Tuning neural circuits and behaviors by microglia in the adult brain

Abstract

Microglia are the primary immune cells of the central nervous system (CNS), contributing to both inflammatory damage and tissue repair in neurological disorder. In addition, emerging evidence highlights the role of homeostatic microglia in regulating neuronal activity, interacting with synapses, tuning neural circuits and modulating behaviors. Here, we review how microglia sense and regulate neuronal activity through synaptic interactions, thereby directly engaging with neural networks and behaviors. We discuss current studies utilizing microglial optogenetic and chemogenetic approaches to modulate adult neural circuits. These manipulations of microglia across different CNS regions lead to diverse behavioral consequences. We propose that spatial heterogeneity of microglia-neuron interaction lays the groundwork for understanding diverse functions of microglia in neural circuits and behaviors.

Microglia dynamically interact with neurons in the adult brain

Microglia dynamically survey the central nervous system (CNS) with highly motile processes [1,2]. As the primary immune cells of the CNS, they are renowned for their rapid response to neuronal injuries, contributing to both inflammatory damage and repair during disease onset and progression [3,4]. In addition, microglial roles extend beyond their immune function in disease. In the healthy brain, homeostatic microglia physically interact with various neuronal compartments, such as neuronal somata, axon initial segment, nodes of Ranvier, and synapses, to sculpt neural structures and modulate neuronal activity [5–7]. Specifically, microglia extend processes to preferentially interact with neuronal somata and form purinergic junctions, which are able to dampen neuronal activity [8,9]. Additionally, microglia interact with the axon initial segment where neurons generate action potentials [10]. This interaction could promote axo-axonic synapse formation between pyramidal neurons and chandelier cells [11]. Furthermore, at the nodes of Ranvier, microglia are capable of sensing neuronal activity and establish contact with the nodes through THIK-1 channels [12].

Neurons form intricate circuits that transmit information through specific synaptic connectivity. In the context of brain development, microglia have garnered attention for their involvement in circuit architecture formation by engaging in synaptic pruning and promoting synaptogenesis [11,13–17]. In the adult CNS, neuronal circuits also undergo substantial tuning and remodeling. However, microglia’s contributions to neural networks in maturity have been relatively understudied compared to microglia’s roles in neural development.

Recent studies have shown that microglia can interact with synapses and thus are an integral part of the adult neural networks and various behaviors [18]. In this review, we focus on the intricate relationship between homeostatic microglia and neuronal synaptic connections in the adult CNS. We begin by providing a concise overview of how microglia sense and regulate neuronal activity. We then discuss current evidence of the impact of homeostatic microglia on neural synaptic connectivity and rodent behaviors. We examine studies employing microglial optogenetic and chemogenetic tools to address microglial functions in neural circuits. Interestingly, region-specific neuronal responses to microglia manipulations were frequently identified in the current literature. Drawing upon this knowledge, we put forth a general hypothesis suggesting that microglial spatial heterogeneity plays a key role in shaping and regulating specific neural circuits in the adult brain and behaviors.

Microglia sense and regulate neuronal activity through synaptic interactions

Microglia play a crucial role in sensing and regulating neuron activity [5,6,19]. When there is an increase in neuronal activity, microglia exhibit heightened process interaction with active neurons. These interactions are characterized by process extension, process convergence, process pouches, and faster movement velocities, leading to increased contact time that can rescue neurons from excitotoxicity [8,9,20–25]. The underlying mechanism involves microglial P2Y12 receptors, which bring microglial processes in close proximity to hyperactive ATP-releasing neurons [8,9,26]. Additionally, the presence of CD39 and CD73 ectoenzymes on microglial membranes allows for rapid hydrolysis of ATP into adenosine to reduce neuronal firing via adenosine receptors [26].

Conversely, when neurons are hypoactive, for example under anesthesia, microglia extend their processes and increase their dynamics as well [27,28]. This response is regulated by the alteration of neuronal norepinephrine (NE) signaling, specifically acting on the microglial β₂-adrenoreceptor, during the transition from a basal state to a hypoactive state [27,28]. How microglial β₂-adrenoreceptor-mediated morphology changes impact hypoactive neuronal networks was also recently investigated [29]. The study showed that microglia enhance neuronal activity following the cessation of isoflurane-induced general anesthesia in mice. During general anesthesia, microglial processes enter the synaptic cleft, shielding GABAergic inputs. Microglia ablation or the loss of microglial β₂-adrenoreceptor prevents the occurrence of post-anesthesia neuronal hyperactivity, indicating that microglial process dynamics enable microglia to transiently enhance neuronal activity by physically shielding inhibitory inputs [29]. Interestingly, another study in mice demonstrated that microglia contribute to the maintenance of pentobarbital-induced general anesthesia and prevent early emergence through P2Y12 receptors [30]. This discrepancy may result from the different anesthetic regimens or the duration of anesthesia. In these studies, isoflurane-induced general anesthesia lasts 30 minutes, whereas pentobarbital-induced anesthesia can extend for several hours. In addition, isoflurane-induced anesthesia increases microglia complexity and process length, whereas pentobarbital administration does not affect microglia morphology [27,28,31]. Hence, microglia may promote neuronal activity via shielding of inhibitory synapses during short-term isoflurane-induced neuronal hypoactivity and, conversely, dampen neuronal activity during prolonged periods of pentobarbital-induced neuronal hypoactivity through P2Y12-mediated mechanisms.

These findings demonstrate the dynamic interplay between microglia and neuronal activity, highlighting a U-shaped pattern of microglial sensing of neuronal activity. Although different mechanisms underlie microglial responses to neuronal hyper- and hypo-activity, microglia are able, overall, to maintain brain homeostasis to achieve a balance of neural activity through their dynamic process interactions with neurons (Figure 1).

Figure 1. A U-shaped pattern of microglia responses for sensing and regulating neuronal activity.

Compared with the basal awake state, hyperactive neurons (upper right) increase ATP release and promote microglial process interaction through microglial P2Y12 receptors. The CD39 and CD73 ectoenzymes on microglia rapidly hydrolyze ATP/ADP into adenosine (ADO), which subsequently reduces neural firing by binding to the adenosine A1 receptor (A1R, bottom right). Under awake condition, adrenergic terminals release norepinephrine (NE) to induce microglia process retraction through microglial β₂-adrenoreceptor (Bottom middle). Concordantly, hypoactive neurons (upper left) increase microglial process dynamics due to the low tonic NE level and disinhibition of β₂-adrenoreceptor-mediated process retraction. Under this condition, microglial processes can enter the synaptic cleft to shield inhibitory inputs and promote neural activity (bottom left).

Microglia tune and remodel adult neural circuits

While microglial roles in circuit formation during development have received significant attention [11,13–17], their contributions to neural networks in adulthood are relatively underexplored. In the adult CNS, neuronal axons and dendrites tend to be stable, but dendritic spines and axonal boutons are highly dynamic, displaying remarkable functional and structural plasticity [32]. Additionally, studies in animal models indicate that adult neurogenesis allows newly generated neurons to integrate into established neural circuits and influence behavioral outcomes [33]. In this section, we address how homeostatic microglia impact functional and structural synaptic plasticity, as well as microglial roles in the integration of adult-born neurons into existing neural networks (Figure 2).

Figure 2. Homeostatic microglia actively shape adult neural circuits.

Microglia modulate synaptic plasticity and adult neurogenesis in the healthy brain. (A) In the cortex, BDNF signaling released by microglia promotes synapse formation and enhances synaptic transmission. (B) Additionally, microglia interact with dendritic spines, thereby increasing synapse activity and enhancing local cortical network synchronization. (C) In the hippocampal CA1 region, microglia physically contact dendritic shafts and postsynaptic spines. Higher contact frequency correlates with more spine formation and elimination. (D) In the dentate gyrus (DG), microglia phagocytose ECM components through interleukin 33 (IL-33) signaling to promote spine formation and functional plasticity. (E) Microglia in the DG actively remove apoptotic newborn neurons but also promote adult neurogenesis. (F) In the olfactory bulb (OB) and DG, microglia prune synapses on adult-born neurons depending on the presence of phosphatidylserine (PS).

Synaptic plasticity

Neuronal circuits undergo substantial tuning and remodeling in the adult CNS, with synaptic plasticity playing a prominent role in shaping neural connectivity [34]. Several studies have suggested the involvement of microglia-derived factors in modulating functional and structural synaptic plasticity. For example, homeostatic microglia can release soluble factors to regulate synaptic plasticity, including brain-derived neurotrophic factor (BDNF), tumor necrosis factor alpha (TNFα), and platelet-derived growth factor B (PDGFB) [35–40]. BDNF signaling from microglia has been implicated in promoting learning-related synapse formation, enhancing synaptic transmission, and spinal long-term potentiation (Figure 2A) [36,37]. Microglia-derived TNFα has been shown to regulate the phosphorylation of sleep-related synaptic proteins, linking microglia to synaptic plasticity in the context of sleep [38]. TNFα can also indirectly alter synaptic connectivity by stimulating microglia. In response to TNFα stimulation, microglia differentially modulate synaptic plasticity through BDNF upregulation in the spinal cord or downregulation in the hippocampus [35], suggesting spatial heterogeneity of microglia in modulating neural circuits. Furthermore, constitutively released microglial PDGFB promotes neural expression of Kv4.3 and increases neural potassium currents to prevent the overactivation of pre-sympathetic neurons in the hypothalamic paraventricular nucleus [40].

The physical interaction between microglia and neurons is an alternative mechanism underlying the regulation of synaptic connectivity. Microglial processes make brief contacts with presynaptic boutons and postsynaptic spines, inducing spine enlargement during contact [14]. The contact frequency is dependent on neuronal activity. Increased neural activity and long-term potentiation (LTP) promote microglial process contact with active dendritic spines [41–44]. Active spines exhibit prolonged contact duration compared to inactive spines [42]. Additionally, these contacts have been correlated with the immediate enhancement of synapse activity and local cortical network synchronization (Figure 2B) [45], and reduced spine stability in the hippocampus (Figure 2C) [43]. Fractalkine signaling has been proposed as one of key mechanisms underlying synaptic alterations mediated by microglia contacts. Inhibition of fractalkine/CX3CR1 mediated neuron-microglia physical interactions increases LTP amplitude [46–48]. These studies demonstrate that microglial contact can promote both functional and structural plasticity. However, how physical contacts by microglia regulates synaptic plasticity is still largely unknown.

Pharmacological elimination of microglia in adult mice has been shown to increase excitatory and inhibitory connectivity in the cortex [49]. Remodeling of the extracellular matrix (ECM) is one of the processes mediating changes in cortical synaptic transmission. For example, microglia depletion was found to result in increased densities of perineuronal nets, the ECM structures surrounding neurons [49]. Increases in perineuronal nets are known to be associated with increased excitatory and inhibitory synaptic transmission [50]. Besides synaptic function, microglia also participate in structural aspects of synaptic plasticity through ECM remodeling [51,52]. Genetic knockout of neuronal interleukin 33 (IL-33) or microglial IL-33 receptors decreased microglial phagocytosis of ECM components and impaired spine formation and functional plasticity in mice (Figure 2D) [51].

Adult neurogenesis

Adult neurogenesis occurs throughout life in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus [53]. Newborn neurons in SVZ can migrate to the olfactory bulb (OB) through the rostral migratory stream (RMS). Adult neurogenesis is dynamically regulated by various stimuli, such as physical exercise, environmental enrichment, and seizures. These newly generated neurons can integrate into circuitry and influence behavior [33].

Microglia were reported to promote adult neurogenesis in the healthy and diseased mouse brain [54–57]. In the healthy brain, microglia play a critical role in promoting beneficial adult neurogenesis and clearing excess adult-born neurons. For example, during the early stages of adult neurogenesis, a significant portion of newborn cells undergo apoptosis. Homeostatic microglia in the adult mouse SGZ niche are actively involved in the clearance of these apoptotic newborn cells through efferocytosis [58,59], suggesting the multifaceted microglial regulation of adult-born neurons (Figure 2E). On the other hand, the depletion of microglia in mouse SVZ decreases the survival and migration of newborn neuroblasts through the RMS to the OB [60]. Furthermore, neuron-microglia signaling via IL-33 promotes environmental-enrichment-induced adult neurogenesis and further affects memory functions in mice [51].

Microglia also regulate the synapses of adult-born neurons. For example, adult-born OB granule cells in microglia-depleted mice have been found to exhibit decreased spine density, reduced turnover rate, and altered odor-evoked responses [61]. These results suggest that microglia contribute to the functional integrity of OB circuitry by regulating the density and turnover of spines in adult-born neurons [61]. Similarly, another study also showed that OB and hippocampal microglia are able to sculpt synapses on adult-born neurons depending on the presence of phosphatidylserine (Figure 2F) [62]. These studies highlight the role of microglia in the formation, pruning, and maintenance of synapses on newborn neurons during adult neurogenesis.

Microglia manipulations modulate neural circuits and behaviors.

Considering the intimate interactions between microglia and neurons, it is not surprising that microglia may contribute to adult neural networks and related behaviors. Utilizing approaches like microglia ablation and microglial conditional gene knockout, studies in animal models have shown that microglia are able to regulate learning and memory [36,51], sleep [38,42,63], anxiety-like behaviors [64], compulsive behaviors [65], and alcohol intake [66]. Mechanistically, studies have shed light on the importance of microglial THIK-1 K⁺ channels, CX3CR1, and P2Y12 in regulating microglial interactions with neuronal circuits [8,9,12,26,47,67].

With this knowledge, several studies have applied optogenetic and chemogenetic tools to unravel the precise functions of microglial ionotropic (channels) and metabotropic (GPCRs) signaling in fine tuning and regulating neural circuitry, as reviewed in the following.

Optogenetic manipulation of microglia

Ionotropic signaling plays a significant role in microglia physiology [3,68]. Homeostatic microglia express various ion channels, including K⁺ channels, proton channels, and purinergic ionotropic receptors, to mediate ionic fluxes [67,69–71]. K⁺ channels are particularly important for microglial process dynamics and ATP-induced chemotaxis [67,72,73]. However, it remains unclear whether the activation of these ion channels or changes in membrane potential affects homeostatic microglia-neuron interactions and neural circuits. Optogenetics approaches allow manipulating the flow of ions into or out of cells using light stimulation [74], and have been extensively employed to locally depolarize or hyperpolarize neurons to dissect complex neural circuits underlying behaviors [75]. More recently, optogenetics approaches have been used to understand the roles of microglial ionotropic signaling in microglial process chemotaxis and neuronal network function [76–80]. For instance, activation of ChETA, an engineered channelrhodopsin (ChR), in microglia resulted in microglial depolarization and slowed the chemotaxis of microglial processes [79].

Further, recent studies showed that optogenetic activation of microglia can enhance neural activity and induce behavioral changes. Optogenetic activation of ReaChR, a red-shifted ChR variant, depolarized spinal microglia and induced Ca²⁺-dependent interleukin-1β (IL-1β) release and synthesis [76]. Interestingly, brief optogenetic activation of spinal microglia in mice increased neuronal activity and mediated mechanical allodynia, observed one hour after stimulation and lasting up to one week (Figure 3A) [76]. These findings suggest the intriguing possibility of "microgliogenic" pain originating from microglial ionotropic signaling. Additionally, optogenetic activation of ChR2 expressed in Hoxb8 microglia in mice was shown to result in distinct behavioral responses, depending on the brain regions being stimulated: optogenetic stimulation of Hoxb8 microglia within the dorsomedial striatum (DMS) or the medial prefrontal cortex (mPFC) induced grooming behaviors, while Hoxb8 microglia activation in the basolateral amygdala (BLA) or central amygdala (CeA) lead to increased anxiety-like behaviors (Figure 3B) [80]. Although further mechanistic investigations are warranted, these studies demonstrate that local optogenetic manipulation of microglial ionotropic signaling can tune neural circuits and behaviors in a region-dependent manner.

Figure 3. Consequences of microglial manipulations on neural circuits and behaviors.

(A) Optogenetic activation of spinal microglia using the red-activatable ChR variant (ReaChR) increases Ca2⁺-dependent IL-1β release. Microglia-released IL-1β promotes neural activity and induces chronic pain in mice [76]. (B) Optogenetic activation of channelrhodopsin 2 (ChR2) on Hoxb8 microglia within the striatum (STR), the medial prefrontal cortex (mPFC), ventral hippocampus (vHIP), or basolateral amygdala (BLA) transiently increases local network activity, inducing grooming or anxiety-like behaviors in mice [80]. (C) Chemogenetic activation of microglial Gq-DREADD in the striatum reduces excitability of medium spiny neurons via microglial-released prostaglandins. Microglial Gq-DREADD activation induces a negative affective state characterized by anhedonia and aversion in mice [107].

Besides ChR2-derived non-selective cation channels, the modern optogenetic toolbox includes light-gated proton pumps, K⁺ channels, and Cl⁻ channels. The functions of the light-activated proton pump, archaerhodopsin (ArchT), have been tested in microglia as well [79]. The light stimulation of ArchT hyperpolarized microglial membrane potential but did not appear to affect microglial chemotaxis responses. However its effects on other microglial functions, such as microglia dynamics and microglia-neuron interactions, are still not fully understood. It is worth noting that microglia highly express a unique voltage-gated proton channel called Hv1, and studies have shown that the knockout of Hv1 significantly affects microglial functions in pathological conditions [70,81–83]. However, the function of microglial Hv1 in the healthy brain remains largely unknown. In this context, the use of ArchT could serve as a valuable tool to mimic and dissect the function of microglial Hv1 channels. In addition, the recent discovery of light-gated K⁺ channels, Kalium channelrhodopsins (HcKCRs), presents an exciting opportunity to investigate the role of K⁺ currents in microglia function [84], given the importance of K⁺ currents in homeostatic microglial surveillance and chemotaxis [67,72,73].

Together, current studies utilizing microglial optogenetics have demonstrated that alterations in microglial ionotropic signaling have profound effects on neural activity and behaviors. Diverse optogenetic tools have been widely used in studying neuronal circuits but have yet to be applied to microglial research. The integration of optogenetics into the larger toolkit for studying microglial function will enable researchers to understand how microglial ionotropic signaling influences adult neural circuits and unravel the complex roles for microglia in behavior.

Chemogenetic manipulation of microglia

GPCR-mediated (metabotropic) signaling is a pivotal component of microglial physiology [85]. Homeostatic microglia express a plethora of GPCRs, including P2Y12, CX3CR1, C3aR1, P2Y6, β₂-adrenoreceptor, and GPR56 [85]. These GPCRs belong to 4 subfamilies based on the α subunits of the heterotrimeric G proteins with which they interact (i.e., Gi, Gq, Gs, G12/13) [86]. In the healthy brain, homeostatic microglia have been recognized to sense and dampen neuronal activity through one of their Gi-GPCRs, P2Y12 [26]. Consistently, perturbation of all microglia Gi-GPCRs using genetically encoded pertussis toxin (PTX) alters microglia morphology, reduces microglia surveillance, and induces neuronal hyperexcitability [87]. Furthermore, in mice, loss of microglial Gi-GPCRs, such as P2Y12, CX3CR1, and C3aR, causes increased innate fear and anxiety-like behaviors [64,88–90], suggesting a critical role for microglial Gi-GPCRs in regulating neural circuits and behaviors.

To gain further insight into microglia GPCR signaling, recent studies have applied the chemogenetic platform Designer Receptors Exclusively Activated by Designer Drugs (DREADD) to manipulate microglial GPCR signaling pathways [77,91]. DREADDs comprise a family of engineered GPCRs that are selectively activated by inert exogenous compounds, such as clozapine N-oxide (CNO), compound 21 (C21), and deschloroclozapine (DCZ) [92]. DREADDs offer the potential to mimic the known functions of endogenous microglial GPCRs and explore previously unknown microglial GPCR-activated signaling pathways. For instance, activation of microglial Gi-GPCRs, such as P2Y12 and C3aR1, has been shown to collapse filopodia and induce large process extension towards the agonist source [93–95]. Similarly, Gi-DREADD activation in microglia elicited process extension towards the CNO source [96], suggesting that Gi-DREADD can recapitulate the general function of microglia endogenous Gi-GPCRs. In addition, acute and chronic activation of Gi-DREADD could inhibit microglial activity, reduce the release of pro-inflammatory cytokines, and alleviate neuropathic pain [97–100]. In the kainic acid-induced mouse seizure model, acute activation of microglial Gi-DREADD protects neurons by reducing neuronal hyperactivity, while prolonged activation results in neuronal death by resting microglia in the homeostatic stage [101]. How microglial Gi-DREADD signaling affects their interactions with neurons, thereby regulating neural circuit and behavioral consequences, will need to be clarified through future studies.

Microglia express a number of Gq-linked GPCRs including the 5-HT-sensitive 5HT_2BR [102], histamine-sensitive H1R-H4R [103], and UDP-sensitive P2Y6R [104]. Loss of the 5HT_2B receptor in mouse microglia similarly results in impaired phagocytotic capability along with deficits in social behavior [105]. Additionally, activation of Gq-GPCR induced the elevation of intracellular calcium signaling, and calcium signaling can be utilized by microglia to sense neuronal activity [106], suggesting microglia may sense and regulate neuronal activity through Gq-GPCRs. Recent studies have investigated the role of Gq-DREADD manipulation in homeostatic microglia [107–109]. Activation of microglial Gq-DREADD increased microglia phagocytotic ability, promoted inflammatory cytokine production, induced pain sensation, and induced a negative affective state in mice [107,108,110]. Interestingly, while the induction of pain sensation by microglial Gq-DREADD implies microglial Gq-GPCR signaling in promoting neuronal activity [108], electrophysiology experiments have demonstrated that microglial Gq-DREADD activation reduces the excitability of striatal medium spiny neurons [107]. This decrease in neural excitability was mediated by the elevation of microglial-released prostaglandins (PGEs). Consequently, mice showed anhedonia-like and aversion behaviors (Figure 3C) [107]. Furthermore, it was shown that in mice, early-life stress induced by limited bedding and nesting caused microglia dysfunction and led to augmented excitatory synapses in the paraventricular hypothalamic nucleus [109]. During early-life stress, activation of microglial Gq-DREADD restored microglial synaptic pruning and reduced stress responses in adulthood [109]. These findings suggest that in different brain regions, microglial Gq-GPCR signaling pathways may exhibit distinct modulatory responses to neural circuits. In addition to Gq-DREADDs, it would be valuable to investigate in future studies the functions of microglial Gs-GPCRs and G12/13-GPCRs by currently available Gs-DREADD and G12/13-DREADD approaches [111–113].

Chemogenetic approaches in microglia can provide insights into the interplay between microglial GPCR signaling and neuronal circuits, shedding light on the complex dynamics of microglia-neuron interactions in the CNS. However, it is important to note that the DREADD ligand CNO could be metabolized to clozapine and elicit related side effects, including sedation [114]. Thus, it is recommended to use improved DREADD ligands and perform appropriate controls [115]. Another caveat to consider is that using the Cx3cr1^CreER/+ to drive the expression of Gi-DREADD on microglia has been associated with transient microglia-independent hypolocomotion, which may potentially interfere with data interpretation [96]. It is recommended therefore to use more selective microglia cre mouse lines (such as Tmem119^CreER/+) for driving the microglial expression of DREADDs to minimize confounding effects on locomotion. In addition to transgenic mice, researchers may use localized viral transfection to study microglial DREADD in particular CNS regions and behaviors, for example manipulating spinal microglia in pain studies, or striatal microglia in studies of aversion behavior [99,100,107].

Diverse microglial functions in neuronal circuits

Region-specific neuronal responses to the microglia manipulations highlight the role of microglial spatial heterogeneity in local neural network modulation. For instance, microglia depletion has been shown to increase synaptic transmission in the cortex while reducing it in the CeA [49,66]. Further, microglia in different regions can exhibit opposite neural circuit modulations in response to the same stimulation. For instance, upon TNFα stimulation, microglia increase BDNF expression in the spinal cord to promote synaptic connectivity, while the opposite occurs in the hippocampus [35]. Additionally, microglia residing in the SVZ have been found to form a functionally distinct class that supports and guides the integration of neurons into the olfactory circuitry [60]. Moreover, optogenetic and chemogenetic manipulations of microglia suggest distinct microglial intracellular signaling pathways account for varying behavioral outcomes, revealing region-specific changes in neural activity and behavior [76,80,107,108]. The mechanisms underlying microglial regulation of neuronal circuits in different CNS regions warrant further investigation.

Microglial spatial heterogeneity may contribute to the region-specific neuronal modulation. Bulk and single-cell analyses of CNS tissues in mice have revealed specific subtypes of microglia that vary in a time- and region-dependent manner [116–118]. Regional neuronal diversity appears to play a role in establishing and maintaining region-specific phenotypes of microglia [119,120]. Even within specific regions of the cortex, microglial states have been shown to be influenced in a layer-specific manner by the pyramidal neurons in the respective layers [121]. These phenomena seem reasonable considering the fact that microglia express a plethora of neurotransmitter receptors, allowing them to potentially sense subtle differences in the CNS microenvironment [3]. Continued research into microglial spatial heterogeneity and how microglia regulate local circuits will deepen our understanding of microglial functions in health and diseases.

Even though microglial spatial heterogeneity is less robust in adulthood than in the developmental mouse brain [122], neuronal heterogeneity may affect the outcome of microglia mediated neuromodulations. Currently, evidence shows the CD39 and CD73 ectoenzymes on microglia rapidly hydrolyze ATP/ADP into adenosine to reduce neural firing by binding to the adenosine A1 receptor. However, adenosine A1 receptors are differently expressed throughout the brain and other adenosine receptor subtypes, such as adenosine A2a receptors, can stimulate neuronal activity upon binding to adenosine [123]. As such, microglia may regulate distinct neural circuits in a differential manner.

Additionally, microglia responses are affected by the microenvironment within local neural circuits. Neurotransmitters are not only released to the synaptic cleft but can also diffuse in the extracellular space, where they might be sensed by microglia. In fact, microglia were reported to express a plethora of neurotransmitter receptors, such as ionotropic/metabotropic glutamate receptors, GABA_B receptors, β₂-adrenoreceptor, 5-HT_2B receptors, α7 nicotinic-acetylcholine-receptor [124]. However, how these receptors function in adult homeostatic microglia in vivo warrants further studies. Notably, norepinephrine has been found to reduce, while serotonin can potentiate microglial process motility as well as ATP-induced microglial chemotaxis [27,28,125,126]. This implies the local concentration of norepinephrine and serotonin may finely tune microglial surveillance to regulate microglial function in neuronal circuits. Furthermore, microglia interact with different types of neurons, such as GABAergic, adrenergic, serotonergic, and cholinergic neurons, whose axonal terminals exhibit spatial differences [27,28,105,127–129]. For example, microglia can extend processes toward the serotonin source through 5-HT_2B receptors and interact with serotonergic axons [102]. Genetic knockout of microglial 5-HT_2B receptors in mice impairs neuronal circuit maturation, causing increased novelty-induced self-grooming, cognitive flexibility deficits, and decreased social interactions [105]. During postnatal development, GABA_B-expressing microglia preferentially interact with inhibitory synapses and sculpt inhibitory circuits [29,127,128]. In mice, the interaction of microglia with GABAergic synapses during development was shown to facilitate phagocytosis of inhibitory synapses [127]. Consequently, microglial GABA_B knockout mice displayed decreased spatial exploration at P30 and hyperactivity in adulthood [127]. ARG1⁺ microglia closely interact with cholinergic fibers [129]. Knockout of microglial ARG1 in mice leads to reduced cholinergic innervation in the hippocampus, and causes impaired long-term memory [129]. Exploring how microglia interact with specific neuronal populations in distinct brain regions may provide novel insights into the diverse roles of microglia in maintaining neuronal homeostasis, synaptic plasticity, and behavioral regulation.

Concluding remarks and future perspectives

Recent efforts in exploring the dynamic interactions between homeostatic microglia and neuronal synapses highlight the multifaceted roles of microglia in modulating neuronal activity, synaptic plasticity, adult neurogenesis, neural circuits, and various behaviors. These studies underscore the key function of microglia in maintaining brain homeostasis. Optogenetic and chemogenetic manipulations have emerged as powerful tools for spatiotemporally investigating microglial ionotropic and metabotropic signaling pathways and their impact on neural circuits and behaviors. In many psychiatric illnesses, neural circuit dysregulation is a common feature [130]. Regulating neuronal receptor activity universally may lead to unwanted side effects by affecting non-targeted neuronal populations. The advantage of microglia mediated neuromodulation lies in its ability to sense neuronal activity, depending on the neuronal inputs. Based on pioneering studies in microglial optogenetics and chemogenetics, we believe that microglia ion channels and GPCRs show the potential as drug targets. Further research may focus on modulating these signaling pathways to enhance the microglial responses to abnormal neuronal activity, thereby facilitating control over brain homeostasis.

As research delves deeper into the mechanisms of how microglia regulate neuronal activity, region-specific neuronal responses to the microglia manipulations were observed.Thus, it is important to gain a comprehensive understanding of how microglia are integrated into various neural circuits (see Outstanding Questions).

Outstanding Questions.

• Microglia constantly survey the brain parenchyma with their extremely motile processes. What external signals control the seemingly random movement of microglial processes? Do these moving processes always interact with other cells (neurons, glia, vasculature cells)? Are all the steps in the movement physiologically relevant, i.e., what is the functional significance of microglia process dynamics under physiological conditions?

• Microglia interact with various neuronal compartments. Do microglia employ different molecular machinery to modulate neuronal functions contingent on the interaction site? This prompts the question of whether functional disparities exist between various sites of microglia-neuron interaction.

• Microglia express a number of neurotransmitter receptors that trigger downstream signaling. How do various neural circuits in the healthy CNS promote and maintain microglia spatial heterogeneity, and what are the epigenetic, transcriptomic, and translational regulatory aspects of this process?

• In the adult brain, how do microglia interact with different neuronal subtypes, such as glutamatergic, GABAergic, adrenergic, serotonergic, and cholinergic neurons? Can multi-photon microscopy be leveraged to image microglia-neuron interactions in deep brain regions to address how heterogeneous microglia sense and modulate specific neural circuits and modify behavior?

• Microglia have the ability to sense hyperactive or hypoactive neurons, contributing to the maintenance of brain homeostasis. Can microglia be targeted to fine-tune neural circuits and harness the beneficial function of microglia to treat brain diseases involving disrupted homeostasis?

In the healthy brain, one characteristic of microglia is their constant and dynamic process movements [1,2]. Microglial surveillance differences and how microglia interact with neurons in various brain regions will be an important area of future study. For instance, a comparison of cortical versus cerebellar microglia motility revealed that cerebellar microglia exhibit less surveillance [131]. More investigations are needed to understand the region-specific surveillance of microglia and their roles in neural circuits and behaviors. Two-photon imaging of microglia has limitations in imaging depth. Typically, microglia are imaged in the superficial layer of the cortex after open skull or thinned skull cranial window surgery [132]. Achieving in vivo imaging of microglia in subcortical regions without highly invasive procedures, such as GRIN lens insertion [133] and cortical tissue aspiration [59], remains challenging. Given the heterogeneity of microglia [121], it is of major importance to investigate microglia in deep cortical or subcortical neural circuits. To this end, recent studies have explored three-photon microscopy for microglia imaging [134,135]. This emerging imaging technique holds promise for non-invasive imaging of microglia through an intact skull [134] and for visualizing microglia in the white matter up to about 1.1 mm below the brain surface [135]. Further, it is important for future studies to conduct real-time imaging of microglia in neuronal circuits in various behavioral contexts. Recent advancements in miniature two-photon microscopy have introduced a promising avenue to study microglia-neuron interactions in freely-moving mice [136].

In addition to microglial interactions with synapses, microglia also engage with different neuronal compartments, including the soma, axon initial segment, and nodes of Ranvier [8,10,12]. Current understanding of how these interactions influence neuronal function in the homeostatic brain remains limited. It also remains to be clarified whether distinct microglial populations are responsible for each of these types of microglia-neuron interactions. Addressing these questions could benefit from a combination of patch-clamp techniques, transcriptome analyses, and multi-photon imaging. Patch-seq allows for the collection of cells for RNA sequencing alongside the recording of membrane activity [137]. By integrating patch-seq with multi-photon imaging of microglia, researchers may distinguish between different types of microglia-neuron interactions, monitor changes in microglial membrane potential during these interactions, and gain insights into their molecular profiles. The application of these and other methodologies will enhance current understanding of the heterogeneous nature of microglia-neuron interactions across various regions of the adult CNS.

Highlights.

• Homeostatic microglia dynamically interact with neuronal synapses to sense and regulate neural activity. They modulate synaptic plasticity by physical interactions as well as the release of soluble factors.

• Homeostatic microglia play a crucial role in adult neurogenesis by regulating the survival of newborn neurons as well as the elimination, formation, and maintenance of synapses.

• Optogenetic and chemogenetic manipulations of microglia provide new avenues to interrogate microglial signaling pathways influencing behavior.

• Spatial heterogeneity of microglia-neuron interaction may contribute to diverse functions of microglia in neural circuits and behaviors in adulthood.