experiments in Fig. 5, the cultured microglia (see Supplementary Figure) that had been preincubated with or without ATP (50 mM) were injected intrathecally in normal rats (see Supplementary Methods for full details). Immunohistochemistry Transverse L5 spinal cord sections (30 mm) were cut and processed for immunohistochemistry with anti-P2X4R antibody (Alomone). Identification of the type of P2X 4 R-positive cells was performed with the following markers: for microglia, OX42 (Chemicon) and iba1 (a gift from S. Kohsaka); for astrocytes, GFAP (Boehringer Mannheim); for spinal cord neurons, NeuN (Chemicon) and MAP2 (Chemicon). To assess immunofluorescence staining of cells quantitatively, we measured the immunofluorescence intensity of the P2X 4 R or OX42 as the average pixel intensity within each cell (see also Supplementary Methods). Western blotting Western blot analysis of P2X 4 R expression in the membrane fraction from L4-L6 spinal cord was performed with anti-P2X4R polyclonal antibody (Oncogene) as described in detail in the Supplementary Methods. Microglial culture Rat primary cultured microglia were prepared in accordance with the method described previously 28. In brief, mixed glial culture was prepared from neonatal Wistar rats and maintained for 10-16 days in DMEM medium with 10% fetal bovine serum. Immediately before experiments, microglia were collected by a gentle shake as the floating cells over the mixed glial culture. The microglia were transferred to coverslips or to Eppendorf tubes for subsequent intrathecal administration. Statistics Statistical analyses of the results were made with Student's t-test, Student's paired t-test or the Mann-Whitney U-test.

A full list of affiliations appears at the end of the paper. 'N euroglia' or 'glia' are collective terms describing cells of neuroepithelial (oligodendrocytes, astrocytes, oligodendrocyte progenitor cells, ependymal cells), neural crest (peripheral glia), and myeloid (microglia) origin. Changes in neuroglia associated with diseases of the CNS have been noted, characterized, and conceptualized from the very dawn of neuroglial research. Rudolf Virchow, in a lecture to students and medical doctors in 1858, stressed that 'this very interstitial tissue [that is, neuroglia] of the brain and spinal marrow is one of the most frequent seats of morbid change... ' 1. Changes in the shape, size, or number of glial cells in various pathological contexts have been frequently described by prominent neuroanatomists 2. In particular, hypertrophy of astrocytes was recognized very early as an almost universal sign of CNS pathology: 'the protoplasmic glia elements [that is, astrocytes] are really the elements which exhibit a morbid hypertrophy in pathological conditions' 3. Neuroglial proliferation was thought to accompany CNS lesions, leading to early suggestions that proliferating glia fully replaced damaged neuronal elements 4. Thus, a historical consensus was formed that a change in 'the appearance of neuroglia serves as a delicate indicator of the action of noxious influences upon the central nervous system, ' and the concept of 'reactionary change or gliosis' was accepted 5. While the origin of 'gliosis' is unclear (glia + osis in Greek means 'glial condition or process'; in Latin the suffix-osis acquired the additional meaning of 'disease'; hence 'astrogliosis'

Microglia, brain immune cells, engage in the clearance of dead cells or dangerous debris, which is crucial to the maintenance of brain functions. When a neighbouring cell is injured, microglia move rapidly towards it or extend a process to engulf the injured cell. Because cells release or leak ATP when they are stimulated or injured, extracellular nucleotides are thought to be involved in these events. In fact, ATP triggers a dynamic change in the motility of microglia in vitro and in vivo, a previously unrecognized mechanism underlying microglial chemotaxis; in contrast, microglial phagocytosis has received only limited attention. Here we show that microglia express the metabotropic P2Y6 receptor whose activation by endogenous agonist UDP triggers microglial phagocytosis. UDP facilitated the uptake of microspheres in a P2Y6-receptor-dependent manner, which was mimicked by the leakage of endogenous UDP when hippocampal neurons were damaged by kainic acid in vivo and in vitro. In addition, systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in messenger RNA encoding P2Y6 receptors that colocalized with activated microglia were observed. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and could function as a sensor for phagocytosis by sensing diffusible UDP signals, which is a previously unknown pathophysiological function of P2 receptors in microglia.

Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8–10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca2+ transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.