Microglia Sculpt Postnatal Neural Circuits in an Activity and Complement-Dependent Manner

“Microglia sampled from P5 CR3 or C3 KO mice had a statistically significant decrease in Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. 697 (A) Immunohistochemistry for the alpha subunit of CR3 (CD11b) reveals that microglia express high levels of CR3/CD11b (left column) in the P5 dLGN (top panels) versus older a…”

“…are the resident CNS immune cells which have long been recognized as rapid responders to injury and disease, play- ing a role in a broad range of processes such as tissue inflammation and clearance of cellular debris (Hanisch and Kettenmann, 2007; Kreutzberg, 1996; Ransohoff and Perry, 2009).”

“Furthermore, microglia can contribute to synaptic plasticity in the adult CNS and, more recently, in the context of the normal developing hippocampus (Paolicelli et al., 2011; Pascual et al., 2012; Roumier et al., 2008).”

“However, recent studies suggest that microglia may play a role in synaptic remodeling and plasticity in the healthy brain (Davalos et al., 2005; Nimmerjahn et al., 2005; Paolicelli et al., 2011; Schafer et al., 2012; Tremblay et al., 2010a; Wake et al., 2009).”

“3D structural illuminationmicroscopy (3D-SIM), a technique enabling 2X the resolution of light microscopy (Gustafsson, 2000), was used to assess the P5 dLGN of CX3CR1+/EGFP mice immunolabeled for VGlut2.”

“…are the resident CNS immune cells which have long been recognized as rapid responders to injury and disease, play- ing a role in a broad range of processes such as tissue inflammation and clearance of cellular debris (Hanisch and Kettenmann, 2007; Kreutzberg, 1996; Ransohoff and Perry, 2009).”

“) (Stevens et al., 2007), suggesting the intriguing possibility that complement cascade activation and function is regulated by neural activity.”

“Microglia-Mediated Engulfment of RGC Inputs Is Regulated by Neural Activity Synaptic pruning is thought to result from competition between neighboring axons for postsynaptic territory based on differences in patterns or levels of activity (Hua and Smith, 2004; Katz and Shatz, 1996; Sanes and Lichtman, 1999).”

“Microglia were labeled using the CX3CR1+/GFP mouse line in which all microglia express EGFP under the control of fractalkine receptor, CX3CR1, expression (Figure 1 and see Figures S1 and S5 available online; Cardona et al., 2006; Jung et al., 2000; Saederup et al., 2010).”

“Taken together, thesedatademonstrate that phagocytic signaling through CR3 and its ligand C3 is one molecular mechanism by which microglia engulf RGC inputs.”

“…work has demonstrated that microglia have the capacity to interact with synaptic elements in response to neurotransmitter release and/or sensory experience (Biber et al., 2007; Fontainhas et al., 2011; Nimmerjahn et al., 2005; Ransohoff and Perry, 2009; Tremblay et al., 2010a; Wake et al., 2009).”

“However, recent studies suggest that microglia may play a role in synaptic remodeling and plasticity in the healthy brain (Davalos et al., 2005; Nimmerjahn et al., 2005; Paolicelli et al., 2011; Schafer et al., 2012; Tremblay et al., 2010a; Wake et al., 2009).”

“To further confirm microglia-mediated phagocytosis of synaptic elements, immunohistochemical electron microscopy (immunoEM) for the microglia marker iba-1 was performed and quantified in the P5 dLGN (Figure 4C; Tremblay et al., 2010b).”

“Microglia-Mediated Engulfment of RGC Inputs Is Regulated by Neural Activity Synaptic pruning is thought to result from competition between neighboring axons for postsynaptic territory based on differences in patterns or levels of activity (Hua and Smith, 2004; Katz and Shatz, 1996; Sanes and Lichtman, 1999).”

“Microglia were labeled using the CX3CR1+/GFP mouse line in which all microglia express EGFP under the control of fractalkine receptor, CX3CR1, expression (Figure 1 and see Figures S1 and S5 available online; Cardona et al., 2006; Jung et al., 2000; Saederup et al., 2010).”

“Furthermore, given that microglia are the only CNS cell that express CR3 in the postnatal dLGN, these data suggest that microglia are mediators of synaptic remodeling in Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. 699 (A) Single plane array tomography images for VGlut2 (green) to label RGC terminals and GluR1…”

“) were completely absent from the P5 dLGN and surrounding brain tissue (Figure S5) (Akiyama and McGeer, 1990; Perry et al., 1985).”

“…havebeen shown tobeactivity dependent, localized to synapses, and colocalized with C1q leaving the possibility that MHC class I molecules may play an upstream role in microglia-mediated pruning of synapses (Corriveau et al., 1998; Datwani et al., 2009; Goddard et al., 2007; Huh et al., 2000).”

“Array Tomography Array tomography was performed as previously described with minor modifications (Greer et al., 2010; Margolis et al., 2010; Micheva and Smith, 2007; Ross et al., 2010; Stevens et al., 2007).”

“…work has demonstrated that microglia have the capacity to interact with synaptic elements in response to neurotransmitter release and/or sensory experience (Biber et al., 2007; Fontainhas et al., 2011; Nimmerjahn et al., 2005; Ransohoff and Perry, 2009; Tremblay et al., 2010a; Wake et al., 2009).”

“Furthermore, microglia can contribute to synaptic plasticity in the adult CNS and, more recently, in the context of the normal developing hippocampus (Paolicelli et al., 2011; Pascual et al., 2011; Roumier et al., 2008).”

“…a competition results in decreased territory of the ‘‘weaker’’ inputs and increased territory of ‘‘stronger’’ inputs within the dLGN (Del Rio and Feller, 2006; Huberman et al., 2008; McLaughlin et al., 2003; Penn et al., 1998; Shatz, 1990; Shatz and Stryker, 1988; Stellwagen and Shatz, 2002;…”

“Using the in vivo phagocytosis assay previously described (Figure 2), engulfmentwas assessed in P5mice lacking functional CR3 (CR3 KO) due to a genetic deletion of the alpha subunit, CD11b (Figure S5B; Coxon et al., 1996) or mice deficient in CR3 ligand, C3 (C3 KO) (Figure S5A).”

“Microglia were labeled using the CX3CR1+/GFP mouse line in which all microglia express EGFP under the control of fractalkine receptor, CX3CR1, expression (Figure 1 and see Figures S1 and S5 available online; Cardona et al., 2006; Jung et al., 2000; Saederup et al., 2010).”

“However, recent studies suggest that microglia may play a role in synaptic remodeling and plasticity in the healthy brain (Davalos et al., 2005; Nimmerjahn et al., 2005; Paolicelli et al., 2011; Schafer et al., 2012; Tremblay et al., 2010a; Wake et al., 2009).”

“…havebeen shown tobeactivity dependent, localized to synapses, and colocalized with C1q leaving the possibility that MHC class I molecules may play an upstream role in microglia-mediated pruning of synapses (Corriveau et al., 1998; Datwani et al., 2009; Goddard et al., 2007; Huh et al., 2000).”

“…of the ‘‘weaker’’ inputs and increased territory of ‘‘stronger’’ inputs within the dLGN (Del Rio and Feller, 2006; Huberman et al., 2008; McLaughlin et al., 2003; Penn et al., 1998; Shatz, 1990; Shatz and Stryker, 1988; Stellwagen and Shatz, 2002; Stellwagen et al., 1999; Torborg and Feller, 2005).”

“(Del Rio and Feller, 2006; Feller, 1999; Penn et al., 1998; Shatz, 1990; Torborg and Feller, 2005).”

“Taken together, thesedatademonstrate that phagocytic signaling through CR3 and its ligand C3 is one molecular mechanism by which microglia engulf RGC inputs.”

“Taken together, thesedatademonstrate that phagocytic signaling through CR3 and its ligand C3 is one molecular mechanism by which microglia engulf RGC inputs.”

“Microglia-Mediated Engulfment of RGC Inputs Is Regulated by Neural Activity Synaptic pruning is thought to result from competition between neighboring axons for postsynaptic territory based on differences in patterns or levels of activity (Hua and Smith, 2004; Katz and Shatz, 1996; Sanes and Lichtman, 1999).”

“Similar to CR3 and consistent with our previous work, immunohistochemistry for CR3 ligand, C3, was enriched in synaptic regions of P5 dLGN and downregulated by P9, an age when pruning is largely complete (Figure 6B; Stevens et al., 2007).”

“To test this possibility, retinogeniculate synapse density was quantified in adult CR3 KOs (P32–P35) using array tomography (AT), a powerful tool for high resolution imaging and quantification of synaptic density in vivo (Greer et al., 2010; Margolis et al., 2010; Micheva and Smith, 2007; Ross et al., 2010).”

“This competition can occur between inputs from the 694 Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. same eye as well as between inputs from both eyes (Chen and Regehr, 2000; Hooks and Chen, 2006; Jaubert-Miazza et al., 2005; Ziburkus and Guido, 2006).”

“) were completely absent from the P5 dLGN and surrounding brain tissue (Figure S5; Akiyama and McGeer, 1990; Perry et al., 1985).”

“, retinal waves) drives the elimination of synapses and segregation of inputs into eye-specific territories in the dLGN (Del Rio and Feller, 2006; Feller, 1999; Huberman et al., 2008; McLaughlin et al., 2003; Penn et al., 1998; Stellwagen and Shatz, 2002; Torborg and Feller, 2005).”

“, eye-specific segregation), resulting in the termination of ipsilateral and contralateral inputs in distinct nonoverlapping domains in the mature dLGN (see Figure 2A; Godement et al., 1984; Guido, 2008; Huberman et al., 2008; Jaubert-Miazza et al., 2005; Sretavan and Shatz, 1986; Ziburkus and Guido, 2006).”

“Consistent with this idea, deficits in complement component C1q result in an increase in the number of presynaptic boutons and exuberant excitatory connectivity in the cortex (Chu et al., 2010).”

“For example, C3 could bind all synapses and only those synapses that are ‘‘stronger’’ ormore active are selectively protected by membrane-bound complement regulatory molecules (Kim and Song, 2006; Song, 2006).”

“…both eyes (i.e., retinal waves) drives the elimination of synapses and segregation of inputs into eye-specific territories in the dLGN (Del Rio and Feller, 2006; Feller, 1999; Huberman et al., 2008; McLaughlin et al., 2003; Penn et al., 1998; Stellwagen and Shatz, 2002; Torborg andFeller, 2005).”

“…of the ‘‘weaker’’ inputs and increased territory of ‘‘stronger’’ inputs within the dLGN (Del Rio and Feller, 2006; Huberman et al., 2008; McLaughlin et al., 2003; Penn et al., 1998; Shatz, 1990; Shatz and Stryker, 1988; Stellwagen and Shatz, 2002; Stellwagen et al., 1999; Torborg and Feller, 2005).”

“…work has demonstrated that microglia have the capacity to interact with synaptic elements in response to neurotransmitter release and/or sensory experience (Biber et al., 2007; Fontainhas et al., 2011; Nimmerjahn et al., 2005; Ransohoff and Perry, 2009; Tremblay et al., 2010a; Wake et al., 2009).”

“In the immune system, C3 is cleaved into anactivated form, iC3b,which covalently binds to the surfaceof cells or debris and targets them for elimination by macrophages via specific phagocytic receptor signaling (e.g., CR3) (Lambris and Tsokos, 1986; van Lookeren Campagne et al., 2007).”

“Microglia were identified by EM using criteria previously described including a small, irregular shaped nucleus containing substantial amounts of coarse chromatin and a cytoplasm rich in free ribosomes, vacuoles, and lysosomes (Mori and Leblond, 1969; Sturrock, 1981).”

“Interestingly, early postnatal microglia also had processes, a morphological characteristic of ‘resting’ microglia which are resident in the healthy adult brain (Figure S2B; Lynch, 2009; Ransohoff and Perry, 2009).”

“…havebeen shown tobeactivity dependent, localized to synapses, and colocalized with C1q leaving the possibility that MHC class I molecules may play an upstream role in microglia-mediated pruning of synapses (Corriveau et al., 1998; Datwani et al., 2009; Goddard et al., 2007; Huh et al., 2000).”

“To further validate that microglia phagocytose RGC inputs, pHrodo-dextran, an anterograde tracer and pH-sensitive dye, was used to label RGC inputs (Figures S1A and S1B; Deriy et al., 2009; Miksa et al., 2009).”

“Particularly during CNS disease, microglia are known scavengers that phagocytose cellular debris (Hanisch and Kettenmann, 2007; Napoli and Neumann, 2009; Ransohoff and Perry, 2009).”

“This competition can occur between inputs from the 694 Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. same eye as well as between inputs from both eyes (Chen and Regehr, 2000; Hooks and Chen, 2006; Jaubert-Miazza et al., 2005; Ziburkus and Guido, 2006).”

“…that engulfed material as well as surrounding dLGN neuropil did not appear to have classic signs of axonal or synaptic degeneration such as multilamellar bodies, electron-dense cytoplasm, lack of synaptic vesicles within presynaptic terminals, etc. (Hoopfer et al., 2006; Perry and O’Connor, 2010).”

“(Del Rio and Feller, 2006; Feller, 1999; Penn et al., 1998; Shatz, 1990; Torborg and Feller, 2005).”

“same eye as well as between inputs from both eyes (Chen and Regehr, 2000; Hooks and Chen, 2006; Jaubert-Miazza et al., 2005; Ziburkus and Guido, 2006).”

“…(Figure 1A) which are undergoing active synaptic remodeling to establish nonoverlapping eye-specific territories (Figure 2A; Godement et al., 1984; Guido, 2008; Huberman et al., 2008; Sretavan and Shatz, 1986; Ziburkus and Guido, 692 Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. 2006).”

“For example, MHC class I molecules, another class of immune molecules demonstrated to play a role in retinogeniculate pruning, have been shown to be activitydependent, localized to synapses, and co-localized with C1q leaving the possibility that MHC class I molecules may play an upstream role in microglia-mediated pru…”

“Consistent with this idea, abnormal microglia function and complement cascade activation have been associated with neurodegeneration of the CNS (Alexander et al., 2008; Beggs and Salter, 2010; Rosen and Stevens, 2010; Schafer and Stevens, 2010; Stephan et al., 2012).”

“Microglia Engulf RGC Inputs during a Period of Active Synaptic Pruning To investigate the functional role of microglia in developmental synaptic remodeling, we used the mouse retinogeniculate system, a classic model for studying activity-dependent developmental synaptic pruning (Feller, 1999; Huberman et al., 2008; Sh…”

“Furthermore, microglia can contribute to synaptic plasticity in the adult CNS and, more recently, in the context of the normal developing hippocampus (Paolicelli et al., 2011; Pascual et al., 2012; Roumier et al., 2008).”

“…of the ‘‘weaker’’ inputs and increased territory of ‘‘stronger’’ inputs within the dLGN (Del Rio and Feller, 2006; Huberman et al., 2008; McLaughlin et al., 2003; Penn et al., 1998; Shatz, 1990; Shatz and Stryker, 1988; Stellwagen and Shatz, 2002; Stellwagen et al., 1999; Torborg and Feller, 2005).”

“To further validate that microglia phagocytose RGC inputs, pHrodo-dextran, an anterograde tracer and pH-sensitive dye, was used to label RGC inputs (Figures S1A and S1B; Deriy et al., 2009; Miksa et al., 2009).”

“Second, previous work using dye tracing or fluorescent protein to label small subsets of RGC afferents in the dLGN demonstrate that RGC axons and arbors within the dLGN undergoing active pruning remain intact and unfragmented (Dhande et al., 2011; Hahm et al., 1999; Snider et al., 1999; Sretavan and Shatz, 1984).”

“To further confirm that microglia were engulfing RGC presynaptic terminals, double immunoEM in P5 dLGN for iba-1 (DAB) and a presynaptic marker specific to RGC terminals, VGlut2 (immunogold; Figures 5E–5G) was performed.”

“…activity, P4 CX3CR1+/EGFP mice were injected with TTX (0.5 mM) to block RGC activity or forskolin to increase activity (10 mM) (Cook et al., 1999; Dunn et al., 2006; Shatz and Stryker, 1988; Stellwagen and Shatz, 2002; Stellwagen et al., 1999) in the left eye and vehicle (saline or DMSO,…”

“(Del Rio and Feller, 2006; Feller, 1999; Penn et al., 1998; Shatz, 1990; Torborg and Feller, 2005).”

“Previous work has suggested that sensory deprivation or pharmacological blockade of neuronal activity (i.e., TTX) results in reduced mitochondria in presynaptic terminals known to undergo subsequent elimination (Hevner and Wong-Riley, 1993; Tieman, 1984).”

“Furthermore, given that microglia are the only CNS cell that express CR3 in the postnatal dLGN, these data suggest that microglia are mediators of synaptic remodeling in Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. 699 (A) Single plane array tomography images for VGlut2 (green) to label RGC terminals and GluR1…”

“Earlier EMwork in the developing mammalian dLGN demonstrated that RGCs transiently synapse within the inappropriate region of the dLGN (Campbell and Shatz, 1992; Campbell et al., 1984).”

“…(Figure 1A) which are undergoing active synaptic remodeling to establish nonoverlapping eye-specific territories (Figure 2A; Godement et al., 1984; Guido, 2008; Huberman et al., 2008; Sretavan and Shatz, 1986; Ziburkus and Guido, 692 Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. 2006).”

“Furthermore, glia are known to engulf axonal material during large-scale developmental pruning of axons in Drosophila and synaptic pruning at the mammalian neuromuscular junction (Bishop et al., 2004; Freeman, 2006; Rochefort et al., 2002).”

“Grid line increments = 5 mm. (D and F) P5 CR3 KO (D) and C3 KO (F) mice (black bars) engulf significantly fewer RGC inputs as compared toWT littermates (white bars).”

“(Del Rio and Feller, 2006; Feller, 1999; Penn et al., 1998; Shatz, 1990; Torborg and Feller, 2005).”

“In this experimental paradigm, an increase in the % overlap between the ipsilateral and contralateral projections within the dLGN is indicative of a deficit in synaptic pruning (Bjartmar et al., 2006; Huh et al., 2000; Pham et al., 2001; Ravary et al., 2003; Stevens et al., 2007).”

“Previous work has suggested that sensory deprivation or pharmacological blockade of neuronal activity (i.e., TTX) results in reduced mitochondria in presynaptic terminals known to undergo subsequent elimination (Hevner and Wong-Riley, 1993; Tieman, 1984).”

“To directly address whether microglia are engulfing RGC presynaptic terminals, immunohistochemistry in P5 dLGN for 696 Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. presynaptic machinery specific to RGCs (i.e., VGlut2) followed by high resolution imagingwas performed.”

“In addition to genetic manipulation of CR3, microglia involvement in eye-specific segregation was further validated by manipulating microglia function pharmacologically using minocycline, an established inhibitor of microglial ‘‘activation’’ (Buller et al., 2009; Figures S6A–S6E).”

“In this experimental paradigm, an increase in the % overlap between the ipsilateral and contralateral projections within the dLGN is indicative of a deficit in synaptic pruning (Bjartmar et al., 2006; Huh et al., 2000; Pham et al., 2001; Ravary et al., 2003; Stevens et al., 2007).”

“…regulated by neural activity, P4 CX3CR1+/EGFP mice were injected with TTX (0.5 mM) to block RGC activity or forskolin to increase activity (10 mM) (Cook et al., 1999; Dunn et al., 2006; Shatz and Stryker, 1988; Stellwagen and Shatz, 2002; Stellwagen et al., 1999) in the left eye and vehicle…”

“same eye as well as between inputs from both eyes (Chen and Regehr, 2000; Hooks and Chen, 2006; Jaubert-Miazza et al., 2005; Ziburkus and Guido, 2006).”

“Furthermore, given that microglia are the only CNS cell that express CR3 in the postnatal dLGN, these data suggest that microglia are mediators of synaptic remodeling in Neuron 74, 691–705, May 24, 2012 ª2012 Elsevier Inc. 699 (A) Single plane array tomography images for VGlut2 (green) to label RGC terminals and GluR1…”

“Earlier EM work in the developing mammalian dLGN demonstrated that RGCs transiently synapse within the inappropriate region of the dLGN (Campbell and Shatz, 1992; Campbell et al., 1984).”

“For example, C3 could bind all synapses and only those synapses that are ‘‘stronger’’ ormore active are selectively protected by membrane-bound complement regulatory molecules (Kim and Song, 2006; Song, 2006).”

“Furthermore, glia are known to engulf axonal material during large-scale developmental pruning of axons in Drosophila and synaptic pruning at the mammalian neuromuscular junction (Bishop et al., 2004; Freeman, 2006; Rochefort et al., 2002).”

“While in at least one other developing system local axonal retraction and synapse elimination appear to occur independent of microglia (Cheng et al., 2010), recent work describes a role for microglia at developing hippocampal synapses (Paolicelli et al., 2011).”

“Two proteins of the complement cascade, C1q and C3, tag unused synapses for microglia recognition via complement receptor 3 (CR3; a heterodimer of CD11b and CD18), or for direct lysis via the complement cascade (68, 69) (Figure 2).”

“In addition, the microglia themselves express activated TrkB, and BDNF released from microglia has been shown to increase their proliferation (Spencer-Segal et al., 2011).”

“Microglia, the brain-resident macrophages, prune synapses during development (Paolicelli et al., 2011; Schafer et al., 2012).”

“Given the recently identified novel role for the complement cascade and microglia in synaptic phagocytosis in normal development and disease (48), we compared expression of relevant genes in microglia and CNS macrophages.”

“Microglia produce copious amounts of C1q, the first protein in the complement activation cascade, and express various complement receptors (Schafer et al., 2012; Stephan et al., 2012).”

“mediate adaptive synaptic remodeling in the healthy developing and adult brain (Schafer et al., 2012; Stevens et al., 2007); (3) TLRs are expressed in neurons, where they play roles in regulating developmental and adult neuroplasticity (Okun et al.”

“Key findings include that (1) transcriptional networks controlling microglial bioenergetic and immunoregulatory functions contribute prominently to heterogeneity in the young adult, (2) immunophenotypic variation suggests a more immune vigilant state of cerebellar microglia, (3) networks of gene co-expression underpin…”

“As microglia participate in the developmental refinement of synaptic networks (28) and elaborate neuromodulatory factors for adult motor learning (29), alterations in the brain microenvironment may lead to pathogenic programs of gene expression in microglia.”

“This behavior may be involved in synaptic pruning (Schafer et al., 2012), a process critical to neural plasticity and learning.”

“, 2013) and other nonneuronal intrinsic regulatory mechanisms including neuroimmune disturbances and astrocyte activation (Garbett et al., 2008; Voineagu et al., 2011; Paolicelli et al., 2011; Schafer et al., 2012; Chung et al., 2013).”

“Using a combination of immune electron microscopy (EM) and high-resolution imaging, microglia were found to engulf RGC presynaptic inputs during a peak pruning period in the developing dLGN (Schafer et al. 2012).”

“Studies have shown that other morphogens, including fibroblast growth factor (FGF) released from neurons (Stork et al., 2014) and bone morphogenetic protein (BMP) (Scholze et al., 2014), can induce structural maturation of astrocytes.”

“Considering CR3 signalling is an essential functional component in synaptic pruning by microglia [93], NOX activation by PRRs is likely a functional inflammatory-related phagocytic response to eliminate dead and dying neurons and aggregated protein, which has been elucidated by 2-photon live imaging [94,95].”

“Recent work has shown that microglia participate in synaptic pruning in the developing postnatal brain (Paolicelli et al., 2011; Schafer et al., 2012), and phagocytose newborn neurons in the subgranular zone of the adult hippocampus (Sierra et al., 2010).”

“Because astrocytes and microglia regulate synaptic development and pruning 226–228 , this may provide another opportunity for therapeutic development.”

“One potential mechanism is through microglia engulfment of synapses [285].”

“In this model, the initiating complement protein , C1q, can opsonize unwanted synapses in the dLGN, along with C3 (Stevens et al. 2007), and these C1q/C3-coated synapses are recognized and engulfed by microglia through C3R- mediated phagocytosis (Schafer et al. 2012).”

“Several previous studies indicate that complement-mediated synaptic pruning by microglia plays a critical role in the refinement of neural circuits during early postnatal development and in the normal aging process (Schafer et al., 2012; Shi et al., 2015; Stephan et al., 2013; Stevens et al., 2007).”

“Synaptic pruning in the dorsal geniculate nucleus of the thalamus is dependent on the expression of the complement protein C1q in the retinal ganglion cells [193, 221], which in turn is regulated by transforming growth factor (TGF)-β secreted by immature astrocytes in the retina [23].”

“, 2007) and recruiting C3, leading to phagocytosis of synapses by microglia (Schafer et al., 2012).”

“C1q or C3 localization to neuronal synapses enhances microglia-dependent synaptic pruning by binding Mac-1 (Schafer et al. 2012; Stevens et al. 2007).”

“Microglia, as one of the major neural cells that contribute to the brain’s development, act to help sculpt neural circuits and are active in synaptic pruning and the support neurogenesis (Paolicelli et al., 2011; Schafer et al., 2012; Xavier et al., 2015).”

“In this respect, TREM2 adds to complement proteins, in particular C1q and C3, which control synaptic pruning in the postnatal retinogeniculate system (Schafer et al., 2012; Stevens et al., 2007) and to MHC class I proteins, which promote the elimination of inappropriate connections during retinal and retinogeniculate…”

“Cx3cr1 is extensively used to characterize microglia dynamics in vivo (Cardona et al., 2006b; Nimmerjahn et al., 2005; Schafer et al., 2012).”

“Importantly, microglia-mediated synaptic elimination depends on neuronal activity, as microglia preferentially phagocytose less active presynaptic inputs [10].”

“They engulf synapse components during early thalamic development and in the hippocampus (Paolicelli et al., 2011; Schafer et al., 2012; Vainchtein et al., 2018; Wang et al., 2020).”

“Additionally, a number of inappropriate synaptic connections are made between neurons during development, and microglia play an important role in the regulation of these contacts via the process of synaptic pruning—the phagocytosis of both pre- and postsynaptic elements in a complement cascade-dependent manner (Salter…”

“However, in contrast to past reports describing microglial expression of complement proteins (Haga et al., 1996; Schäfer et al., 2000; Griffin et al., 2007; Rutar et al., 2014), we detected little C3 protein in microglia of APP transgenic mice.”

“cells [6], thereby potentially degrading healthy synapses and contributing to the ongoing degenerative process [44].”

“In mice lacking components of the complement cascade, there is an excess convergence of connections from the retinal ganglion cells of the eye onto neurons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus that is maintained until later in development than normal [8, 9].”

“…in the meninges and choroid plexus, which contain perivascular APCs, or by direct drainage of CNS antigens in the cerebrospinal fluid from the subarachnoidal space to channels in the cribiform plate and ultimately into deep cervical lymph nodes (Galea et al., 2007; Ransohoff and Engelhardt, 2012).”

“…and dissect molecular mechanisms underlying these interactions, recent studies have utilized mice harboring deletions of genes specific to microglia in the context of the healthy developing CNS (Paolicelli et al., 2011; Pascual et al., 2011; Roumier et al., 2004, 2008; Schafer et al., 2012).”

“Microglia have been reported to prune or sculpt developing synaptic connections, and a role for complement has been proposed (Stevens et al , 2007; Schafer et al , 2012).”

“pses as a mechanism of synaptic pruning (Schafer et al., 2012).”

“…adolescence, adulthood and normal ageing, within the thalamus, cerebral cortex and hippocampus (Tremblay et al. 2010, 2012; Paolicelli et al. 2011; Schafer et al. 2012; Milior et al. 2015), suggesting a crucial role in synaptic plasticity and behavioural adaptation to the environment (Tremblay et…”

“Besides their roles in these immune-related processes, microglia are emerging as important contributors to normal brain development (Graeber, 2010; Hanisch and Kettenmann, 2007; Marı́n-Teva et al., 2004; Paolicelli et al., 2011; Parkhurst and Gan, 2010; Ransohoff and Perry, 2009; Sierra et al., 2010; Stevens et al., 2…”

“One key microglial function during development is the coordination of synaptic pruning via the classical complement cascade 556,557 .”