A new approach for ratiometric in vivo calcium imaging of microglia

Abstract

Microglia, resident immune cells of the brain, react to the presence of pathogens/danger signals with a large repertoire of functional responses including morphological changes, proliferation, chemotaxis, production/release of cytokines, and phagocytosis. In vitro studies suggest that many of these effector functions are Ca²⁺-dependent, but our knowledge about in vivo Ca²⁺ signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely resisted all attempts of in vivo labelling with Ca²⁺ indicators. Here, we introduce a novel approach, utilizing a microglia-specific microRNA-9-regulated viral vector, enabling the expression of a genetically-encoded ratiometric Ca²⁺ sensor Twitch-2B in microglia. The Twitch-2B-assisted in vivo imaging enables recording of spontaneous and evoked microglial Ca²⁺ signals and allows for the first time to monitor the steady state intracellular Ca²⁺ levels in microglia. Intact in vivo microglia show very homogenous and low steady state intracellular Ca²⁺ levels. However, the levels increase significantly after acute slice preparation and cell culturing along with an increase in the expression of activation markers CD68 and IL-1β. These data identify the steady state intracellular Ca²⁺ level as a versatile microglial activation marker, which is highly sensitive to the cell's environment.

Figure 1

Expression of target genes (GFP or Twitch-2B) in LV.miR-9.T vector-transduced brain tissue. (a) Scheme of the LV.PGK.GFP.miR-9.T construct. (b) Maximum intensity projection (MIP) images (5–15 µm, step 1 µm) illustrating the identity of LV.PGK.GFP.miR-9.T-transduced cells (left) through immunofluorescent staining with an antibody against the microglial marker Iba1 (middle). The colour merged image is shown on the right. (c) Two-photon MIP in vivo image (18–27 µm, step 2 µm) of the transduced cells showing typical microglial morphology. Inset is a MIP image (3–21 µm, step 1 µm) showing a zoom-in of a rectangular square in the middle of the image. (d) Scheme of the LV.PGK.Twitch-2B.miR-9.T construct. (e,f) MIP images ((e) 0–19 µm, (f) 0–20 µm, step 1 µm) showing either native fluorescence of the cells transduced with LV.PGK.Twitch-2B.miR-9.T construct (left) or fluorescence of the same area labelled with an anti-GFP antibody recognizing Twitch-2B (right).

Figure 2

Optimization of the LV.Twitch-2B.miR-9.T vector. (a) Illustration of the experimental approach to test the efficacy of LV.Twitch-2B.miR-9.T vectors with different promoters. Lentiviral vectors were first compared in HEK293 cells in vitro and then tested in the brain in vivo. (b–d) Epifluorescence single-plane images of HEK293 cells transduced with different viral vectors shown in (a), taken with the same excitation light intensity and exposure time. Insets show the contrast-enhanced display of the same image. Numbers in the lower left corner show the range of display for each image. (e,f) Single-plane images of the fixed striatal (e) or cortical (f) tissue transduced in vivo with LV.CMV.Twitch-2B.miR-9.T viral vector. Note that Twitch-2B-expressing cells (left) are positive for CD11b (e, middle) and Iba1 (f, middle) and therefore are microglial cells. The colour merged images are shown on the right.

Figure 3

Post hoc analyses of activation markers expressed in Twitch-2B labelled microglia. (a,b) MIP images of fixed cortical tissue, transduced in vivo with LV.CMV.Twitch-2B.miR-9.T viral vector, taken in the epicentre (a; 7–59 µm, step 1 µm) and at the periphery (b; 4–44 µm, step 1 µm) of the injection site. Left: Twitch-2B positive microglial cells labelled with an anti-GFP antibody. Middle: the same field of view labelled with anti-CD68 antibody. Merged images are shown on the right. (c,d) MIP images of fixed cortical tissue (as above) taken in the epicentre (c; 2–28 µm, step 1 µm) and at the periphery (d; 1–33 µm, step 1 µm) of the injection site. Left: Twitch-2B positive microglial cells labelled with an anti-GFP antibody. Middle: the same field of view labelled with anti-IL-1β antibody. Merged images are shown on the right. Inset in (d) shows a rare Twitch-2B and IL-1β positive cell at the periphery of the injection site. (e) Summary box plot illustrating distributions of background-subtracted CD68 fluorescence per microglial cell in the epicentre (n = 100 cells in 3 mice) and at the periphery (n = 86 cells in 3 mice) of the injection site. Note that CD68 fluorescence is significantly higher in microglia in the epicentre compared to periphery (p < 0.001, Mann-Whitney test). (f) Summary bar graph showing the fraction of IL-1β positive cells in the population of Twitch-2B positive microglia in the epicentre (n = 31 cells in 4 mice) and at the periphery (n = 54 cells in 5 mice) of the injection site.

Figure 4

LV.CMV.Twitch-2B.miR-9.T vector enables ratiometric calcium imaging of microglia in vivo. (a) MIP images (left: 18–33 µm depth; right: 17–36 µm depth, step 1 µm) showing combined fluorescence of mCerulean3 and cpVenus^CD of cortical microglia in vivo. (b) Representative traces, recorded from the region of interest delineated in the inset (dashed line), showing mCerulean3 (top) and cpVenus^CD (middle) channels for three pressure applications of a P2Y receptor agonist UDP (1 mM in the application pipette, 200 ms). Inset: MIP image (117–153 µm depth, step 1 µm) of the recorded microglial cell. Bottom: trace showing the ΔR/R signal. Arrowheads indicate time points of UDP applications. Similar results were obtained in n = 5 cells (summarized in Fig. 5b).

Figure 5

Versatility of the LV.CMV.Twitch-2B.miR-9.T vector for in vivo Ca²⁺ imaging of microglia. (a) Representative microglial Ca²⁺-transient evoked by pressure-application of a P2X receptor agonist Bz-ATP (1 mM in the application pipette, 100 ms). (b) Summary box plot showing peak amplitudes evoked by pressure-applications of UDP (1 mM, n = 5 cells) and Bz-ATP (1 mM, n = 5 cells) in microglial cells in vivo. (c) Bar graph showing the fraction of microglia with and without spontaneous Ca²⁺-activity over a 10-min-long imaging period (n = 10 cells). (d) Representative microglial Ca²⁺-transient evoked by damaging a neuron in cell’s vicinity (cell-to-cell distance 18 µm). (e) Box plot showing distribution of peak amplitudes of microglial Ca²⁺ transients evoked by damaging cells located 15–38 µm apart from the recorded microglia (n = 7 cell damage-induced transients, each caused by damaging a separate cell, recorded from 4 cells in 4 mice). The cell damage-induced Ca²⁺ transients were observed in 4 out of 5 microglial cells tested. (f) MIP image (56–84 µm depth, step 1 µm), taken 60 min after a laser induced injury of the cortical parenchyma, showing a Twitch-2B positive microglia (green, combined fluorescence of mCerulean3 and cpVenus^CD) responding to tissue damage. Note that tissue autofluorescence (yellow) is increased in the laser-damaged area (indicated by a broken white line). (g) Box plot illustrating a significant increase in basal cpVenus^CD/mCerulean3 fluorescence ratio in microglia after laser-induced damage compared to control conditions (median: 1.43, IQR: 1.19–1.93, p < 0.05; Wilcoxon Signed-rank test; n = 6 cells in 3 mice). The laser induced injury increased cpVenus^CD/mCerulean3 fluorescence ratios in all microglial cells tested.

Figure 6

Use of the LV.Twitch-2B.miR-9.T vector enables estimation of basal Ca²⁺ levels in microglia. (a) MIP image (6–15 µm depth, step 1 µm; mCerulean3 channel) showing Twitch-2B-positive ramified microglial cells in culture. (b) MIP images of the same preparation after fixation and labelling with an anti-Iba1 antibody. Left: native fluorescence of Twitch-2B (combined signal from mCerulean3 and cpVenus^CD); middle: fluorescence of Alexa Fluor 594 bound to anti-Iba1 antibody; right: a colour merged image of the two channels. (c) Box plot showing cpVenus^CD/mCerulean3 fluorescence ratios of cortical microglia and neurons under different experimental conditions. When compared to ramified microglia in acute in vivo preparations (n = 32 cells), microglia in primary cell culture (n = 50 cells) and acute brain slices (n = 25 cells) show elevated cpVenus^CD/mCerulean3 ratios (p < 0.0001 for acute in vivo vs. brain slice and 0.007 for acute in vivo vs. cell culture, Kruskal-Wallis test followed by post hoc Dunn-Sidak test). The same is true for comparison with microglia in chronic in vivo preparations (n = 15 cells; p < 0.0001 for chronic in vivo vs. brain slice and 0.006 for chronic in vivo vs. cell culture, Kruskal-Wallis test with post hoc Dunn-Sidak correction). In acute preparations, microglia exhibit significantly higher Ca²⁺-levels than cortical layer 2/3 neurons labelled with a AAV.synapsin1.Twitch-2B vector (n = 147 cells; p < 0.0001, Mann-Whitney test). TTX (2 µM) has no effect on basal cpVenus^CD/mCerulean3 ratios of cortical neurons (n = 202 cells; p = 0.343, Kruskal-Wallis test with post hoc Dunn-Sidak correction). Note that neurons in acute brain slices (n = 601 cells) have slightly but significantly higher Ca²⁺-levels compared to those in acute in vivo preparations (p < 0.0001, Kruskal-Wallis test with post hoc Dunn-Sidak correction).