Impact of TLR4 on hippocampus-dependent contextual fear learning and memory In addition to spatial learning, the hippocampus is involved in contextual fear learning [18]. Fear conditioning is a Pavlovian task shown to involve the hippocampal formation, the amygdala and the prefrontal cortex [19]. To test the hypothesis that hippocampal TLR4 has a role in contextual fear learning, TLR42/2 mice were tested in the fear-conditioning paradigm. TLR42/2 mice dis- played an inability to associate the tone-shock pair compared with TLR4+/+ mice (F(1,123) = 12.97, p = 0.0008) (Figure 3A). In addition, TLR42/2 mice showed significantly lower freezing during contextual fear conditioning compared with TLR4+/+ mice (F(1,164) = 20.41, p,0.0001) (Figure 3B, C). Similarly, TLR42/2 mice showed significantly lower freezing during cued fear conditioning compared with TLR4+/+ mice, which could be attributed to initial lower freezing behavior in TLR42/2 mice (F(1,74) = 23.27, p,0.0001) (Figure 3D). Interestingly, inhibition of TLR4 in adult mice using an antagonist had no effect on acquisition (F(1,60) = 0.06, p = 0.98) (Figure 4A), contextual memory (F(1,64 = 0.52, p = 0.48) (Figure 4B, C) or cued memory (F(1,34) = 0.51, p = 0.47) (Figure 4D) in the fear conditioning paradigm. CREB levels are upregulated in brains of TLR42/2 mice To understand what could contribute to the altered cognitive ability of TLR42/2 mice in spatial and contextual learning and memory paradigms, we compared cortices and hippocampi from TLR42/2 and TLR4+/+ mice for expression of proteins previously found by us to be altered in response to TLR3 deficiency in the brain and that were correlated with cognitive behavioral changes [10]. In contrast to our observations in hippocampi of TLR3- deficient mice, the glutamate receptor (GluR1) – ERK pathway TLR4 in Learning was not upregulated in the hippocampi of TLR42/2 mice. Levels of CREB, the master-regulator of synaptic plasticity [20] and its phosphorylated form were elevated in hippocampi (Figure 5) but not cortices (Figure S4) of TLR42/2 mice compared to TLR4+/+ mice. Discussion TLR4, an innate immune receptor involved in the detection of gram-negative bacteria [3] and in tissue damage responses [21], has recently been suggested to play roles in CNS plasticity [6,22]. Here we describe several roles for TLR4 in hippocampus- dependent learning and memory. We utilized mice devoid of TLR4, as well as adult mice infused with a TLR4 antagonist into the lateral ventricles in the brain, and a battery of behavioral tests to dissect the roles of TLR4 in hippocampus-dependent spatial as well as contextual learning and memory. To the best of our knowledge, the TLR4 antagonist we used throughout this study antagonizes TLR4 with a high affinity and specificity; however, it is possible that other, unknown signaling molecules, also bind this antagonist. Our findings suggest that TLR4 has a developmental role in shaping spatial and contextual learning and memory. Developmental TLR4 deficiency, as exhibited in TLR42/2 mice, conferred the strongest effect on hippocampus-dependent cognitive spatial and contextual learning and memory compared to pharmacological blockade of TLR4 in the adult brain. In a spatial reference memory task, TLR42/2 mice showed enhanced performance compared to TLR4+/+ mice. On the other hand, pharmacological inhibition of TLR4 in adult mice did not alter spatial reference memory. Although TLR42/2 mice swam faster in the reference memory variant of the MWM compared to TLR4+/+ mice, their shorter latency is attributed to better spatial navigation skills due to shorter mean distance from Figure 3. Developmental TLR4 deficiency impairs fear-learning and memory. (A) TLR42/2 (n = 19) mice show impaired association curves in the fear-conditioning paradigm compared with TLR4+/+ (n = 24) mice. (B) TLR42/2 mice exhibit significantly impaired hippocampus dependent contextual fear compared to TLR4+/+ mice, as measured by time freezing during 5 minutes of exposure to the original context. (C) Average freezing during contextual fear. (D) TLR42/2 mice exhibit reduced freezing compared with TLR4+/+ mice in the presence of tone, indicating impaired fear response. doi:10.1371/journal.pone.0047522.g003 PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47522