showed significantly improvement in motor coordination on the second day of training (Figure 7a; 2-way ANOVA, trial, p<0.0001; treatment, p<0.0001; interaction p= 0.69). As demonstrated in the first study (Figure 2a), all wild-type mice performed significantly better on trial 8 compared to trial 1 than did CX3CR1−/− control group (Bonferroni posttest, WT ACSF, heat-inact. IL-1ra, WT IL-1ra vs CX3CR1−/− p< 0.05). CX3CR1−/− IL-1ra - treated mice motor performance was not different from CX3CR1−/− control group (CX3CR1−/− vs CX3CR1−/− IL-1ra, p>0.05, data not shown), suggesting that IL-1ra does not modulate the motor learning impairment. There were no significant differences on the first trial of testing between all experimental groups, which suggests that there is no difference in baseline motor skills (data not shown). Taken together, these data suggest that IL-1β does not modulate the deficits in motor learning observed in the CX3CR1−/− mice.
To address the role of IL-1β in the cognitive impairment observed in CX3CR1 deficient mice, we trained wild-type mice (3 subgroups: ACSF, heat-inactivated IL-1ra, IL-1ra) and CX3CR1−/− mice (2 subgroup: ACSF, IL-1ra) on contextual fear conditioning and auditory cue fear conditioning as described above. During training, all groups showed similar levels of freezing in response to the first and second pairing of tone and mild foot shock (Figure 7B). This indicates that all groups have the capability of freezing and that the acquisition of fear memory is indistinguishable between the two groups. When placed in the training context 24 hours following training, CX3CR1−/− mice showed a significant reduction in the amount of freezing compared to wild-type mice (Figure 7c; p<0.0005; Bonferroni post-hoc = WT ACSF vs CX3CR1−/− p < 0.05; wild-type heat-inact. IL-1ra vs CX3CR1−/− p < 0.001). Interestingly, IL-1ra completely reversed the deficit in contextual memory in CX3CR1−/− mice (Bonferroni post-hoc = p< 0.001), suggesting that IL-1β modulates the deficit in contextual memory due to the loss the loss of CX3CR1. Wild-type mice treated with IL-1ra were not significantly different from CX3CR1−/− control or from wild-type controls. In contrast, all groups exhibited identical responses to the presentation of the auditory CS when presented in a different context (data not shown). These data suggest that IL-1β action is specific for hippocampal-dependent cognitive function. To control for possible differences in response to the foot shock, a shock threshold test was performed. No differences were observed in the perception of the foot shock delivery as assessed by determining the stimulus amplitude necessary to evoke a flinching, jumping, or vocalization (data not shown).
Following the observation that IL-1 directly modulated the impairment in contextual memory in CX3CR1 deficient mice, we sought to determine if IL-1β also modulated the deficit previously observed in spatial learning (Figure 3). To answer this question, 8 days following fear conditioning the same animals were trained to locate a submerged platform in a circular pool filled with opaque water as described in the first study, a probe trial was performed after 9 days of training matching the data shown from study 1. No difference were observed between groups in the latencies to find the platform during training, indicating that all animals have normal acquisition of spatial information (Figure 7D). We then analyzed memory retention with a probe trial where the platform was removed and mice were allowed to free-swim. CX3CR1 −/− control mice had a significant decrease in the number of target platform crossings during the probe trial compared to wild-type control mice (Figure 7e; Two-way ANOVA, P = <0.0001 followed by Bonferroni post-hoc: WT ACSF, WT heat-inactivated IL-1ra vs CX3CR1−/− control p<0.05). Importantly IL-1ra was able to completely reverse the deficit in spatial memory observed in CX3CR1 deficient mice (CX3CR1−/− control vs CX3CR1−/− IL-1ra, p < 0.05). No differences were observed between experimental groups on the visible platform test (data not shown). In addition we analyzed the time spent in the target platform zone. CX3CR1−/− mice spent significantly less time in the target zone compared to wild-type control (Figure 7F, Two-way ANOVA, p<0.0001 followed by Bonferroni post-hoc: wild-type ACSF, wild-type heat-inactivated IL-1ra vs CX3CR1−/− control IL-1ra p<0.05). CX3CR1−/− mice treated with IL-1ra spent the same amount of time in the target zone when compared to control (CX3CR1−/− control vs CX3CR1−/− IL-1ra p< 0.001). No difference was observed in the number of crossing and in the time spent in the target quadrant between CX3CR1−/− control mice and wild type mice treated IL-1ra. These data further support the role of the CX3CR1 receptor to modulate hippocampal-dependent learning and memory and that IL-1β modulates the impairment in hippocampus dependent cognitive function.
For the first time the present studies test the hypothesis that CX3CL1signaling through CX3CR1 may be necessary for normal synaptic function. We demonstrated a physiological role of the chemokine receptor CX3CR1 to regulate cognitive function and synaptic plasticity. Furthermore, we show that genetic disruption of CX3CR1 impairs hippocampal neurogenesis, motor learning, associative memory, spatial memory, and the induction of LTP. Importantly, these deficits appear to be gene-dose dependent and related to increased levels of IL-1β.
These novel discoveries provide important insight to understanding the involvement of the CX3CR1 receptor under physiological conditions. Previously, we showed that CX3CR1−/− mice have decrease hippocampal neurogenesis when compared to heterozygote littermates (Bachstetter et al., 2009). We now demonstrate a gene-dose-dependent effect of CX3CR1 deficiency as demonstrated by heterozygous CX3CR1 mice that exhibit an intermediate phenotype. This effect was observed in associative and spatial memory formation, which indicates that the loss of a single allele is sufficient to impair the receptor function.
Recently, a gene-dose effect of CX3CR1 receptor was reported that related to fibrillar Aβ deposition in APP-PS1 mice crossed with CX3CR1−/−, CX3CR1+/− or CX3CR1+/+ (Lee et al., 2010). Reduction in fibrillar Aβ deposition in the APP-PS1 mouse model of AD was gene-dose dependent on CX3CR1 deficiency, with CX3CR1+/− mice displaying an intermediate phenotype. The present studies are in agreement with those previous results and underscore the profound implication of this discovery toward interpretation of all previous studies using only CX3CR1−/− and CX3CR1+/− mice.
We report a novel physiological role for the CX3CR1 receptor in the regulation of hippocampal-dependent memory formation. The impairment of LTP and neurogenesis likely represent the mechanism responsible for the defect observed in hippocampal-dependent associative and spatial memory formation. However, multiple mechanisms can account for the impairment in cognitive function and synaptic plasticity observed in the CX3CR1-defient mice.
CX3CL1 has been implicated as a endogenous neuronal modulator, which controls the over-production of iNOS, IL-1β, TNFα and IL-6 (Biber et al., 2007). CX3CL1 levels are down-regulated with neuronal injury, which results in microglial recruitment, activation and increase production of TNFα, IL-1β and p38 MAP kinase. IL-1β is a pro-inflammatory cytokine, which is constitutively expressed in the CNS, particularly in the hippocampus, and is synthesized by neurons and glial cells following neuronal injury (Rothwell et al., 1997;Dinarello, 1998). Binding studies demonstrate that the hippocampus contains the highest density of IL-1β binding sites (Farrar et al., 1987;Takao et al., 1990). These findings suggest that the effects of IL-1β might be specific to hippocampus, which may affect hippocampal-dependent learning and memory processes. Furthermore, there may be a causal relationship between the decrease of LTP and increase of IL-1β expression.
In the present study, we demonstrate that hippocampal protein levels of IL-1β and phospho-p38 are significantly increased in mice lacking CX3CR1 receptor. This increase was accompanied by a change in morphology, consistent with the activation of hippocampal microglia cells, observed as increased area of Iba-1 staining. Furthermore, the increase of these protein levels was accompanied by a decrease in LTP in CX3CR1 deficient mice. The deficit in LTP was gene-dose dependent, with CX3CR1+/− mice that demonstrate an intermediate phenotype. Importantly, both the deficits in LTP and cognitive function were rescued with administration of IL-1ra, which blocked the IL-1 receptor I. Under physiologic circumstances IL-1β seems to be required for normal learning and memory processes. This assertion derived from the observation that mice with targeted deletion of IL-1RI exhibit impairments in memory and synaptic plasticity (Hirsch et al., 1996).. Similarly, the administration of IL-1ra impairs fear-conditioning (Pugh et al., 1999;Rachal et al., 2001) and performance in the Morris water maze in young rats (Yirmiya et al., 2002). In agreement with the literature, our results show that wild-type mice treated with IL-1ra are not different from CX3CR1−/− deficient mice in the response to contextual freezing and Morris water maze task. Thus, physiologic levels of IL-1β might be critical for normal memory formation in the hippocampus and the increased IL-1β levels observed with age might impair hippocampal-dependent learning and memory.
In addition to the impairment of memory function, CX3CR1-deficient mice have impairments in motor learning. Interesting, cerebellar levels of TNFα were significantly increased in mice lacking CX3CR1 receptor. This result is in agreement with previous reports that show TNFα modulates age-dependent deficits in motor learning (Cartford et al., 2002). Also, we demonstrate an important, physiological role of the CX3CR1 receptor in normal memory formation and synaptic plasticity. Furthermore, our results demonstrated that disruption of CX3CR1 signaling triggers a downstream increase in IL-1β, which appear to be the key modulator responsible for the detrimental effect of loss of CX3CL1/CX3CR1 signaling on cognitive function.
We clearly demonstrate that under non-pathological conditions, disruption of CX3CL1/CX3CR1 signaling results in increased expression of IL-1β and p38, which is accompanied by the loss of synaptic plasticity and memory formation. Furthermore, these effects appear to be gene-dose dependent. This observation needs to be considered when interpreting data associated with disease conditions or under conditions where heterozygous mice were used as controls for CX3CR1−/− mice. However, this discovery does not negate all of the important findings to date with regard to the role of CX3CR1/CX3CL1 signaling in many disease conditions.
There is substantial literature in the area of CX3CR1/CX3CL1 signaling, yet no consensus to the role of this pathway as either neuroprotective or neurodegenerative has been agreed upon. In AD mouse models, CX3CR1−/− microglia have increased phagocytosis, which helps to clear Aβ (Lee et al., 2010). Deletion of the CX3CR1 gene in a triple transgenic (3xTg) mouse model of AD is protective against neuronal loss (Fuhrmann et al., 2010). However, CX3CR1-deficient 3xTg mice were examined at an age prior to the development of either extracellular Aβ deposition or intracellular microtubule-associated protein tau (MAPT) aggregation. Furthermore, CX3CR1−/− mice crossed with the AD mouse model CRND8 had lower brain levels of Aβ40 and Aβ42 and reduced amyloid deposits, which are related to increased microglia proliferation and phagocytosis (Liu et al., 2010). Finally, CX3CR1−/− mice crossed with mice overexpressing hTau have increased hyperphosphorylated tau and increased toxicity, likely related to the increase p38 and IL-1β (Bhaskar et al., 2010) expression. Taken together, the mechanisms that underlie CX3CR1 signaling which lead to neurotoxicity remains unclear. In conclusion, this study demonstrated that CX3CR1 receptors play a physiological role in normal hippocampal-dependent cognitive function and synaptic plasticity. In addition, the present study provides mechanistic links between CX3CR1 and the release of IL-1β and phospho-p38, ultimately implicating an increase in inflammation with the deficits observed in synaptic plasticity and cognition.
P.C.B. Supported by USPHS grant AG-04418 and the VA Medical Research Service. Amgen (Thousand Oaks CA) provide the IL-1Ra as a kind gift.
AUTHOR CONTRIBUTIONS J. T. R. performed the experimental design of the electrophysiology experiments and assisted with manuscript preparation. J. M. M. carried out the ELISA and Western Blot experiments for IL-1β, p38 and TNFα and performed neuronal counting. A. D. B. assisted in the design of the experiments. M. M. P designed and supervised all the behavioral experiments and assisted with manuscript preparation. C. E. H. assisted in the maintenance of the mouse colony and immunostaining. B. G performed part of the behavioral analysis. E. J. W. supervised and assisted in the electrophysiological and behavioral experiments, and edited the entire manuscript. P.C.B assisted with the experimental design and manuscript preparation and provided the basis for the development of the experimental design. C.G. conceived and designed the entire study, carried out behavioral experiments, stereological analysis and analyzed data, C.G. and P.C.B. interpreted the results and prepare the manuscript
COMPETING INTERESTS STATEMENT The authors declare that they have not competing financial interests.