Introduction

During development, neurons construct neural circuitry through the seamless progression of a discrete series of steps; neurons differentiated from progenitor cells migrate to specific areas, extend their axons toward potential target cells, and connect via synapse formation. These formation events occur in excess; neurons initially form an inordinate number of branches and synapses. Inappropriate connections are removed by axon pruning and/or synapse elimination (Fig. 1). In addition to neuron-intrinsic mechanisms, the interactions of neurons with various cell types, such as glial cells and vascular cells, are also involved in these steps [1, 2]. Recent studies revealed that microglia, which are the resident immune cells of the central nervous system (CNS), play key roles in these processes to establish neural circuits in the developing brain.

Fig. 1
figure 1

Schematic model illustrating the refinement of neural circuits and the effect of microglia. Initially, neurons form excessive trial branches and synapses, which are concurrently or sequentially eliminated, resulting in proper network formation

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Microglia have long been studied for their roles in pathological conditions since they rapidly respond to pathology via changes in their morphology and functions, such as releasing inflammatory cytokines and exhibiting active phagocytic properties [3,4,5]. More recent studies utilizing new genetic and functional approaches have investigated microglial functions in physiological conditions, particularly in the development of the CNS. Microglia continuously scan the surrounding environment using their processes under physiological conditions [6,7,8,9]. Advances in microscopy indicate that microglia contact neurons at various points, such as the cell body, dendrites, axons, and synapses. These interactions are involved in CNS surveillance, and are required for the construction of neural circuitry. In this review, we will provide an overview of neuron–microglia interactions in CNS development, highlighting the signaling and significance of microglia–axon interactions. Moreover, the disturbance of these interactions has been observed in diseases and brain injury conditions, and may contribute to the pathogenesis of neurodevelopmental diseases. Therefore, we will also discuss how microglia–neuron interactions may be affected in disease conditions, and how such knowledge may lead to further advances in the therapeutic strategies for diseases and injuries of the CNS.

The role of microglia in CNS development

Regarding the origin of microglia, del Rio-Hortega demonstrated the concept of a mesodermal origin of microglia based on Cajal’s silver carbonate staining [10,11,12]. There has since been much debate regarding the origin of microglia [13,14,15,16,17]. There are three main theories regarding this point: (1) mesodermal/mesenchymal tissues [18, 19]; (2) the neuroectoderm (similar to neurons, astrocytes, and oligodendrocytes) [20,21,22,23,24]; and (3) hematopoietic cells (circulating blood monocytes), which produce peripheral macrophages [25, 26]. Accumulating evidences support the notion that microglia are derived from myeloid progenitors in the mouse embryonic yolk sac at embryonic day (E) 7.5 [27,28,29,30] before infiltrating the brain at E9.5 [13, 27,28,29, 31, 32]. Microglia then accumulate in clusters close to the white matter, where they actively proliferate. This typical distribution disappears in the adult brain, indicating the specific roles of microglia in the developing brain.

During development, regressive events, such as cell death [33, 34] and synaptic elimination [35,36,37], are necessary along with constructive events including proliferation, differentiation, axonal elongation, and synaptogenesis to establish proper neural circuitry. Microglia have various functions in these steps. For instance, microglia regulate developmental cell death and survival via their bidirectional functions. Microglia are attracted by apoptotic cells expressing “find-me” signals [38], and “eat-me” signals in the membrane of the apoptotic cell allows microglia to engulf and phagocytose the cell [39]. Uridine diphosphate, the product of uridine-5′-triphosphate hydrolysis, acts on the P2Y6 receptor in microglia to facilitate phagocytosis [40]. Microglia-derived nerve growth factor triggers cell death in the chick retina during early embryonic development [41], suggesting another causal effect of microglia on programmed cell death. In contrast, microglia also express a variety of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2, macrophage colony-stimulating factor, osteopontin, and insulin growth factor 1 (IGF-1), which are known to support the proliferation or survival of neurons [42,43,44].

The details of the various functions and mechanisms of neuron-microglia contact and the history of these topics have been reviewed elsewhere. In the following section, we focus on microglia–axon interactions and discuss how these interactions are coordinated and their role in CNS development.

Axon–microglia interactions in physiological condition

The construction of neural circuits is a dynamic process mediated by both formation and elimination events [35, 45,46,47]. Initially, neurons form axonal branches and synapses in excessive numbers, and these trial connections are not always maintained in the mature brain. Inappropriate connections are subsequently or concurrently removed. In the following section, we summarize the microglial functions in these constructive and deconstructive processes, and discuss how they affect neural circuit formation (Fig. 1). Similar to other fundamental developmental processes, such as cell death/survival, and synapse formation/elimination, microglia also exert both constructive and deconstructive effects on axons with a certain amount of common molecular mechanisms.

Microglia–axon interactions support neuronal survival and early network wiring

In the developing brains of various species, including humans, microglia exhibit a specific and heterogeneous distribution [48,49,50,51]. In particular, microglia accumulate at a higher density at the developing axonal tract during a specific period of brain development, and these microglia demonstrate a unique morphology (Fig. 2) [52,53,54]. The accumulation of microglia around axons was initially described in the early 1900s [10, 11] and is now widely recognized in both rodents and humans [48,49,50,51, 55,56,57]. Further, in vivo two-photon imaging studies provided evidence that microglia can directly contact neurons [58].

Fig. 2
figure 2

Various interactions between microglia and axons during brain development. a Microglia (green) accumulate around dopaminergic axons (red) in the subpallium at embryonic day (E)14.5. Pharmacological [colony-stimulating factor 1 receptor (CSF-1R) blocking] or genetic (Pu.1 −/− mutants) depletion of microglia promotes dopaminergic axonal outgrowth into the striatum, whereas maternal immune activation (MIA) reduces the extension of dopaminergic axons. b Microglia accumulate around axonal fibers in the corpus callosum (CC) and mediate its fasciculation at E17.5 via a mechanism involving DAP12 signaling. The loss of DAP12, depletion of microglia in Pu1 −/− mice, and MIA lead to defasciculation of the CC. c Microglia accumulate around the axons from layer V neurons, peaking at postnatal day (P)3–7, and support the survival of layer V neurons via the release of insulin-like growth factor 1 (IGF-1). Pharmacological inactivation (minocycline treatment), and genetic inactivation or elimination of microglia (Cx3cr1 −/− mouse or CD11b-DTR transgenic mice) increases the rate of apoptosis in the layer V neurons. NetirnG1 expressed in neurons and its ligand netrin-G ligand-1 (NGL1) expressed in microglia are involved in this interaction

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An early study using primary culture systems reported that conditioned media from primary microglia cultures isolated from embryonic rat brains enhances neurite outgrowth [59]. Blockade of the extracellular matrix protein thrombospondin by antibodies inhibits this effect, suggesting that microglia mediate neurite growth by releasing thrombospondin. Conditioned media from microglial cultures also increase the proliferation of cultured cerebellar granule cells via mitogen-activated protein kinase, phosphatidylinositol-3-kinase/Akt, and delta-Notch signaling pathways [60]. These findings suggest that embryonic microglia support the processes of neural circuit formation.

The accumulation of microglia is notably observed in dopaminergic axons during the embryonic stage (Fig. 2a) [52]. Microglial depletion or dysfunction via disruption of microglial signaling or maternal immune activation (MIA) modulates the outgrowth of dopaminergic axons. Microglial depletion via colony-stimulating factor 1 receptor (CSF-1R) blockade in Cx3cr1 green fluorescent protein (GFP)/ + or Pu.1 −/− mutants results in the exuberant extension of tyrosine hydroxylase (TH)-positive dopaminergic axons into the subpallium of the embryonic ventral telencephalon, whereas MIA induces a mild but robust reduction in the extension of TH-positive axons into the subpallium at E14.5. Three-dimensional reconstruction of confocal images and electron microscopy demonstrated direct contact between microglia and TH-positive axons, and TH-positive axon fragments were detected inside the cytoplasms of GFP-positive microglia. These findings suggest that microglia might phagocytose fragments of dopaminergic axons in physiological conditions. Interestingly, no defects in the neighboring serotoninergic fibers or internal capsule were observed, suggesting that microglial regulation of axon outgrowth is rather specific to dopaminergic fibers in the embryonic brain.

Microglia also contact developing axonal fibers in the corpus callosum and mediate its fasciculation in the embryonic brain (Fig. 2b). The loss of DAP12, a key microglial-specific signaling molecule; depletion of microglia in Pu1 −/− mice; and an MIA model demonstrated the defasciculation of dorsal callosal axons as detected by the increased ratio of neuropilin 1-positive dorsal tract width normalized to the width of L1-CAM-positive callosal fibers at E17.5 [54]. Remarkably, the time-course of microglial accumulation assessed in this study slightly differed from that in dopaminergic axons [52].

In the early postnatal brain, microglia accumulate around the axons of layer V neurons and support the survival of these neurons (Fig. 2c) [53]. Microglia begin to accumulate in the white matter, including the subcortical white matter, internal capsule, cerebral peduncle, and cerebellar white matter, where the axons of corticospinal motor neurons in layer V pass, from postnatal day (P)1–3. Their levels peaked in these areas at P3–7. Apoptosis of layer V neurons was observed in the three different models involving the inhibition of microglial activation with minocycline and transient genetic depletion of microglia with Cd11b-DTR mice and CX3CR1 knockout mice. Both in vitro and in vivo observations demonstrate that this effect of microglia on neuronal survival is due to the release of IGF-1, which enhances axon outgrowth of corticospinal motor neurons [61]. Further, microglial accumulation at this axonal tract is mediated by netrin-G1-netrin-G ligand-1 (NGL1) signaling in the postnatal brain [62]. Netrin-G1 is specifically expressed in corticospinal neurons and at high levels in the postnatal period [63]. Netrin-G1 is structurally related to the netrin family of axon guidance molecules [64,65,66]. However, unlike classic netrins, it is linked to the plasma membrane surface by a glycosylphosphatidylinositol anchor and lacks affinity for the known netrin receptors, DCC and UNC-5. Instead, netrin-G1 binds to a cell adhesion molecule, NGL1. Deletion of netrin-G1 or NGL1 expression reduces the accumulation of microglia along the axons of layer V neurons.

The inactivation of microglia by minocycline treatment from P3 to P4 induces the death of cortical neurons [53]. However, an increased rate of apoptosis was not observed in layer V or other layers when minocycline was administered to mice at E16–17 or P14–15 when amoeboid microglia were not clustered in the white matter. Consistently, another group reported that the death of layer V neurons in the somatosensory cortex was not observed when mice were treated with minocycline from P8 to P10 [67]. These observations suggest that the time-point of the effect of microglial inhibition is finely restricted.

Collectively, microglia accumulate at different axons at different time-windows during development, and play a key role in supporting axons and neurons. The organization of the dynamic alteration of temporal and spatial microglial accumulation and the link to the modulation of certain axonal tracts would be clarified in the future studies.

Axons in the CNS can be supported by myelin sheaths created by oligodendrocytes, which may also be associated with the accumulation of microglia along axons. Indeed, crosstalk between microglia and oligodendrocytes has been noted in both physiological and pathological conditions [68,69,70]. Similar to how neurons construct excess axons and synaptic networks and are selectively eliminated, myelin sheaths are first overproduced and pruned to refine myelination during development [71]. A study using in vivo imaging of zebrafish has revealed that microglia phagocytose myelin sheaths lead to myelin refinement [68]. Neuronal activity attracts microglia towards neuronal cell bodies, whereas less neuronal activity promotes microglia to associate with axons and phagocytose myelin. Thus, microglia–axon interactions and myelin phagocytosis are bidirectionally regulated by neuronal activity.

Effects on axon pruning

The excess branches formed by unconstrained growth of axons in the initial phase are pruned in a degenerative or retractive manner [46, 47, 72]. The impairment of axon pruning leads to behavior deficits [73, 74], indicating that this deconstruction process is also critical for CNS development as well as the processes involved in the early wiring of the CNS, such as axon outgrowth.

Axon pruning in drosophila mushroom body γ neurons has been widely used as a model to assess the mechanism of pruning, since it occurs in a defined set of temporally-regulated morphological changes, hallmarked by the initial extension of excess axons during the larval stage, and following fragmentation of axons between 8 and 12 h after puparium formation [75, 76].

In this model, glial cells surrounding the axon processes engulf degenerating axons [77, 78]. Interestingly, the invasion of glia at these axons precedes axon fragmentation, suggesting that glial invasion occurs rather independently of activation in response to axon fragmentation [77]. These findings indicate that glia may play an initiative role in triggering axon fragmentation. The glial proteins draper and ced-6, which are essential for the clearance of apoptotic cells in C. elegans, are required in the glial engulfment of axons in the larval stage [79]. Since ecdysone signaling, which mediates a cell-autonomous genetic program for axon pruning [80, 81], induces draper expression, this signaling may link the intrinsic and extrinsic programs for axon pruning during Drosophila metamorphosis [79].

Although it should be noted that these processes could involve neuroectodermally derived cells with immune functions that are different from microglia cells and but more like macroglial cells, their ability to engulf degenerating neurons reflects the phagocytic aspect of microglia in mammals. Microglia engulf axon debris via the p38 mitogen-activated protein kinase pathway in the axons of rodent cortical explants [82]. However, the role and underlying mechanisms of microglia in axon pruning remain unclear. The mechanism underlying the clearance of axon debris is more evident in pathological conditions, which is described in the following Sect. (“Alteration of microglia–axon interactions in the pathological conditions”).

Interaction of microglia with the axon initial segment

In addition to these diverse interactions of microglia and axons, microglia preferentially interact with the axon initial segment (AIS) located at the base of axons, which is responsible for action potential generation and is a major regulator of neuronal excitability [83]. A subset of microglia that are intimately associated with the neuronal cell body extend a single process and contact the AIS [84]. Microglia–AIS interactions occur early in the postnatal stage (from P9) and are maintained throughout adulthood (8–12 weeks in mice).

Further, these interactions exhibit specificity with regard to brain regions. AIS-associated microglia are more prominently observed in the cortex than in the thalamus and striatum. Furthermore, AIS-associated microglia preferentially associate with non-GABAergic neurons than GABAergic neurons, and more frequently associate with pyramidal neurons in layer V of the cortex compared to the total cortical neurons, suggesting that AIS-associated microglia favor excitatory neurons. Neuron-specific knockout of ankyrin G reduced the number of neurons with AIS-associated microglia, suggesting that, although microglia can contact various parts of neurons, a certain subset of microglia may recognize the AIS via ankyrin G expression. Since microglia–AIS interactions are lost following microglial activation 3–72 h after traumatic brain injury, these interactions may be more conspicuously involved in controlling healthy brain functions than in modulating response to injury. Although the function of AIS-associated microglia remains unclear, the anatomical and physiological properties of the AIS suggest that those microglia may be to modulate the excitable properties of the AISs they contact or monitor AIS synapses.

The role of microglia in the refinement of synaptic circuits

To establish appropriate synaptic connections, neural circuits are refined and reinforced by the removal of exuberant synapses. There has been increasing interest in the role of microglia in synaptic refinement, particularly regarding the activity-dependent mechanism [9, 85]. In line with neural network formation, we briefly summarize the functions of interactions between microglia and synapses.

As well as intrinsic programs, extrinsic cues resulting in neural activity are critical for the proper maturation and maintenance of the neural network [86]. In vivo two-photon imaging has been used to determine the role of microglia in the regulation of neuronal excitability. Resting microglia in the mouse visual cortex can contact dendritic spines via their dynamic processes [87, 88]. Microglial processes make brief and direct contacts with neuronal synapses within minutes at a frequency of approximately one per hour. [88]. Global downregulation of neural activity following the injection of the voltage-gated sodium channel blocker tetrodotoxin (TTX) reduced the frequency of such contacts, indicating that microglia-synapse contacts are activity-dependent in physiological conditions. During alterations in visual experience, microglial processes changed their morphology and microglial behavior was altered, such as increased phagocytosis of synaptic elements and interaction with subsets of structurally dynamic and transient synapses, suggesting that microglia contribute to the activity-dependent modification or elimination of synapses for experience-dependent plasticity in the healthy brain [87]. In vivo time-lapse imaging of both microglial morphology and neuronal activity in zebrafish larvae indicated the reciprocal regulation of microglia and neuronal activity [89]. Neuronal activity induces resting microglia to make contact with active neurons, resulting in the selective reduction of contacted neuron activity.

When the repetitive stimulation of individual layer II/III pyramidal neurons induces neuronal hyperexcitability and elicits swelling of axons, microglial processes migrate to and wrap these damaged axons following the release of both ATP, known to be a potent attractant of microglia, and glutamate via volume-activated anion channels (VAACs) [90]. This microglia–axon contact induces rapid repolarization in the neuron, which reverses the hyperexcitability to resting levels. Inhibition of microglial migration by pharmacological blockage of VAACs sustains the activity-induced depolarization until cell death, indicating that the microglia–axon interaction contributes to the protection of the neuronal soma from damage due to pathological activity and to the maintenance of neuronal viability.

Taken together, these observations suggest that microglia can control the proper window for neuronal activity, which is essential for the maturation and maintenance of neural circuitry. Therefore, they contribute to brain functions, such as learning and memory, in the healthy brain.

Microglia eliminate synapses through interactions and phagocytosis via signaling pathways involving the chemokine fractalkine (Cx3cl1) and its receptor, Cx3cr1 [92, 93], and complement pathways [91], which are involved in microglial migration and phagocytic functions, respectively.

Stimulated emission depletion microscopy and a combination of electron microscopy with immunolabeling can detect both pre- and post-components of excitatory neurons in the cytoplasm of microglial cells in the hippocampus, indicating the engulfment of synaptic materials by microglia [92]. Cx3cr1-knockout mice showed a transient reduction in the number of microglia and an increase in the number of dendritic spines. The impairment of synaptic elimination in these mice leads to weak synaptic transmission, reduced functional brain connectivity, and behavior deficits associated with neurodevelopmental and neuropsychiatric disorders [93].

Intensive studies revealed that neural activity modulates elimination processes during CNS development [94,95,96]. The retino-genicular circuit system is a classical model for studying activity-dependent synaptic elimination [97,98,99]. Complement component C3 expressed in immature synapses and its receptor complement receptor 3 (CR3/CD11b-CD18/macrophage antigen-1) expressed on the surface of microglia are necessary for the elimination of retino-geniculate synapses at the postnatal stage [91, 100]. Blockade of retinal ganglion cell (RGC) activity by TTX increased the engulfment of its connection by microglia, whereas increased RGC activity by forskolin reduced the engulfment of its connection by microglia. These observations suggest that microglia contribute to the elimination of the retino-genicular connection, preferentially at less active (weaker) synapses.

In addition to elimination, microglia are also involved in synapse formation via direct contact with dendrites in layer II/III pyramidal neurons in the developing somatosensory cortex at P8-10 when intense synaptogenesis occurs [101]. Microglia contact with dendrites induces filopodia formation, which typically occurs at dendrites with contact-induced Ca2+ elevation.

Depletion of microglia impaired the turnover of spines on pyramidal neurons of the motor cortex, with an effect on both the elimination and the formation of spines in the layer V pyramidal neurons of late postnatal (P19) and young adult (P30) mice [102]. Microglia-derived BDNF is a key mediator of this synaptic plasticity since microglia-specific knockout of BDNF largely recapitulated the altered structural plasticity in the spines and performance deficits in microglia-depleted mice.

Alteration of microglia–axon interactions in the pathological conditions

Following neural insults or diseases, the pathological state is progressively altered [103]. Axonal damage causes axon degeneration and generates myelin debris derived from broken myelin sheaths, which contain myelin-derived inhibitors to axon growth [104, 105]. This debris not only constitutes obstructions and inhibitory extrinsic cues for axonal regrowth/regeneration, but also triggers undesirable inflammation, leading to further damage to axons and neurological dysfunction [106, 107].

Following pathological events, such as injury, inflammation, infection, and neurodegeneration, microglia quickly respond and can accumulate at the lesion site accompanied by morphologic alteration from a resting state referred to as “ramified” to an active state. Microglia are involved in neurotrophic support and the removal of damaged cells and debris, which is important for reconstructing the neural network (Fig. 3) [108, 109]. The following section discusses the bidirectional functions of the microglia–axon interaction in favor of and in opposition to the reconstruction of neural circuits.

Fig. 3
figure 3

Schematic representation of the role of microglia–axon interactions in pathological conditions. a Microglia have both beneficial and detrimental effects in disease and injury, and are involved in inflammation following axonal damage. b The active phagocytic activity of microglia following axonal damage contributes to debris clearance and arranges the environment into a favorable condition for regeneration. TNF-α tumor necrosis factor-α, iNOS inducible nitric oxide synthase, IL-1 interleukin-1, NO nitric oxide, ROS reactive oxygen species

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Inflammation

In physiological conditions, microglia continuously monitor the brain environment, but are prominently activated in response to neural insult. Abnormal microglial activation contributes to secondary damage to neural circuits [110,111,112,113,114,115]. Reactive microglia abundantly express or release various inflammatory proteins, such as tumor necrosis factor-α, inducible nitric oxide synthase, or interleukin-1, which leads to the production of nitric oxide and reactive oxygen species (Fig. 3a) [116,117,118].

However, the activation of microglia might be important as it facilitates their phagocytic activity, which arranges the environment into a favorable condition for regeneration, such as the removal of myelin debris or damaged cells (Fig. 3b), and proper stripping of the synapse to protect neurons from excessive neural excitation. Regardless, the excessive or continuous activation of microglia could shift the microglia into a detrimental-dominant role, and contribute to acute and chronic neuropathology. This is evidenced by a mouse model of Alzheimer’s disease (AD). TDP-43 is a DNA–RNA binding protein that regulates microglial phagocytosis in the Aβ42 oligomers-injected AD model mouse [119]. Mice lacking TDP-43 in microglia show enhanced phagocytosis in AD model mouse, but at the same time, exacerbate synaptic loss even in the absence of amyloid. These observations indicate the causative role of microglia in the pathogenesis of neurodegenerative disorders, and suggest that controlling phagocytic activity is a good strategy to regulate disease progression.

Debris clearance to allow a favorable environment for axon regeneration

In rat ischemia models, activated microglia /macrophages rapidly migrated to the infarct area and displayed phagocytic features at day 1 after ischemia [120, 121]. Resident microglia, rather than in filtrating macrophages, likely contribute to debris clearance in the transient focal cerebral ischemia model induced by middle cerebral artery occlusion using GFP-transgenic bone marrow chimeric mice [122].

In a dog spinal cord injury (SCI) model, the number of microglia with a phagocytic phenotype increased in the later stage of injury. However, these microglia exhibited reduced phagocytic activity [123].

In a zebrafish traumatic brain injury model, microglia rapidly accumulated at the lesion site and removed damaged cells by phagocytosis after the injury. In this model, two distinct phases of cell death occur: primary cell death as detected by propidium iodide (PI)-positive cells peaked immediately after injury, and secondary cell death as detected by pyknotic nuclei peaked at 6 h post injury. Live imaging of p2y12:GFP;mpeg1:mCherry double transgenic zebrafish larvae revealed that p2y12+/mpeg1+ microglia rather than p2y12−/mpeg1+ macrophages infiltrated the brain at the lesion site after the injury. Microglia rapidly phagocytosed PI-positive cells 0.5 h after brain injury. The pharmacological inhibition of phosphatidylserine-dependent phagocytosis by O-phospho-l-serine or CRISPR/Cas9-mediated gene knockdown of microglial phagocytosis by injection of gRNA targeting adgrb1a and adgrb1b [124] suppressed the engulfment of PI-positive damaged cells. Remarkably, this increased secondary cell death 6 h after injury, indicating that the rapid clearance of cellular debris by microglial phagocytosis reduces secondary cell death after brain injury.

These findings suggest that the rapid phagocytic activity of microglia following injury largely contributes to the removal of damaged axons, which is beneficial to induce efficient reconstruction of neural circuits.

Accumulating studies demonstrate that regressive events are also involved in the progressive process of neural circuit reconstruction following injury [125,126,127]. In rodent SCI models, injured corticospinal tract axons above the injury site form a large number of new collaterals and synaptic contacts to interneurons in the spinal cord at the initial phase. The numbers of these connections are reduced in the later phase that contribute to the refinement and reinforcement of a detour circuit to the below target area that avoids the lesion site, leading to functional recovery. These processes following axonal damage are similar to the formation and deconstruction processes during de novo construction of neural circuits at the developmental stage. Although there is no solid evidence that microglia are involved in these processes, accumulating evidences on their active phagocytic abilities following neural injury or diseases suggest the potential role of microglia in such processes.

A recent study revealed that microglia are involved in scar-free wound healing after crush injuries to the spinal cord in neonatal mice [128]. Similar to fish and amphibians, neonatal mice are capable of scar-free wound healing and spontaneous axon regrowth, although this is not the case in adult mammals [129]. Single-cell RNA-seq analysis revealed the features of repair-promoting microglia populations with a significant enrichment for genes related to the extracellular matrix. Transplantation of neonatal microglia or proteinase inhibitor treatment help improve wound healing and axon regeneration after SCI in adult mice. Thus, neonatal microglia can have a protective function even in the adult injury, and it would be intriguing to develop a potential application that enables a shift in the microglia to its neonatal state during disease conditions.

Additional roles of microglia in pathophysiological conditions.

In line with the importance of microglial phagocytosis in normal brain development, disruption of this function is also linked to neurodevelopmental diseases. Microglia from MECP2-null mice, a mouse model of Rett syndrome, exhibited reduced phagocytic activity and replacement with wild-type microglia arrested disease progression (Derecki NC, 2012, Nature). Thus, the phagocytic properties of microglia are indispensable for normal brain development and function, and deficits in phagocytosis might be associated with neurodevelopmental diseases.

In addition to their phagocytic activity, microglia can support neurons via mechanisms involving the release of neuroprotective factors [130,131,132]. In a mouse SCI model, microglia induced astrocyte proliferation via IGF-1 and supported neuroprotective scar formation. Depletion of microglia after the injury caused disruption of glial scar formation, enhanced immune infiltration, exacerbated axonal damage, and impaired locomotor recovery [132, 133].

Thus, reactive microglia can also have beneficial effects in pathological conditions via phagocytosis and the production of factors associated with neuroprotective functions.

Conclusion and perspectives

The diverse functions of microglia have further been demonstrated in physiological and pathological conditions. As featured in this review, microglia–axon interactions have long been known to elicit various effects in the construction or reconstruction of neural circuits during development and following injury and diseases. The molecular mechanisms of these interactions have been progressively unveiled. In the rapidly expanding research area on the interaction between the nervous system and the immune system in the CNS, more questions have been raised.

For instance, how microglia communicate with axons remains far from clear. Microglia distribute ubiquitously in the brain, but accumulate at specific axons at high levels, and this preferential association can be altered in a fine time-window of development. One prominent limitation that makes it difficult to analyze these spatio-temporal specific interactions is the challenge of tracking both microglia and developing axons in the wide field of brain with both spatial and temporal information. Advances in the techniques used to generate genetically modified animal models expressing cell type-specific and inducible reporters, and in vivo imaging analysis will certainly help us to surpass these limitations.

In addition, although remarkable progress has been made toward understanding the molecular mechanisms underlying microglia–axon interactions, we need to understand how best to apply these findings to develop novel diagnostic and therapeutic strategies for diseases and injuries of the CNS. Interestingly, microglia have the potential to alter their identity even in the adult brain by forced expression of a single transcription factor, NeuroD1. NeuroD1 is an essential transcription factor for the generation of neurons in the developing brain, and can directly converts mouse microglia into induced neuronal cells that express mature neuronal markers and form synaptic networks with primary cultured cortical neurons [134]. These induced neuronal cells are accompanied by global remodeling of the microglial epigenetic signature. Furthermore, the induced neuronal cells were functionally integrated into brain circuits via synaptic connections with other neurons in the adult mouse brain. Strategies for reprogramming pathological microglia that accumulate at lesion sites into neurons or inducing the neuroprotective character would be beneficial for the development of novel therapeutic approaches for nerve injury and disease.

This is the era that enables us to address the molecular features of cells at the single-cell level [135,136,137,138,139] Microglia demonstrate greater diversity during development and disease or in the aging brain than in the normal, healthy adult brain [135]. To specify microglial heterogeneity and decipher how transcription is regulated in such unique developmental microglia–axon interactions would be of great benefit in answering these fundamental questions regarding development and disease and in the development of novel therapeutic approaches.