RESEARCH ARTICLE Microglial Polarization and Plasticity: Evidence from Organotypic Hippocampal Slice Cultures Maria Antonietta Ajmone-Cat, Melissa Mancini, Roberta De Simone, Piera Cilli, and Luisa Minghetti Increasing evidence indicates that “functional plasticity” is not solely a neuronal attribute but a hallmark of microglial cells, the main brain resident macrophage population. Far from being a univocal phenomenon, microglial activation can originate a plethora of functional phenotypes, encompassing the classic M1 proinflammatory and the alternative M2 anti-inflammatory phenotypes. This concept overturns the popular view of microglial activation as a synonym of neurotoxicity and neurogenesis failure in brain disorders. The characterization of the alternative programs is a matter of intense investigation, but still scarce information is available on the course of microglial activation, on the reversibility of the different commitments and on the capability of preserving molecular memory of previous priming stimuli. By using organotypic hippocampal slice cultures as a model, we developed paradigms of stimulation aimed at shedding light on some of these aspects. We show that persistent stimulation of TLR4 signaling promotes an anti-inflammatory response and microglial polarization toward M2-like phenotype. Moreover, acute and chronic preconditioning regimens permanently affect the capability to respond to a later challenge, sug- gesting the onset of mechanisms of molecular memory. Similar phenomena could occur in the intact brain and differently affect the vulnerability of mature and newborn neurons to noxious signals. GLIA 2013;61:1698–1711 Key words: brain macrophages, microglia, endotoxin tolerance, M1, M2 inflammation Introduction T he recently recognized existence of a wide range of func- tional outcomes of microglial activation, strictly stimuli- dependent and related to the pathological conditions examined (Perry et al., 2010; Saijo and Glass, 2011), urges the need to define the many features of “activation” and dis- close the immunological role of different microglial phenotypes. The knowledge gained over the past decade on peripheral mac- rophages has been only recently extended to their nervous sys- tem counterpart, that is, microglia. The nomenclature adopted for defining the pro- and anti-inflammatory extremes of macro- phage activation (M1 for the proinflammatory, M2 for the anti-inflammatory state) has been modeled on that used to cat- egorize polarized T lymphocytes. Unlike classical M1 activation induced by Toll like receptor (TLR) agonists and Th1- cytokines, alternative or M2 activation, elicited by Th2- cytokines IL-4 or IL-13, is characterized by induction of genes associated with resolution of inflammation. M2 proper- ties are not necessarily associated with positive outcomes as inappropriate down-regulation of inflammatory response can result in pathological conditions, such as induction of fibrosis, subversion of adaptive immunity, and promotion of tumor growth and metastasis (Mantovani et al., 2002). The label M2 is also widely used to define a state of “acquired deactivation,” such as that induced by uptake of apoptotic cells, oxidized lipids, exposure to the anti-inflammatory cyto- kines IL-10 and TGF-b or immune complexes, although such phenotype should be better referred as to “M2b” as the immu- nological profile induced is not completely overlapping that of alternative M2 macrophages (Gordon, 2003; Gordon and Taylor, 2005). Other activation states, typified by specific combinations of molecular markers, have been described either in peripheral organs (Biswas and Mantovani, 2010 and references therein), View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22550 Published online Aug 5, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received Sep 25, 2013, Accepted for publication June 17, 2013. Address correspondence to Maria Antonietta Ajmone-Cat; Istituto Superiore di Sanit a, Viale Regina Elena 299, 00185 Rome, Italy. E-mail: mariaantonietta.ajmone-cat@iss.it From the Department of Cell Biology and Neuroscience, Istituto Superiore di Sanit a, Rome, Italy. 1698 V C 2013 Wiley Periodicals, Inc.