Progress in Structural and Functional In Vivo Imaging of Microglia and Their Application in Health and Disease.

The first line of defense for the central nervous system (CNS) against injury or disease is provided by microglia. Microglia were long believed to stay in a dormant/resting state, reacting only to injury or disease. This view changed dramatically with the development of modern imaging techniques that allowed the study of microglial behavior in the intact brain over time, to reveal the dynamic nature of their responses. Over the past two decades, in vivo imaging using multiphoton microscopy has revealed numerous new functions of microglia in the developing, adult, aged, injured, and diseased CNS. As the most dynamic cells in the brain, microglia continuously contact all structures and cell types, such as glial and vascular cells, neuronal cell bodies, axons, dendrites, and dendritic spines, and are believed to play a central role in sculpting neuronal networks throughout life. Following trauma, or in neurodegenerative or neuroinflammatory diseases, microglial responses range from protective to harmful, underscoring the need to better understand their diverse roles and states in different pathological conditions. In this chapter, we introduce multiphoton microscopy and discuss recent advances in structural and functional imaging technologies that have expanded our toolbox to study microglial states and behaviors in new ways and depths. We also discuss relevant mouse models available for in vivo imaging studies of microglia and review how such studies are constantly refining our understanding of the multifaceted role of microglia in the healthy and diseased CNS.

Calcium Signalling in Microglia.

Intracellular Ca2+ signalling represents the substrate of microglial excitability. Spatially and temporally organised changes in the free cytoplasmic Ca2+ concentration ([Ca2+]i) are generated in response to physiological and pathological stimuli. Parameters of these intracellular Ca2+ signals are defined by Ca2+ signalling toolkits that may change with age or context therefore increasing adaptive capabilities of microglia. Main Ca2+ signalling pathways in microglial cells are associated with dynamic endoplasmic reticulum Ca2+ stores and with plasmalemmal Ca2+ entry mediated by several sets of Ca2+-permeable channels including transient receptor potential (TRP) channels, ORAI channels and P2X4/7 purinoceptors. Microglial Ca2+ dynamics is also linked to TREM2 signalling cascade, contributing to neuroprotection in neurodegenerative diseases. Microglial Ca2+ signals act as reliable and precise sensors of brain dyshomeostasis and pathological insults.

Impairment of brain function in a mouse model of Alzheimer's disease during the pre-depositing phase: The role of α7 nicotinic acetylcholine receptors.

Alzheimer's disease (AD) is an age-dependent incurable neurodegenerative disorder accompanied by neuroinflammation, amyloid accumulation, and memory impairment. It begins decades before the first clinical symptoms appear, and identifying early biomarkers is key for developing disease-modifying therapies. We show now in a mouse model of AD that before any amyloid deposition the brains of 1.5-month-old mice contain increased levels of pro-inflammatory cytokines IL-1ß and IL-6, decreased levels of nicotinic acetylcholine receptors (nAChRs) in the brain and brain mitochondria and increased amounts of α7 nAChR-bound Aß1-42, along with impaired episodic memory and increased risk of apoptosis. Both acute (1-week-long) and chronic (4-month-long) treatments with α7-selective agonist PNU282987, starting at 1.5 months of age, were well tolerated. The acute treatment did not affect the levels of soluble Aß1-42 but consistently upregulated the α7 nAChR expression, decreased the level of α7-Aß1-42 complexes, and improved episodic memory of 1.5-month-old mice. The chronic treatment, covering the disease development phase, strongly upregulated the expression of all abundant brain nAChRs, reduced both free and α7-coupled Aß1-42 within the brain, had anti-inflammatory and antiapoptotic effects, and potently upregulated cognition, thus identifying α7 nAChRs as both early biomarker and potent therapeutic target for fighting this devastating disease.

Morphotype-specific calcium signaling in human microglia.

BACKGROUND: Key functions of Ca2+ signaling in rodent microglia include monitoring the brain state as well as the surrounding neuronal activity and sensing the danger or damage in their vicinity. Microglial Ca2+ dyshomeostasis is a disease hallmark in many mouse models of neurological disorders but the Ca2+ signal properties of human microglia remain unknown. METHODS: We developed a novel genetically-encoded ratiometric Ca2+ indicator, targeting microglial cells in the freshly resected human tissue, organotypically cultured tissue slices and analyzed in situ ongoing Ca2+ signaling of decades-old microglia dwelling in their native microenvironment. RESULTS: The data revealed marked compartmentalization of Ca2+ signals, with signal properties differing across the compartments and resident morphotypes. The basal Ca2+ levels were low in ramified and high in ameboid microglia. The fraction of cells with ongoing Ca2+ signaling, the fraction and the amplitude of process Ca2+ signals and the duration of somatic Ca2+ signals decreased when moving from ramified via hypertrophic to ameboid microglia. In contrast, the size of active compartments, the fraction and amplitude of somatic Ca2+ signals and the duration of process Ca2+ signals increased along this pathway.

Impact of caloric restriction on oxidative stress and key glycolytic enzymes in the cerebral cortex, liver and kidney of old and middle-aged mice.

Caloric restriction (CR) is proposed as a strategy to prevent age-related alterations like impaired glucose metabolism and intensification of oxidative stress. In this study, we examined effects of aging and CR on the activities of glycolytic enzymes and parameters of oxidative stress in the cerebral cortex, liver, and kidney of middle-aged (9 months old) and old (18 months old) C57BL6/N mice. Control middle-aged and old mice were fed ad libitum (AL groups), whereas age-matched CR groups were subjected to CR (70% of individual ad libitum food intake) for 6 and 12 months, respectively. There were no significant differences in the activities of key glycolytic and antioxidant enzymes and oxidative stress indices between the cortices of middle-aged and old AL mice. The livers and kidneys of old AL mice showed higher activity of glucose-6-phosphate dehydrogenase, an enzyme that produces NADPH in the pentose phosphate pathway, compared to those of middle-aged mice. CR regimen modulated some biochemical parameters in middle-aged but not in old mice. In particular, CR decreased oxidative stress intensity in the liver and kidney but had no effects on those parameters in the cerebral cortex. In the liver, CR led to lower activities of glycolytic enzymes, whereas its effect was the opposite in the kidney. The results suggest that during physiological aging there is no significant intensification of oxidative stress and glycolysis decline in mouse tissues during the transition from middle to old age. The CR regimen has tissue-specific effects and improves the metabolic state of middle-aged mice. This article is part of the Special Issue on "Ukrainian Neuroscience".

Live imaging of microglia during sleeping sickness reveals early and heterogeneous inflammatory responses.

Introduction: Invasion of the central nervous system (CNS) is the most serious consequence of Trypanosoma brucei infection, which causes sleeping sickness. Recent experimental data have revealed some more insights into the disease during the meningoencephalitic stage. However, detailed cellular processes befalling the CNS during the disease are poorly understood. Methods: To further address this issue, we implanted a cranial window on the cortex of B6.129P2(Cg)-Cx3cr1tm1Litt/J mice, infected them with Trypanosoma brucei expressing RFP via intraperitoneal injection, and monitored microglial cells and parasites longitudinally over 30 days using in vivo 2-photon imaging. We correlated the observed changes with histological analyses to evaluate the recruitment of peripheral immune cells. Results and discussion: We uncovered an early involvement of microglia that precedes invasion of the CNS by the parasite. We accomplished a detailed characterization of the progressive sequence of events that correlates with microglial morphological changes and microgliosis. Our findings unveiled a heterogeneous microglial response in places of initial homeostatic disruption near brain barriers and pointed out an exceptional capability of microglia to hamper parasite proliferation inside the brain. We also found early signs of inflammation in the meninges, which synchronize with the microglial response. Moreover, we observed a massive infiltration of peripheral immune cells into the parenchyma as a signature in the final disease stage. Overall, our study provides new insights into the host-pathogen immune interactions in the meningeal and parenchymal compartments of the neocortex.

Plaque vicinity as a hotspot of microglial turnover in a mouse model of Alzheimer's disease.

Microglia, the major immune cells of the brain, are functionally heterogeneous but in vivo functional properties of these cells are rarely studied at single-cell resolution. By using microRNA-9 regulated viral vectors for multicolor labeling and longitudinal in vivo monitoring of individual microglia, we followed their fate in the cortex of healthy adult mice and at the onset of amyloidosis in a mouse model of Alzheimer's disease. In wild-type mice, microglia were rather mobile (16% of the cells migrated at least once in 10-20 days) but had a low turnover as documented by low division and death rates. Half of the migratory events were tightly associated with blood vessels. Surprisingly, basic migration properties of microglia (i.e., fraction of migrating cells, saltatory migration pattern, speed of migration, translocation distance, and strong association with blood vessels) were preserved in amyloid-depositing brains, despite amyloid plaques becoming the major destination of migration. Besides, amyloid deposition significantly increased microglial division and death rates. Moreover, the plaque vicinity became a hotspot of microglial turnover, harboring 33% of all migration, 70% of death and 54% of division events.

Neuronal silence as a prosurvival factor for adult-born olfactory bulb interneurons.

Adult-born cells, arriving daily into the rodent olfactory bulb, either integrate into the neural circuitry or get eliminated. However, whether these two populations differ in their morphological or functional properties remains unclear. Using longitudinal in vivo two-photon imaging, we monitored dendritic morphogenesis, odor-evoked responsiveness, ongoing Ca2+ signaling, and survival/death of adult-born juxtaglomerular neurons (abJGNs). We found that the maturation of abJGNs is accompanied by a significant reduction in dendritic complexity, with surviving and subsequently eliminated cells showing similar degrees of dendritic remodeling. Surprisingly, ∼63% of eliminated abJGNs acquired odor responsiveness before death, with amplitudes and time courses of odor-evoked responses similar to those recorded in surviving cells. However, the subsequently eliminated cell population exhibited significantly higher ongoing Ca2+ signals, with a difference visible even 10 days before death. Quantitative supervised machine learning analysis revealed a relationship between the abJGNs' activity and survival probability, with low neuronal activity being supportive for survival.