Intracerebroventricular cannulation and TLR4 antagonist infusion. A chronic indwelling cannula was im- planted into the lateral ventricles of mice to allow intracerebro- ventricular (ICV) application of TLR4 antagonist (inhibitory LPS from R. Sphaeroides, Invivogen, San Diego, CA, USA) or artificial cerebrospinal fluid (aCSF). When applying this antagonist at concentrations 10–100 fold higher than LPS, effective competition over the binding site on MD-2 is achieved. We chose a concen- tration of 1 mg/ml because it was previously shown by Rodgers KM [13] and colleagues that an even lower concentration of 0.2 mg/ml successfully antagonizes LPS in the brain in-vivo. Cannulae (brain infusion kit 3) and micro-osmotic minipumps (model 1004) were purchased from Alzet (DURECT). At 2 months of age, mice were anesthetized with isoflurane, and a cannula was implanted and fixed on the surface of the skull using dental cement. The tip of the cannula was located in the lateral ventricle (distance from the bregma: anteroposterior, 20.25 mm; lateral, 1 mm; depth, 2.5 mm, according to [14]) for ICV infusion. aCSF (n = 10) or TLR4 antagonist [1 mg/mL], dissolved in aCSF (n = 10)] was delivered using a subcutaneous micro-osmotic pump (model 1004) attached to the ICV cannula that provided a 0.1 mL/ h flow rate. Immunoblots. Lysates of cultured cortical neuronal cells were obtained by washing cells in ice-cold PBS and resuspending them in a modified RIPA lysis buffer (150 mM sodium chloride, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1% Triton-X100, 1% sodium deoxicholic acid, 0.1% sodium dodecylsulfate, 5 mg/ml aprotinin, 5 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluo- ride). Protein concentration was determined using the Bradford reagent (BioRad, Hercules, Canada). Protein samples (50 mg) were prepared and electrophoresed on a 12% Bis-Tris gel. Proteins in the gel were then transferred to a PVDF membrane. Membranes were blocked in 5% non-fat milk for 1 h at room temperature (RT), followed by an overnight incubation at 4uC with primary antibodies against: CREB, (1:5000, Cat # 9197, Cell signaling), phospho-CREB (1:2000, Cat # 9198, Cell Signaling), ERK1/2 (1:400, Cat # 9102, Cell signaling), phospho-ERK1/2 (1:500, Cat # 9106, Cell signaling), GluR1 (1:10000, Cat # AB1504, Millipore). Phospho-Ser945 GluR1 (1:1000, Cat # AB5849, Millipore), in 0.05% sodium azide and 1% bovine serum albumin in TBS with 0.1% Tween-20 at RT for 1 h. Protein bands were visualized using a chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ, USA). Statistical analysis. Analyses of the MWM (except probe trials), fear-conditioning and rotarod experiments were performed using two-way ANOVA repeated measures with a Bonferroni post- hoc test. ANOVA statistical analysis was performed using Prizm 5 (Graphpad, USA). Correlation matrices were generated for Latency to reach the hidden platform and mean distance from the platform versus swim speed. An analysis of covariance (ANCOVA) was used to determine whether the observed effects were still significant after controlling for the variance contributed by the correlated variables. Analysis of co-variance (ANCOVA) and Pearson’s correlation was performed using SPSS (IBM, USA). Probe trials in the MWM test were analyzed using one-way ANOVA repeated measures, with a Bonferroni post-hoc test to verify that the time mice spent in the target quadrant where the platform was located was significantly longer than in all 3 other non-targeted quadrants. All other data in this study were analyzed using unpaired two-tailed Student’s t-test. Results are expressed as mean 6 S.E.M. TLR4 in Learning Results TLR4 deficiency improves spatial memory acquisition To determine whether TLR4 affects cognitive behavior, we first compared long-term spatial memory acquisition abilities between TLR42/2 and TLR4+/+ mice using a reference memory variant of the MWM task [15]. TLR4 deficiency resulted in significantly shorter mean distance from the hidden platform (F(1,164) = 12.01, p = 0.0013) (Figure 1A) and shorter latency to reach the hidden platform (F(1,164) = 7.36, p = 0.0097) (Figure S1A) compared with TLR4+/+ mice. While both strains swam similar distances (F(1,164) = 2.10, p = 0.1550) (Figure S1B), TLR42/2 mice exhibited significantly faster swim speed (F(1,164) = 9.94, p = 0.0141) (Figur- e S1C), correlated with poorer path efficiency (the ratio of the shortest possible path length to actual path length; higher path efficiency pertains to better performance) during trials (F(1,164) = 7.29, p = 0.0076) (Figure S1D). This is potentially due to their faster swim speed, which results in a greater area covered in a trial. To test the possible effect of swim speed on the performance of mice in this test, a correlation matrix including all measures with significant main effects (swim speed, latency to reach the hidden platform, mean distance from the platform) was performed. Results revealed that both latency to reach the hidden platform and mean distance from the hidden platform were correlated with swim speed (r = 0.57, p,0.0001 and r = 0.31, p,0.0001 respectively) (Figures S1E, F respectively). Because performance in the MWM test strongly depends on swim speed, the enhanced performance of TLR42/2 mice compared with TLR4+/+ mice in the MWM task may have been the result of the higher swim speed of TLR42/2 mice. To control for this possibility, an analysis of covariance (ANCOVA) was performed, in which swim speed was co-varied out of the latency to reach the hidden platform and mean distance from the hidden platform effects. The difference in mean distance from the hidden platform was still significant (F(1,215) = 5.4, p,0.021) while the difference in latency was no significant (F(1,215) = 0.336, p,0.563). The higher swim speed of TLR42/2 mice was also correlated with a significantly enhanced motor performance on the rotarod (F(1,72) = 7.75, p = 0.008) (Figure S1G). Spatial learning and memory tasks are comprised of several stages, including memory acquisition and memory extinction [16]. In probe trials to measure retention of the memory of the platform location, TLR42/2 mice swam a shorter mean distance from the platform in probe trials conducted 24 and 48 hours after training compared with TLR4+/+ mice (p,0.05) (Figure 2A). These results could not be attributed to altered anxiety or exploratory levels as performance of these mice in the elevated plus maze task was similar (F(1,43) = 0.28, p = 0.6) (Figure S1H), and no differences were observed in the open field arena (F(1,340) = 0.03, p = 0.87) (Figure S1I). Because TLR4 deficiency might affect development of the hippocampal circuits involved in cognition, we assessed the effect of pharmacological inhibition of TLR4 in mature mice to determine whether TLR4 mediates physiological hippocampal synaptic plasticity after the adult neural circuitry is established. To accomplish this, we infused either aCSF (vehicle control) or an antagonistic variant of LPS from Rhodobacter Sphearoides [17] into the lateral ventricles of adult TLR4+/+ mice (referred herein as TLR4 antagonist infused mice). This antagonistic LPS directly competes with hexa-acylated LPS for the same binding site on myeloid differentiation factor 2 (MD-2), as well as inhibits hexa- acylated endotoxin: MD-2 complexes function at TLR4 [13]. The infusion lasted 4 week, during which the first week was dedicated for animals’ recovery and during the last 3 weeks the mice were PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47522