Microglial Interactions with Synapses Are Modulated by Visual Experience

Special issue editorial: Glial plasticity in health and disease.

In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile processes. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimination of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit reorganization.

BackgroundAlzheimer’s disease (AD) is the most common neurodegenerative disease, characterized by the deposition of extracellular fibrillar amyloid β (fΑβ) and the intracellular accumulation of neurofibrillary tangles. As AD progresses, Aβ drives a robust and prolonged inflammatory response via its recognition by microglia, the brain’s immune cells. Microglial reactivity to fAβ plaques may impair their normal surveillance duties, facilitating synaptic loss and neuronal death, as well as cognitive decline in AD.MethodsIn the current study, we performed correlative light, transmission, and scanning electron microscopy to provide insights into microglial structural and functional heterogeneity. We analyzed microglial cell bodies and processes in areas containing fAβ plaques and neuronal dystrophy, dystrophy only, or appearing healthy, among the hippocampus CA1 of 14-month-old APPSwe-PS1Δe9 mice versus wild-type littermates.ResultsOur quantitative analysis revealed that microglial cell bodies in the AD model mice were larger and displayed ultrastructural signs of cellular stress, especially nearby plaques. Microglial cell bodies and processes were overall less phagocytic in AD model mice. However, they contained increased fibrillar materials and non-empty inclusions proximal to plaques. Microglial cell bodies and processes in AD model mice also displayed reduced association with extracellular space pockets that contained debris. In addition, microglial processes in healthy subregions of AD model mice encircled synaptic elements more often compared with plaque-associated processes. These observations in mice were qualitatively replicated in post-mortem hippocampal samples from two patients with AD (Braak stage 5).ConclusionTogether, our findings identify at the ultrastructural level distinct microglial transformations common to mouse and human in association with amyloid pathology.

Immunohistochemical electron microscopy (EM) allows the identification of microglial cell bodies and processes, which are otherwise difficult to recognize based on their ultrastructural features. The technique has been essential in defining, at high spatial resolution, microglial interactions with neurons and synapses, thus providing, among other discoveries, important insights into their roles in synaptic pruning and stripping. In this protocol, we describe the preparation of mouse brain tissue for EM, the immunocytochemical staining against ionized calcium binding adaptor molecule 1, the imaging of microglial cell bodies and processes, and the analysis of microglial relationships with the synaptic neuropil.

BackgroundHuntington’s disease (HD) is a dominantly inherited neurodegenerative disorder that affects cognitive and motor abilities by primarily targeting the striatum and cerebral cortex. HD is caused by a mutation elongating the CAG repeats within the Huntingtin gene, resulting in HTT protein misfolding. Although the genetic cause of HD has been established, the specific susceptibility of neurons within various brain structures has remained elusive. Microglia, which are the brain’s resident macrophages, have emerged as important players in neurodegeneration. Nevertheless, few studies have examined their implication in HD.MethodsTo provide novel insights, we investigated the maturation and dysfunction of striatal microglia using the R6/2 mouse model of HD. This transgenic model, which presents with 120+/-5 CAG repeats, displays progressive motor deficits beginning at 6 weeks of age, with full incapacitation by 13 weeks. We studied microglial morphology, phagocytic capacity, and synaptic contacts in the striatum of R6/2 versus wild-type (WT) littermates at 3, 10, and 13 weeks of age, using a combination of light and transmission electron microscopy. We also reconstructed dendrites and determined synaptic density within the striatum of R6/2 and WT littermates, at nanoscale resolution using focused ion beam scanning electron microscopy.ResultsAt 3 weeks of age, prior to any known motor deficits, microglia in R6/2 animals displayed a more mature morphological phenotype than WT animals. Microglia from R6/2 mice across all ages also demonstrated increased phagocytosis, as revealed by light microscopy and transmission electron microscopy. Furthermore, microglial processes from 10-week-old R6/2 mice made fewer contacts with synaptic structures than microglial processes in 3-week-old R6/2 mice and age-matched WT littermates. Synaptic density was not affected by genotype at 3 weeks of age but increased with maturation in WT mice. The location of synapses was lastly modified in R6/2 mice compared with WT controls, from targeting dendritic spines to dendritic trunks at both 3 and 10 weeks of age.ConclusionsThese findings suggest that microglia may play an intimate role in synaptic alteration and loss during HD pathogenesis.

Fetal alcohol spectrum disorder patients suffer from many cognitive disabilities. These include impaired auditory, visual, and tactile sensory information processing, making it more difficult for these patients to learn to navigate social scenarios. Rodent studies have shown that alcohol exposure during the brain growth spurt (BGS) can lead to acute neuronal apoptosis and an immunological response by microglia in the somatosensory cortex. Since microglia have critical physiological functions, including the support of excitatory synapse remodeling via interactions with dendritic spines, we sought to understand whether BGS alcohol exposure has long-term effects on microglial or dendritic spine dynamics. Using in vivo two-photon microscopy in 4-5 week old mice, we evaluated microglial functions such as process motility, the response to tissue injury, and the dynamics of physical interactions between microglial processes and dendritic spines. We also investigated potential differences in the morphology, density, or dynamics of dendritic spines in layer I/II primary sensory cortex of control and BGS alcohol exposed mice. We found that microglial process motility and contact with dendritic spines were not altered after BGS alcohol exposure. While the response of microglial processes toward tissue injury was not significantly altered by prior alcohol exposure, there was a trend suggesting that alcohol early in life may prime microglia to respond more quickly to secondary injury. Spine density, morphology, stability, and remodeling over time were not perturbed after BGS alcohol exposure. We demonstrate that after BGS alcohol exposure, the physiological functions of microglia and excitatory neurons remain intact in early adolescence.

Remodeling of brain circuits, including the formation, modification, and elimination of synaptic structures, occurs throughout life as animals adapt to their environment. Until very recently, known mechanisms for experience-dependent synaptic plasticity had placed neurons and their structural interactions with astrocytes in the spotlight. However microglia, the immune cells of the brain, are very active even in the absence of pathological insults and their processes periodically contact dendritic spines and axon terminals in vivo.1-3 This intriguing behavior prompted us to explore, using electron microscopy and two-photon in vivo imaging in the primary visual cortex of juvenile mice, a possible role for quiescent microglia in the modification of synaptic structures.4 Our work uncovered subtle changes in the behavior of microglia during manipulations of visual experience including regulation of perisynaptic extracellular spaces, contact with subsets of structurally dynamic and transient dendritic spines, and phagocytic engulfment of intact synapses. Based on these results, here we further discuss three means of synapse modification or elimination that could be mediated by microglia in the context of normal experience-dependent plasticity.

Clustered Dynamics of Inhibitory Synapses and Dendritic Spines in the Adult Neocortex

The hippocampal CA1 field integrates a wide variety of subcortical and cortical inputs, but its synaptic organization in humans is still unknown due to the difficulties involved studying the human brain via electron microscope techniques. However, we have shown that the 3D reconstruction method using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) can be applied to study in detail the synaptic organization of the human brain obtained from autopsies, yielding excellent results. Using this technology, 24,752 synapses were fully reconstructed in CA1, revealing that most of them were excitatory, targeting dendritic spines and displaying a macular shape, regardless of the layer examined. However, remarkable differences were observed between layers. These data constitute the first extensive description of the synaptic organization of the neuropil of the human CA1 region.

In recent decades, mounting evidence has shown that microglia play a vital role in maintaining synapses throughout life. This maintenance is done via numerous microglial processes, which are long, thin, and highly motile protrusions from the cell body that monitor their environment. However, due to the brevity of the contacts and the potentially transient nature of synaptic structures, establishing the underlying dynamics of this relationship has proven difficult. This article describes a method of using rapidly acquired multiphoton microscopy images to track microglial dynamics and microglia:synapse interactions and the fate of the synaptic structures following those interactions. First, we detail a method for capturing multiphoton images at 1-min intervals for approximately 1 hr and how that process can be done at multiple time points. We then discuss how best to prevent and account for any drifting of the region of interest that can occur during the imaging session and how to remove excessive background noise from those images. Finally, we detail the annotation process for dendritic spines and microglial processes using plugins in MATLAB and Fiji, respectively. These semi-automated plugins allow tracking of individual cell structures, even if both microglia and neurons are imaged in the same fluorescent channel. This protocol presents a method of tracking both microglial dynamics and synaptic structures, in the same animal, at multiple time points, giving the user information on process speed, branching, tip size, location, and dwell time, as well as any dendritic spine gains, losses, and size changes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Rapid multiphoton image capture Basic Protocol 2: Image preparation using MATLAB and Fiji Basic Protocol 3: Dendritic spine and microglial processes annotation using ScanImage and TrackMate.

Microglia dynamically interact with neurons influencing the development, structure, and function of neuronal networks. Recent studies suggest microglia may also influence neuronal activity by physically interacting with axonal domains responsible for action potential initiation and propagation. However, the nature of these microglial process interactions is not well understood. Microglial-axonal contacts are present early in development and persist through adulthood, implicating microglial interactions in the regulation of axonal integrity in both the developing and mature central nervous system. Moreover, changes in microglial-axonal contact have been described in disease states such as multiple sclerosis (MS) and traumatic brain injury (TBI). Depending on the disease state, there are increased associations with specific axonal segments. In MS, there is enhanced contact with the axon initial segment and node of Ranvier, while, in TBI, microglia alter interactions with axons at the site of injury, as well as at the axon initial segment. In this article, we review the interactions of microglial processes with axonal segments, analyzing their associations with various axonal domains and how these interactions may differ between MS and TBI. Furthermore, we discuss potential functional consequences and molecular mechanisms of these interactions and how these may differ among various types of microglial-axonal interactions.

To date, numerous mathematical models have been proposed on the basis of some types of Hebbian synaptic learning to account for the activity-dependent development of orientation maps as well as neuronal orientation selectivity. These models successfully reproduced orientation map-like spatial patterns. Nevertheless, we still have questions: (1) How does synaptic rewiring occur in the visual cortex during the formation of orderly orientation maps in early life? (2) How does visual experience contribute to the maturation of orientation selectivity of visual cortical neurons and reorganize orientation maps? (3) How does the sensitive period for orientation plasticity end? In this study, we performed animal experiments and mathematical modeling to understand the mechanisms underlying synaptic rewiring for experience-dependent formation and reorganization of orientation maps. At first, we visualized orientation maps from the intrinsic signal optical imaging in area 17 of kittens reared under single-orientation exposure through cylindrical-lens-fitted goggles. The experiments revealed that the degree of expansion of cortical domains representing the experienced orientation depends on the age at which the single-orientation exposure starts. As a result, we obtained the sensitive period profile for orientation plasticity. Next, we refined our previously proposed mathematical model for the activity-dependent self-organization of thalamo-cortical inputs on the assumption that rewiring is caused by the competitive interactions among transient synaptic contacts on the same dendritic spine. Although various kinds of molecules have been reported to be involved in such interactions, we attempt to build a mathematical model to describe synaptic rewiring focusing on brain-derived neurotrophic factor (BDNF) and its related molecules. Performing computer simulations based on the refined model, we successfully reproduced orientation maps reorganized in kittens reared under single-orientation exposure as well as normal visual experience. We also reproduced the experimentally obtained sensitive period profile for orientation plasticity. The excellent agreement between experimental observations and theoretical reproductions suggests that the BDNF-induced competitive interaction among synaptic contacts from different axons on the same spine is an important factor for the experience-dependent formation and reorganization of orientation selectivity and orientation maps.

Microglia, the resident immune cells of the central nervous system (CNS), were traditionally believed to be set into action only in case of injury or disease. Accordingly, microglia were assumed to be inactive or resting in the healthy brain. However, recent studies revealed that microglia carry out active tissue sampling in the intact brain by extending and retracting their ramified processes while periodically contacting synapses. Microglial morphology and motility as well as the frequency and duration of physical contacts with synaptic elements were found to be modulated by neuronal activity, sensory experience and neurotransmission; however findings have not been straightforward. Microglial cells are the most morphologically plastic element of the CNS. This unique feature confers them the possibility to locally sense activity, and to respond adequately by establishing synaptic contacts to regulate synaptic inputs by the secretion of signaling molecules. Indeed, microglial cells can hold new roles as critical players in maintaining brain homeostasis and regulating synaptic number, maturation and plasticity. For this reason, a better characterization of microglial cells and cues mediating neuron-to-microglia communication under physiological conditions may help advance our understanding of the microglial behavior and its regulation in the healthy brain. This review highlights recent findings on the instructive role of neuronal activity on microglial motility and microglia-synapse interactions, focusing on the main transmitters involved in this communication and including newly described communication at the tripartite synapse.

Sickness behavior is a set of behavioral changes induced by infections and mediated by pro-inflammatory cytokines. It is characterized by fatigue, decreased appetite and weight loss, changes in sleep patterns, cognitive functions, and lost interest in social activity. It can expedite recovery by conserving energy to mount an immune response involving innate immunity. To provide insights into microglial implication in sickness behavior with special focus on cognitive and social impairment, we investigated changes in their ultrastructure and interactions with synapses using a toxemia mouse model. Adult mice were injected with 1 mg/kg lipopolysaccharide (LPS) or saline, and assayed for signs of sickness behavior. LPS treated mice displayed reduced activity in open-field tests 24 h post-injection, while social avoidance and weight gain/loss were not significantly different between treatment groups. Microglia were investigated using electron microscopy to describe changes in their structure and function at nanoscale resolution. Microglial cell bodies and processes were investigated in the hippocampus CA1, a region responsible for learning and memory that is often impacted after peripheral LPS administration. Microglia in LPS treated animals displayed larger cell bodies as well as less complex processes at the time point examined. Strikingly, microglial processes in LPS injected animals were also more likely to contact excitatory synapses and contained more phagocytic material compared with saline injected controls. We have identified at the ultrastructural level significant changes in microglia-synapse interactions shortly after LPS administration, which draws attention to studying the roles of microglia in synaptic rewiring after inflammatory stimuli.

BackgroundMicroglia play crucial roles in maintaining neuronal homeostasis but have been implicated in contributing to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the role of microglia in ALS/FTD remains incompletely understood.MethodsHere, we generated highly enriched cultures of VCP mutant microglia derived from human induced pluripotent stem cells (hiPSCs) to investigate their cell autonomous and non-cell autonomous roles in ALS pathogenesis. We used RNA-sequencing, proteomics and functional assays to study hiPSC derived VCP mutant microglia and their effects on hiPSC derived motor neurons and astrocytes.ResultsTranscriptomic, proteomic and functional analyses revealed immune and lysosomal dysfunction in VCP mutant microglia. Stimulating healthy microglia with the inflammatory inducer lipopolysaccharide (LPS) showed partial overlap with VCP mutant microglia in their reactive transformation. LPS-stimulated VCP mutant microglia displayed differential activation of inflammatory pathways compared with LPS-stimulated healthy microglia. Conserved gene expression changes were identified between VCP mutant microglia, SOD1 mutant mice microglia, and postmortem ALS spinal cord microglial signatures, including increased expression of the transmembrane glycoprotein GPNMB. While knockdown of GPNMB affected inflammatory and phagocytosis processes in microglia, this was not sufficient to ameliorate cell autonomous phenotypes in VCP mutant microglia. Secreted factors from VCP mutant microglia were sufficient to activate the JAK-STAT pathway in hiPSC derived motor neurons and astrocytes.ConclusionsVCP mutant microglia undergo cell autonomous reactive transformation involving immune and lysosomal dysfunction that partially recapitulate key phenotypes of microglia from other ALS models and post mortem tissue. These phenotypes occur independently of GPNMB. Additionally, VCP mutant microglia elicit non cell autonomous responses in motor neurons and astrocytes involving the JAK-STAT pathway.