TLR4 in Learning Figure 1. Developmental TLR4 deficiency enhances spatial reference memory. (A) TLR42/2 (n = 19) and TLR4+/+ (n = 24) mice were trained in the spatial reference memory hidden platform variant of the MWM during 5 consecutive days with 4 trials per day. TLR42/2 mice demonstrated improved spatial navigation capabilities compared with TLR4+/+ mice as indicated by significantly shorter mean distance from the platform. (B) Mice (C57BL/6) were implanted with an osmotic pump containing either aCSF or a TLR4 antagonist (n = 10 per group). The pump was connected via tubing to a cannula, which was positioned to the lateral ventricle. Mice were trained in the spatial reference memory hidden platform variant of the MWM over 5 days with 4 trials per day. No difference was measured in mean distance from the platform. *p,0.05 (Two-way RM-ANOVA). doi:10.1371/journal.pone.0047522.g001 tested. A concentration of 1 mg/ml was chosen for its ability to compete with saturated levels of exdotoxin or comparable ligand. Initially, the mice were tested in the reference memory variant of the MWM. The TLR4 antagonist did not affect mean distance from the platform (F(1,68) = 0.08, p = 0.08) (Figure 1B). While swim distance or latency to reach the hidden platform were not different between the two groups (F(1,68) = 0.01, p = 0.92 and F(1,68) = 1, p = 0.3313 respectively) (Figures S2A and S2B respectively), swim speed was significantly lower in TLR4 antagonist infused mice (F(1,68) = 9.73, p = 0.0063) (Figure S2C), which correlated with higher path efficiency during training (p,0.05) (Figure S2D). Retention of the memory of the platform location was not significantly different between the control and TLR4 antagonist- treated groups (Figure 2B). Despite no differences in spatial learning, anxiety levels were significantly different between the two treatment groups, as TLR4-antagonist infused mice showed significantly altered anxiety (more time spent in the open arms) as tested using the elevated plus maze (p = 0.0418) (Figure S2E) compared to aCSF infused mice. In addition, open field exploration analysis showed treatment differences in time spent in both periphery and center: TLR4 antagonist infused mice spent Figure 2. Developmental TLR4 deficiency, but not pharmaco- logical TLR4 antagonism, enhances retention of spatial refer- ence memory. (A) TLR42/2 (n = 19) and TLR4+/+ (n = 24) mice were tested in probe trials at 24, 48, 72 and 96 hours following training for retention of spatial reference memory. Tests were done after all experimental groups exhibited loss of memory of the platform location. Mean distance from the platform was measured and used to indicate efficiency in locating the hidden platform. TLR42/2 mice showed shorter mean distance from the platform at 24 and 48 hours after training compared with TLR4+/+ mice, indicating a more accurate swim toward the platform quadrant (B) Mice (C57BL/6) were implanted with an osmotic pump containing either aCSF (n = 10) or a TLR4 antagonist (n = 10). The pump was connected via tubing to a cannula, which was positioned to the lateral ventricle. Following training in the MWM task, mice were tested in probe trials at 24 and 48 hours following training for retention of spatial reference memory. Both experimental groups exhibited similar performance during probe trials, as measured by mean distance from the platform. doi:10.1371/journal.pone.0047522.g002 significantly more time in the periphery and less time in the center compared with aCSF infused mice (F(1,180) = 7.29, p = 0.0076) (Figure S2F). During the 4 week period between cannulation and the end of behavioral tests, mice consistently gained weight and had overall good health (Figure S2G). In a visible platform variant, aimed at assessing the impacts of motivation, visual abilities or motor function of the mice during performance in the MWM tasks described above, all four groups exhibited similar performance (TLR4+/+ vs. TLR42/2, F(1,42) = 0.82, p = 0.37, aCSF vs. TLR4 antagonist infused mice, F(1,34) = 0.01, p = 0.9) (Figure S3A and B). PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47522