A genetically targeted optical sensor to monitor calcium signals in astrocyte processes

A genetically targeted optical sensor to monitor calcium signals in astrocyte processes 1 Eiji Shigetomi, 1 Sebastian Kracun, 2 Michael V. Sofroniew & 1,2 *Baljit S. Khakh Ψ 1 Departments of Physiology and 2 Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, USA Supplementary Information Supplementary Fig. 1: TIRF images of astrocyte footprints from cells expressing Lck GCaMP2 and Lck GFP as well as those labeled with FM1 43. Histograms of fluorescence intensity were well fit by a single Gaussian function. In no case was the distribution multimodal or skewed, implying that regions of high fluorescence were not detected. There was no significant difference in coefficient of variance between the three groups. 1

Supplementary Fig. 2: GFAP staining. The images show GFAP immunostaining in control/untreated cells (left) and those treated with db camp treatment (5 mm, 48 hrs; right panel). Note that db camp caused the appearance of extended processes in astrocytes. 2

Supplementary Fig. 3: The effect of GPCR agonists on microdomain calcium signals. a. Shows the normalized frequency of the microdomain calcium signals (relative to control) for ADPβS (30 μm; n = 11), TFLLR (30 μm; n = 15) and both agonists together (n = 25). Agonists were applied for 15 s. Microdomain calcium signals (like those reported in the main manuscript; Figs 6 8) were measured before and after application of the agonists for 5 min. There was no significant difference in the frequency of microdomains triggered by GCPR activation. b. Shows peak of df/f for global elevations of cytosolic calcium for the agonists, proving that they did elevate calcium levels in these cells. 3

Supplementary Fig. 4: Calcium signals in neurons measured by Lck GCaMP2: a. An example of a representative Lck GCaMP2 expressing neuron imaged with EPI microscopy with three ROIs (1 3). In (i) the traces show the df/f of Lck GCaMP2 fluorescence plotted over time for the regions shown as 1 3. In (ii) another example is shown for three ROIs. These ROIs were chosen to show the greatest changes in fluorescence, but no signals indicative of calcium microdomains were observed in 9 of 10 neurons tested (see also Supplementary Video 7). b. EFS evoked (30 Hz for 3 s) calcium signals in neurons. Even for 90 APs neurons displayed brief calcium increase during stimulation. On the other hand, astrocytes displayed long lasting calcium increase (see Fig. 5). EFS evoked calcium signals in neurons were reproducible as evidenced by the 1 st and 2 nd stimulation (stim) periods. EFS evoked responses in neurons were blocked by TTX (1 μm; n = 3). We could not track a single AP in neurons with Lck GCaMP2, because our camera does not posses the requisite acquisition speed. However, we could detect the signals due to 9 APs; the data are presented in Table 1 of the manuscript. c. Hippocampal neurons displayed calcium signals that were evoked by glutamate (1 mm; n = 6), but not robustly by ATP (30 100 μm; n = 5/6; only one neuron responded to ATP at 100 μm). On the other hand the gray traces show that astrocytes displayed calcium signals in response to both glutamate and ATP (see main manuscript for details). 4

Supplementary Fig. 5: Microdomain calcium signals were not due to the movement of vesicles which express Lck GCaMP2. a. Cartoons illustrating that if vesicles expressing Lck GCaMP2 move toward the plasma membrane (i ii) and then quickly move away again from the plasma membrane (ii iii), this may cause Lck GCaMP2 fluorescence to apparently increase as shown in the cartoon in the bottom panel. However, the empirical data shown in panels b-e, suggest that this is not the case. b. An image of a Lck GFP expressing astrocyte captured with EPI microscopy. An arrow indicates a vesicle expressing Lck GFP (see Supplementary video 7 to see this vesicle move near the membrane). c. Traces of df/f of Lck GFP fluorescence over time for vesicles selected post hoc to show significant movements into ROIs. No clear fluorescence increase above background was observed. d. Shows the histogram of df/f at the peak of Lck GCaMP2 (n = 515) and Lck GFP (n = 36), providing strong evidence that the fluorescence signal due to Lck GFP movement can not account for the signals measured with Lck GCaMP2. e. High magnification image of an Lck GFP expressing vesicle. The graph indicates the full width half maxima (FWHM) of the vesicles (n = 8). The size of the vesicle is at the limit of resolution of our microscope (~ 400 nm) and far smaller than the size of the microdomain calcium signals which are ~5 μm (see Fig 6). 5

Supplementary Fig. 6: Microdomain calcium signals do not occur in areas with higher/accumulated Lck GCaMP2 fluorescence. a. An image of an Lck GCaMP2 expressing astrocyte acquired with EPI microscopy with ten ROIs indicated to display calcium microdomains. In the traces on the right the df/f of Lck GCaMP2 fluorescence was plotted over time for the regions shown as 1 10 in a. c. The bar graph shows the resting fluorescence intensity of Lck GCaMP2 at the center of a microdomain ROI as well as in ROIs 2 and 5 μm away (n = 55 microdomains). Note: the resting intensity was measured before a calcium microdomain signal occurred. The fact that the resting signal is identical in all three ROIs indicates that the microdomains do not occur in regions of differing fluorescence intensity. 6

Supplementary Fig. 7: Analysis of Lck GCaMP2 expression and mobility. a. EPI and TIRF images of an astrocyte expressing Lck GCaMP2. The TIRF image shows only the basement membrane of the astrocyte (see Fig. 1b; the footprint ), whereas the EPI image shows the cell in the optical axis. b. We normalised the EPI and TIRF intensity measurements with respect to 100 nm beads imaged with both TIRF and EPI microscopy. The average intensity of several hundred beads is shown in the images in arbitrary units of fluorescence, with the mean for each representing 1 bead unit. c. Cartoon illustrating the relationship between the intensity of ROIs in the EPI image (F EPI ; the whole cell in the optical axis) and the intensity of the basement 7

membrane (F TIRF ). The equations show how F EPI may be related to the bottom and top membranes alone (top equation), or in addition to the intensity of intracellular fluorescence (F IC ). d. We expressed the intensity of Lck GCaMP2 over time, before, during and after 30 μm ATP for images from EPI and TIRF microscopy in bead units. The lower bar graphs show the intensity from EPI and TIRF microscopy before and during ATP. The bars are the experimental data (n = 6), which closely matches that expected if there was no fluorescence from inside the cell (shown as dashed lines; i.e. the upper equation in panel c). These analyses suggest that the intensity of astrocytes imaged by EPI microscopy can be accounted for almost entirely by the robust expression of Lck GCaMP2 in the top and bottom plasma membranes. e. Fluorescence intensity of cytosolic GCaMP2 and Lck GCaMP2 measured with EPI microscopy. f. Fluorescence intensity of cytosolic GCaMP2 and Lck GCaMP2 measured with TIRF microscopy. g. Fluorescence intensity in TIRF scaled to account for differences in collection depth for cytosolic GCaMP2 (~ 100 nm in the optical axis) and for Lck GCaMP2 (~ 7 nm for the thickness of the plasma membrane). h. FRAP curves for cytosolic GCaMP2 (light blue) and Lck GCaMP2 (pink). The y axis indicates fluorescence intensity as a percentage of the pre-bleach control value. The FRAP experiments were carried out in HEK 293 cells because this allowed us to easily identify near membrane regions of interest. Explanatory text accompanying this figure: Why is Lck GCaMP2 better than GCaMP2 for reporting astrocyte calcium signals? Using EPI microscopy with Lck GCaMP2 we could detect spontaneous signals that were never detected with cytosolic GCaMP2. In this section we report experiments that point to at least three reasons why this was the case. First, we found that Lck GCaMP2 is mainly expressed in the plasma membrane (although it does also enter recycling vesicles). This was suggested by the confocal images shown in Figure 3c, but also by subsequent calibrations of the intensity of astrocyte membrane footprints imaged with TIRF 5 in relation to the intensity of the entire astrocyte imaged with EPI microscopy (Supplementary Fig. 7a). To compare EPI and TIRF images we corrected for differences in illumination power (laser for TIRF v monochromator for EPI; Fig. 1) by normalising the pixel intensities from EPI and TIRF images with respect to fluorescent beads (Supplementary Fig. 7a,b). For small regions of interest in the centre of an EPI image the total fluorescence signal can arise from two plasma membranes (top and bottom; i.e. F EPI 2.F TIRF ) or from two plasma membranes as well as intracellular fluorescence (i.e. F EPI = 2.F TIRF + F IC ; where F IC is some value representing the contribution of intracellular fluorescence in Supplementary Fig. 7c). We found that the intensities of the EPI images and the peak ATP evoked responses were well accounted for (to within 1%) by the sum of the signals expected from two plasma membranes (i.e. the top and the bottom of the cell; Supplementary Fig. 7c,d), providing excellent evidence that there was little cytosolic fluorescence in astrocytes expressing Lck GCaMP2 and that most of the signals arose from the plasma membrane (i.e. F IC was negligible). Second, by analysing TIRF images of astrocytes expressing cytosolic GCaMP2 and Lck GCaMP2 we estimate that the latter is ~ 14 fold more abundant near the plasma membrane. The EPI intensity of GCaMP2 expressing astrocytes were higher than those expressing Lck GCaMP2 (Supplementary Fig. 7e), but the TIRF intensities were equal (Fig. 8e). When interpreting these data it is important to recall that EPI reports on the entire cell in the optical axis, whereas TIRF reports only on near membrane regions 5 (Fig. 1a). Therefore, for cytosolic GCaMP2 the TIRF intensity will represent fluorescence collected within ~ 100 nm of the plasma membrane, but for membrane targeted Lck GCaMP2 the intensity will represent collection from ~ 7 nm (approximately the thickness of the plasma membrane). Thus by scaling for these differences in collection depth the data suggest that Lck GCaMP2 is ~ 14 fold 8

more concentrated in the plasma membrane as compared to near membrane GCaMP2 (Supplementary Fig. 7g), although there is more GCaMP2 overall in the cytosol of astrocytes. Third, using published fluorescence recovery after photo bleaching methods 3 we find that the fluorescence recovery kinetics of membrane targeted Lck GCaMP2 are significantly slower than those of cytosolic GCaMP2 (Supplementary Fig. 7h; estimated diffusion coefficient D = ~ 0.05 μm 2 /s for Lck GCaMP2 and > 8 μm 2 /s for GCaMP2). Even in the simplest case, if we assume diffusion in the plane of the membrane, cytosolic GCaMP2 would diffuse ~ 10 μm in ~ 3 s (i.e. the half duration of a microdomain), whereas Lck GCaMP2 would diffuse < 1 μm over the same time frame. Because spotty astrocyte calcium signals are localised (FWHM ~ 5 μm) and slow (T 0.5 ~ 3 s) a diffusible cytosolic GCaMP2 reporter would substantially degrade the signal. Thus, our experiments suggest that the combination of high and selective plasma membrane expression of a slowly diffusible indicator renders Lck GCaMP2 ideally suited to measure the localised/spotty and seconds time scale spontaneous microdomain calcium signals that occur near the membrane and in restricted volumes of astrocyte processes. 9

Supplementary Fig. 8: Local calcium signaling in astrocyte processes. a. The image shows a hippocampal astrocyte from the stratum radiatum region in a P15 rat hippocampal brain slice that has been loaded by Fluo 4/M. Four regions of interest in the astrocyte are shown. The intensity in these regions is plotted in the black traces. The coloured traces show the same records superimposed. The stars indicate localised calcium signals that occurred in processes. Similar data were gathered for 47/66 astrocytes from 29 brain slices from 10 rats. 10

Supplementary Table 1: Summary of data gathered with Lck-GCaMP2 for astrocytes and neurons. Astrocytes Neurons Fluorescence T 0.5 (s) n Fluorescence T 0.5 (s) n Φ Baseline signal (a.u.) 642 ± 56 30 699 ± 68 25 Ψ ATP evoked (df/f) signal 0.84 ± 0.04 2.9 ± 0.2 39 0 0 6 Glutamate evoked signal (df/f) 1.1 ± 0.1 2.1 ± 0.1 10 0.76 ± 0.23 8.6 ± 4.0 6 * EFS evoked signal (df/f) 1 AP 9 APs 90 APs 0.36 ± 0.08 0.59 ± 0.18 0.98 ± 0.13 17 ± 4.7 24 ± 6.3 21 ± 14 8 11 14 ND 1 0.23 ± 0.05 0.96 ± 0.18 ND 1 0.37 ± 0.06 3.0 ± 0.3 5 5 7 Microdomain (df/f) All pooled Somatic In processes In extended processes 0.50 ± 0.01 0.46 ± 0.01 0.68 ± 0.04 0.73 ± 0.04 3.9 ± 0.2 4.0 ± 0.2 3.7 ± 0.4 2.7 ± 0.2 515 427 88 70 0 0 0 ND 2 0 0 0 ND 2 9 9 9 Key to Table 1 The values are shown as mean ± s.e.m. Φ Background fluorescence was subtracted from these values to give the Lck GCaMP2 signal alone on a 16 bit scale (images taken with an Andor ixon DV887DCS EMCCD camera) indicates not relevant as these parameters could not be measured a.u. means arbitary units of fluorescence on a 12 bit scale Ψ Response evoked by 30 μm ATP Responses evoked by 300 μm glutamate for astrocytes and 1 mm glutamate for neurons. * Response evoked by APs at 30 Hz ND 1 means we could not determine this because our camera does not have the ability to acquire images at fast rates ND 2 means we did not determine the effect of db camp on hippocampal neurons Using imaging settings identical to those used for astrocytes, we did not observe microdomains in 9/10 neurons and have thus entered a value of zero in the table. In 1/9 neurons we observed spotty calcium signals in 2 locations on dendrites (in 5 min), these were abolished by TTX and are thus different to the astrocyte calcium signals which were resistant to TTX (see text). These two neuronal spotty calcium signals had an average df/f of 1.1 and T 0.5 of 3.5 s, thus they were also of larger amplitude than the microdomains observed in astrocytes. 11

Supplementary Videos Supplementary Video 1: Video of two astrocytes expressing cyotsolic GCaMP2: no obvious spontaneous events were seen. No obvious spontaneous calcium signal was observed. Frame rate = 1 Hz, duration of video = 300 s. Representative of 8 similar experiments. Supplementary Video 2: Video of astrocytes challenged with ATP. The video is of the same two astrocytes as in video 1, but this time challenged with 30 μm ATP (indicated by the dot). Note the large increase in fluorescence from cytosolic GCaMP2. Supplementary Video 3: Video of an astrocyte expressing Lck GCaMP2. Also see Fig. 6 of manuscript; video shown here as a df/f video. Note the appearance of spotty spontaneous signals, which we call microdomains. Frame rate = 1 Hz, duration of video = 130 s. Representative of 159 similar microdomains from 56 cells. Supplementary Video 4: Original video of the astrocyte shown in Supplementary video 3. In this case the data are shown as raw fluorescence; microdomains were readily visible without any data processing. Supplementary Video 5: Video of an astrocyte expressing Lck GCaMP2 imaged with EPI microscopy. For these specific experiments astrocytes were pre-incubated with db camp (5 mm, 48 hr). Note the spotty calcium signals at fine processes. Frame rate = 1 Hz, duration of video = 270 s. Representative of 5 similar experiments. 12

Supplementary Video 6: Video of a neuron expressing Lck GCaMP2 imaged with EPI microscopy. No obvious spontaneous microdomain calcium signals were seen. Frame rate = 1 Hz, duration of video = 270 s. Representative of 9 similar experiments. Supplementary Video 7: Video of an astrocyte expressing Lck GFP imaged with EPI microscopy. Small vesicles are seen moving around the cell. However, these movements are clearly different from calcium microdomains observed with Lck GCaMP2 with respect to size and fluorescence changes. Frame rate = 1 Hz, duration of video = 300 s. Representative of 9 similar experiments. References for Supplementary Information 1. Bers, D.M., Patton, C. W. & Nuccitelli, R. A practical guide to the preparation of Ca2+ buffers. Methods. Cell. Biol 40, 3-29 (1994). 2. Shigetomi, E. & Khakh, B.S. Measuring near plasma membrane and global intracellular calcium dynamics in astrocytes. JoVE 26, http://www.jove.com/index/details.stp?id=1142, doi: 10.3791/1142 (2009). 3. Richler, E., Chaumont, S., Shigetomi, E., Sagasti, A. & Khakh, B.S. An approach to image activation of transmitter-gated P2X receptors in vitro and in vivo. Nat. Methods 5, 87-93 (2008). 4. Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell. Biol 2, 444-56 (2001). 5. Jaiswal, J.K. & Simon, S.M. Imaging single events at the cell membrane. Nat. Chem. Biol 3, 92-8 (2007). 13