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Ca2+ signaling in astrocytes from Ip3r2−/− mice in brain slices and during startle responses in vivo

Nature Neuroscience volume 18, pages 708–717 (2015)Cite this article

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Abstract

Intracellular Ca2+ signaling is considered to be important for multiple astrocyte functions in neural circuits. However, mice devoid of inositol triphosphate type 2 receptors (IP3R2) reportedly lack all astrocyte Ca2+ signaling, but display no neuronal or neurovascular deficits, implying that astrocyte Ca2+ fluctuations are not involved in these functions. An assumption has been that the loss of somatic Ca2+ fluctuations also reflects a similar loss in astrocyte processes. We tested this assumption and found diverse types of Ca2+ fluctuations in astrocytes, with most occurring in processes rather than in somata. These fluctuations were preserved in Ip3r2−/− (also known as Itpr2−/−) mice in brain slices and in vivo, occurred in end feet, and were increased by G protein–coupled receptor activation and by startle-induced neuromodulatory responses. Our data reveal previously unknown Ca2+ fluctuations in astrocytes and highlight limitations of studies that used Ip3r2−/− mice to evaluate astrocyte contributions to neural circuit function and mouse behavior.

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Figure 1: Ca2+ fluctuations in hippocampal astrocytes from WT and Ip3r2−/− mice.
Figure 2: Ca2+ fluctuations within astrocyte processes are largely intact in brain slices from Ip3r2−/− mice.
Figure 3: Effect of nominally Ca2+-free buffer applications on astrocyte Ca2+ fluctuations in WT mice.
Figure 4: GPCR-mediated Ca2+ fluctuations in astrocyte processes are largely intact in hippocampal slices from Ip3r2−/− mice.
Figure 5: Abundant Ca2+ fluctuations persist in astrocyte processes from WT and Ip3r2−/− mice in vivo.
Figure 6: Ca2+ fluctuations persist in vivo within end feet of cortical astrocytes from Ip3r2−/− mice.
Figure 7: Endogenously evoked astrocyte process Ca2+ fluctuations recorded during in vivo startle responses reveal early and late components.
Figure 8: Prazosin-sensitive and insensitive components of the endogenously evoked astrocyte process Ca2+ fluctuations recorded during in vivo startle responses.

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Acknowledgements

The authors are grateful to current and past members of the laboratories for discussions and comments. Thanks also to M.V. Sofroniew for sharing equipment and to J. Chen for sharing mice. Special thanks to M.D. Haustein who helped with the initial testing of GECIquant and made valuable suggestions on its development. We thank C. Octeau for comments on the paper. Most of the work was supported by the US National Institutes of Health (NIH, NS060677). B.S.K., R.S. and S.V. were also supported by NIH grants (MH099559A, MH104069) and the CHDI Foundation. P.G. and B.S.H. were supported by NIH MH101198-1 and a Simon's Foundation Circuits Grant.

Author information

Author notes

  1. Rahul Srinivasan and Ben S Huang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Rahul Srinivasan, Sharmila Venugopal, April D Johnston, Hua Chai & Baljit S Khakh

  2. Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Ben S Huang & Peyman Golshani

  3. Allen Institute for Brain Science, Seattle, Washington, USA

    Hongkui Zeng

  4. Integrative Center for Learning and Memory, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Peyman Golshani

  5. West Los Angeles VA Medical Center, Los Angeles, California, USA

    Peyman Golshani

  6. Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Baljit S Khakh

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Contributions

R.S. carried out the molecular biology, hippocampal stereotaxic injections and most of the slice experiments with help from A.D.J. and H.C. B.S.H. performed all of the cortical virus injections and cranial window implantations for the in vivo experiments. B.S.H. and R.S. did the in vivo imaging together. S.V. wrote the GECIquant software and R.S. tested it. P.G. shared expertise on in vivo calcium imaging. H.Z. made and shared GCaMP6f knock-in mice. R.S. and B.S.K. analyzed data. B.S.K. directed the experiments, assembled the figures and wrote the paper. All of the authors contributed to the final version.

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Correspondence to Baljit S Khakh.

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Integrated supplementary information

Supplementary Figure 1 Unbiased semi-automated Ca2+ fluctuationdetection within astrocytes using GECIquant.

a. Schematic showing the sequence of steps involved in isolating the objects of interest. The confocal t-stack (left) is represented as a series of x-y frames with time t as the third dimension and the pixel intensities are given by I(x,y,t) for 0 ≤ x < imagewidth, 0 ≤ y < imageheight, and, 0 < t ≤ stacklength. As a first step, the t-stack is reduced to a two-dimensional maximum intensity projection image (middle) with I (x,y)max representing the maximum intensities at each x-y pixel coordinate of the t-stack. This image is thresholded to generate a binary image (right) to isolate the objects I(x,y)thresh (black) from background (white). b-d. Pixel operations involved in each of the above steps. For background subtraction, I(t)avg_bck is the average of n pixel intensities in a user selected area at a given time frame t and I(x,y,t)total represents the raw pixel intensities before background subtraction. For object thresholding, AO and AB refer to averages of object and background pixels respectively and T is the threshold intensity estimated using an iterative algorithm (Isodata) in ImageJ. e. Shows the steps involved in object tracing. The resulting object’s area is calculated by summing the areas of all the pixels (1:Np) covered by the detected object as given by the object area equation, where Wp and Hp represent the pixel width and height respectively in microns. f. Pixel calculation for ROI centroids is given in the equationwhere, Xk, Yk is the centroid of the kth ROI, (xi, yi) are the pixel coordinates of the kth ROI, NP is the number of pixels in the ROI and M represents the total number of ROI. Pixel calculation for the Euclidean distance between two ROI centroids are shown in equation (ii) where, Xreference, Yreference represents the centroid of the reference somatic ROI. g. Representative maximum projection image of a hippocampal astrocyte expressing GCaMP6f from a 300 s movie. This image was subjected to semi-automated data analyses using GECIquant, which separated somatic, wave and microdomain compartments.

Supplementary Figure 2 Testing ofGECIquant to measure the size of fluorescent beads.

Fluorescence images of 4 and 1 μm diameter beads (white) (Tetraspeck fluorescent microspheres size kit, Life Technologies catalog number T14792) along with their semi-automated detection images from GECIquant (red). All the beads were reliably detected and the measured areas of the beads in cross section were almost identical to the calculated areas, as shown in the graph.

Supplementary Figure 3 Testing ofGECIquant to draw around cellular shapes.

Fluorescence image of bovine pulmonary artery endothelial cells (Molecular Devices Fluocells prepared Slide Set catalog number F36924), which were subjected to semi-automated rendering with GECIquant (yellow). The program faithfully captured much of the complexity and accurately outlined the cells and their shapes, as shown in the black-and-white image and in the line profile graph across a region of the cell.

Supplementary Figure 4 Testing ofGECIquant to detect blinking quantum dots.

For the panels on the left, the distance scale bars represent 10 μm. Image of quantum dots acquired with a CCD camera. The lower image shows that each dot was faithfully captured as an ROI with GECIquant. A selection of these dots (in red) was used to generate plots of their intensity over time (traces in red). Clear fluctuations in quantum dot intensity due to blinking behavior were detected using GECIquant. The details of the quantum dots and imaging conditions have been reported already (Richler, E., Shigetomi, E. & Khakh, B. S. 2011 J Neurosci 31, 16716-16730).

Supplementary Figure 5 Average areas for somatic Ca2+ fluctuations as well as wave and microdomain Ca2+ fluctuations in WT and IP3R2–/–mice.

Supplementary Figure 6 Ca2+ waves and microdomains detected in astrocyte processes from WT and IP3R2–/– mice were located at similar distances when their centroid distances were measure relative to the centroid of the somata.

Supplementary Figure 7 Representative example of an isolated astrocyte expressing GCaMP6f from a specific set of experiments where we searched for and only recorded from astrocytes that were not adjacent to other GCaMP6f expressing astrocytes. Representative traces for the cell shown are plotted to the right.

Supplementary Figure 8 Studies with GCaMP6fflx mice crossed with GLAST-Cre/ERT2 mice.

a. Cartoon of the experiment, whereby GCaMP6fflx mice (JAX #024105) were crossed with GLAST-Cre/ERT2 mice. The GCaMP6fflx mice were donated to JAX by Dr. Hongkui Zeng (Allen Institute for Brain Sciences). All of the information on the genetics and genotyping is available at JAX by searching online for the mouse identification number, 024105. b. Two representative images of GCaMP6f immuno expression (green) in relation to S100β. GCaMP6f was expressed in ~40% of S100β positive astrocytes and of these ~33% were isolated from other GCaMP6f expressing astrocytes. We exploited this feature to image isolated astrocytes like that shown in c. The traces for astrocyte shown in c are shown on the right.

Supplementary Figure 9 Average data for Ca2+ fluctuation frequencies for somatic fluctuations as well as fluctuations in processes (waves and microdomains).

We analyzed the frequency of the three types of fluctuations using our original data set (Fig 1 of paper) and for isolated astrocytes from AAV2/5 microinjections (Supplementary Fig. 7) and from GCaMP6fflx mice (Supplementary Fig. 8). The frequencies were not statistically different for any type of fluctuation across these three data sets, indicating that our original data set (Fig. 1) contained an undetectable number of Ca2+ fluctuations from adjacent GCaMP6f expressing astrocytes (see Main text for details).

Supplementary Figure 10 Traces and average data for waves and microdomains from astrocyte processes from WT mice before, during and after applications of nominally Ca2+ free buffer.

These data are for ROIs that did not show changes upon applying Ca2+ free buffers. Data are from the same cells depicted in Fig. 3 of the main manuscript but are from regions in these cells that did not show changes in fluctuation frequency in Ca2+ free buffers. Data for regions that did show changes in fluctuation frequency in Ca2+ free buffers are shown in Fig 3 of the paper.

Supplementary Figure 11 Summary data for startle-evoked Ca2+ fluctuations in cortical astrocyte somata and processes showing a persistent late component in IP3R2–/–astrocyte processes.

a. The bar graphs show data at the indicated time epochs relative to baseline from experiments such as those shown in Fig. 7. The mice were startled at 225 s. Graphs are shown for WT and IP3R2–/– mice. b. As in a, but for data gathered from processes. The P value listed above each bar indicates the result of statistical comparison of that data set with the baseline period. The statistical test employed was a paired non-parametric Mann-Whitney test.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 1 and 2 (PDF 858 kb)

Supplementary Methods Checklist (PDF 388 kb)

Supplementary Code

GECIquant code and instructions (PDF 1807 kb)

Representative movie for spontaneous Ca2+ fluctuations in hippocampal astrocytes from WT mice. (AVI 14733 kb)

Representative movie for spontaneous Ca2+ fluctuations in hippocampal astrocytes from Ip3r2-/- mice. (AVI 19340 kb)

Representative movie for endothelin-evoked Ca2+ fluctuations in hippocampal astrocytes from WT mice. (AVI 21006 kb)

41593_2015_BFnn4001_MOESM118_ESM.avi

Representative movie for endothelin-evoked Ca2+ fluctuations in hippocampal astrocytes from Ip3r2-/- mice. (AVI 46045 kb)

Representative movie for spontaneous Ca2+ fluctuations in cortical astrocytes in vivo from WT mice. (AVI 16847 kb)

41593_2015_BFnn4001_MOESM120_ESM.avi

Representative movie for spontaneous Ca2+ fluctuations in cortical astrocytes in vivo from Ip3r2-/- mice. (AVI 17588 kb)

41593_2015_BFnn4001_MOESM121_ESM.wmv

Representative movie for behavioral startle response of a WT mouse in response to an air puff to the face. (WMV 1481 kb)

Representative movie for startle-evoked Ca2+ fluctuations in cortical astrocytes in vivo from WT mice. (AVI 43429 kb)

41593_2015_BFnn4001_MOESM123_ESM.avi

Representative movie for startle-evoked Ca2+ fluctuations in cortical astrocytes in vivo from WT mice after administration of prazosin. (AVI 44444 kb)

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Srinivasan, R., Huang, B., Venugopal, S. et al. Ca2+ signaling in astrocytes from Ip3r2−/− mice in brain slices and during startle responses in vivo. Nat Neurosci 18, 708–717 (2015). https://doi.org/10.1038/nn.4001

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