Deficiency of the Microglial Receptor CX3CR1 Impairs Postnatal Functional Development of Thalamocortical Synapses in the Barrel Cortex

“…be that MGCs, once inside TCA clusters, could contribute to the selection of synapses with a mature signature of their glutamate receptors (Yashiro and Philpot, 2008) by pruning immature synapses through phagocytosis (Paolicelli et al., 2011) in a complement-dependent manner (Schafer et al., 2012).”

“In the diseased adult CNS, several signaling pathways control the recruitment of microglia at sites of compromised homeostasis (Hanisch and Kettenmann, 2007; Ransohoff and Perry, 2009).”

“In few experiments heterozygous CX3CR1 /eGFP mice of either sex were obtained by crossing CX3CR1 eGFP/eGFP (Jung et al., 2000) with wild-type C57BL/6J mice (Janvier).”

“In the diseased adult CNS, several signaling pathways control the recruitment of microglia at sites of compromised homeostasis (Hanisch and Kettenmann, 2007; Ransohoff and Perry, 2009).”

“This chemokine is expressed by neurons; its receptor CX3CR1 is only expressed by MGCs and controls their migration and functions (Harrison et al., 1998; Maciejewski-Lenoir et al., 1999; Cardona et al., 2006; Ruitenberg et al., 2008; Liang et al., 2009; Paolicelli et al., 2011).”

“…local role of fractalkine in which cellular elements localized within the barrel hollows, possibly thalamic fibers, overexpress the chemokine at P5–P7 and thus favor directed migration of microglia (Harrison et al., 1998; Maciejewski-Lenoir et al., 1999) toward maturating thalamocortical synapses.”

“In rodents, experimental evidence shows that MGCs contribute to developmentally regulated neuronal apoptosis but also to synapse pruning (Mallat et al., 2005; Pont-Lezica et al., 2011; Schlegelmilch et al., 2011; Tremblay et al., 2011).”

“Although macrophages are known to influence the development of peripheral organs, the idea that brain resident macrophages could play a role during CNS development is relatively new (Erblich et al., 2011; Pont-Lezica et al., 2011; Schlegelmilch et al., 2011).”

“Several studies on adult rodents have reported that CX3CL1/R1 signaling, probably by controlling the activation state of MGCs, has an impact on functional properties and plasticity of hippocampal glutamate synapses (Ragozzino et al., 2006; Maggi et al., 2011; Rogers et al., 2011).”

“In parallel, several functional parameters of thalamocortical synapses evolve between P4 and the end of the second postnatal week (López-Bendito and Molnár, 2003; Daw et al., 2007; Inan and Crair, 2007).”

“…local role of fractalkine in which cellular elements localized within the barrel hollows, possibly thalamic fibers, overexpress the chemokine at P5–P7 and thus favor directed migration of microglia (Harrison et al., 1998; Maciejewski-Lenoir et al., 1999) toward maturating thalamocortical synapses.”

“In humans, description of the spatiotemporal organization of microglia in the embryonic and fetal CNS suggests that these cells play active roles in developmental processes (Verney et al., 2010).”

“This chemokine is expressed by neurons; its receptor CX3CR1 is only expressed by MGCs and controls their migration and functions (Harrison et al., 1998; Maciejewski-Lenoir et al., 1999; Cardona et al., 2006; Ruitenberg et al., 2008; Liang et al., 2009; Paolicelli et al., 2011).”

“In tangential sections through layer 4 at P4 and P5, the first stages at which barrels can be identified (Rebsam et al., 2002), we observed that MGCs remained mostly outside clusters of TCAs (Fig.”

“Several studies on adult rodents have reported that CX3CL1/R1 signaling, probably by controlling the activation state of MGCs, has an impact on functional properties and plasticity of hippocampal glutamate synapses (Ragozzino et al., 2006; Maggi et al., 2011; Rogers et al., 2011).”

“In parallel, several functional parameters of thalamocortical synapses evolve between P4 and the end of the second postnatal week (López-Bendito and Molnár, 2003; Daw et al., 2007; Inan and Crair, 2007).”

“In these mice, however, this modification which results from a transient impairment of microglia-neuron interactions during embryonic life is observed at excitatory synapses of mature animals (Roumier et al., 2008).”

“In rodents, experimental evidence shows that MGCs contribute to developmentally regulated neuronal apoptosis but also to synapse pruning (Mallat et al., 2005; Pont-Lezica et al., 2011; Schlegelmilch et al., 2011; Tremblay et al., 2011).”

“Several studies on adult rodents have reported that CX3CL1/R1 signaling, probably by controlling the activation state of MGCs, has an impact on functional properties and plasticity of hippocampal glutamate synapses (Ragozzino et al., 2006; Maggi et al., 2011; Rogers et al., 2011).”

“Thalamocortical (TC) slices (400 m) from P5 to P33 mice were prepared as previously described (Laurent et al., 2002) in an ice-cold solution containing (in mM): 215 sucrose, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 20 glucose, 5 pyruvate, 1 CaCl2 and 7 MgCl2, bubbled with carbogene, pH 7.4, 310 mOsm.”

“In rodents, experimental evidence shows that MGCs contribute to developmentally regulated neuronal apoptosis but also to synapse pruning (Mallat et al., 2005; Pont-Lezica et al., 2011; Schlegelmilch et al., 2011; Tremblay et al., 2011).”

“In rodents, experimental evidence shows that MGCs contribute to developmentally regulated neuronal apoptosis but also to synapse pruning (Mallat et al., 2005; Pont-Lezica et al., 2011; Schlegelmilch et al., 2011; Tremblay et al., 2011).”

“Alternatively, signaling molecules such as interleurkin-1 , tumor necrosis factor- or brain-derived neurotrophic factor, known to be released by microglia, can modulate the expression and the function of glutamate receptors (Beattie et al., 2002; Chao, 2003; Zhang et al., 2010; Zhong et al., 2010).”

“In parallel, several functional parameters of thalamocortical synapses evolve between P4 and the end of the second postnatal week (López-Bendito and Molnár, 2003; Daw et al., 2007; Inan and Crair, 2007).”

“It resembles that of microglia around glomeruli of the developing olfactory bulb (Fiske and Brunjes, 2000) and indicates that MGC colonization of layer 4 does not proceed randomly.”

“Finally, MGCs probably also influence other aspects of barrel maturation such as the rearrangement of dendrites of layer 4 cells and the removal of subplate cell neurites which occurs at the end of the first postnatal week (Hoerder-Suabedissen and Molnár, 2012).”

“This chemokine is expressed by neurons; its receptor CX3CR1 is only expressed by MGCs and controls their migration and functions (Harrison et al., 1998; Maciejewski-Lenoir et al., 1999; Cardona et al., 2006; Ruitenberg et al., 2008; Liang et al., 2009; Paolicelli et al., 2011).”

“Alternatively, signaling molecules such as interleurkin-1 , tumor necrosis factor- or brain-derived neurotrophic factor, known to be released by microglia, can modulate the expression and the function of glutamate receptors (Beattie et al., 2002; Chao, 2003; Zhang et al., 2010; Zhong et al., 2010).”

“CX3CR1-CX3CL1 interactions are also required for functional maturation of thalamocortical synapses (72).”

“Recent work has shown that mice lackingCX3CR1 have delayed invasion of the cortical barrel fields by microglia, and a transient delay in the normally observed AMPA receptor subunit switching in thalamocortical barrel cortex synapses (Hoshiko et al., 2012).”

“Lack of CX3CR1 has been associated with reduced synaptic pruning in other brain regions [9,14].”

“It is necessary for the timely recruitment of microglial cells into the early postnatal hippocampus and cerebral cortex (Hoshiko et al. 2012; Ueno et al. 2013; Paolicelli et al. 2014).”

“In CX3CR1 null mice, the invasion of microglial cells to the barrel centers is delayed by several days, as is maturation of the thalamocortical synapses (Hoshiko et al., 2012).”

“CX3CR1−/− mice have a transiently decreased microglia density in the hippocampus and delayed microglia entry into the barrel cortices and social behavior (11, 12, 62).”

“In more recent studies, transient reductions in microglia numbers in the hippocampus or barrel cortex due to a genetic deletion of CX3CR1 (CX3CR1 KO) resulted in delayed maturation of structural and functional synapses [55, 56].”

“In addition, another study reported that CX3CR1-deficient animals exhibited an inhibition in microglial recruitment to forming synapses in the barrel field of the somatosensory cortex, leading to an abnormal synapse formation in this area (Hoshiko et al., 2012).”

“However, the defect was transient, without any remaining difference in the decay time of NMDAR-mediated responses at P27– P33 (Hoshiko et al., 2012).”

“Because emerging data suggest that microglia as the brain resident immune cells play crucial roles in neural development (Eyo and Dailey, 2013; Hoshiko et al., 2012; Paolicelli et al., 2011; Paolicelli and Gross, 2011; Pont-Lezica et al., 2011; Schafer et al., 2012; Schlegelmilch et al., 2011; Squarzoni et al., 2014),…”

“However, our study does not exclude the possibility that CX3CR1 might play a role during development where CX3CR1 deficiency has been demonstrated to result in reduced microglial numbers, impaired switching of GluN2B-to-GluN2A, and delays in synaptic maturation (Paolicelli et al., 2011; Hoshiko et al., 2012).”

“CX3CL1-CX3CR1 is a critical signaling pathway during development as shown by the delay of glutamatergic synapse maturation (Paolicelli et al., 2011; Hoshiko et al., 2012) and functional consequences in adult synapses (Basilico et al.”

“Synaptic refinement At postnatal stages, microglial cells contribute to the refinement of synaptic networks and to the functional maturation of synapses (Hoshiko et al., 2012; Paolicelli et al., 2011; Schafer et al., 2012; Zhan et al., 2014).”

“Moreover, microglial cell infiltration into the barrel centers of the developing somatosensory cortex, that normally occurs around postnatal day 5, is delayed by a few days in Cx3cr1-/- GFP/GFP mice, even if no differences are detected at postnatal day 9 [143].”

“…as THIK-1, that regulate microglial membrane potential and receptor signaling; mechanisms that allow ion exchange between the cell and the extracellular matrix (e.g., via P2Y2R); and anchoring structures such as b1-integrin (Haynes et al., 2006; Hristovska and Pascual, 2016; Madry et al., 2018a).”

“Recent reports have contributed to broaden our understanding that microglia capacities in the CNS include several essential features such as synaptic pruning (Stevens et al., 2007; Paolicelli et al., 2011; Hoshiko et al., 2012; Schafer et al., 2012), influencing functions of activated and/or over-activated neurons (Pa…”

“Hoshiko et al. (2012) showed that the lack of the Cx3cr1 was associated with delayed maturation of the developing synapses in the barrel cortex, specifically affecting the NMDAR composition.”

“functions, including synaptic remodeling, during the development of central nervous systems [12,13,14].”

“…immaturity (Paolicelli et al., 2011; Rogers et al., 2011; Zhan et al., 2014), the maturation of glutamatergic thalamocortical synapses was delayed during postnatal development (Hoshiko et al., 2012), while the positioning of neocortical interneurons was altered prenatally (Squarzoni et al., 2014).”

“During development, CX3CL1/CX3CR1 signaling contributes to refinement of synaptic elements in varied CNS regions during the postnatal “critical period” (Tremblay et al., 2010; Paolicelli et al., 2011; Hoshiko et al., 2012).”

“During development, CX3CL1-CX3CR1 signaling influences microglial regulation of neuronal survival (Sierra et al., 2010; Ueno et al., 2013), and synaptic maturation and refinement (Hoshiko et al., 2012; Paolicelli et al., 2011) in the brain.”

“…reports on defective neuronal development in the hippocampus of fractalkine receptor-deficient mice were extended to the cortex in another study where proper maturation of thalam-ocortical circuits in the barrel cortex of developing mice was shown to require functional fractalkine signaling [44].”

“The fractalkine receptor is only expressed on microglia in the CNS, and CX3CR1:GFPKO/KO mice are one of the most widely used tools to study modulation of synaptic engulfment during early postnatal development (Paolicelli et al., 2011; Zhan et al., 2014; Hoshiko et al., 2012).”

“…bi-directional communication between synapses and microglia is best heralded by the synaptic dysfunction observed upon genetic manipulation of microglia function (Roumier et al., 2004; Costello et al., 2011; Hoshiko et al., 2012), which can be direct or involve astrocytes (Pascual et al., 2012).”

“The CX3CL1-CX3CR1 axis is already well established as a major pathway of neuron-microglia communication that is involved in synaptic pruning and development [38, 269, 270] as well as microglial mobility, motility, and activation [270–272] and therefore seems to be a feasible candidate in the regulation of this process.”

“Moreover, CX3CR1-deficient microglia demonstrate delayed recruitment to the developing barrel cortex, with a corresponding delay in maturation of the postsynaptic compartment in terms of the AMPA/NMDA receptor ratio as well as the subunit composition of NMDA receptors (Hoshiko et al. 2012).”

“Neurons and microglia both express its receptor, CX3CR1 (Hatori et al., 2002), which regulates memory formation and synaptic plasticity via direct effects on glutamatergic synapses (Hoshiko et al., 2012; for an extensive review, see Sheridan and Murphy, 2013).”

“Microglia play a key role in synaptic pruning in the developing visual and somatosensory systems (Hoshiko et al., 2012; Miyamoto et al., 2016; Schafer et al., 2012; Sipe et al., 2016), as well as in developing hippocampus (Paolicelli et al.”

“…signaling was shown previously to be involved in neuron–microglia communication, synaptic pruning by microglia, microglia migration, hippocampal neurogenesis, and cognitive function (Sierra et al., 2010; Bachstetter et al., 2011; Paolicelli et al., 2011; Rogers et al., 2011; Hoshiko et al., 2012).”

“However, the same functional parameters of thalamocortical synapses do not differ between adult CX3CR1 deficient and wild type mice [48] (Arnoux & Audinat, unpublished).”

“For instance, in the mouse, the chemokines CXCL12 and CX3CL1 have been shown to be involved in the recruitment of microglia to specific brain areas, such as the sub-ventricular zone and the barrel centers, respectively (Arnò et al., 2014; Hoshiko et al., 2012).”

“…cerebellar microglia contribute to the developmental pruning of synapses as circuits develop, much like microglial roles elsewhere in the brain (Stevens et al., 2007; Paolicelli et al., 2011; Hoshiko et al., 2012; Schafer et al., 2012; Zhan et al., 2014; Pagani et al., 2015; Sipe et al., 2016).”

“In the developing barrel cortex, CX3CR1-deficient mice exhibit delayed developmental switch of the glutamate receptor subunits GluN2B to GluNA, one of the main features of barrel cortex development [54].”

“Phagocytosis of overproduced neurons and weak synapses by microglia, processes that govern brain connectivity, plays key role in organizing brain circuits during the postnatal period (Paolicelli et al., 2011; Hoshiko et al., 2012; Schafer et al., 2012; Bialas and Stevens, 2013; Zhan et al., 2014).”

“…signaling pathway also appears to be involved in synaptic maturation since CX3CR1 deficiency leads to a delay in the maturation of glutamatergic thalamocortical synapses, as well as a transient immature connectivity in the developing hippocampus (Paolicelli et al., 2011; Hoshiko et al., 2012).”

“In the brain, soluble fractalkine is released by the metalloprotease ADAM10 on neurons, and may drive migration of microglia to developing neurites for synaptic pruning during circuitry development (19, 20, 35, 36).”

“More generally, these results suggest that, in some neuropsychiatric disorders in which no marked neurodegeneration occurs, microglial dysregulation may constitute a failure of neuroprotective functions, which may create a vulnerability to neuroinflammation.”

“Fractalkine receptor deficiency leads to a transient reduction of microglial number during early postnatal development (12, 13), microglia-mediated neurotoxicity (65– 67), and impaired phagocytosis of apoptotic newborn neurons, thus causing reduced neurogenesis and deficits in synaptic pruning associated with weaker s…”

“, 2012), and maturation of synapses (Hoshiko et al., 2012; Zhan et al., 2014; Sipe et al., 2016).”

“Overall, these data suggest that microglial depletion is associated with the appearance of immature synaptic features (Basilico et al., 2019; Hoshiko et al., 2012).”

“Microglia have been demonstrated to modulate neurotransmission and selective activation of microglia enhances excitatory neurotransmission (53, 54).”

“In mouse model lacking CX3CR1, synaptic pruning was reduced, which led to the formation of immature brain and synapses (Paolicelli et al., 2011; Hoshiko et al., 2012).”

“, the barrel centers) after P5 and that a deficit in this recruitment is associated with an impairment of the functional maturation of these glutamatergic synapses (Hoshiko et al., 2012).”

“For example, it has been suggested that CX3CL1 may mediate microglia–neuron communication and synaptogenesis (Hoshiko et al. 2012), and several chemokines have recently been identified as potential regulators for OL development and myelination (Patel et al. 2010).”

“ORIGINAL RESEARCH published: 31 March 2015 doi: 10.3389/fncel.2015.00111 Edited by: Silvia Alboni, University of Modena and Reggio Emilia, Italy Reviewed by: Claudia Verderio, Consiglio Nazionale delle Ricerche, Italy Claudia Eder, University of London, UK Etienne Audinat, Paris Descartes University, France *Correspon…”

“It is important to point out that a similar reduction in AMPA/NMDA ratio has been reported in thalamo-cortical synapses in the developing barrel cortex of Cx3cr1 KO mice, where it was associated with a delay in the migration of microglia into the cortical barrels (Hoshiko et al., 2012).”

“As a result, glutamatergic synapse maturation is delayed (Hoshiko et al., 2012).”

“In addition, elimination or reduction of microglia activity impaired maturation of synapses in the hippocampus and thalamocortical synapses of the barrel cortex as evaluated during the first two weeks of life in mice (Hoshiko et al., 2012; Paolicelli et al., 2011).”

“…traditional roles in inflammation and disease (Hopkins and Rothwell, 1995; Rothwell and Hopkins, 1995; Reichenberg et al., 2001; Pollmächer et al., 2002; Wilson et al., 2002; Adler and Rogers, 2005; Adler et al., 2006; McAfoose and Baune, 2009; Hoshiko et al., 2012; Williamson and Bilbo, 2013).”

“However, there was a change in AMPA/NMDA ratio because of an impairment in the normal developmental switch of NMDAR subunits from GluN2B to GluN2A (Hoshiko et al., 2012).”