effects of TLR4 signaling on cognitive function are partially due to altered neurogenesis in the adult dentate gyrus. The observed differences between the relative lack of effects on behavior of pharmacological inhibition of TLR4 in adult mice, compared to the effects of developmental TLR4 deficiency strongly suggests a developmental role for TLR4 in shaping the cellular and molecular substrates of learning and memory. TLR4 is expressed in NPC and neurons during development [6,10], and could therefore affect the formation of neural circuits involved in cognition. Interestingly, TLR4 inhibition in adult mice, but not de- velopmental TLR4 deficiency, resulted in altered anxiety levels, as evident in performance in the open field arena and elevated plus maze task, suggesting that physiological TLR4 signaling may also be involved in anxiety/exploratory behaviors. Possible explana- tions for the latter results include; (1) chronic infusion of the TLR4 antagonist into the lateral ventricles results in diffusion of the antagonist to regions other than the hippocampus. (2) Anxiety is mediated by several brain structures such as the amygdala, cerebral cortex and locus coeruleus, as well as the hippocampus. When applying a TLR4 antagonist, the activation levels of TLR4 are altered, while in mice with developmental deficiency for TLR4, TLR4 is completely absent from the tissue. Moreover, while our results indicate that antagonizing TLR4 causes mice to spend a higher amount of time in the periphery of the open field arena, and decreases the amount of time they spend in the closed arms of the elevated plus maze, opposite effects are observed when TLR4 is activated using LPS in a rat model [23]; The rats spend a higher amount of time in the closed arms of the elevated plus maze and a higher amount of time in the center of the open field arena. This suggests that TLR4 is important for regulating normal anxiety responses. It is possible that during embryonic development, different brain sub-regions important for fear conditioning are affected by developmental TLR4 deficiency, resulting in impaired fear learning in TLR4-deficient mice. The fact that only developmen- tal TLR4 deficiency caused impairment in the fear-related learning and memory could also suggest that other brain sub- regions, such as the amygdala or the prefrontal cortex involve physiological rather than developmental effects by TLR4, which raises the need to study the role of TLR4 in these sub-regions as well. Similarly, as motor function, tested using the rotarod task, was altered in TLR42/2 mice but not mice with TLR4 antagonist infusion, brain regions important for coordination may develop differently under developmental TLR4 deficiency but are not affected by physiological TLR4 signaling. It remains to be studied whether TLR42/2 mice exhibit structural anomalies in brain regions important for coordination such as the cerebellum. Elevated CREB levels, correlated with the behavioral pheno- type exhibited by TLR42/2 mice, but were not correlated with several candidate pathways for CREB activation including ERK and GluR1. The molecular cascade from TLR4 to CREB therefore remains to be established. We hypothesize that increased CREB levels in TLR42/2 mice have a functional role during development on synapse formation and brain structure. Further studies on the role of TLR4 on brain development during embryogenesis are needed to address the functional role of increased CREB activity in TLR42/2 mice. The data presented here expand our view of the role of TLR4 in learning and memory, and anxiety behaviors. However, further work will be required to understand the cellular and molecular mechanisms by which TLR4 signaling influences the formation and plasticity of neuronal circuits. It is unclear whether TLR4- TLR4 in Learning dependent neurogenesis in the DG is responsible for the effects of TLR4 deficiency on spatial reference memory, and/or contextual fear learning and memory. As the most pronounced effects were conferred by developmentally TLR4 deficiency (improved motor ability (higher swimming speed and rotarod), improved spatial reference, impaired fear-learning), further examination of the roles of TLR4 in different brain regions during and following embryonic development is necessary. It is interesting to compare the behavioral phenotype exhibited by mice lacking TLR3 to that of TLR4-deficient mice. Despite the structural and immunological functional similarities between the two receptors, the functional behavioral differences between the two receptors are striking. In contrast to TLR32/2 mice, TLR42/ 2 mice have enhanced acquisition of spatial reference memory. However, in contrast to TLR42/2 mice, TLR32/2 mice have slower spatial memory extinction. In fear-learning paradigms, TLR32/2 mice show no impairment in memory acquisition, but show enhanced hippocampal and impaired amygdala component, while TLR42/2 mice show impaired memory acquisition, impaired hippocampal and intact amygdala components. Finally, In contrast to TLR4 deficiency, TLR3-deficient mice exhibit impaired anxiety responses. These cognitive and anxiety differ- ences are marked, and suggest inherent and critical roles for TLR- related genes during embryonic brain development. It will be interesting to see whether other members of this family of innate- immune receptors play roles in developmental neuroplasticity, learning and memory, and other behaviors. Supporting Information TLR42/2 Figure S1 mice exhibit enhanced spatial reference memory, motor function and normal anxi- ety-like behavior. TLR4+/+ (n = 24) and TLR42/2 (n = 19) mice were trained for 5 days in the MWM with 4 trials per day. (A) Latency to reach the platform was significantly lower in TLR42/2 mice compared with TLR4+/+ mice, (B) swimming distance was not different between the experimental groups and (C) mean swimming speed was significantly higher in TLR42/2 mice compared with TLR4+/+ mice (D) Path efficiency was significantly lower in TLR42/2 mice compared with TLR4+/+ mice (E) Pearson’s correlation between swim speed and latency to reach the platform (F) Pearson’s correlation between swim speed and mean distance from the platform (G) TLR4+/+ (n = 24) and TLR42/2 (n = 19) mice were tested in a Rota-Rod apparatus (Med- associates, St. Albans, VT, USA). Rota-rod acceleration was set to 4–40 revolutions per minute (RPM). Mice were placed on the Rota-rod for 3 trials of 5 minutes each with 15 minutes rest between trials. Time spent on the rod was measured. TLR42/2 mice showed superior performance in this task compared with TLR4+/+ mice (H) TLR4+/+ (n = 24) and TLR42/2 (n = 19) mice were tested in an elevated plus maze. Mice were placed in the maze for 5 minutes, and time spent in the open and closed arms was measured. No difference was observed between the experi- mental groups (I) TLR4+/+ (n = 24) and TLR42/2 (n = 19) mice were tested in an open field arena. Mice were place in the center of the arena for 15 minutes, and time spent in the center versus the periphery of the arena was measured. No difference was observed between the experimental groups. (TIF) Figure S2 CNS TLR4 inhibition affects anxiety but not spatial reference memory. Mice implanted with osmotic pumps that infuse either aCSF (n = 10) or TLR4 antagonist (n = 10) were trained for 5 days in the MWM with 4 trials per day. (A) Latency to reach the hidden platform was not significantly PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47522