Postnatal day 80 male Wistar rats (330–380 g) were used for behavioral studies and were obtained from the Biomedical Facility at University College Dublin, Ireland. All experimental procedures were approved by the Animal Research Ethics Committee of the Biomedical Facility at UCD and were carried out by individuals who held the appropriate license issued by the Minister for Health and Children. Animals were housed in groups of four and given ad libitum access to food and water. The experimental room was kept on a 12 h light/dark cycle at 22 ± 2°C. The behavior of each animal was assessed in an open field apparatus (620 mm long, 620 mm wide, and 150 mm high) both 48 and 24 h prior to commencement of training. The base of the open field box was demarcated into an 8 × 8 grid. The animals’ locomotion, rearing, and grooming behavior was monitored over a 5 min period and deemed normal prior to water maze training (data not shown). Their weights were also recorded immediately following the open field. Behavioral assessment was conducted in a quiet room under low-level red light illumination.

On postnatal day 80, animals were trained in the Morris water maze spatial learning task. Briefly, the water maze apparatus consists of a large circular pool (150 cm diameter, 80 cm deep) and a hidden platform (11 cm diameter). Both were constructed from black polyvinyl plastic, offering no intramaze visual cues that may help guide escape behavior. The platform was submerged 1.5 cm below the water surface (temperature 26 ± 1°C) and positioned 30 cm from the edge of the maze wall. The platform remained in the same position throughout the training session. The experimental room contained several extra-maze visual cues. The rat was lowered into the water facing the wall of the maze (30 cm high) at one of three locations which were alternated with each trial. Trials lasted a maximum of 90 s and the length of time taken for the rat to find the hidden platform was recorded. Rats failing to find the platform within the 90 s were placed on it for 10 s and allowed orient themselves. The training session consisted of five trials with an inter-trial interval of 300 s. Each trained animal was assigned a corresponding passive control animal that spent the same lengths of time swimming in the pool, minus the platform. After training, the rats were dried-off and placed back into their home cages. They were then killed by cervical dislocation at specific time-points post-training, i.e. 1, 2, or 3 h after commencement of the third trial. Brains used for immunofluorescent labeling procedures were quickly dissected out, covered in optimal cutting temperature (OCT compound; Agar Scientific) and snap frozen in N-hexane cooled to –80°C with CO₂.

Coronal cryosections of whole brain were taken at –3.3 mm with respect to Bregma in order to examine the dorsal hippocampus (Paxinos and Watson, 2005). The 12 μm sections were adhered to glass slides coated with poly-L-lysine. Sections were fixed in 70% ethanol for 25 min and then washed in phosphate-buffered saline (PBS). Sections were then incubated for 18 h at room temperature in primary antibody solution. The primary antibodies used were: (1) AF537 (R&D Systems; 1:250 dilution), a goat IgG polyclonal antibody that labels recombinant rat CX₃CL1 and; (2) MAB377 (Millipore; 1:500 dilution), a mouse IgG monoclonal antibody that detects the neuronal marker NeuN. The primary antibody solution consisted of 1% bovine serum albumin (BSA) and 1% normal rabbit serum in PBS. Following two 10 min washes in PBS, sections were incubated for 3 h with a rabbit anti-goat IgG secondary antibody conjugated to FITC (Sigma; 1:1000 dilution) which detected the CX₃CL1 antibody. The NeuN primary antibody was detected by a rabbit anti-mouse IgG TRITC-labeled secondary antibody (Sigma; 1:1000 dilution). Where applicable, nuclei were visualized using either propidium iodide or Hoechst 33258 (Invitrogen). Sections were mounted in Citifluor glycerol PBS solution (Agar Scientific), cover-slipped and stored in darkness at 4°C until imaged. To minimize any potential confounder effects from the immunohistochemical technique, trained sections were prepared, stained and imaged at the same time as their relevant passive control.

Post-natal day 21–25 male Wistar rats (60–100 g) were obtained from the Biomedical Facility, University College Dublin, Ireland. All experimental procedures were approved by the Animal Research Ethics Committee of the Biomedical Facility at UCD. Animals were anesthetized using isoflurane (Abbott Laboratories Ireland Ltd.) and decapitated by guillotine. The brain was rapidly removed and placed into ice-cold artificial cerebro-spinal fluid (aCSF) bubbled with 95% O₂ and 5% CO₂ (aCSF composition: 120 mM NaCl, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, 2.5 mM KCl, 2 mM MgSO₄⋅7H₂0, 2 mM CaCl₂ and 10 mM D-glucose). This high magnesium aCSF facilitates slice viability and recovery through greater NMDA receptor blockade. Transverse hippocampal slices (400 μm) were cut from both hemispheres using a vibroslice (Campden Instruments). They were then transferred to a submerged incubation chamber containing bubbled, room temperature aCSF and allowed to recover for 90 min. Following this recovery period, slices were transferred to a recording chamber perfused with aCSF at a flow rate of 4–5 ml/min at 31 ± 0.5°C. The composition of aCSF used for recording was a modified version of that used during slice recovery, i.e., the MgSO₄⋅7H₂O content was reduced to 1.3 mM to decrease NMDA receptor blockade and facilitate LTP induction. Extracellular field excitatory post-synaptic potentials (fEPSPs) were elicited by stimulation of the medial perforant path of the DG by a monopolar glass electrode at a frequency of 0.05 Hz. Responses were recorded using a glass electrode placed in the middle third of the molecular layer and stimulus strength was adjusted to give a response 35% of maximal. The effect of 500 pM rCX₃CL1 (recombinant rat CX₃CL1, Peprotech EC) on LTP in the DG was investigated both in the presence and absence of 100 μM of the GABA_A receptor/chloride channel inhibitor, picrotoxin (Sigma–Aldrich). Stable baseline recordings were made for at least 20 min prior to application of drugs. LTP was induced by theta-burst stimulation (TBS) consisting of eight trains (40 ms duration) of eight pulses at 200 Hz with 2 s intervals between trains and at a stimulus strength corresponding to 70% of maximal. Following TBS, the stimulus voltage was returned to that of baseline levels and fEPSPs were recorded every 20 s for a further 60 min.

Slices were coated in OCT (Agar UK) and snap-frozen in n-hexane cooled to –80°C with compressed CO₂. Hippocampal slices were cryosectioned into 12 μm sections that were adhered to glass slides and immunofluorescently stained for CX₃CL1 (as above). Nuclei were counterstained with propidium iodide.

Organotypic hippocampal cultures were prepared according to Stoppini et al. (1991). Briefly, post-natal day 7 male Wistar rats were decapitated without anaesthetic and their brains quickly dissected out and placed into ice-cold Earle’s balanced salt solution (EBSS) for 1 min. Both hippocampi were removed and cut into 400 μm slices using a McIlwain tissue chopper. Slices were separated and arranged onto organotypic inserts (three per insert, Millicell PICMORG50). The inserts were housed in standard six-well cell culture plates which were kept in an incubator at 35°C and 5% CO₂ in air. The slices were grown using an interface method with 1 ml organotypic medium supplying the under-surface of the slice. The organotypic medium consisted of 50% minimum essential medium (MEM, Gibco), 25% EBSS (Gibco), 25% heat-inactivated horse serum (Sigma) and supplemented with 2 mM glutamine, 28 mM D-glucose, 100 U/ml penicillin/streptomycin, and 25 mM HEPES. The first medium change was conducted 24 h following slice preparation with subsequent medium changes occurring every 2 days. Slices were maintained for 21 days in vitro (DIV) prior to experimentation.

All confocal images used for quantitative analysis of immunofluorescence (12-bit; 1024 × 1024 pixels) were captured using a 40×/0.8 W water-dipping lens (Zeiss Achroplan). Images of hippocampal sections from water maze-trained animals were captured from three defined regions of the hippocampus, i.e., CA1, CA3, and the apex of the DG. Images taken from acute slice preparations were captured from the upper (unstimulated) and lower (TBS-stimulated) blades of the DG. The specific areas of the hippocampal neuronal circuit captured were kept consistent between sections. Three sections from each rat brain and acute hippocampal slice were used for analysis.

Image analysis was conducted using EBImage; a package for the R programming environment (Pau et al., 2010). Analysis of the combined nuclear and surrounding cell soma expression of CX₃CL1 fluorescence was calculated for every cell in each image. Briefly, taking the DG confocal image in Figure S1 as a typical example, the red, green and blue channels were first separated for each image and every pixel within the images (1024 × 1024) was assigned an intensity value between 0 and 1. Using the blue channel as a nuclear reference, size and fluorescence intensity thresholds were set in order to select only those pixels likely to represent Hoechst-labeled nuclei. The nuclei were then ‘dilated’ using morphological kernel expansion. This step allowed the designation of a soma region surrounding each nucleus. A distance map was then generated for the image which calculates the distance of each foreground pixel (white) to the nearest background pixel (black). The watershed segmentation algorithm is then employed which accurately separates clusters of nuclei that are very close together, or touching, into individual cells. Minimum distance between objects and minimum radius criteria are written into the analysis scripts which further refines object separation. CX₃CL1 immunofluorescence is calculated for every cell as the average pixel fluorescence intensity.

At 21 days in vitro, organotypic hippocampal cultures were prepared for calcium imaging experiments by transferring inserts to room-temperature BSS (buffered salt solution) composed of 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 2 mM MgSO₄, 5.5 mM D-glucose, and 20 mM HEPES, pH 7.3. The insert membranes were cut using a scalpel and individual slices were transferred to 35 mm Petri dishes containing 2 ml of 3 μM fluo-4 AM calcium indicator (Invitrogen) in BSS and allowed incubate in the dark for 30 min. Similarly, cover-slips containing mixed neuronal-glial cultures were individually transferred to standard six-well plates, containing 3 μM fluo-4 AM in BSS, for 20 min.

Time-series calcium imaging experiments in organotypic slices were conducted in the CA1 pyramidal cell layer of the hippocampus using an upright LSM V Pascal confocal microscope (Zeiss). The field of view on the 10× water immersion lens was halved allowing us to monitor calcium responses at a rate of two frames per second. We measured baseline calcium levels in the first 20 s (i.e. average of 40 frames). After 20 s a solution of glutamate (30 μM) in BSS was washed onto the slice and filled the slice chamber. This glutamate solution remained in the chamber for 105 s. The calcium response of each cell over the 125 s time-course was calculated using EBImage software. The concentrations of rCX₃CL1 (500 pM for 15 min) and picrotoxin (100 μM for 15 min) used for live-cell calcium imaging experiments in organotypic slices was the same as those used in acute slice electrophysiology experiments.

For live-cell calcium imaging experiments in primary mixed neuron-glial cultures, cells were loaded with 3 μM fluo-4 AM calcium indicator (Invitrogen) in BSS as above, and were then transferred to a custom-built imaging chamber containing fresh BSS. The imaging chamber allowed for wash-in/out of CX₃CL1 and glutamate solutions (Pickering et al., 2008). Experiments were conducted at room temperature using an upright LSM V Pascal confocal microscope and a 10×/0.3 W Ph1 water-dipping lens so as to capture several hundred cells per experiment. Time-series confocal images were captured at frame-rate of 1 Hz. Cells were pre-treated for 15 min with either 500 pM or 2 nM rCX₃CL1. Baseline calcium levels for every cell were monitored for 20 s prior to a 30 μM glutamate exposure. This glutamate solution remained in the chamber for 90 s before being washed out with fresh BSS solution and frames were captured for a further 40 s. The calcium response of each cell was calculated every second for 150 s using EBImage software.

All raw data was imported into GraphPad Prism 6 software where statistical analyses were performed. Kruskal–Wallis non-parametric analysis of variance (ANOVA) tests were performed in conjunction with Dunn’s multiple comparisons post hoc analyses in order to determine statistical significance (p < 0.01) for: (1) CX₃CL1 regulation post-spatial learning in rats; (2) CX₃CL1 regulation post-theta burst stimulation in acute hippocampal slices and; (3) glutamate-mediated calcium responses in primary mixed neuron-glial cell cultures. One-way ANOVA tests were performed in conjunction with Bonferroni post hoc analyses in order to determine statistical significance (p < 0.05) for: (1) the reduction in swim-times during water maze training; and (2) the effect of rCX₃CL1 on LTP and paired pulse ratio. Mann–Whitney U tests were performed in order to determine statistical significance (p < 0.001) for the effects of rCX₃CL1 and picrotoxin on glutamate-mediated calcium responses in organotypic hippocampal slice cultures. The relationship between NeuN expression and CX₃CL1 expression in the rat hippocampus was analyzed by Pearson correlation and linear regression.

We characterized the expression and distribution of CX₃CL1 in the rat dorsal hippocampus using immunofluorescence. CX₃CL1 expression was predominately restricted to the glutamatergic pyramidal and granule neurons of the hippocampus (Figure 1A). At higher magnification, it is clear that CX₃CL1 expression co-localizes with the neuronal marker NeuN in CA1 and CA3 pyramidal neurons (Figures 1B,C; yellow arrowheads), dentate granule cells (Figure 1D) and presumptive interneurons outside the primary cell layers in all three hippocampal subfields (Figures 1B–D; white arrowheads). In all cases, the chemokine appears to be predominantly expressed on the plasma membrane of the cell soma. We assigned expression intensity values for NeuN and CX₃CL1 to each cell in the image dataset (see Figure S1 for image analysis method) and then analyzed the relationship between expression of the mature neuronal marker, NeuN, and CX₃CL1 by Pearson correlation in each sub-region of the hippocampus. A high degree of correlation was evident between CX₃CL1 and NeuN expression in all three regions of the hippocampus (Figure 1E). The CA3 region showed the strongest linear regression coefficients of determination for correlation between CX₃CL1 and NeuN expression (r² = 0.70, p < 0.05). These data confirm previous reports that mature NeuN + hippocampal neurons in the adult rat hippocampus express high levels of CX₃CL1 under naïve resting conditions.

Rats were trained in a five-trial water maze session (n = 12 in total; n = 4 per time point) and the latencies to find the platform were recorded. Latency-to-platform times decreased significantly (one-way ANOVA, p < 0.001) over the five trials of the training session indicating that the animals acquired the task (Figure 2A). We quantified CX₃CL1 expression in the hippocampus of these rats using immunofluorescence. Total CX₃CL1 expression was very similar in trained and passive animals 1 h following training in all hippocampal regions (Figures 2B–D). At the 2 h time-point, CX₃CL1 expression was significantly higher in trained animals compared to passive controls in all three hippocampal regions analyzed (Kruskal–Wallis ANOVA, Dunn’s multiple comparisons post hoc test, p < 0.001). At the 3 h time-point in the DG, CX₃CL1 expression in trained animals again matched that of passive controls, highlighting the temporal specificity and transient nature of the 2 h up-regulation in dentate granule cells of rats that learned the spatial task. In the CA3, however, there was a significant, training-specific down-regulation in CX₃CL1 expression whereas levels of CX₃CL1 remained elevated in the CA1 region of trained animals compared to passive control counterparts at the 3 h time-point (Kruskal–Wallis ANOVA, Dunn’s multiple comparisons post hoc test, p < 0.01). It should be noted that passive control animals exhibited an up-regulation in CX₃CL1 at the 2 h post-swim time-point indicating that, in addition to the learning-specific regulations described above, this chemokine is also responsive to the general stressors associated with the paradigm.

We investigated a possible functional role for CX₃CL1 up-regulation in modulating synaptic transmission during memory-associated synaptic plasticity. Previous studies have shown that CX₃CL1 inhibits LTP and mimics long-term depression (LTD) at the CA3–CA1 synapses in acute hippocampal slices (Bertollini et al., 2006; Ragozzino et al., 2006; Maggi et al., 2009). Given that we observed memory-associated increases in CX₃CL1 levels in CA1, CA3, and DG regions of the hippocampus following spatial learning, we next asked what effect CX₃CL1 exerts on LTP in the dentate granule cell synaptic field. Pre-treatment with CX₃CL1 (500 pM) significantly reduced induction and completely prevented maintenance of LTP in the DG (Figure 4A).

Consistent with previous work on pyramidal hippocampal neurons (Ragozzino et al., 2006), the paired-pulse ratio of DG neuron responses evoked by two successive stimuli (50 ms apart) was unaffected by CX₃CL1 treatment either at baseline or following TBS (Figure 4B). While not ruling out a presynaptic contribution, these data support a post-synaptic action for CX₃CL1-mediated inhibition of fEPSP amplitude in the DG following TBS.

Recent evidence has shown that CX₃CL1 reduces the activity of serotonergic neurons in the Raphe nucleus through enhanced GABA_A receptor-mediated inhibition (Heinisch and Kirby, 2009). We next investigated if CX₃CL1-mediated control of glutamatergic neuroplasticity in the hippocampal DG requires GABAergic inhibitory transmission. LTP was induced in the presence of GABA_A receptor/chloride channel blocker picrotoxin (100 μM) using the same TBS protocol as in Figure 4A. As expected, the degree of potentiation of fEPSP amplitude was substantially greater in the presence of GABA_A receptor blockade (117 ± 5% versus 200 ± 3% of baseline average in first 10 min post-TBS in the absence and presence of picrotoxin, respectively; Figures 4A,C; Arima-Yoshida et al., 2011). Interestingly, CX₃CL1 did not prevent LTP in the presence of picrotoxin (Figure 4C). In fact, when picrotoxin was present, the magnitude of LTP was enhanced by CX₃CL1 during the initial 20 min following the TBS (p < 0.05). CX₃CL1 again had no effect on paired-pulse depression in the presence of picrotoxin (Figure 4D) suggesting a post-synaptic action for CX₃CL1-mediated short-term enhancement of fEPSP amplitude following TBS.

Long-term potentiation of excitatory synaptic transmission in the hippocampus is heavily dependent on post-synaptic intracellular calcium rise (Bliss and Collingridge, 1993). We next assessed if CX₃CL1 exerts differential effects on glutamate-induced intracellular calcium rise in the presence and absence of GABA_A receptor blockade. Live-cell calcium imaging was performed in organotypic hippocampal slice cultures pre-treated with CX₃CL1 (500 pM) and/or picrotoxin (100 μM) for 15 min. Slices were then exposed to glutamate (30 μM) and intracellular calcium was monitored for 1 min 45 s in the CA1 pyramidal cell layer of the hippocampus. Pre-treatment with CX₃CL1 prior to glutamate application resulted in an attenuated intracellular calcium rise in cells in the CA1 region of organotypic slice cultures (Figure 4E). While the peak amplitude of the calcium response in CX₃CL1-treated slices was not different than in controls (Figure S2A), the total calcium entry in the 60 s post-glutamate exposure was reduced, as calculated by the area under the curve (AUC) in Figure 4E. In contrast, in the presence of GABA_A receptor/chloride channel blockade, CX₃CL1 enhanced glutamate-induced calcium influx as measured by AUC (Figure 4F) or peak intracellular calcium (Figure S2B). These findings are consistent with the opposing actions of CX₃CL1 on LTP studies in the presence and absence of picrotoxin. They suggest that neuronal responses to CX₃CL1 can vary depending on the balance between excitation and inhibition in the hippocampal network. Therefore, during periods of enhanced excitatory activity in the hippocampus, CX₃CL1 may act as a neuroprotective dampener of excessive glutamatergic neurotransmission and this action appears to be dependent on GABA-mediated inhibition.

In order to assess if physiological concentrations of CX₃CL1 exert equivalent effects on glutamate-induced calcium responses in both neurons and non-neuronal cell populations, we used hippocampal mixed cell culture preparation. Primary hippocampal cells were pre-treated with CX₃CL1 (500 pM or 2 nM) for 15 min prior to glutamate (30 μM) challenge. Importantly, we have shown previously that using this mixed hippocampal cell culture preparation, we can discriminate between neurons and non-neuronal cells based on the shapes of their respective glutamate-mediated calcium response curves (Pickering et al., 2008). This allowed us to simultaneously evaluate the effects of different concentrations of CX₃CL1 on intracellular calcium dynamics in neurons and non-neuronal cells following glutamate exposure (Figures 5A–C). This method of distinguishing between neurons and non-neuron cell types was shown to be as accurate as traditional methods of immunocytochemistry staining for neuronal and glial cell markers (NeuN and GFAP, respectively; Pickering et al., 2008). Neurons were identified by their substantial and prolonged increase of intracellular calcium following glutamate administration while non-neurons exhibited a sharp rise and fall back to plateaux (Figures 5D,E, respectively). In non-neuronal cells, both low (500 pM) and higher (2 nM) physiologically relevant concentrations of CX₃CL1 attenuated glutamate-induced calcium influx, in a dose-dependent manner (Kruskal–Wallis ANOVA, Dunn’s multiple comparisons post hoc test, p < 0.05; Figures 5E,G). In neurons, however, the lower concentration of CX₃CL1 (500 pM) had little or no effect on glutamate-induced calcium influx (Figures 5D,F; and Figure S2C). Pre-treatment of hippocampal cultures with a higher concentration (2 nM) of CX₃CL1 caused a substantial attenuation of glutamate-mediated calcium responses in neurons (Kruskal–Wallis ANOVA, Dunn’s multiple comparisons post hoc test, p < 0.001; Figures 5D,F), in addition to causing further dose-dependent attenuations in calcium influx in non-neuronal cell types (Figures 5E,G; and Figure S2D). The differential responses of CX₃CL1 on neurons and non-neurons at the lower concentration (500 pM) may relate to variations in absolute levels of CX₃CR1 receptor expression on distinct cell populations in the hippocampus.