Monitoring in vivo function of cortical microglia

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Review

Monitoring in vivo function of cortical microglia Bianca Brawek, Olga Garaschuk ∗ Institute of Physiology, Department of Neurophysiology, Eberhard Karls University Tübingen, Keplerstraße 15, 72074 Tübingen, Germany

a r t i c l e

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Article history: Received 8 February 2017 Accepted 8 February 2017 Available online xxx Keywords: In vivo Ca2+ imaging Intact microglia Microglial dynamics Two-photon microscopy

a b s t r a c t Microglia, the innate immune cells of the brain, are becoming increasingly recognized as an important player both in the context of physiological brain function and brain pathology. To fulfill their executive functions microglia can modify their morphology, migrate or move their processes in a directed fashion, and modify the intracellular Ca2+ dynamics leading to modifications in gene expression, phagocytosis, release of cytokines and other inflammation markers, etc. Here we describe the recently developed tools enabling in vivo monitoring of morphology and Ca2+ signaling of microglia and show how these techniques may be used for examining microglial function in healthy and diseased brain. © 2017 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 In vivo imaging of microglial morphology and dynamics in transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Other labeling approaches for in vivo imaging of microglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Imaging of microglial Ca2+ signals in the intact brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Imaging in vivo microglial Ca2+ signals in the aging and diseased brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Microglia are the resident macrophages of the central nervous system (CNS). They constitute about 5–12% of all cells in the mouse brain, but their density varies between different brain regions being highest in the gray matter [1,2]. Under physiological conditions they have small cell somata and long, elaborate, and highly motile processes which enable them to constantly monitor their microenvironment without the need of cell body translocation [3,4]. Being equipped with a plentitude of receptors and surface molecules they closely monitor their surroundings and react to any alteration of tissue homeostasis. Therefore, the functional state of microglial cells strongly depends on the state of their local environment. Injury of the surrounding tissue detected, for example, by the presence

∗ Corresponding author. E-mail addresses: [email protected] (B. Brawek), [email protected] (O. Garaschuk).

of so-called damage- or pathogen-associated molecular patters (DAMPs or PAMPs), changes microglial morphology and function. The cells move to the site of injury, insulate it from the intact brain, engulf and eliminate neuronal debris [5]. This phenotype is commonly referred to as the ‘activated’ state of microglia. In analogy to T lymphocytes, microglial activation states have previously been subdivided into M1 and M2 phenotypes, with the M1 phenotype, characterized by release of pro-inflammatory cytokines, regarded as neurotoxic and the M2 phenotype, characterized by release of anti-inflammatory cytokines, regarded as neuroprotective. Nowadays, however, it is recognized that the isolation of the two activation states only partially describes the activation profile of microglia. In reality, activated microglia traverse a continuum of different activation states often combining classical features of M1 and M2 phenotypes [5]. Due to their ability to detect perturbations in tissue homeostasis, microglia are involved in virtually all pathological processes in the CNS, from infection and neurodegenerative diseases to mental disorders. Interestingly, the properties

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of microglia depend not only on the local microenvironment in the CNS but also on the state of the entire body. In this context, it has been demonstrated that the maintenance and maturation of brain microglia is dependent on the gut microbiome [6]. Furthermore, microglia are able to communicate with peripheral immune cells, which is especially important during peripheral inflammation, when the peripheral and central systems have to cooperate in order to fight the disease [7]. In addition to their innate immune functions, microglia were recently shown to contribute to the correct wiring and the maintenance of neural networks during brain development and adulthood. In the developing brain, they are actively involved in synaptic remodeling influencing both synapse formation [8] and elimination [9,10]. In addition, microglia have been shown to actively promote programmed neuronal cell death during postnatal development [11], phagocyte apoptotic but also viable neurons in the neurogenic niche [12,13] and promote survival of cortical layer 5 neurons by a CX3 CR1-dependent release of insulin-like growth factor [14]. Furthermore, activated microglia support neurogenesis and oligodendrogenesis in the perinatal subventricular zone by releasing pro-inflammatory cytokines [15]. In the mature brain, microglia are similarly important for the maintenance and refinement of synaptic connections. They are able to monitor the activity state of the neural network and react to its alterations [16–18] and regularly contact neuronal elements with their processes in an activity-dependent manner [16,19–21]. As non-excitable cells, microglia depend on changes in the intracellular Ca2+ concentration ([Ca2+ ]i ) for communication with each other and with other cell types like neurons and astrocytes. [Ca2+ ]i of microglia is tightly regulated both in vitro and in vivo [22–24] and sustained as well as transient elevations of [Ca2+ ]i have been implicated in microglia’s executive functions like release of cytokines and nitric oxide [25–30] and phagocytosis [30]. However, so far microglial Ca2+ signaling has mainly been studied in cultured cells. Although cell culture methods offer an easy and rapid access to cells under study, cultured microglial cells differ dramatically from their counterparts in the intact tissue. As the functional state of microglia is regulated by their micromilieu, they inevitably react to the isolation process. In the healthy brain, microglial cells are kept in a downregulated state due to the presence of so-called ‘OFF’ signals (e.g. TREM-2/DAP12, CD200R/CD200, CX3 CR1/fractalkine, transforming growth factor-␤, etc.) [31]. Some of them represent receptor-ligand pairs enabling intimate interactions between microglia and surrounding neurons, whereas the others are microglia-located receptors for molecules released during physiological neuronal activity. Naturally, the vast majority of these cues are lost when isolating microglia. Consistently, the microglia-specific gene expression pattern that can be found in freshly isolated cells is lost after 7 days in culture [32]. Additionally, serum and other factors present in the medium used for cell culturing but never seen by microglia in vivo, further modify the functional state of microglia. Finally, for primary cell cultures microglia are often isolated from newborn animals, in which these cells are still in an immature, amoeboid state characterized by a different gene expression profile compared to mature microglia [33]. Therefore, the resilient knowledge about functional properties of these cells and their role in mediating immune and homeostatic responses of the brain can only be obtained under in vivo conditions. However, technical challenges have so far hampered the analysis of microglial function and especially its Ca2+ signaling in vivo. First, the multi cell bolus loading technique using small molecule indicator dyes like Oregon Green BAPTA-1 (OGB-1; [24]), which over the last decade was routinely used to study other cell types under in vivo conditions [34–36], failed to label microglia [24]. Second, many genetic targeting strategies cannot differentiate between microglial cells and monocyte-derived macrophages, a

fact that is less important in the healthy brain tissue, but becomes critical in the diseased brain, when peripheral cells might enter the CNS. Here we are going to discuss the modern tools enabling monitoring of morphology and Ca2+ signaling of microglia in vivo and to show how these techniques may be used for examining microglial function in health and disease.

2. In vivo imaging of microglial morphology and dynamics in transgenic mice The development of several mouse lines in which microglia are specifically labeled with green fluorescent protein (GFP) for the first time enabled observation of these cells in the intact in vivo brain by means of two-photon microscopy. In the most widely used mouse line the receptor for the chemokine fraktalkine (CX3 CR1) is replaced by a gene encoding GFP under the CX3 CR1 promoter [37]. In these mice, microglia, the only cell type expressing CX3 CR1 in the healthy brain, are brightly labeled enabling in vivo visualization of the complete microglial cell morphology up to the tips of the fine processes. The first seminal studies using these mice changed our view on microglial cells, which were historically regarded as being rather static. These studies, in contrast, revealed that microglia in the living brain are highly dynamic cells constantly scanning/surveying their environment and reacting to danger signals like ATP with a rapid extension of their highly branched processes [3,4]. From this time point, heterozygous CX3 CR1GFP/+ mice were extensively used to study microglia morphology and dynamics in the healthy brain [2,20,24,38], as well as in the context of disease. The latter studies comprise analyses of mouse models of Alzheimer’s disease (AD) [39–42], malignant tumors [43,44], ischemia/stroke [45,46], encephalopathy [47], systemic inflammation [48,49], or traumatic brain injury [50]. In addition to microglial motility, CX3 CR1GFP/+ mice enabled live monitoring of microglial fate under in vivo conditions [2]. When imaged every day by means of two-photon microscopy (Fig. 1), population of microglial cells turned out to be much less stable than assumed previously based at [3 H]thymidine incorporation studies [51]. Both cell division and cell elimination events were easily observed. The proliferation rate of resident microglia amounted to 0.79% per day (Fig. 1A, B). After a cell division event, two daughter cells were moving apart from each other reaching the mean distance between the resident microglia in 3–4 days (Fig. 1C, D). The cell death of resident microglia (monitored as a selective disappearance of a single cell with all surrounding cells remaining in place, see Fig. 5A in [2] was found to be 1.23% per day. Interestingly, the death of a single microglia triggered an increase in the proliferation activity in its vicinity during the next 5 days. This locally increased proliferation rate was most probably counterbalanced by apoptosis, as the death rate of newborn microglia was highest during the first 5 days after division (5.0% ± 3.5% per day, significantly higher than the death rate in the resident adult cell population, see above). These in vivo data, together with complementary immunohistochemical analyses of mouse and human brain samples [2] suggest that the microglial turnover in the mammalian brain is approximately an order of magnitude higher than assumed previously. Therefore, instead of being life-long living cell microglia is likely to turn over several times during a lifetime of an organism and the microglial self-renewal is probably maintained by coupling between the proliferation and apoptosis. Appreciating an unprecedented insight into microglial physiology enabled by the use of CX3 CR1GFP/+ mice, it is important, however, to keep in mind that in these mice the CX3 CR1 gene is replaced by GFP, leading to a functional knockout of this receptor. On the one hand, this can be advantageous, because homozygous

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Fig. 1. In vivo imaging of the fate of microglia. (A) Maximum intensity projection (MIP) images of the same field of view in a CX3 CR1GFP/+ mouse taken at different time points. Arrows point to a proliferating microglial cell and to its progeny. (B) Box plot illustrating the proliferation rate of microglia 0.79 ± 0.81% per day (median ± interquartile range, IQR; n = 669 cells, 9 fields of view (FOV), 4 mice). One data point represents a mean proliferation rate in one field of view. (C) A bar graph illustrating the mean distance between the centers of two neighboring cells for resident cells and for newborn cells during the first 24 h of their life. The distance between the resident microglia is 42.40 ± 1.73 ␮m (mean ± SEM; n = 587 cells, 10 FOVs, 4 mice) and between the newly generated cells 15.31 ± 1.20 ␮m (mean ± SEM; n = 62 cells, 9 FOVs, 4 mice; *p < 0.001; Student’s t-Test). (D) A graph illustrating the distance between the twin microglial cells as a function of their age. Note that the distance between the twin cells increases over time. On the fifth day after birth, the average distance is 37.02 ± 17.71 ␮m (median ± IQR; n = 31 pair of twin cells, 8 FOVs, 4 mice), and is not significantly different from the distance between the resident cells (p = 0.06, Student’s t-Test). Modified from [2].

mice can be used as a knockout model for analyses of the receptor function. On the other hand, the partial CX3 CR1 knockout in CX3 CR1GFP/+ mice, which are commonly used for in vivo studies of microglia, might also lead to functional alterations. As already mentioned above, in the CNS CX3 CR1 is exclusively expressed in microglia whereas its ligand CX3 CL1/fractalkine is expressed by specific neurons. The CX3 CR1/CX3 CL1 interaction between these cell types plays a critical role for regulating the activation state and recruitment of microglia and is essential for the maturation and maintenance of neuronal circuits (reviewed in [52,53]). Microglia in CX3 CR1GFP/+ mice produce a reduced level of CX3 CR1 and under some circumstances may differ from their wild type counterparts both under physiological [54] and pathological conditions [55]. Therefore, results obtained using heterozygous CX3 CR1 mice should be double checked using other experimental techniques. In the second mouse line that is routinely used for the in vivo study of microglia, GFP is expressed under control of the ionized Ca2+ -binding protein-1 (Iba-1) promoter [56]. This mouse line was used for deciphering the role of microglia for shaping developing neural networks [8], for studying communication between neurons and microglia in the mature brain [16], for understanding microglial function during normal aging [57], and in mouse models of AD [58–60]. In contrast to CX3 CR1 mice, Iba-1-GFP mice do not carry the risk of potential deficits due to knockout of the endogenous protein. However, microglial GFP expression in Iba-1-GFP mice is weaker than in CX3 CR1GFP/+ mice and the intensity of labeling is more heterogeneous with dim and bright cells located within the same field of view. Additionally, in individual cells the Iba-1 expression is not stable over time, as microglial activation leads to an upregulation of Iba-1 protein [61]. In consequence, also the strength of GFP expression depends on the activation state of microglia. Unfortunately, in both Iba-1-GFP and CX3 CR1GFP/+ reporter mouse strains the peripheral myeloid cell populations are also labeled. This fact is especially critical under pathological conditions (e.g. ischemia/stroke or traumatic brain injury), when infiltration of peripheral immune cells into the brain might play

a role. Development of the CX3 CR1CreER mouse strain expressing tamoxifen-inducible Cre-recombinase in CX3 CR1 positive cells in combination with reporter lines for fluorescent proteins seem to overcome this problem [21,62,63]. Although initially all CX3 CR1 positive cells including peripheral myeloid cells are targeted by tamoxifen treatment, some 6 weeks after the end of the treatment all short-lived blood cells (e.g. monocytes) are gone, leaving microglia as the only labeled population [63]. In addition to labeling microglia with fluorescent markers, this approach enables microglia-specific Cre-dependent manipulations of gene expression and will therefore facilitate functional differentiation between microglia and peripheral myeloid cells in future studies. Other transgenic mouse lines in which microglia express GFP or cyan fluorescent protein (CFP) under control of the receptor for colony stimulating factor-1 (MacGreen mice [64] or MacBlue mice [65]) or the CD68 promoter (CD68-GFP [66]), have so far not been used for in vivo imaging of brain microglia.

3. Other labeling approaches for in vivo imaging of microglia To avoid time- and resource-consuming breeding of transgenic animals our group developed a technique enabling in vivo visualization of microglia by using commercially available plant lectins conjugated to fluorophores [67]. The major advantage of this approach is its simple and fast applicability to any mouse strain at any developmental stage. In our studies, we used Tomato lectin conjugated to DyLight 594 or fluorescein (obtained from Vector Laboratories, Burlingame, CA, USA) as well as Isolectin IB4 conjugated to Alexa 594 (obtained from Invitrogen, Eugene, OR, USA). For in vivo labeling of cortical microglia a glass micropipette (Ø = 1 ␮m) filled with a solution containing Tomato lectin (25 ␮g/ml) or Isolectin IB4 (75 ␮g/ml) was inserted into the brain parenchyma via a craniotomy using a micromanipulator and the dye was injected into the cortical tissue by pressure-application (60 kPa for 30–90 s). This protocol resulted in a labeled tissue volume with a diam-

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Fig. 2. Tomato Lectin as a versatile marker for in vivo labeling of microglia. (A) MIP images of a microglial cell labeled in vivo with Tomato lectin conjugated to DyLight 594 (red; left) and imaged after tissue fixation using formaldehyde and post hoc immunostaining with an anti-Iba1 antibody (green; middle). Merged image is shown on the right. Note that Tomato lectin labels the entire microglial cell including the fine processes (white arrows). (B) In vivo MIP image of Tomato lectin-labeled microglial cells (red) in the vicinity of amyloid depositions in a mouse model of AD (APP23/PS45/CX3 CR1GFP/+ , 10 months). Amyloid depositions are visualized using Thioflavin-S (blue). Note that only blue and red channels are merged. (C) In vivo MIP image of Tomato lectin-labeled cortical microglial cells (red) in an aged CX3 CR1GFP/+ mouse (23 months). Fluorescence of GFP is shown in green. Note that here and in (D) recordings were performed at an excitation wavelength of 800 nm; therefore GFP is not optimally excited and restricted to microglial cell bodies. (D–F) Microglial process extension in response to ATP. (D) In vivo MIP image showing microglia labeled with Tomato lectin conjugated to DyLight 594 (red) and a glass micropipette containing ATP (5 mM) and Alexa 488 in a CX3 CR1GFP/+ mouse. GFP and Alexa 488 are shown in green. The image shows the time point of final process convergence at the tip of the pipette. Ø symbol and white broken lines label the diameter of the spherical containment formed by microglial processes. (E) Changes in the diameter of the spherical containment in the presence of a point source of ATP formed by microglial processes labeled with GFP (black) and Tomato lectin (red) over time. Traces are aligned to the time point of final convergence (black arrow). (F) Box-and-whisker plot showing the average extension rate of processes during convergence of GFP and Tomato lectin-labeled microglia, respectively. Modified from [67].

eter of approximately 100–150 ␮m. If necessary, the size of the labeled volume could be increased by multiple injections of the dye. Within the labeled volume both endothelial cells surrounding blood vessels and microglia were labeled but blood vessels could be easily distinguished from microglia based on morphology (Fig. 2). In our hands, the imaging quality of microglia labeled with Tomato lectin conjugated to DyLight 594 was comparable to imaging quality of GFP-expressing microglia in transgenic mice. Conveniently, the labeling was preserved after formaldehyde fixation and isolation of the brain allowing further post hoc characterization of the microglial cells previously studied in vivo (Fig. 2A). Noteworthy, the labeling quality depended on the use of a given type of plant lectin. Whereas both Isolectin IB4 and Tomato lectin could in principle be used to label microglia in wild type mice, the fine microglial processes were better visualized using Tomato lectin, whereas Isolectin IB4 staining appeared less continuous. Importantly, Isolectin IB4 resulted in a massive background labeling in mouse models of AD precluding the identification of microglial cells in these preparations [67]. This was not the case for Tomato lectin (Fig. 2B), which we therefore recommend for in vivo visualization of microglia in AD mice. Moreover, Tomato lectin provided high quality labeling of cortical microglia in aged mice (Fig. 2C). Tomato lectin labeling also enables in vivo monitoring of microglial process motility. The processes can easily be identified in Tomato lectinlabeled cells and their movement (here in response to an insertion of an ATP-containing pipette) can be followed over prolonged periods of time (Fig. 2F, G). The speed of the process extension in the

direction of the ATP pipette was comparable between GFP-labeled microglia in transgenic mice and Tomato lectin labeled microglia in wild type mice, suggesting that both techniques are applicable for measuring process motility in vivo. Importantly, the lectin-based labeling technique can easily be applied to transgenic mice expressing fluorescent proteins in specific types of cells (e.g. neurons or astrocytes), thereby enabling the study of interactions between those cells and microglia. An obvious disadvantage of Tomato lectin labeling compared to expression of GFP in reporter lines is that it requires an acute experiment and cannot be used for longitudinal imaging studies. To overcome this problem, a further alternative is the delivery of viral vectors inducing a stable expression of fluorescent proteins in microglia. In contrast to acute labeling using plant lectins, this method allows long-term imaging of brain microglia under the implanted cranial window. An elegant way to specifically label microglia by a viral approach is the use of microRNA-9-regulated vectors [68]. MicroRNAs are small non-coding RNAs that play an important role in post-transcriptional control of gene expression. Incorporation of complementary microRNA target sites into the transgene cassette leads to degradation of the transgene messenger RNA specifically in those cells expressing the respective microRNA. In the intact in vivo brain microglia are the only cell type lacking activity of microRNA-9. Therefore, the transgene is degraded in all cell types except microglia. Åkerblom et al. used this approach to induce microRNA-9-regulated expression of GFP in the brain parenchyma of rats and mice. The approach yielded a bright and

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specific GFP-expression in microglial cells, suggesting that it is well suited for in vivo studies. Importantly, because the viral vector is directly released into the brain parenchyma labeling of peripheral myeloid cells is excluded. When using this approach to visualize microglia in the intact brain, it is important to keep in mind that the strength of the GFP expression may vary depending on the actual strength of microRNA-9 activity. For example, the intraperitoneal injections of LPS were found to upregulate microglial microRNA-9 expression [69], raising some concerns about the applicability of this approach in disease models. However, in their original publication Åkerblom et al. showed that microglia activated by an acute brain lesion can be labeled using a microRNA-9 regulated vector. Future studies have to prove whether this is also true for chronically activated microglial cells, like the ones present in the brain in conditions of neurodegenerative diseases.

4. Imaging of microglial Ca2+ signals in the intact brain The first data about in vivo Ca2+ signaling in microglia came from single-cell electroporation experiments [24]. In analogy to the approach established previously for neurons [70–72], GFP- or lectin-labeled microglial cells were approached with a thin glass micropipette (Ø < 1 ␮m) filled with the hexapotassium salt of the small molecule Ca2+ indicator OGB-1 (10 mM inside the pipette). As soon as the pipette touched the membrane, a single short current pulse (-600 nA for 10 ms) was applied to transport the dye into the cell of interest (Fig. 3A). Electroporated cells preserved their ramified morphology hours after electroporation, did not evoke any phagocytic or chemotactic responses in the surrounding intact GFP-labeled microglia, and were themselves able to react to signals accompanying tissue damage with a directed process extension [24]. This suggested that electroporated cells remained viable and enabled us to study their in vivo Ca2+ signaling. In vivo microglia vividly responded to low concentrations (1–5 ␮M in the pressureapplication pipette) of purinergic receptor agonists ATP or UDP (Fig. 3B). Taking into account the dilution of substance that occurs during its diffusion in the extracellular space, the effective concentration reaching the microglial cell in this case is estimated to be in the nanomolar range. Such high sensitivity is unique to microglia in vivo and has never been observed in cultured microglial cells. The latter do not respond to ATP applied at a concentration lower than 10 ␮M [73]. Also in contrast to cultured cells known to respond with Ca2+ transients to activation of the variety of different receptors (reviewed in [74]), in vivo microglia responded with Ca2+ transients almost exclusively to ATP and its analogs (Fig. 3C). Further experiments have shown that in the intact in vivo brain microglia show little spontaneous Ca2+ signaling, but very reliably respond with large Ca2+ transients to a damage of an individual cell in their microenvironment. Taken together, these data allowed us to conclude, that in vivo transient elevations of [Ca2+ ]i in microglia reflect pathological rather than physiological events in their microenvironment. Although in vivo electroporation preceded by lectin-mediated visualization of intact cells (see above) enables analyses of individual microglia in different mouse strains (and probably also in other animal species) at any experimental age, this labeling technique is tedious, requires direct access to the brain and therefore can only be performed in acute experiments on anesthetized animals. Moreover, analyses of microglial networks are hardly possible using single cell electroporation technique. The other techniques, as for example a cell-type specific expression of a genetically encoded Ca2+ indicator (GECI), seem much better suited for this kind of recordings. Over the recent years several different approaches for labeling microglia with GECIs were considered. Seifert et al., 2011, for

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example, expressed a genetically encoded Ca2+ indicator GCaMP2 in microglia using a retroviral gene transfer [75]. However, since retroviral constructs only transduce proliferating cells, proliferation of microglia had to be triggered by a stab wound injury 2 days prior to virus injection into the brain. The authors did not observe any transduction of microglia when they applied the virus simultaneously with the induction of stab wound injury. This labeling technique yielded brightly labeled microglial cells (as assured by the post hoc immunocytochemistry using an anti-Iba1 antibody), but also labeled some 20–30% of Iba1-negative (i.e. non-microglial) cells which also proliferated at the time of virus injection. Shortly after the viral injection (3–6 days after injury) there were many GCaMP2-positive cells with amoeboid morphology (see Fig. 5 in [75]). The morphological and the electrophysiological properties of labeled cells started resembling that of ramified microglia 42 days after injury, but by that time the density of labeled cells decreased significantly from initially 60–80 (3–6 days after injury) down to approximately 10 cells per 130 ␮m thick brain slice. This suggests that the population of microglial cells labeled by this technique is transient and largely disappears after resolution of inflammation accompanying stab wound injury. By crossing a mouse line expressing a genetically encoded Ca2+ indicator GCaMP5G as well as red fluorescent dye tdTomato in a Cre-recombinase dependent manner (PC:G5-tdT mouse) with a microglia-specific Cre driver line, the groups of Petr Tvrdik and Mario Capecchi succeeded in expressing GCaMP5G in virtually all microglial cells in the brain [76]. However, most probably due to by now famous resistivity of microglial cells to labeling with Ca2+ indicators [24,75], the expression levels of the indicator still remained low. Nevertheless, simultaneous expression of tdTomato enabled easy detection of cells with dim GCaMP5 fluorescence. Using Hoxb8-IRES-Cre driver mouse (Hoxb8 is important for the maintenance and differentiation of hematopoietic cells), it was possible to study a small subpopulation of bone marrow derived microglia in acute brain slices [77]. The use of Aif1(Iba1)-IRES-Cre driver line (Allograft inflammatory factor 1 (Aif1), also known as Iba-1 (see above), is a specific marker of brain microglia) enabled analyses of microglial Ca2+ signaling in vivo [76]. However, due to the fast bleaching of the dye imaging of a given cell was limited to 20 min in total. In vivo Ca2+ imaging of genetically labeled intact microglia in transgenic mice [76] largely confirmed the data obtained in electroporated microglial cells. Thus, genetically labeled microglial cells rarely showed spontaneous Ca2+ transients, but vividly responded with large Ca2+ transients to applications of ATP analogs as well as to cell/tissue damage in their vicinity. Importantly, when monitoring microglial networks, Pozner et al. [76] have never detected any synchronized wave-like Ca2+ transients in intact microglia.

5. Imaging in vivo microglial Ca2+ signals in the aging and diseased brain The in vivo techniques outlined above were also used for Ca2+ imaging of microglial cells during normal aging as well as in conditions of peripheral or amyloid-induced inflammation [60,76]. Normal aging was accompanied by the doubling of the fraction of microglial cells with spontaneous Ca2+ transients ([60]; Fig. 4B). The fraction of spontaneously active cells was even higher (up to 80%) in two different mouse models of AD, especially in cells dwelling in the immediate vicinity of amyloid plaques (Fig. 4A, C). Similarly, the fraction of spontaneously active microglia increased (to approximately 34%) in a mouse model of acute peripheral inflammation induced by a subcutaneous injection of bacterial lipopolysaccharide [76]. Also epileptiform activity, evoked by topical application of a GABAA receptor blocker bicuculline, substantially increased the

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Fig. 3. Single-cell electroporation technique for in vivo Ca2+ imaging of microglia. (A) MIP image of GFP-positive microglial cells in a CX3 CR1GFP/+ mouse (red; left). (B) One of the microglial cells is labeled with the Ca2+ indicator OGB-1 (green) using single-cell electroporation (middle). Merged image is shown on the right. (B) Dose-response curves for UDP (red) and ATP (black) pressure-applied to electroporated microglial cells. Amplitudes are normalized to maximum amplitudes of Ca2+ transients evoked by 100 ␮M ATP or UDP, respectively. Solid lines are fits using the Hill equation. Inset shows representative Ca2+ transients evoked by the indicated concentrations of pressureapplied ATP. (C) Bar graph showing the fraction of microglial cells responding to pressure application of ATP␥S (Adenosine 5 -O-(3-thiotriphosphate), 5 mM); 2-MeSATP (2-methylthio-ATP, 5 mM,); Bz-ATP (Benzoylbenzoyl-ATP, 5 mM,); ␣-␤-MeATP (␣,␤-Methylene-ATP, 10 mM); 2-MeSADP (2-methylthio-ADP, 500 ␮M); glutamate (10 mM), trans-ACPD ((±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid, 5 mM); carbachol (10 mM)/oxotremorine (5 mM); fractalkine (chemokine domain, 250 ␮g/ml) and KCl (80 mM). Receptors targeted by the different agonists are indicated in the respective bar. Modified with permission from [24].

incidence of spontaneous Ca2+ transients in microglia. Moreover, under bicuculline spontaneous activity of microglia transformed from asynchronous Ca2+ transients in individual cells into synchronized Ca2+ waves, sometimes spreading across the entire imaging field [76]. Such synchronized Ca2+ waves were occasionally recorded also in lipopolysaccharide-treated, but never in intact microglia (see above). Mechanistically all damage-induced or spontaneous microglial Ca2+ transients in the intact or diseased brain tested so far, required activation of purinergic receptors and Ca2+ release from the intracellular stores, suggesting that they are mediated by metabotropic P2Y receptors [24,60,76]. In the amyloid-depositing mice, spontaneous microglial Ca2+ transients were not induced by astrocytic or neuronal activity, but neuronal silencing selectively increased the frequency of Ca2+ transients in plaque-associated microglia [60]. This finding is consistent with an assumption that physiological levels of ongoing neuronal activity serve as an OFF signal for microglia. Together all these data suggest that microglial activation is accompanied by transient increases in microglial [Ca2+ ]i [60,76]. 6. Conclusions In summary, the use of transgenic mice harboring GFPexpressing microglia enabled for the first time in vivo observation

of these cells in their intact environment. These studies provided a wealth of knowledge about microglia motility, and revealed an unexpected role of microglia for normal brain development. Several recently developed complementary approaches will further facilitate and accelerate in depth characterization of microglia. These are (i) inducible expression of fluorophores using microglia-specific Cre driver lines (e.g. CX3 CR1CreER ) and different reporter mouse lines, (ii) acute labeling using plant lectins, and (iii) microRNA-9regulated expression of fluorophores using viral vectors. Whereas the use of CX3 CR1CreER enables genetic expression of fluorophores in the whole microglial population, lectins and microRNA-9 based approaches produce a more localized labeling. In contrast to lectin labeling, genetically encoded expression of fluorophores enables long-term observation of microglial cells. However, lectin labeling is immediately applicable to any experimental animal and therefore represents a versatile and easy technique complementing the currently available toolkit for analyses of microglial morphology and dynamics in vivo. Concerning Ca2+ imaging of microglia, there currently exist two complementary labeling techniques: (i) labeling of individual cells with small molecule indicators by means of electroporation, (ii) labeling of cell populations with GECIs using GECI-expressing reporter mice crossed with microglia-specific Cre driver lines. Single cell electroporation technique is laborious and applicable only in acute preparations, but provides robust and bright labeling last-

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Fig. 4. In vivo Ca2+ imaging of microglia in the aging and diseased brain. (A; left panel) In vivo MIP image showing cortical GFP-labeled microglial cells (green) in the immediate vicinity of an amyloid plaque in a mouse model of AD (APPPS1/Iba1-GFP, 9 months). Amyloid plaque is visualized using Thioflavin-S (blue). One of the microglial cells is loaded with a Ca2+ sensor OGB-1 (red) by means of single-cell electroporation. (A; right panel) Representative Ca2+ transients recorded from the microglial cell labeled in the image on the left (cell 1) as well as other plaque-associated microglial cells (cell 2–4). (B) Bar graph illustrating the fraction of microglial cells showing a spontaneous Ca2+ transient over a 15-min imaging period in 2–4 and 8–15 months old control and CX3 CR1 knockout mice. (C) Bar graph illustrating the fraction of microglial cells showing a spontaneous Ca2+ transient over a 15-min imaging period in two mouse models of AD. Note that different microglial phenotypes were separated into two different groups (ramified and hypertrophic/amoeboid). Reproduced with permission from [60].

ing till the end of an acute experiment. This technique allows the use of green as well as red or orange Ca2+ indicators, provides immediate results and is applicable to different mouse mutants at any developmental age. Moreover, when combined with lectin-based cell visualization techniques, it is likely to be directly applicable in other experimental animals like rats, guinea pigs or new world monkeys (e.g. marmosets). The use of transgenic mice enabled for the first time observation of Ca2+ dynamics within the microglial network and clearly will become the method of choice as soon as robust bright labeling of microglia with genetically encoded Ca2+ sensors will be achieved.

Conflict of interest statement The authors declare no conflict of interest.

Author contributions B.B. and O.G. made the figures and wrote the manuscript. Acknowledgements This work was partially supported by the Alzheimer Forschung Initiative e.V. (grant no. 14812) and VolkswagenStiftung (grant no. 90233) to O.G. References [1] L.J. Lawson, V.H. Perry, P. Dri, S. Gordon, Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain, Neuroscience 39 (1990) 151–170. [2] K. Askew, K. Li, A. Olmos-Alonso, F. Garcia-Moreno, Y. Liang, P. Richardson, T. Tipton, M.A. Chapman, K. Riecken, S. Beccari, A. Sierra, Z. Molnar, M.S. Cragg, O. Garaschuk, V.H. Perry, D. Gomez-Nicola, Coupled proliferation and

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Please cite this article in press as: B. Brawek, O. Garaschuk, Monitoring in vivo function of cortical microglia, Cell Calcium (2017), http://dx.doi.org/10.1016/j.ceca.2017.02.011