The research into synaptic development using depletion models has prompted further investigations to elucidate the physiological function of microglia in refining neural circuits in development. Fundamental insights have come from studies in which the physiological microglial activity was perturbed either by environmental or genetic approaches.
Further investigations have attempted to dissect the differential contribution of systemic vs. brain immune response. Indeed, recent neuropathological studies in human post-mortem brains have revealed strong microglial activation in neurodevelopmental disorders, such as schizophrenia (de Baumont et al., 2015; Sekar et al., 2016), and ASD (Voineagu et al., 2011; Gupta et al., 2014; Takano, 2015).
While the above studies provided only correlative evidence, data from genetically engineered animal models have demonstrated a causative role for microglia in the pathogenesis of neurodevelopmental disorders. Genetic manipulation affecting specific microglial function resulted in behavioral alterations reminiscent of obsessive-compulsive disorder (Chen et al., 2010; Zhan et al., 2014) and Rett Syndrome (Maezawa and Jin, 2010; Derecki et al., 2012), and in impaired functional brain connectivity reminiscent of autism and other neurodevelopmental disorders (Zhan et al., 2014).
Therefore, the collective evidence from human studies and microglial perturbation animal models (using both environmental and genetic approaches) indicate that altering microglial function during development contributes to the pathogenesis of neurodevelopmental disorders. Given the role of microglia in refining synaptic connectivity, an impaired cross-talk between microglia and synapses might represent a crucial mechanism linking microglial perturbation in early life to mental diseases susceptibility. Below, we review the current literature showing that environmental and genetic perturbations of microglial function during development have acute and long-lasting effects on synapses and behavior.
Environmental Factors Modulating Microglial Activity
Environmental factors, such as infections, stress and dietary intake are associated with synaptic dysfunction and are linked to increased risk for neurodevelopmental/psychiatric disorders (Marques et al., 2013, 2015; Knuesel et al., 2014; Ajdacic-Gross et al., 2016; Estes and McAllister, 2016). Importantly, all of the above factors have been shown to activate the immune system during early brain development. Recent studies also showed that microglia–synapse contacts are strongly modulated by neural activity, highlighting sensory deprivation as a further regulator of microglial activity. Burgeoning evidence suggests that microglia substantially contribute to the environmental-mediated synaptic defects.
Infections
Both viral and bacterial infections have been shown to alter behavior and cognition in experimental animals when administered during critical developmental periods (Boksa, 2010; Meyer, 2014), suggesting profound synaptic remodeling. Importantly, the initial systemic immune response elicited by the infection is followed by a microglial-mediated inflammatory reaction in the brain (Turrin et al., 2001; Puntener et al., 2012). Therefore, there is a growing interest in the role of microglia in infection-mediated synaptic remodeling during development.
Viral infection
In a seminal paper, maternal infection of experimental rodents with the influenza virus resulted in deficits in a variety of behavioral displays, including open-field, prepulse inhibition, object recognition and social behavior in the adult offspring (Shi et al., 2003). This study was the first to establish a causal relationship between prenatal viral infection and psychiatric and neurological symptoms in adulthood, which had been previously suggested by a plethora of epidemiological studies (Brown, 2012).
The most widely used model in experimental rodents is the prenatal injection of viral mimetic polyriboinosinic-polyribocytidilic acid (Poly I:C). Poly I:C, a synthetic analog of double-stranded RNA that is recognized as foreign by the rodent immune system, efficiently stimulates an immune response via TLR3 activation. In the mouse species, prenatal immunological stimulation with Poly I:C in early/middle gestation leads to a spectrum of behavioral abnormalities in adulthood, strongly depending on the timing of the infection. Mid-gestational (E9) Poly I:C exposure leads to impairments in pre-pulse inhibition of the acoustic startle reflex and in latent inhibition (Meyer et al., 2005), considered relevant to schizophrenia and ASD. On the other hand, impairments in cognition are elicited following Poly I:C exposure at late gestation (E17) (Meyer et al., 2006). At the molecular level, changes in both pre-and post-synaptic elements, with altered NMDA receptor composition (Forrest et al., 2012), reduced PSD95 and SynGAP density (Giovanoli et al., 2016b), as well as reduced synpatophysin elements (Oh-Nishi et al., 2010; Giovanoli et al., 2016b) have been described in the adult offspring of Poly I:C infected dams. In addition, prenatal Poly I:C injection has been linked to the onset of white matter abnormalities in corticostriatal areas, including myelin-related transcriptional and epigenetic changes, and alterations of myelin water fraction in adulthood (Richetto et al., 2016).
Both acute and chronic immune responses are observed following prenatal Poly I:C injection. Acutely, Poly I:C causes a transient increase in inflammatory mediators in maternal blood and fetal brain (Gilmore et al., 2005; Forrest et al., 2012; Smolders et al., 2015). The cellular origin of these inflammatory mediators is still a matter of debate. Indeed, Pratt et al. (2013) have reported an acute increase in cytokine expression by fetal microglia, suggesting that microglia might be the source of inflammatory cytokines in the brain of prenatally Poly I:C injected mice. However, other authors did not observe acute changes in microglial density or expression of inflammatory markers following prenatal exposure to Poly I:C, in spite of systemic inflammation in the dams (Smolders et al., 2015).
While the inflammatory response in the fetal brain is well-established, its persistence in the post-natal brain is highly controversial. In some studies inflammation was shown to almost completely subside in adulthood, with normal cytokine plasma levels, unaltered microglial density and moderate increase in IL-1β levels in the hippocampus of adult offspring of infected dams at E9 (Giovanoli et al., 2016b) and E14 (Mattei et al., 2014). In others, Poly I:C injections led to sustained cytokine level changes that lasted into adulthood. A comprehensive study by Garay et al. (2013) reported long-lasting, region-specific inflammatory changes in offspring of Poly I:C injected dams (E12.5), across five post-natal ages. In cortical regions, most cytokines were altered at all of the ages analyzed, following a pattern of elevation at birth, decreasing during the early post-natal weeks (PND7-PND30), and elevation again in adulthood (P60) (Garay et al., 2013). Interestingly, cytokine levels in the hippocampus showed a distinct pattern, with mixed direction-switches across ages (Garay et al., 2013). All of the changes in the brain occurred independently of serum cytokine alterations, in the absence of immune cell infiltration, and with no increase in microglia density (Garay et al., 2013).
Importantly, the Poly I:C-induced inflammatory response during development is sufficient to induce long-lasting changes in microglial expression profile and function, but not in microglial numbers. Indeed, recent global transcriptional data have revealed that Poly I:C injection at E14.5 interferes with development of early microglia, shifting them into a more advanced developmental stage (Matcovitch-Natan et al., 2016).
Such early interference has long-lasting effects on microglial function, producing ‘primed’ microglia, with increased susceptibility to activation. In fact, a ‘second hit’ (i.e., sub chronic unpredictable stress) can elicit an exacerbated hippocampal microglial response (with increased soma size, CD68 and IL-1β expression) in the offspring of Poly I:C injected mothers as compared to controls (Giovanoli et al., 2016a).
The molecular mediators of Poly I:C effects on both the structure and the function of the developing CNS are not fully elucidated; however, inflammatory cytokines, including IL-6 and IL-1β, are likely to be crucial players. IL-6 release has been shown to mediate the behavioral effects of viral prenatal infections. Injection of IL-6 into pregnant dams at E12 induced pre-pulse inhibition and latent inhibition deficits in adulthood, similar to Poly I:C treatment. Importantly, genetic or pharmacological blockade of IL-6 fully prevented Poly I:C-induced gene expression changes and behavioral alterations (Smith et al., 2007). In addition, recent evidence suggests that IL-1β might be involved in the effects of prenatal Poly I:C on white matter in adulthood. Intraperitoneal injections of IL-1β in newborn mice (PND1-PND4) is sufficient to largely disrupt the developmental program of white matter (Favrais et al., 2011), in line with the observations of Richetto et al. (2016) in Poly I:C-challenged offspring. Recent studies found that the anti-inflammatory drug minocycline prevented the effect of prenatal Poly I:C administration on behavior in adulthood (Mattei et al., 2014; Zhu et al., 2014; Giovanoli et al., 2016a), further indicating a causative relationship between inflammation and synaptic dysfunction.
Bacterial infections
Systemic injections into pregnant dams or pups of the gram-negative bacterial component lipopolysaccharide (LPS), or inactivated Escherichia coli, are commonly used models to study central effects of pre- and early post-natal bacterial infections.
Maternal immune activation with LPS resulted in increased anxiety (Hava et al., 2006) and decreased social behavior in the male offspring (Kirsten et al., 2010), phenotypes relevant to ASD. The behavioral alterations were accompanied by glutamatergic remodeling in the hippocampus, with increased AMPAR contribution to excitatory neurotransmission (Roumier et al., 2008) and increased spine density (Fernandez de Cossio et al., 2016).
A prenatal LPS injection caused acute and long-lasting effects on microglia. LPS administration at E15–16 resulted in immediate up-regulation of iNOS expression in microglia in the embryos (Cunningham et al., 2013), indicating acute activation. Such alterations persist during early post-natal periods, with increased microglial density in hippocampus (Roumier et al., 2008) and substantia nigra (Ling et al., 2006) of prenatally LPS-injected mice analyzed at PND0. Importantly, up-regulation of OX-6+ microglial cells in the substantia nigra was shown to be sustained until PND84, and to be further exacerbated by a second hit of LPS injection in adulthood, suggesting enduring effects on prenatal infection on microglia (Ling et al., 2006).
Several reports support the notion that prenatal bacterial infection interferes with specific physiological functions of microglia during development. Interestingly, synaptic remodeling induced by LPS prenatal injection is phenocopied in models of microglial depletion, pointing to a loss of homeostatic function.
Roumier et al. (2004, 2008) have shown that mice carrying a loss of function mutation in the DAP12 gene (a signaling protein transiently expressed by microglia at birth) recapitulate the glutamatergic remodeling seen with LPS injection. More recently, Squarzoni et al. (2014) have shown that maternal immune activation with LPS induces alterations in the laminar positioning of LHX-6+ interneurons similar to what was observed via depletion of microglia. The exact mechanisms by which prenatal bacterial infection interferes with synapse–microglial interaction are still unclear, but recent evidence suggests a role for CX3CR1 signaling. In fact, prenatally LPS-infected rats display reduced hippocampal CX3CR1 expression along with increased spines in the dentate gyrus (Fernandez de Cossio et al., 2016). It could be speculated that the LPS induced-downregulation of the CX3CR1 receptor might alter the physiological pruning of synapses. However, further work is required to test this hypothesis.
Early post-natal infection with LPS has been shown to induce anxiety-like behaviors (Sominsky et al., 2012) and impaired cognitive function in adult rats (Pang et al., 2016). LPS injection in rat pups at PND5 results in acute microglial activation with increased numbers of Iba1+ cells in the hippocampus and up-regulation of both M1 and M2 markers (Smith et al., 2014; Pang et al., 2016). Iba1+ cell numbers normalized by PND21, but M1 marker expression was still subtly altered (Smith et al., 2014). Furthermore, increased Iba1 immunoreactivity was observed in the dentate gyrus of PND85 neonatally infected rats (Sominsky et al., 2012). These results suggest that behavioral deficits might be due to both acute and long-lasting effects of neonatal LPS injection on microglia.
Escherichia coli administration in PND4 rats induced mild behavioral perturbations in adulthood, including hippocampal-dependent deficits in the reversal phase of the Morris Water Maze task (Williamson and Bilbo, 2014) and impaired motor coordination (Lieblein-Boff et al., 2013). Effects on inflammatory markers are short and transient in this model, with up-regulation of IL-1β and CD11b expression in the hippocampus that normalizes by 72 h post-injection (Bilbo et al., 2005). However, the immune system of post-natally E. coli-infected mice has been shown to be primed, and displays an exacerbated response to a ‘second hit’ with a low-dose of LPS, in adulthood (Bilbo et al., 2005; Williamson et al., 2011). Importantly, the immune alterations caused by the ‘second hit’ with LPS were accompanied by severe memory impairments, indicating a crucial role for primed microglia in mediating cognitive deficits. Importantly, blocking microglial activation with minocycline or caspase1 inhibitor immediately before a ‘second hit’ of LPS in adulthood prevented the appearance of cognitive deficits in neonatally infected rats (Bilbo et al., 2005; Williamson et al., 2011). It is crucial to note that E. coli injections at later post-natal time points, such as PND30, do not result in exacerbated microglial response or altered behavioral display following a ‘second hit’ with LPS in adulthood (Bilbo et al., 2006). These findings further support the notion that the acquired vulnerability to the second hit is not a general sensitizing event, but it is strictly dependent on the developmental stage in which the first infection occurs (Bilbo and Schwarz, 2009, 2012).
Stress
Intermittent and unpredictable maternal separation, as well as stressful manipulations of pregnant dams (via restraint, sleep deprivation, or light exposure), are common experimental models of early-life adverse events and developmental stress. In striking similarity with human data, the early-stressed pups go on to develop syndromes later in life that resemble depression (Mintz et al., 2005; Ruedi-Bettschen et al., 2005), and anxiety (Vallee et al., 1997); for a review (Nishi et al., 2014). In recent years an increased interest has arisen on the role of microglia in early-stress-induced remodeling of cortical and hippocampal synapses. Indeed, different models of perinatal stress have been shown to induce both acute and long-lasting effects on microglia activity.
Prenatally stressed mice displayed higher numbers of ramified microglia in several brain regions analyzed at PND1, including parietal, entorhinal and frontal cortices, septum, basal ganglia, thalamus, medulla oblongata, and internal capsula (Gomez-Gonzalez and Escobar, 2010). Primary cultures of microglia isolated from prenatally stressed rats showed increased release of pro-inflammatory cytokines, including IL-1β, IL-18, TNF-α and IL-6, and reduction of IGF-1 (Slusarczyk et al., 2015). Such cytokines, and in particular IL-18 and TNF-α, were shown to directly increase neuronal excitability via up-regulation of voltage-activated Na+ currents (Igelhorst et al., 2015; Klapal et al., 2016). The microglial effects have been shown to be mostly transient, with microglia morphology returning to normal by PND10 (Gomez-Gonzalez and Escobar, 2010). However, prenatally stressed mice, when examined in adulthood, showed hippocampal microglial alterations, with a higher density of Iba1+ cells, enlargement of soma, increased pro-inflammatory expression and heightened response to a second hit with LPS injection (Diz-Chaves et al., 2012, 2013). Similar findings were reported in rats, with adult prenatally stressed subjects displaying increased expression of microglial activation markers, and increased Iba1+ microglial density in the hippocampus and frontal cortex (Slusarczyk et al., 2015; Zhao et al., 2015). These results suggest that prenatal stress can induce long-lasting effects on microglial function in the hippocampus and cortex.
More subtle effects have been observed in models of early post-natal stress. Maternal deprivation during the first 2 weeks of life was associated with acute effects on hippocampal microglia, including larger soma size, increased release of IL-1β (Roque et al., 2016) and increased microglial surface area (Delpech et al., 2016). Delpech et al. (2016), however, reported that alterations in microglial morphology were transient and parameters normalized later at PND28. Analysis of PND28 hippocampi from mice that experienced early stress revealed that, in spite of apparently normal morphology, microglia had altered transcriptional activity of PU1, Creb and Sp1, resulting in gene expression changes and increased phagocytic activity (Delpech et al., 2016). Long-lasting effects of early-life stress were also reported in other brain areas. Increased microglia motility was observed in the sensorimotor cortex of adult mice that had experienced maternal deprivation (Takatsuru et al., 2015). In this study, the authors observed a positive correlation between microglial motility and nociceptive threshold, suggesting a functional link between altered microglia activity during development and synaptic plasticity in adulthood.
Minocycline treatment can efficiently prevent behavioral deficits that follow prenatal stress, normalizing, in parallel, microglial expression of pro- and anti-inflammatory markers in the hippocampus (Zhao et al., 2015). These findings suggest that long-lasting microglial alterations might mediate the behavioral consequences of early stress.
The link between microglia and stress has been convincingly shown in adult mice. Anti-inflammatory treatment ameliorated chronic stress-induced behaviors (Hinwood et al., 2012, 2013; Kreisel et al., 2014; Fuertig et al., 2016). Furthermore, mice lacking the microglial-specific CX3CR1 receptor showed resilience to stress-induced depressive behavior (Hellwig et al., 2016; Milior et al., 2016). Therefore, microglia can play a crucial role in mediating vulnerability to stress and its downstream effects on synaptic circuits. However, the molecular mechanisms by which microglia mediate synaptic and behavioral changes in early-stress models are far from been understood.
Dietary Intake
Imbalance in dietary intake has been convincingly associated with cognitive function. High-fat diet (HFD) consumption is associated with cognitive impairment, increased aggression and altered social- and reward-related behaviors in mice (Hilakivi-Clarke et al., 1996; Pistell et al., 2010; Takase et al., 2016). A mouse model of n-3 polyunsaturated fatty acids (PUFAs) deficiency resulted in defective spatial learning and memory tasks, with increased depressive-like behavior (Larrieu et al., 2012; Moranis et al., 2012).
Both the imbalance in PUFAs content and the long-term exposure to HFD lead to dysregulation of inflammatory processes (Ortega et al., 1997; Pistell et al., 2010; Sharma et al., 2012; Valdearcos et al., 2014; Baufeld et al., 2016; Guillemot-Legris et al., 2016; Gzielo et al., 2016). Interestingly, docosahexaenoic acid (DHA), one of the most abundant PUFAs in the brain and a potent immunomodulator, was shown to prevent LPS-induced cytokine production in microglia, by directly inhibiting the surface presentation of CD14 and TLR4 (De Smedt-Peyrusse et al., 2008). A growing literature provides evidence for a direct role of DHA in regulating microglial activity (Antonietta Ajmone-Cat et al., 2012; Chen et al., 2014; Chang et al., 2015; Baufeld et al., 2016; Hao et al., 2016; Hopperton et al., 2016; Tremblay et al., 2016).
The effects of dietary imbalance during development have been recently investigated. Dietary intake was shown to act as a modulator of microglial behavior in the perinatal brain in studies where female mice were fed throughout gestation and lactation with n-3 PUFAs deficient or a control diet. Mice raised under n-3 PUFAs deficiency exhibited altered levels of microglial phenotypic markers and defective microglia motility at PND21. This defective microglial activity was associated with increased pro-inflammatory cytokine expression, and altered expression of plasticity-related genes in neurons (Madore et al., 2014). Overall these findings indicate that dietary intake, specifically in relation to PUFA content, might drastically impact on microglial properties. The consequent microglial-mediated increase in inflammation, when occurring early in development, may contribute to neurodevelopmental disorders (Bolton and Bilbo, 2014).
The mechanisms linking dietary intake to microglial activation are beginning to be elucidated. Erny et al. (2015) have recently shown that gut microbiota control maturation and function of microglia, possibly via release of short-chain fatty acids (SCFAs: microbiota-derived bacterial fermentation products). Germ-free mice and antibiotic treated mice displayed altered microglia expression and morphology consistent with immature phenotype; the results were phenocopied in mice deficient for the SCFA receptor FFAR2 (Erny et al., 2015). Hence, evidence exist that dietary intake can modulate microglial activity, and at least some of this interaction might be mediated via host microbiota (Smith, 2015; Campos et al., 2016; Erny et al., 2017).
Neural Activity-Dependent Modulation of Microglia
Increasing neural activity by pharmacological blocking of inhibition, by AMPAR or by NMDAR activation (Fontainhas et al., 2011; Dissing-Olesen et al., 2014), but not by electrical stimulation (Wu and Zhuo, 2008), results in enhanced microglial motility and extension of microglial processes. Conversely, GABAergic activation leads to a reduction in microglial motility (Fontainhas et al., 2011). Many studies support the theory that microglia–synapse contacts and engulfment of synaptic material are strongly modulated by neural activity. Apposition between microglia and synaptic terminals increase following neuronal activity, and correlate with higher rate of synaptic elimination upon ischemia (Wake et al., 2009) and in an experience-dependent manner (Tremblay et al., 2010). Similarly, microglia-mediated engulfment of synaptic inputs in the early developing dorsal lateral geniculate nucleus is regulated by neural activity: increasing upon inhibition with tetrodotoxin or stabilizing with forskolin activation (Schafer et al., 2012).
Indeed, environmental manipulations that either increase or decrease neuronal activity have been shown to impact on the microglial phenotype and its interaction with dendritic spines. Environmental enrichment (EE) is a well-established experimental paradigm of neuronal stimulation, resulting in a brain derived neurotrophic factor (BDNF)-dependent increase in the number of spines, physical rewiring of the brain and improvement in several cognitive domains (van Praag et al., 2000). Exposure to an enriched environment has also been shown to promote microglial branching (Xu et al., 2016) and profoundly affect neuroimmune functions in the hippocampus (Williamson et al., 2012). Importantly, LPS injection abrogated the EE-induced increase in BDNF production, suggesting that microglia are not simply bystanders, but are actively involved in the beneficial effects of EE (Williamson et al., 2012). On the other hand, deprivation of a specific sensory input (e.g., vision) during critical developmental periods also results in dramatic neuronal remodeling at both the synaptic and circuit levels. Microglia have been shown not only to respond to sensory deprivation, but also to mediate some of its neuronal effects. Following dark adaptation (a paradigm resulting in increased spine motility and turnover in primary visual cortex, Majewska and Sur, 2003), Tremblay et al. (2010) found subtle changes in the behavior of microglia in the primary visual cortex of juvenile mice. The observed changes included an expansion of microglial processes, an increased occurrence of cellular inclusions (suggesting phagocytosis), an increased frequency of contact with synaptic clefts, and an increased apposition with synaptic elements, with overall reduced motility (Tremblay et al., 2010). These results are largely confirmed by Sipe et al. (2016) in a model of monocular deprivation (MD), where sensory inputs are abrogated from one eye only. In this model, the authors observed an increase in the contacts between microglia and spines, occurring at day 1 post MD. This increased interaction was followed by active engulfment of synaptic material by microglia 4 days after MD. These data suggest that microglia are capable of sensing the altered neuronal activity and can respond by remodeling synapses (Sipe et al., 2016). Importantly, in MD models, neuron–microglia interaction was shown to significantly contribute to the functional plasticity that follows sensory deprivation. In fact, MD normally results in the full shift of ocular dominance from the closed eye to the open eye within 2 days (Wiesel and Hubel, 1963). However, this effect was abrogated in mice either lacking the P2Y12 receptor, or receiving a specific P2Y12 inhibitor (Sipe et al., 2016). Since this receptor is exclusively expressed in CNS microglia, crucially mediating the neuron–microglia cross-talk (Sasaki et al., 2003; Haynes et al., 2006), the above results indicate that sensory deprivation requires microglia to rewire the visual cortex. How P2Y12 modulates ocular dominance, and whether this is mediated via spine remodeling, remains to be elucidated.
A reciprocal regulation between microglia and neural activity has also been shown in the zebrafish optic tectum, where microglia contacts with active neurons are more frequent, and result in neuronal silencing in a contact-dependent mechanism (Li et al., 2012). These findings indicate that neural activity is shaping brain connectivity also through the critical contribution of microglia.
Genetic Factors Modulating Microglial Activity
The use of genetically modified mouse models and the recent advent of Cre lines selective for microglia have been instrumental in dissecting the role of specific genes in modulating microglial activity. Altering the function of genes involved in the regulation of key executive microglial tasks, such as motility, migration, phagocytosis and cytokine release, affects a broad spectrum of microglial-mediated cellular processes, which occur in the developing brain. Several such alterations are linked to the onset of behavioral symptoms reminiscent of neurodevelopmental and psychiatric disorders in humans, indicating a key role for microglia in such diseases.
DAP12 is a transmembrane protein associated with the TREM2 receptor, with specific microglial expression, and is up-regulated in the developing brain (Aoki et al., 2000; Roumier et al., 2008). Mouse models expressing a mutated form of DAP12 lacking the expression of the functional protein were one of the first examples of selective microglia manipulation significantly impacting on synaptic function. DAP12 KO mice displayed defective mIPSC (miniature inhibitory post-synaptic currents), impaired AMPAR accumulation at the post-synaptic site, and showed a drastic reduction in BDNF-specific TrkB receptor. Such impairments translated into defective synaptic transmission, thus likely to affect brain wiring (Kaifu et al., 2003; Roumier et al., 2004). In addition, the positioning of Lh6-expressing interneurons in the cortical plate of E18.5 embryos and P7 mice was impaired in the DAP12 KO models, suggesting that microglia may support the functioning of neural circuits by contributing to the maintenance of inhibitory/excitatory balance (Squarzoni et al., 2014). Adult DAP12 KO mice displayed phenotypes relevant to schizophrenia and ASD, such as reduced startle reflex in response to acoustic stimuli and reduced prepulse inhibition, suggesting an impairment of sensorimotor gating (Kaifu et al., 2003).
CX3CR1 is a Gi-protein coupled receptor, encoded by the Cx3cr1 gene, expressed mostly by microglia in the brain (Jung et al., 2000; Mizutani et al., 2012). Its ligand CX3CL1, also known as fractalkine, is on the other hand largely expressed in neurons (Tarozzo et al., 2003). The fractalkine signaling has been extensively investigated in recent years, with accumulating evidence supporting its role in controlling fundamental microglia functions (Limatola and Ransohoff, 2014; Paolicelli et al., 2014; Arnoux and Audinat, 2015). The loss of the fractalkine signaling significantly affects basic microglial properties, such as voltage-dependent K+ currents, processes ramification and ATP-induced migration (Pagani et al., 2015). The brains of Cx3cr1 KO embryos displayed extensions of TH-positive dopaminergic neurons, selectively in the subpallium, but not in the midbrain, and showed defective positioning of Lh6-expressing interneurons in the neocortical plate (Squarzoni et al., 2014). Cx3cr1 KO brains at early post-natal stages also showed a significant increase in cell death in the cortical layer V, phenocopying the effects of microglia depletion via DTR expression (Ueno et al., 2013). 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. These findings confirmed previous studies reporting causal effects on synaptic plasticity with modulation of Cx3Cl1/Cx3cr1 signaling (Ragozzino et al., 2006; Maggi et al., 2011; Rogers et al., 2011). Cx3cr1 KO mice displayed a transient reduction in microglial numbers in the developing hippocampus, and showed defective synaptic pruning, resulting in synaptic deficits and long-lasting impairments in social behavior and brain connectivity (Paolicelli et al., 2011; Zhan et al., 2014). The lack of fractalkine signaling was associated with reduced levels of free Insulin-like Growth Factor 1 (IGF1) in the mouse cortex at PND5, ultimately affecting the survival of corticospinal and callosal neurons (Ueno et al., 2013). Overall, these observations indicate that the defective microglial function induced by the lack of Cx3cl1/Cx3cr1 signaling significantly affects brain development, with long-lasting consequences on neural circuits and brain function.
BDNF Microglial contribution to synaptic plasticity and function has been elegantly demonstrated through an inducible conditional knockout (cKO) mouse line, where BDNF was selectively depleted from microglia (Parkhurst et al., 2013). BDNF influences a large variety of cellular processes, ranging from survival, to apoptosis, cellular morphology, and synaptic plasticity (Hohn et al., 1990; Lu et al., 2005; Park and Poo, 2013). Along with neurons, microglia also represent a source of neurotrophin in the brain (Elkabes et al., 1996; Elkabes et al., 1998; Coull et al., 2005; Trang et al., 2011). Mice lacking microglial BDNF displayed altered synaptic protein levels and impaired spine formation during motor learning tasks, which resulted in the absence of training-induced improvement in motor behavior performance, suggesting an important role for microglial derived BDNF in modulating synaptic plasticity (Parkhurst et al., 2013).
The Complement system is a complex innate immune surveillance system, with important functions in inflammation, pathogen defense and host homeostasis (Zabel and Kirsch, 2013; Merle et al., 2015). Complement molecules such as C1q and C3, are up-regulated in the early post-natal brain, and have been implicated in synaptic remodeling during the development of the retinogeniculate system (Stevens et al., 2007). C1q and C3, closely apposed to synapses, work as ‘eat-me’ signals for the synaptic terminals to be removed. Loss of function approaches, such as genetic knockout mouse lines, resulted in increased synapse number, suggesting that the absence of such molecules could impair synaptic pruning by microglia, leading to enhanced connectivity and epilepsy (C1q KO model, Chu et al., 2010) and decreased synaptic engulfment (C3 KO or Complement Receptor 3, CR3 KO, Schafer et al., 2012). Recently, microglial CR3 activation was found to trigger long term synaptic depression (LTD), upon the combination of hypoxia and inflammatory stimuli, via NADPH oxidase. This finding suggests that microglial CR3-triggered LTD may underlie synaptic dysfunction (Zhang et al., 2014). Further investigations are required to elucidate the possible occurrence of such mechanisms during early brain development.
Progranulin (PGRN) is a protein encoded by the progranulin gene (GRN), and it is implicated in the regulation of phagocytosis and the release of pro-inflammatory cytokines in microglia and macrophages (Yin et al., 2010; Kao et al., 2011; Martens et al., 2012). GRN is associated with frontotemporal lobar degeneration, and microglia lacking GRN are shown to have an aberrant expression profile at 18 but not at 4 months of age (Lui et al., 2016). However, the same study showed that primary microglia isolated from neonatal GRN KO mice expressed more abundant levels of C1q and C3 complement molecules, described to mediate synaptic pruning (see above). In vitro systems assessing the functional implication of GRN KO in neonatal microglia, based on primary neuronal–microglia co-culture, revealed a significant increase in synaptic pruning, and provided evidence for C1qa-tagged synaptic puncta within microglial phagocytic structures (Lui et al., 2016). These findings indicate that an aberrant complement expression by microglia, combined with an enhanced phagocytosis, is detrimental for the synaptic function.
ATG7 (autophagy related gene 7) is an E1-like activating enzyme required for cytoplasm – vacuole transport, and is essential for autophagy. Kim et al. (2016) recently showed that microglial autophagy is involved in synaptic refinement. Microglia lacking Atg7 displayed impaired degradation of synaptosomes in vitro. Deletion of Atg7 from myeloid cell-specific lysozyme M-Cre mice resulted in increased dendritic spines and synaptic markers, and was associated with long-lasting deficits in sociability, with no changes in social recognition. In addition, increased repetitive behaviors were observed in mice lacking microglial Atg7, indicating that deficient microglial function in the developing brain might lead to ASD-like behaviors (Kim et al., 2016).
Overall these findings, based on selective manipulation of microglia, indicate that genes regulating microglial functions can have a critical impact on synaptic refinement and might be involved in neurodevelopmental disorders.