Evidence for a Developmental Role for TLR4 in Learning and Memory Eitan Okun1*, Boaz Barak2, Ravit Saada-Madar1, Sarah M. Rothman3, Kathleen J. Griffioen3, Nicholas Roberts3, Kamilah Castro3, Mohamed R. Mughal3, Mario A. Pita3, Alexis M. Stranahan4, Thiruma V. Arumugam5, Mark P. Mattson3,6 1 The Mina and Everard Goodman Faculty of Life Sciences, The Leslie and Susan Gonda Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat Gan, Israel, 2 Department of Neurobiology, Life Sciences Faculty and the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel, 3 Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, Maryland, United States of America, 4 Department of Physiology, Georgia Health Sciences University, Augusta, Georgia, United States of America, 5 School of Biomedical Sciences, The University of Queensland, Queensland, Australia, 6 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America Abstract Toll-like receptors (TLRs) play essential roles in innate immunity and increasing evidence indicates that these receptors are expressed in neurons, astrocytes and microglia in the brain where they mediate responses to infection, stress and injury. Very little is known about the roles of TLRs in cognition. To test the hypothesis that TLR4 has a role in hippocampus- dependent spatial learning and memory, we used mice deficient for TLR4 and mice receiving chronic TLR4 antagonist infusion to the lateral ventricles in the brain. We found that developmental TLR4 deficiency enhances spatial reference memory acquisition and memory retention, impairs contextual fear-learning and enhances motor functions, traits that were correlated with CREB up-regulation in the hippocampus. TLR4 antagonist infusion into the cerebral ventricles of adult mice did not affect cognitive behavior, but instead affected anxiety responses. Our findings indicate a developmental role for TLR4 in shaping spatial reference memory, and fear learning and memory. Moreover, we show that central TLR4 inhibition using a TLR4 antagonist has no discernible physiological role in regulating spatial and contextual hippocampus-dependent cognitive behavior. Citation: Okun E, Barak B, Saada-Madar R, Rothman SM, Griffioen KJ, et al. (2012) Evidence for a Developmental Role for TLR4 in Learning and Memory. PLoS ONE 7(10): e47522. doi:10.1371/journal.pone.0047522 Editor: Lena Alexopoulou, Centre d’Immunologie de Marseille-Luminy, CNRS-Inserm, France Received May 9, 2012; Accepted September 12, 2012; Published October 11, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This study was supported in part by the Intramural Research Program of the National Institute on Aging and in part by the Gonda Multidisciplinary Center for Brain Research and the Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: eitan.okun@biu.ac.il Introduction Toll like receptors (TLRs) are type-I trans-membrane receptors that are best known as sensors of microbe-associated molecular patterns (MAMPs) by cells of the innate immune system [1]. In addition, TLRs recognize damage-associated molecular patterns (DAMPs), also termed ‘endogenous ligands’, generated in response to traumatic tissue injury or as a by-product of inflammation [2]. Binding of MAMPs or DAMPs to TLRs typically activates signaling cascades that result in production of inflammatory cytokines/chemokines by effector cells, and may also stimulate a peripheral immune response [3]. TLR4, a widely studied TLR, is activated by bacterial lipopolysaccharide (LPS), a constituent of the outer membrane of gram-negative bacteria. Central activation of TLR4 by LPS has been thoroughly studied and was shown to reduce hippocampal pyramidal neuron dendrite length and to impair hippocampal-dependent spatial reference memory in an inflammation-dependent manner implying a neuroinflammatory role for TLR4 following activation with bacterial-derived ligands [4,5]. Increasing evidence indicates that TLRs located in the central nervous system are involved in developmental and adult neuroplasticity even in the absence of activation by infectious agents or tissue damage [6]. TLR3 for example, is a negative regulator of embryonic neural progenitor cell (NPC) proliferation [7]. TLR2 and TLR4 are expressed in adult NPCs [8] and have distinct and opposing functions in NPC proliferation and differentiation; TLR2 deficiency impairs hippocampal neurogen- esis, whereas TLR4 deficiency enhances proliferation and neuronal differentiation [8]. Further, TLRs 2, 3 and 4 are expressed in hippocampal neurons [9], and we showed recently that TLR3-deficient mice exhibit enhanced hippocampus-de- pendent working (but not reference) memory, coupled with extended retention of spatial reference memory [10]. In contrast, TLR3-deficient mice demonstrate impaired amygdala- and anxiety-related behavior [10]. Because TLR3 is involved in neurogenesis, NPC proliferation and cognitive learning and memory, we sought to determine the involvement of TLR4 in these processes. The hippocampus, which possesses a well-defined neuroanato- my, is involved in various types of learning, which are governed by different strategies [11,12]. While TLR4 is involved in CNS plasticity processes such as NPC proliferation, the impact of TLR4s on various aspects of hippocampus-dependent learning PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47522