Cien Años de Microglía: Milestones in a Century of Microglial Research
Authors: Amanda Sierra, Rosa C. Paolicelli, Helmut Kettenmann
Journal: Trends in Neurosciences (2019)

Microglia were first described by the Spanish researcher Pío del Río-Hortega in 1919.Río-Hortega's discoveries identified and defined the three types of glial cells of the CNS: astrocytes, oligodendrocytes, and microglia.The field stagnated until the 1960s, when Georg Kreutzberg discovered the role of microglia in synaptic stripping in pathology.Microglia were subsequently characterized as pathologic sensors in essentially all brain diseases and orchestrators of the neuroinflammatory response.In the last two decades, after the discovery of their extraordinary motility and their unique origin in the yolk sac, microglial research has grown exponentially.Physiological roles of microglia include synapse monitoring and pruning, as well as modulating neurogenesis, myelination, and vasculogenesis. The year 2019 marks the 100-year anniversary of the discovery of microglia by Pío del Río-Hortega. We will recount the state of neuroscience research at the beginning of the 20th century and the heated scientific dispute regarding microglial identity. We will then walk through some of the milestones of microglial research in the decades since then. In the last 20 years, the field has grown exponentially. Researchers have shown that microglia are unlike any other resident macrophages: they have a unique origin and distinguishing features. Microglia are extraordinarily motile cells and constantly survey their environment, interacting with neurons, astrocytes, oligodendrocytes, neural stem cells, and infiltrating immune cells. We finally highlight some open questions for future research regarding microglia's identity, population dynamics, and dual (beneficial and detrimental) role in pathology. The year 2019 marks the 100-year anniversary of the discovery of microglia by Pío del Río-Hortega. We will recount the state of neuroscience research at the beginning of the 20th century and the heated scientific dispute regarding microglial identity. We will then walk through some of the milestones of microglial research in the decades since then. In the last 20 years, the field has grown exponentially. Researchers have shown that microglia are unlike any other resident macrophages: they have a unique origin and distinguishing features. Microglia are extraordinarily motile cells and constantly survey their environment, interacting with neurons, astrocytes, oligodendrocytes, neural stem cells, and infiltrating immune cells. We finally highlight some open questions for future research regarding microglia's identity, population dynamics, and dual (beneficial and detrimental) role in pathology. In memoriam, Georg Kreutzberg (1932–2019), father of modern microglial research. 'As a result of our first observation, we are convinced that this new cell type is a glial cell and we call it microglia due to the tiny size of their soma, which is considerably smaller than that of fibrous and protoplasmic astrocytes. Nonetheless, the subsequent assumption that microglia have a mesodermal origin inspired us also to name them mesoglia' (Río-Hortega, El "Tercer Elemento" de los Centros Nerviosos. I. La Microglía en Estado Normal, 1919). With these words, the scientific field of microglia research was born 100 years ago, amidst a scientific and personal dispute between its scientific father, Pío del Río-Hortega, and the Spanish Nobel awardee Santiago Ramón y Cajal. In the next sections we will describe the origin of the clash between Cajal and Río-Hortega, the state-of-the-art of neuroscience during the early years of the 20th century, and the main findings of Río-Hortega, many of which have only been recently further advanced. We will then recount some of the major findings in the last 100 years of microglial research and we will finish by discussing key topics to be addressed in the future. The early years of the 20th century were a thriving cultural and scientific period in Spain, when Ramón y Cajal created a school of neuroscience research in Madrid, to which many international researchers contributed (Figure 1). In the late decades of the 19th century, neurons and their connections had been established by Cajal as the basis of brain function, in what was called the 'neuron doctrine', defining neurons as the building blocks of the brain. In contrast, Camillo Golgi promoted the 'reticular theory', that considered the brain as a continuous network. For those studies, both Cajal and Golgi were awarded the Nobel prize in 1906. In addition to neurons, 'neuroglia' had been identified by the German pathologist Rudolf Virchow in 1856 [1Virchow R. Gesammelte Abhandlungen zur Wissenschaftlichen Medizin, Meidinger.1856Google Scholar] and the term 'astrocyte' was introduced by the Hungarian researcher Mihaly Lenhossék in 1895 [2Lenhossek M. Bau des Nervensystems, Fischer Verlag.1895Google Scholar]. In the early 1900s, Cajal became interested in astrocytes, inspired by Nicolás Achúcarro, who joined his laboratory after working with Alois Alzheimer in Munich, Germany. Cajal developed a novel method to stain the brain (the gold chloride sublimate method) that allowed him to obtain superb images of protoplasmic and fibrous astrocytes. The rest of the parenchymal cells remained poorly stained and showed no processes and Cajal named them 'apolar cells' or 'third element' [3Ramón y Cajal S. Contribución al Conocimiento de la Neuroglía del Cerebro Humano, Trabajos del Laboratorio de Investigaciones Biológicas de la Universidad de Madrid XI.1913: 215-315Google Scholar]. At this time, Río-Hortega, a young researcher from a remote Castilian village in Spain who professed the highest admiration for Cajal, joined Achúcarro's lab. Río-Hortega's systematic research led him to develop novel methods to stain the brain (ammoniacal silver carbonate method) and he was struck to find that the apolar cells consisted in fact of two different cell types, which he named microglia and interfascicular glia (he later renamed them as oligodendrocytes). In his 1919 series of four seminal papers The 'Third Element' of the Neural Centers, published in Spanish in Boletín de la Sociedad Española de Biología, the scientific journal edited by Cajal, Río-Hortega opposed Cajal's view of the 'third element' and stated that microglia are the phagocytic cells of the brain and are of mesodermal origin [4Río-Hortega P. El "Tercer Elemento" de los Centros Nerviosos. I. La Microglía en Estado Normal, Boletín de la Sociedad Española de Biología VIII.1919: 67-82Google Scholar, 5Río-Hortega P. El "Tercer Elemento de los Centros Nerviosos". II. Intervención de la Microglía en los Procesos Patológicos (Células en Bastoncito y Cuerpos Gránuloadiposos), Boletín de la Sociedad Española de Biología VIII.1919: 91-103Google Scholar, 6Río-Hortega P. El "Tercer Elemento" de los Centros Nerviosos. III. Naturaleza Probable de la Microglía, Boletín de la Sociedad Española de Biología VIII.1919: 108-121Google Scholar, 7Río-Hortega P. El "Tercer Elemento de los Centros Nerviosos". IV. Poder Fagocitario y Movilidad de la Microglía, Boletín de la Sociedad Española de Biología VIII.1919: 154-171Google Scholar] (for an English translation see [8Sierra A. et al.The "big-bang" for modern glial biology: translation and comments on Pio del Rio-Hortega 1919 series of papers on microglia.Glia. 2016; 64: 1801-1840Crossref PubMed Scopus (48) Google Scholar]). To fully appreciate the relevance of these statements, it is necessary to explain the complexity of neuroscience research during that period. In those days, each laboratory had its own staining method and animal/disease model and, as a result, researchers faced a confounding nomenclature of brain cells that, in addition to neurons and astrocytes, included 'foam cells', 'rod cells', 'scavenger cells', and 'granuloadipose cells', among others [9Tremblay M.E. et al.From the Cajal alumni Achucarro and Rio-Hortega to the rediscovery of never-resting microglia.Front. Neuroanat. 2015; 9: 45Crossref PubMed Scopus (0) Google Scholar]. Adding to the confusion in the nomenclature was the mix of languages used in the scientific communication at the time (primarily German, French, or Spanish). Sharing reagents, recipes, and microscopy preparations was not easy and, in many cases, researchers had to rely on published drawings of the cells rather than photography to compare each others' findings. Río-Hortega's staining, beautiful drawings, and systematic description of the cells under different experimental conditions unified the literature and provided a clear and systematic identification of the three major glial cell types in the brain: astrocytes, oligodendrocytes, and microglia. Río-Hortega encountered, however, a strong opposition by Cajal. The personal origin of the feud is somewhat obscure but could be related, at least to some extent, to the fact that Río-Hortega's growing laboratory was occupying Cajal's own laboratory, leading to a clash between their members. At the scientific level, the problem was related to the superior staining of Río-Hortega's method, which allowed one to see profusely ramified microglia where Cajal had only seen 'apolar cells' [3Ramón y Cajal S. Contribución al Conocimiento de la Neuroglía del Cerebro Humano, Trabajos del Laboratorio de Investigaciones Biológicas de la Universidad de Madrid XI.1913: 215-315Google Scholar]. Further complication arose from Río-Hortega's choice to use the term 'mesoglia' to refer to microglia, which stemmed from his (correct) conjuncture that they are of mesodermal origin, unlike the rest of the brain cells. It is somewhat unfortunate that the term 'mesoglia' had been used a few years earlier by the Scottish researcher William Ford Robertson to refer to small phagocytic cells of mesodermal origin [10Robertson W. A microscopic demonstration of the normal and pathological histology of mesoglia cells.J. Ment. Sci. 1900; 46: 724Crossref Google Scholar]. Cajal, having read a recount of Robertson's paper by the Italian researcher Ugo Cerletti, assigned Robertson the priority, and named the cells 'Robertson-del Río' cells [11Ramón y Cajal, S Algunas Consideraciones Sobre la Mesoglía de Robertson y Río Hortega, Trabajos del Laboratorio de Investigaciones Biológicas de la Universidad de Madrid XVIII.1920: 129-141Google Scholar]. The dispute between Río-Hortega and Cajal evolved over a few years and it was only in 1924 that the American neurosurgeon Wilder Penfield had the opportunity to directly inspect Robertson's preparations and assess that in fact Robertson had not labeled microglia, but rather oligodendrocytes, which are neither phagocytic nor mesodermal [12Penfield W. Oligodendroglia and its relation to classical neuroglia.Brain. 1924; 47: 430-452Crossref Scopus (0) Google Scholar]. Since then, Río-Hortega's nomenclature and major findings have been widely recognized to hold true. In the following, we list Río-Hortega's key findings, each followed by related quotes from his 1919 papers [4Río-Hortega P. El "Tercer Elemento" de los Centros Nerviosos. I. La Microglía en Estado Normal, Boletín de la Sociedad Española de Biología VIII.1919: 67-82Google Scholar, 5Río-Hortega P. El "Tercer Elemento de los Centros Nerviosos". II. Intervención de la Microglía en los Procesos Patológicos (Células en Bastoncito y Cuerpos Gránuloadiposos), Boletín de la Sociedad Española de Biología VIII.1919: 91-103Google Scholar, 6Río-Hortega P. El "Tercer Elemento" de los Centros Nerviosos. III. Naturaleza Probable de la Microglía, Boletín de la Sociedad Española de Biología VIII.1919: 108-121Google Scholar, 7Río-Hortega P. El "Tercer Elemento de los Centros Nerviosos". IV. Poder Fagocitario y Movilidad de la Microglía, Boletín de la Sociedad Española de Biología VIII.1919: 154-171Google Scholar, 8Sierra A. et al.The "big-bang" for modern glial biology: translation and comments on Pio del Rio-Hortega 1919 series of papers on microglia.Glia. 2016; 64: 1801-1840Crossref PubMed Scopus (48) Google Scholar]: The 'apolar' cell defined by Cajal consists of two cell types, microglia and interfascicular cells (later termed oligodendrocytes), both of which are different from astrocytes: '(our staining method) convinced us about two things, both equally important: that the "third element" is not, as it is presumed, without processes; and that the "third element" of the white and grey matter are different. Our research revealed the existence of two different cell types. Many perineuronal satellite cells, which are not astrocytes, are scattered in the grey matter and some of those which are in the white matter are identical in nature and have branched appendages, constituting, for us, the microglia. The «apolar» cells of the white matter, grouped into packages or columns between the bundles of axons, also possess more or less developed processes, but dramatically differ from those of the microglia and in fact are a different cell type distinct from astrocytes. We shall call them interfascicular glia.' Microglia are of mesodermal origin and related to leukocytes; oligodendrocytes are more related to astrocytes: 'While microglia derive from the mesoderm and are able to phagocytose debris, interfascicular glia (which also have processes) are more alike to astrocytes in their origin and function.' Microglia phagocytose dendritic spines and cells during development: 'It is very likely that during the development of the complex neuronal network, before young neurons and embryonic neuroglia acquire their final location and shape, some delicate neuronal processes become fragmented and some cells disintegrate or die. These phenomena would require the intervention of microglial macrophages to gather and degrade the cellular debris.' Microglia are plastic, interact with all other cells of the brain parenchyma, and undergo a morphological activation after damage: 'The nomadic nature of microglia is best observed in neurodegenerative processes, during which the apparent rest they enjoyed in the normal state turns into migratory and phagocytic activity.' Microglia proliferate in response to damage: 'Microglial proliferation can be easily observed in pathological conditions, during which mitotic and amitotic forms of division coincide with microglial hypertrophy.' Microglia phagocytose neuronal debris during pathological conditions: 'Microglia acts as a voracious macrophage. A few hours after the injury is produced, they already enclose granulations and thick corpuscles in their cytoplasm (…). The gathering and degradations of cell debris occur, then, in a short period of time.' After Río-Hortega's definition of microglia, their presence was verified in different brain regions, animal species, and in a variety of pathologies. Wilder Penfield promoted Río-Hortega's findings and analyzed glioma tissue, describing the presence of microglia [13Penfield W. Microglia and the process of phagocytosis in glioma.Am. J. Pathol. 1925; 1: 77-90PubMed Google Scholar]. Some authors named microglia 'Hortega cells' [14Metz A.U.S.H. Die Hortega'schen Zellen, das sogenannte "dritte Element" und uber ihre funktionelle Bedeutung.Z. Neur. 1924; 100 (in German): 428-449Google Scholar]. In the first review on microglia, Río-Hortega summarized the field in the book series Cytology and Cellular Pathology of the Nervous System, issued by Penfield, and this review article in English remained the state-of-the-art for many decades to come [15Río- Hortega P. Microglia.in: Penfield W. Cytology of the Nervous System. Hafner Publishing, 1932: 482-534Google Scholar]. The origin of microglia in the human brain was first analyzed by John Kershman from the Montreal Neurological Institute, who observed the infiltration of microglia during embryonic human development and described hot spots of migrating cells, such as at the choroid plexus, coining the term 'microglia fountains' for these sites [16Kershman K. Genesis of microglia in the human brain.Arch. Neurol. Psychiatr. 1939; 41: 24-50Crossref Scopus (64) Google Scholar]. Thereafter, the field of microglial research stagnated for many decades (Figure 2). Over the 23-year span between 1945 and 1968, only 38 publications with the word 'microglia' in their title or abstract were published, based on a PubMed search. In sharp contrast, in 2018, 38 such papers were published, on average every 4 days. The field was slowly revived, starting with the studies performed by Georg Kreutzberg's group in Munich in 1968. Kreutzberg passed away earlier this year, in this centennial anniversary of the discovery of microglia, and we dedicate our publication to him. In the 1960s and 1970s, Kreutzberg's group developed a novel model to study microglial activation in the absence of peripheral monocyte invasion. By lesioning the facial nerve in the periphery, they observed an activation of microglia in the facial nucleus. These cells were involved in the reorganization of the circuitry of the nucleus, which was associated with removal of synaptic terminals on the motoneurons or 'synaptic stripping' by microglia [17Blinzinger K. Kreutzberg G. Displacement of synaptic terminals from regenerating motoneurons by microglial cells.Z. Zellforsch. Mikrosk. Anat. 1968; 85: 145-157Crossref PubMed Scopus (644) Google Scholar]. Another key historical finding was the establishment of novel methods to identify microglia beyond Río-Hortega's silver staining, which works well in rabbit but is not as efficient in mouse or rat, animal models that by the 1960s and 1970s were more widely used. A histochemical identification by ATPase activity was developed, based on the enzymatic activity of ATP degrading enzymes, which are highly expressed by microglia [18Ibrahim M.Z. et al.The histochemical identification of microglia.J. Neurol. Sci. 1974; 22: 211-233Abstract Full Text PDF PubMed Scopus (0) Google Scholar]. While the morphologic inspection of microglia in the different healthy or pathologic tissues showed some of their functional features such as migration or phagocytosis, further studies were difficult to perform in situ. The establishment of the first cell culture system by Dana Giulian and Timothy Baker in 1986 to grow isolated and purified microglia was an important step to manipulate and study functional parameters, such as cytokine release. In these studies, microglia were typically isolated from the early postnatal rat brain and showed some of the properties as those of microglia found in situ [19Giulian D. Baker T.J. Characterization of ameboid microglia isolated from developing mammalian brain.J. Neurosci. 1986; 6: 2163-2178Crossref PubMed Google Scholar]. This culture method rapidly spread among researchers world-wide and resulted in a first wave of functional studies on microglia. To provide some context for the more junior scientists among our readers, at that time, only a few molecules had been cloned, monoclonal antibodies had just started to spread, and modern techniques of molecular biology were not yet available. Using this culture system, it became evident that microglia produce superoxide after stimulation with the yeast-derived glucan zymosan [20Colton C.A. Gilbert D.L. Production of superoxide anions by a CNS macrophage, the microglia.FEBS Lett. 1987; 223: 284-288Crossref PubMed Scopus (500) Google Scholar]. Cell culture studies also revealed that microglia are capable of expressing molecules for immune interaction, such as MHC-I, which participates in antigen presentation to T lymphocytes [21Suzumura A. et al.MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by gamma-interferon.J. Neuroimmunol. 1987; 15: 263-278Abstract Full Text PDF PubMed Scopus (0) Google Scholar]. MHC-I was also found in parenchymal cells in postmortem tissue of multiple sclerosis patients using a pan macrophage marker that did not distinguish between intrinsic microglia and monocytes invading from the periphery, but nonetheless suggested that microglia could be the partners of invading T cells in this disease [22Hayes G.M. et al.Microglia are the major cell type expressing MHC class II in human white matter.J Neurol Sci. 1987; 80: 25-37Abstract Full Text PDF PubMed Google Scholar]. At that time, in 1988, the Kreutzberg group published the first review (to our knowledge) on microglia since the one by Río-Hortega in 1932, and organized the first conference devoted to these cells (Methods in Microglial Research, 1992) summarizing their known functions and providing a vision of where the field should be going [23Streit W.J. et al.Functional plasticity of microglia: a review.Glia. 1988; 1: 301-307Crossref PubMed Scopus (794) Google Scholar]. Microglia emerged as central elements in the pathologic production and release of chemokines and cytokines in the brain. Using bacterial lipopolysaccharide (LPS) as a stimulus, which had been adopted from the macrophage field as an efficient activator, it became evident that microglia are the predominant producer of the cytokine interleukin-1 beta (IL-1β) in neuroinflammation [24Hetier E. et al.Brain macrophages synthesize interleukin-1 and interleukin-1 mRNAs in vitro.J Neurosci Res. 1988; 21: 391-397Crossref PubMed Google Scholar]. Cultured microglia were soon also identified as the major source of another major cytotoxic cytokine, tumor necrosis factor alpha (TNF-α), after stimulation with LPS [25Sawada M. et al.Production of tumor necrosis factor-alpha by microglia and astrocytes in culture.Brain Res. 1989; 491: 394-397Crossref PubMed Scopus (0) Google Scholar]. At that time, a recently developed electrophysiology technique to study ionic currents in isolated cells, patch-clamp, was applied to study cultured microglia. It was found that microglia express a unique membrane current pattern that distinguishes them from macrophages [26Kettenmann H. et al.Cultured microglial cells have a distinct pattern of membrane channels different from peritoneal macrophages.J Neurosci Res. 1990; 26: 278-287Crossref PubMed Scopus (186) Google Scholar] and it has become evident now that K+ channel activity regulates microglial functions [27Madry C. et al.Microglial ramification, surveillance, and interleukin-1beta release are regulated by the two-pore domain K(+) channel THIK-1.Neuron. 2018; 97: 299-312Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar]. Another major development was the introduction of BV-2 cells, a stable microglial cell line generated by infecting cultured murine microglia with a v-raf/v-myc recombinant retrovirus [28Bocchini V. et al.An immortalized cell line expresses properties of activated microglial cells.J Neurosci Res. 1992; 31: 616-621Crossref PubMed Scopus (284) Google Scholar]. BV-2 cells share many properties with cultured microglia, but over the years it became evident that in vitro and ex vivo microglia differ in many functional aspects. Despite these limitations, this cell line is still in use today (over 400 publications in PubMed using BV-2 cells in 2018). Physiological studies in vitro were also extended to demonstrate that microglia respond to neuroactive substances. Among them stand out purinergic receptors, which bind to extracellular purines such as ATP and were identified using the patch-clamp technique [29Walz W. et al.Extracellular ATP activates a cation conductance and a K+ conductance in cultured microglial cells from mouse brain.J Neurosci. 1993; 13: 4403-4411Crossref PubMed Google Scholar]. In vivo studies of microglia were pushed forward by the development of microglial-preferring ligands, such as PK11195, which binds microglial peripheral benzodiazepine receptors (PBR, also known as TSPO, or 18 kDa translocator protein). Using PK11195, the first positron emission tomography (PET) studies in which microglia were labeled in humans came to light, showing that 'activated' microglia were present in areas of demyelination of two multiple sclerosis patients [30Vowinckel E. et al.PK11195 binding to the peripheral benzodiazepine receptor as a marker of microglia activation in multiple sclerosis and experimental autoimmune encephalomyelitis.J Neurosci Res. 1997; 50: 345-353Crossref PubMed Scopus (231) Google Scholar]. Nonetheless, TSPO also labels astrocytes and, thus, novel and more specific markers needed to be developed [31Best L. et al.New and old TSPO PET radioligands for imaging brain microglial activation in neurodegenerative disease.Curr Neurol Neurosci Rep. 2019; 19: 24Crossref PubMed Scopus (2) Google Scholar]. In 1998, a novel calcium-binding protein, the ionized calcium-binding adaptor molecule 1 (Iba1), was cloned. When antibodies were generated against this protein, a novel and important tool had appeared to reliably identify microglia in tissue, and the Iba1-label has become one of the gold standards to identify microglia [32Ito D. et al.Microglia-specific localisation of a novel calcium binding protein, Iba1.Brain Res Mol Brain Res. 1998; 57: 1-9Crossref PubMed Scopus (793) Google Scholar], although it also labels perivascular, meningeal, and choroid plexus macrophages [33Goldmann T. et al.Origin, fate and dynamics of macrophages at central nervous system interfaces.Nat Immunol. 2016; 17: 797-805Crossref PubMed Scopus (219) Google Scholar]. Until 2000, the field was dominated by studies of microglia in cell culture and this period was essential to establish many microglial functional parameters (Figure 2). It was evident, however, that cultured cells would never reflect the complexity of signals faced by microglia in the brain environment. A novel labeling method, based on tomato-lectin staining of living microglia in acute brain slices, eventually allowed recapitulation of the physiological properties of microglia in the tissue [34Streit W.J. An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4).J Histochem Cytochem. 1990; 38: 1683-1686Crossref PubMed Google Scholar]. This further confirmed that microglia in a normal tissue environment (as normal as a brain slice can be) express a membrane current pattern that is distinct from the cultured microglia. Under pathological conditions, the cells in the tissue soon acquire the membrane current pattern typical of cultured cells, indicating that culture conditions represent a pathologic environment [35Boucsein C. et al.Electrophysiological properties of microglial cells in normal and pathologic rat brain slices.Eur J Neurosci. 2000; 12: 2049-2058Crossref PubMed Scopus (105) Google Scholar]. In the same year, a knockout mouse line for the chemokine fractalkine receptor (also known as CX3CR1), which in the brain is selectively expressed in microglia, was established [36Jung S. et al.Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol Cell Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (1385) Google Scholar]. The deletion construct also had a GFP reporter, and the scientific community soon recognized the potential use of heterozygous mice as a microglial-specific reporter line, although some effects due to the lack of one copy of the endogenous locus have been reported [37Rogers J.T. et al.CX3CR1 deficiency leads to impairment of hippocampal cognitive function and synaptic plasticity.J Neurosci. 2011; 31: 16241-16250Crossref PubMed Scopus (276) Google Scholar]. Using this mouse line, two independent studies combined it with in vivo imaging techniques and found that microglia rapidly scan the environment with their processes. In response to a laser injury, these processes engulf the damaged tissue within tens of minutes [38Davalos D. et al.ATP mediates rapid microglial response to local brain injury in vivo.Nat Neurosci. 2005; 8: 752-758Crossref PubMed Scopus (1953) Google Scholar, 39Nimmerjahn A. et al.Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.Science. 2005; 308: 1314-1318Crossref PubMed Scopus (2725) Google Scholar]. With these imaging techniques, the field took off, and in the past 20 years has evolved rapidly. The last decades of microglial research have been focused on figuring out the contributions of microglia to brain physiology and pathology. It is beyond the scope of this article to review thoroughly all functions of microglia in health and disease and we apologize to all our colleagues whose work could not be cited due to space constraints. We will now summarize three major areas of research in the past 20 years: brain physiology, brain pathologies, and microglial identity (Figure 3). The first evidence that microglia actively participate in brain development came from in vitro studies in the cerebellum, where it was found that microglia promoted the elimination of Purkinje neurons via superoxide ions produced by microglial respiratory bursts, thereby actively shaping the neuronal circuitry [40Marin-Teva J.L. et al.Microglia promote the death of developing Purkinje cells.Neuron. 2004; 41: 535-547Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Since then, microglia have also been shown to limit the production of cortical neurons in rodents as well as in primates [41Cunningham C.L. et al.Microglia regulate the number of neural precursor cells in the developing cerebral cortex.J Neurosci. 2013; 33: 4216-4233Crossref PubMed Scopus (314) Google Scholar] and to support survival of layer V neurons in postnatal development through the release of insulin growth factor 1 (IGF1) [42Ueno M. et al.Layer V cortical neurons require microglial support for survival during postnatal development.Nat Neurosci. 2013; 16: 543-551Crossref PubMed Scopus (288) Google Scholar]. In the adult hippocampal neurogenic cascade, microglia remove the excess of newborn cells [43Sierra A. et al.Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis.Cell Stem Cell. 2010; 7: 483-495Abstract Full Text Full Text PDF Pub