A genetically targeted optical sensor to monitor calcium signals in astrocyte processes - PubMed (original) (raw)

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

Eiji Shigetomi et al. Nat Neurosci. 2010 Jun.

Abstract

Calcium signaling is studied as a potential form of astrocyte excitability that may control astrocyte involvement in synaptic and cerebrovascular regulation. Fundamental questions remain unanswered about astrocyte calcium signaling, as current methods can not resolve calcium in small volume compartments, such as near the cell membrane and in distal cell processes. We modified the genetically encoded calcium sensor GCaMP2 with a membrane-tethering domain, Lck, increasing the level of Lck-GCaMP2 near the plasma membrane tenfold as compared with conventional GCaMP2. Using Lck-GCaMP2 in rat hippocampal astrocyte-neuron cocultures, we measured near-membrane calcium signals that were evoked pharmacologically or by single action potential-mediated neurotransmitter release. Moreover, we identified highly localized and frequent spontaneous calcium signals in astrocyte somata and processes that conventional GCaMP2 failed to detect. Lck-GCaMP2 acts as a genetically targeted calcium sensor for monitoring calcium signals in previously inaccessible parts of astrocytes, including fine processes.

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Figures

Figure 1

Figure 1

Simultaneous imaging of global and near-membrane calcium in astrocytes. (a) Diagram illustrates the principal of TIRF, whereby an evanescent field illuminates a restricted area of the cell. Fluo-4 was excited by an argon laser for TIRF (488 nm) or a monochromator for EPI microscopy (488 nm). (b) Representative images of an astrocyte loaded with Fluo-4 under EPI and TIRF microcopy, where the calcium transients were synchronous between these imaging modalities. The arrowhead indicates an ROI, which corresponds to the traces (d_F_/F over time) on the right. All calcium transients observed were synchronized between EPI and TIRF, resulting in a flat trace when the normalized TIRF traces were subtracted from the normalized EPI traces. (c) Data are presented as in b for an astrocyte that showed nonsynchronized events between EPI and TIRF microscopy. In this case, subtraction resulted in a peaky trace. Arrows indicate calcium signals that were observed in EPI. In both b and c, ATP caused a uniform increase in calcium.

Figure 2

Figure 2

Quantification of spontaneous and pharmacologically evoked calcium signals measured by TIRF and EPI microscopy. (a) Representative traces of calcium transients observed using Fura-2; 30 µM ATP was applied for the duration indicated by the bar (∼30 s). (b) Relationship between d_F_/F measured with Fluo-4 and estimated calcium measured with Fura-2. (c) Agonist-induced calcium transients observed in EPI and TIRF (top, averaged EPI traces; middle, averaged TIRF traces; bottom, normalized traces). We used 30 µM ATP (n = 54), 300 µM glutamate (n = 54), 10 µM DHPG (group I mGluR agonist, n = 65), 30 µM TFLLR (PAR1 agonist, n = 59), 100 nM endothelin-1 (ET-1, n = 45) and 5 µM FLRF amide (MrgA1 receptor agonist, n = 13). As a result of spectral overlap, we used Fura-Red for calcium imaging of MrgA1-EGFP–transfected astrocytes (2 d after transfection); thus, the traces are downward for these experiments. Error bars represent s.e.m.

Figure 3

Figure 3

Design and characterization of Lck-GCAMP2. (a) Schematic representation of cytosolic GCaMP2 and membrane targeted Lck-GCaMP2. The membrane-tethering Lck domain was added to N terminus of GCaMP2. (b) Representative image and line profile of two HEK-293 cells expressing cytosolic GCaMP2; the inset cartoon shows cytosolic GCaMP2 in relation to the plasma membrane (PM, representative of n = 5 fields of view). The colors in the cartoon correspond to the colors in a. (c) Data are presented as in b for Lck-GCaMP2; note that the fluorescence was strongly located at the edges of the cells, near the membrane (representative of n = 5 fields of view). (d) HEK cells permeablized with 0.1% Triton X-100 for 15–30 s, before, during and after application of buffered solutions containing 10 µM free calcium ions. Right, calcium sensitivity of Lck-GCaMP2 measured in this way (n = 9). Error bars represent s.e.m.

Figure 4

Figure 4

ATP-evoked calcium signals in astrocytes measured with Lck-GCaMP2. (a) Upper panels show a Lck-GCaMP2–expressing astrocyte imaged with EPI microscopy. The lower panels show the footprint of the same cell imaged 10 ms later with TIRF. The EPI images are brighter than the TIRF images because this mode of illumination reports on the entire cell and because the EPI excitation was brighter. When these factors are corrected for, the expression of Lck-GCaMP2 is extremely robust in the plasma membrane (by TIRF; Supplementary Fig. 7). Representative images are shown before, during and after applications of 30 µM ATP. (b) Representative traces for ATP-evoked changes in fluorescence measured in astrocytes with EPI and TIRF microscopy. Right, superimposed traces measured with EPI and TIRF from the same astrocyte. (c) Dose-response curves for ATP measured with EPI (n = 10) and TIRF (n = 10) microscopy from astrocytes expressing Lck-GCaMP2. (d) ATP dose-response curve for astrocytes loaded with Fluo-4 (n = 28–54). Error bars represent s.e.m.

Figure 5

Figure 5

Responses of astrocytes expressing Lck-GCaMP2 during EFS of neurons. (a) Traces from 11 astrocytes (gray), their averages superimposed (black), showing the Lck-GCaMP2 response during EFS. The number of action potentials applied is indicated in brackets. (b) The graphs plot the relationship between the change in fluorescence of Lck-GCaMP2 and the number of action potentials for individual cells (gray) and their average (black). (c) Representative images of an astrocyte expressing Lck-GCaMP2 gathered with EPI microscopy, before and during EFS under control conditions (top) and in the presence of 1 µM TTX (bottom). (d) Representative traces for five cells when EFS was applied twice (top) and when the second period of EFS was applied in the presence of 1 µM TTX. (e) Average data for experiments such as those shown in c and d. In these experiments, the second period of EFS was preceded by 10-min incubation with 1 µM TTX, 100 µM Cd2+, 30 µM PPADS, 20 µM MRS2179 and 20 µM CPA. EFS consisted of 90 action potentials triggered in 3 s at 30 Hz. Error bars represent s.e.m. **P < 0.01.

Figure 6

Figure 6

Spontaneous calcium signals measured with Lck-GCaMP2. (a) Time series of an astrocyte expressing Lck-GCaMP2. Numerous spotty calcium signals were observed. The gray cartoons show the outline of the astrocyte with the location of the spots superimposed. (b) Intensity versus time profile of 11 ROIs from images such as those shown in a. Note, calcium signals can be measured repeatedly in the same ROI. (c) Time course of ten spotty signals and the average in black. (d) The FWHM of the events (n = 6) and 100-nm beads (n = 10). Error bars represent s.e.m.

Figure 7

Figure 7

Spotty calcium signals measured in astrocyte processes. (a) Image of an astrocyte with its outline superimposed and three regions of interest indicated as 1–3. Right, the d_F_/F of Lck-GCaMP2 fluorescence was plotted over time for 1–3. The lower right images give three further examples of localized calcium signals occurring in processes, independent of those in the cell body; note, processes were readily visible. (b) Image of an astrocyte treated with 5 mM db-cAMP for 48 h to extend processes; note the longer processes as compared to the image in a (Supplementary Fig. 2). Right, d_F_/F over time for seven microdomain signals.

Figure 8

Figure 8

Microdomain signals are a result of transmembrane calcium flux. (a) Representative d_F_/F traces of microdomains measured with Lck-GCaMP2. TTX (1 µM) was applied at the bar for 5 min. (b,c) Data are presented as in a for CPA (20 µM, b) or calcium-free buffer (c). (d) Top, summary data for microdomain frequency in cells treated with the indicated drugs. Bottom, peak ATP responses relative to control for the same drugs. Error bars represent s.e.m. ***P < 0.001.

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