The cellular mechanisms of neuronal swelling underlying cytotoxic edema.

Cytotoxic brain edema triggered by neuronal swelling is the chief cause of mortality following brain trauma and cerebral infarct. Using fluorescence lifetime imaging to analyze contributions of intracellular ionic changes in brain slices, we find that intense Na(+) entry triggers a secondary increase in intracellular Cl(-) that is required for neuronal swelling and death. Pharmacological and siRNA-mediated knockdown screening identified the ion exchanger SLC26A11 unexpectedly acting as a voltage-gated Cl(-) channel that is activated upon neuronal depolarization to membrane potentials lower than -20 mV. Blockade of SLC26A11 activity attenuates both neuronal swelling and cell death. Therefore cytotoxic neuronal edema occurs when sufficient Na(+) influx and depolarization is followed by Cl(-) entry via SLC26A11. The resultant NaCl accumulation causes subsequent neuronal swelling leading to neuronal death. These findings shed light on unique elements of volume control in excitable cells and lay the ground for the development of specific treatments for brain edema.

Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes.

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states-including chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

Neuron-Glia Signaling in Synapse Elimination.

Maturation of neuronal circuits requires selective elimination of synaptic connections. Although neuron-intrinsic mechanisms are important in this process, it is increasingly recognized that glial cells also play a critical role. Without proper functioning of these cells, the number, morphology, and function of synaptic contacts are profoundly altered, resulting in abnormal connectivity and behavioral abnormalities. In addition to their role in synaptic refinement, glial cells have also been implicated in pathological synapse loss and dysfunction following injury or nervous system degeneration in adults. Although mechanisms regulating glia-mediated synaptic elimination are still being uncovered, it is clear this complex process involves many cues that promote and inhibit the removal of specific synaptic connections. Gaining a greater understanding of these signals and the contribution of different cell types will not only provide insight into this critical biological event but also be instrumental in advancing knowledge of brain development and neural disease.

Microglia: Dynamic Mediators of Synapse Development and Plasticity.

Neuronal communication underlies all brain activity and the genesis of complex behavior. Emerging research has revealed an unexpected role for immune molecules in the development and plasticity of neuronal synapses. Moreover microglia, the resident immune cells of the brain, express and secrete immune-related signaling molecules that alter synaptic transmission and plasticity in the absence of inflammation. When inflammation does occur, microglia modify synaptic connections and synaptic plasticity required for learning and memory. Here we review recent findings demonstrating how the dynamic interactions between neurons and microglia shape the circuitry of the nervous system in the healthy brain and how altered neuron-microglia signaling could contribute to disease.

Cognitive flexibility and long-term depression (LTD) are impaired following ß-catenin stabilization in vivo.

The cadherin/ß-catenin adhesion complex is a key mediator of the bidirectional changes in synapse strength which are believed to underlie complex learning and memory. In the present study, we demonstrate that stabilization of ß-catenin in the hippocampus of adult mice results in significant impairments in cognitive flexibility and spatial reversal learning, including impaired extinction during the reversal phase of the Morris water maze and deficits in a delayed nonmatch to place T-maze task. In accordance with these deficits, ß-catenin stabilization was found to abolish long-term depression by stabilizing cadherin at the synaptic membrane and impairing AMPA receptor endocytosis, while leaving basal synaptic transmission and long-term potentiation unaffected. These results demonstrate that the ß-catenin/cadherin adhesion complex plays an important role in learning and memory and that aberrant increases in synaptic adhesion can have deleterious effects on cognitive function.

Ca2+ transients in astrocyte fine processes occur via Ca2+ influx in the adult mouse hippocampus.

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca2+ -sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two-photon imaging of Ca2+ transients in adult CA1 hippocampal astrocytes using a new single-cell patch-loading strategy to image Ca2+ -sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca2+ transients whereas astrocyte soma rarely showed spontaneous Ca2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L- or T-type voltage-gated calcium channels. Removal of extracellular Ca2+ almost completely and reversibly abolished the spontaneous signals while IP3 R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca2+ . The Ca2+ influx dependency of the spontaneous signals in patch-loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093-2103.

Activation of neuronal NMDA receptors triggers transient ATP-mediated microglial process outgrowth.

Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron-microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron-microglial communication mediated by ATP.

Cytokine-producing microglia have an altered beta-amyloid load in aged APP/PS1 Tg mice.

Beta-amyloid (Aß) plaques and chronic neuroinflammation are significant neuropathological features of Alzheimer's disease. Microglial cells in aged brains have potential to produce cytokines such as TNF and IL-1 family members (IL-1α, IL-1ß, and IL-1Ra) and to phagocytose Aß in Alzheimer's disease, however the inter-relationship between these processes is poorly understood. Here we show that % Aß plaque load followed a sigmoidal trajectory with age in the neocortex of APPswe/PS1ΔE9 Tg mice, and correlated positively with soluble Aß40 and Aß42. Aß measures were moderately correlated with mRNA levels of CD11b, TNF, and IL-1Ra. Cytokine production and Aß load were assessed in neocortical CD11b(+)(CD45(+)) microglia by flow cytometry. Whereas most microglia in aged mice produced IL-1Ra, relatively low proportions of microglia produced TNF, IL-1α, and IL-1ß. However, microglial production of these latter cytokines was generally increased in APP/PS1 Tg mice. Microglia that phagocytosed endogenously-produced Aß were only observed in APP/PS1 Tg mice. Differences in phagocytic index and total Aß load were observed in microglia with specific cytokine profiles. Both phagocytic index and total Aß load were higher in IL-1α(+) and IL-1Ra(+) microglia, than microglia that did not produce these cytokines. In contrast, total Aß load was lower in IL-1ß(+) and TNF(+) microglia, compared to IL-1ß(-) and TNF(-) microglia, and TNF(+) microglia also had a lower phagocytic index. Using GFP bone marrow chimeric mice, we confirmed that the majority of neocortical CD11b(+)(CD45(+)) microglia were resident cells (GFP(-)) in APP/PS1 Tg mice, even after selectively analysing CD11b(+)CD45(high) cells, which are typically considered to be infiltrating cells. Together, our data demonstrate that cytokine expression is selectively correlated with age and Aß pathology, and is associated with an altered Aß load in phagocytic microglia from APP/PS1 Tg mice. These findings have implications for understanding the regulation of microglial cytokine production and phagocytosis of Aß in Alzheimer's disease.

FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins.

Although engulfment is a hallmark of microglia function, fully validated platforms that facilitate high-throughput quantification of this process are lacking. Here, we present FEAST (Flow cytometric Engulfment Assay for Specific Target proteins), which enables interrogation of in vivo engulfment of synaptic material by brain resident macrophages at single-cell resolution. We optimize FEAST for two different analyses: quantification of fluorescent material inside live cells and of engulfed endogenous proteins within fixed cells. To overcome false-positive engulfment signals, we introduce an approach suitable for interrogating engulfment in microglia from perfusion-fixed tissue. As a proof-of-concept for the specificity and versatility of FEAST, we examine the engulfment of synaptic proteins after optic nerve crush and of myelin in two mouse models of demyelination (treatment with cuprizone and injections of lysolecithin). We find that microglia, but not brain-border associated macrophages, engulf in these contexts. Our work underscores how FEAST can be utilized to gain critical insight into functional neuro-immune interactions that shape development, homeostasis, and disease.

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

Microglial depletion disrupts normal functional development of adult-born neurons in the olfactory bulb.

Microglia play key roles in regulating synapse development and refinement in the developing brain, but it is unknown whether they are similarly involved during adult neurogenesis. By transiently depleting microglia from the healthy adult mouse brain, we show that microglia are necessary for the normal functional development of adult-born granule cells (abGCs) in the olfactory bulb. Microglial depletion reduces the odor responses of developing, but not preexisting GCs in vivo in both awake and anesthetized mice. Microglia preferentially target their motile processes to interact with mushroom spines on abGCs, and when microglia are absent, abGCs develop smaller spines and receive weaker excitatory synaptic inputs. These results suggest that microglia promote the development of excitatory synapses onto developing abGCs, which may impact the function of these cells in the olfactory circuit.

The brain has its own population of resident immune cells known as microglia, which defend against infections and are involved in conditions such as Alzheimer's, Parkinson's and other diseases. In the last decade, new studies have suggested that these cells also sculpt brain circuits during early development. They can 'eat' weak connections between neurons, and help strong ones to mature. Most of brain 'wiring' happens during development, when the majority of neurons is born and connects together. However, a few brain areas can incorporate new neurons during adulthood into existing circuits. In mice for example, this process takes place in the olfactory bulb, the area that first processes smells: it is believed that new neurons connecting to existing ones helps to detect new odors. It is unclear, however, whether microglia also help to shape these connections, or if their role is confined to early development. To investigate this question, Wallace et al. gave adult mice a drug that kills only microglia, and then examined how the neurons respond when the animals are exposed to smells. The results show that the new neurons that developed without microglia responded to fewer odors. These neurons also formed weaker connections and had physical features that indicated they might not have been properly incorporated into the circuit. It may be possible to encourage new neurons to be born in brain areas that normally do not produce these cells in adulthood. Ultimately, this could potentially help to repair the damages of age or disease, but this will rely on understanding exactly how new neurons are integrated into existing brain circuits. Future work, however, is still necessary to figure out how much these new neurons could compensate for cells damaged by injury or disease.

Nanoscale Surveillance of the Brain by Microglia via cAMP-Regulated Filopodia.

Microglia, the brain's immune cells, maintain homeostasis and sense pathological changes by continuously surveying the parenchyma with highly motile large processes. Here, we demonstrate that microglia also use thin actin-dependent filopodia that allow fast nanoscale sensing within discrete regions. Filopodia are distinct from large processes by their size, speed, and regulation mechanism. Increasing cyclic AMP (cAMP) by activating norepinephrine Gs-coupled receptors, applying nitric oxide, or inhibiting phosphodiesterases rapidly increases filopodia but collapses large processes. Alternatively, Gi-coupled P2Y12 receptor activation collapses filopodia but triggers large processes extension with bulbous tips. Similar control of cytoskeletal dynamics and microglial morphology by cAMP is observed in ramified primary microglia, suggesting that filopodia are intrinsically generated sensing structures. Therefore, nanoscale surveillance of brain parenchyma by microglia requires localized cAMP increases that drive filopodia formation. Shifting intracellular cAMP levels controls the polarity of microglial responses to changes in brain homeostasis and alters the scale of immunosurveillance.

Microglia Express Insulin-Like Growth Factor-1 in the Hippocampus of Aged APPswe/PS1ΔE9 Transgenic Mice.

Insulin-like growth factor-1 (IGF-1) is a pleiotropic molecule with neurotrophic and immunomodulatory functions. Knowing the capacity of chronically activated microglia to produce IGF-1 may therefore show essential to promote beneficial microglial functions in Alzheimer's disease (AD). Here, we investigated the expression of IGF-1 mRNA and IGF-1 along with the expression of tumor necrosis factor (TNF) mRNA, and the amyloid-ß (Aß) plaque load in the hippocampus of 3- to 24-month-old APPswe/PS1ΔE9 transgenic (Tg) and wild-type (WT) mice. As IGF-1, in particular, is implicated in neurogenesis we also monitored the proliferation of cells in the subgranular zone (sgz) of the dentate gyrus. We found that the Aß plaque load reached its maximum in aged 21- and 24-month-old APPswe/PS1ΔE9 Tg mice, and that microglial reactivity and hippocampal IGF-1 and TNF mRNA levels were significantly elevated in aged APPswe/PS1ΔE9 Tg mice. The sgz cell proliferation decreased with age, regardless of genotype and increased IGF-1/TNF mRNA levels. Interestingly, IGF-1 mRNA was expressed in subsets of sgz cells, likely neuroblasts, and neurons in both genotypes, regardless of age, as well as in glial-like cells. By double in situ hybridization these were shown to be IGF1 mRNA+ CD11b mRNA+ cells, i.e., IGF-1 mRNA-expressing microglia. Quantification showed a 2-fold increase in the number of microglia and IGF-1 mRNA-expressing microglia in the molecular layer of the dentate gyrus in aged APPswe/PS1ΔE9 Tg mice. Double-immunofluorescence showed that IGF-1 was expressed in a subset of Aß plaque-associated CD11b+ microglia and in several subsets of neurons. Exposure of primary murine microglia and BV2 cells to Aß42 did not affect IGF-1 mRNA expression. IGF-1 mRNA levels remained constant in WT mice with aging, unlike TNF mRNA levels which increased with aging. In conclusion, our results suggest that the increased IGF-1 mRNA levels can be ascribed to a larger number of IGF-1 mRNA-expressing microglia in the aged APPswe/PS1ΔE9 Tg mice. The finding that subsets of microglia retain the capacity to express IGF-1 mRNA and IGF-1 in the aged APPswe/PS1ΔE9 Tg mice is encouraging, considering the beneficial therapeutic potential of modulating microglial production of IGF-1 in AD.

The function of the human interferon-beta 1a glycan determined in vivo.

Recombinant human interferon-beta (rhIFN-beta) is the leading therapeutic intervention shown to change the cause of relapsing-remitting multiple sclerosis, and both a nonglycosylated and a significantly more active glycosylated variant of rhIFN-beta are used in treatment. This study investigates the function of the rhIFN-beta1a glycan moiety and its individual carbohydrate residues, using the myxovirus resistance (Mx) mRNA as a biomarker in Mx-congenic mice. We showed that the Mx mRNA level in blood leukocytes peaked 3 h after s.c. administration of rhIFN-beta1a. In addition, a clear dose-response relationship was confirmed, and the Mx response was shown to be receptor-mediated. Using specific glycosidases, different glycosylation analogs of rhIFN-beta1a were obtained, and their activities were determined. The glycosylated rhIFN-beta1a showed significantly higher activity than its deglycosylated counterpart, due to a protein stabilization/solubilization effect of the glycan. It is interesting to note that the terminating sialic acids were essential for these effects. Conclusively, the structure/bioactivity relationship of rhIFN-beta1a was determined in vivo, and it provided a novel insight into the role of the rhIFN-beta1a glycan and its carbohydrate residues. The possibilities of improving the pharmacological properties of rhIFN-beta1a using glycoengineering are discussed.

Toll-like receptor 2 signaling in response to brain injury: an innate bridge to neuroinflammation.

Reactive gliosis is a prominent feature of neurodegenerative and neuroinflammatory disease in the CNS, yet the stimuli that drive this response are not known. There is growing appreciation that signaling through Toll-like receptors (TLRs), which is key to generating innate responses to infection, may have pathogen-independent roles. We show that TLR2 was selectively upregulated by microglia in the denervated zones of the hippocampus in response to stereotactic transection of axons in the entorhinal cortex. In mice lacking TLR2, there were transient, selective reductions in lesion-induced expression of cytokines and chemokines. Recruitment of T cells, but not macrophages, was delayed in TLR2-deficient mice, as well as in mice lacking TNFR1 (tumor necrosis factor receptor 1). TLR2 deficiency also affected microglial proliferative expansion, whereas all of these events were unaffected in TLR4-mutant mice. Consistent with the fact that responses in knock-out mice had all returned to wild-type levels by 8 d, there was no evidence for effects on neuronal plasticity at 20 d. These results identify a role for TLR2 signaling in the early glial response to brain injury, acting as an innate bridge to neuroinflammation.

New insights on the role of microglia in synaptic pruning in health and disease.

Recent genome-wide association studies implicate microglia in Alzheimer's disease (AD) pathogenesis; however, their biological significance remains poorly understood. Synapse loss is a significant correlate of cognitive decline that serves as a critical hallmark of AD and other neurodegenerative diseases; however, mechanisms underlying synaptic vulnerability remain elusive. Emerging research on microglia function in the healthy brain is providing new insight into fundamental roles of microglia and immune molecules in brain wiring. Among their many roles, microglia prune developing synapses and regulate synaptic plasticity and function. Here, we review and discuss how this emerging work may provide new insight into how disruptions in microglia-synapse interactions could contribute to synapse loss and dysfunction, and consequently cognitive impairment, in AD.

Fixation and Immunolabeling of Brain Slices: SNAPSHOT Method.

Acute brain slices are widely used in neuroscience because this preparation enables pharmacological interventions in a timely manner, similar to what is currently done in cultured cell studies, while preserving the natural cytoarchitecture. However, compared with cells in culture and thin cryostat sections, acute brain slices are not commonly used for immunolabeling because of poor fixation and antibody penetration. Thus, we have established a novel protocol to overcome these issues. We named this protocol SNAPSHOT (StaiNing of dynAmic ProcesseS in HOt-fixed Tissue) because it describes a simple approach for preserving the morphology of fine dynamic cellular processes at the exact time of fixation and for improving the penetration of antibodies. We have previously shown that SNAPSHOT preserves the ultrastructure of the tissue and allows for a uniform immunolabeling throughout a 300 µm thick slice. SNAPSHOT has recently proven to be beneficial in addressing several unique biological questions.

Microglial CR3 activation triggers long-term synaptic depression in the hippocampus via NADPH oxidase.

Complement receptor 3 (CR3) activation in microglia is involved in neuroinflammation-related brain disorders and pruning of neuronal synapses. Hypoxia, often observed together with neuroinflammation in brain trauma, stroke, and neurodegenerative diseases, is thought to exacerbate inflammatory responses and synergistically enhance brain damage. Here we show that when hypoxia and an inflammatory stimulus (lipopolysaccharide [LPS]) are combined, they act synergistically to trigger long-term synaptic depression (LTD) that requires microglial CR3, activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), and GluA2-mediated A-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization. Microglial CR3-triggered LTD is independent of N-methyl-D-aspartate receptors (NMDARs), metabotropic glutamate receptors (mGluRs), or patterned synaptic activity. This type of LTD may contribute to memory impairments and synaptic disruptions in neuroinflammation-related brain disorders.

Population control of resident and immigrant microglia by mitosis and apoptosis.

Microglial population expansion occurs in response to neural damage via processes that involve mitosis and immigration of bone marrow-derived cells. However, little is known of the mechanisms that regulate clearance of reactive microglia, when microgliosis diminishes days to weeks later. We have investigated the mechanisms of microglial population control in a well-defined model of reactive microgliosis in the mouse dentate gyrus after perforant pathway axonal lesion. Unbiased stereological methods and flow cytometry demonstrate significant lesion-induced increases in microglial numbers. Reactive microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations.