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Microglia perform diverse functions in both the healthy and diseased brains. In a recent study published in Cell Research, Wang et al. show that a subpopulation of microglia in the visual cortex interacts with the axon initial segment of excitatory neurons to modulate neuronal excitability and contribute to visual perception.

Microglia are the resident immune cells of the central nervous system, with highly motile processes that extend and retract to surveil the local environment. In addition to immunosurveillance, studies over the past two decades revealed additional key roles for microglia in neuronal synapse development and refinement in the healthy brain1,2,3 (Fig. 1). Distinct subpopulations of microglia exist throughout the brain, varying by function, brain region, stage of life, and injury or disease states.4 One unique subpopulation comprising ~10%–20% of microglia extend a single process to contact the axon initial segment (AIS) of excitatory pyramidal neurons (PNs) in the cortex.5 The AIS is a specialized subcellular compartment that is located at the proximal axon and functions as the site of neuronal action potential initiation due to its high density of ion channels.6 Axon initial segment-associated (AXIS) microglia have been observed across mammalian species including mice, rats, and primates.5 Notably, the interaction between AXIS microglia and PNs is lost after injury, suggesting that these cells contribute to healthy brain function, and change their state when responding to brain injury or disease.5 Consistent with this notion, AXIS microglia reportedly participate in axo-axonic synapse assembly between chandelier neurons and PNs during early postnatal development.7 Furthermore, it was speculated that in the healthy adult brain, AXIS microglia may modulate the excitable properties of the neurons they contact, perhaps through buffering K+ through inwardly rectifying channels.5 However, for more than a decade the physiological contribution of AXIS microglia to healthy nervous system function has remained a mystery.

Fig. 1: Microglia play diverse roles in the healthy brain.

Axon initial segment-associated microglia (AXIS MG) modulate neuronal activity through direct interaction with the AIS. Mechanistically, this involves microglial depolarization mediated by muscarinic acetylcholine (ACh) receptors (MR) and local K+ release via THIK-1. Created in BioRender. https://BioRender.com/5zr1vf5.

Wang and colleagues have solved the mystery of AXIS microglia function and demonstrate for the first time that AXIS microglia can promote the activity of PNs.8 A notable feature of this work is the many independent experimental approaches used by the authors to arrive at their conclusions. For example, using both patch clamp and optogenetic approaches, the authors show that brief depolarization of AXIS microglia leads to increased action potential firing of their associated neurons. Mechanistically, they demonstrate that the increased neuronal activity is caused by the local release of K+ from microglia through the outward rectifying K+ channel THIK-1, which mediates K+ efflux to maintain microglial resting potential.9 The local increase in K+ concentration at the AIS could lower threshold for PN action potential initiation. Pharmacological inhibition of THIK-1 or microglia-specific knockout of THIK-1 abolishes the increased action potential firing frequency observed after depolarization of AXIS microglia. Calcium imaging to measure neuronal activity reveals that PNs associated with AXIS microglia respond more robustly to visual stimulation than nearby PNs lacking AIS–microglia contact. Importantly, loss of THIK-1 activity reduces the calcium responses of PNs associated with AXIS microglia. Moreover, THIK-1 inhibition impairs performance in a visual discrimination task. Thus, K+ release via THIK-1 enables AXIS microglia to modulate AIS and neuronal activity, which in turn contributes to visual perception.

What depolarizes AXIS microglia in the first place? Two-photon imaging of awake mice shows that visual stimulation with drifting gratings induces transient depolarization of AXIS microglia in layer 2/3 of the visual cortex. Pharmacological screens of G protein-coupled receptors reveal that muscarinic acetylcholine receptors are required for AXIS microglia depolarization. In addition, the non-selective sodium leak channel NALCN is required to depolarize microglia in response to visual stimulation. Thus, neuromodulatory acetylcholine depolarizes AXIS microglia, leading to increased PN activity.

How do AXIS microglia form such precise contacts with the AIS? In addition to the physiological studies performed, RNA sequencing experiments of different microglial populations reveal that AXIS microglia have unique transcriptomic profiles. Among the specific genes upregulated in AXIS microglia is Igtb1, which encodes the cell adhesion molecule integrin β1 (ΙΤGΒ1). Immunostaining showed that ITGB1 is specifically enriched in microglial processes that contact the AIS. To determine the necessity of ITGB1 for the interaction between AXIS microglia and the AIS, the authors ablate Itgb1 from microglia. Loss of ITGB1 significantly reduces the percentage of microglia that interact with the AIS in the cortex. Furthermore, mice with ITGB1-deficient microglia have reduced neuronal calcium responses to visual stimulation compared to wild-type mice. Finally, mice with ITGB1-deficient microglia perform worse than wild-type mice in a visual discrimination task. Thus, microglial ITGB1 is required for the physical and functional interactions between AXIS microglia and the AIS of PNs in the visual cortex that ultimately affect visual discrimination behavior.

Taken together, Wang and colleagues significantly expand our understanding of the non-immune functions of microglia. They demonstrate for the first time that AXIS microglia can modulate neuronal activity through their direct interaction with the AIS. Their work supports a model (Fig. 1) where visual stimulation activates microglial muscarinic receptors, which in turn activate the cation channel NALCN to transiently depolarize AXIS microglia. Depolarization facilitates K+ efflux through THIK-1 directly onto the AIS, leading to increased excitability of the subpopulation of neurons contacted by AXIS microglia. The coordinated activity of this subpopulation of neurons, located in layer 2/3 of the visual cortex, ultimately influences higher-order brain functions like visual discrimination.

Although the authors focused their study on the visual cortex, since AXIS microglia are present across the entire cortex,5 it will be interesting to determine whether the mechanisms revealed by Wang et al. are generalizable and contribute to other brain circuits. Interestingly, K+ flux by THIK-1 was reported to be necessary for the interaction between microglia and nodes of Ranvier, another excitable axonal domain composed of many of the same proteins as the AIS.10 Finally, the defining feature of AXIS microglia is their precise interaction with the AIS. While the study here shows that microglial ITGB1 is required for this interaction, its neuronal ligand is currently unknown. Previous studies showed that loss of AnkyrinG, the master scaffolding protein of the AIS, disrupted AXIS microglia.5 Interestingly, in addition to high densities of ion channels, the AIS also contains several cell adhesion molecules, including Neurofascin-186, NrCAM, and Contactin-1.6,11 One or more of these neuronal molecules may interact with microglial ITGB1 to link the AIS to the microglial process.

Overall, this work profoundly broadens our knowledge of microglia in the healthy brain, highlighting that microglial subpopulations exhibit distinct functional properties and raising fascinating questions about how microglia–neuron interactions might alter neuronal networks in injury or disease states.