Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps - PubMed (original) (raw)

Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps

Alexander M B Reeves et al. J Neurosci. 2011.

Abstract

Calcium signaling has been studied in astrocyte cell bodies using bulk loading of calcium indicator dyes, and astrocytes are known to display intracellular calcium transients. An assumption in recent data on the neuronal impact of somatic astrocyte calcium transients has been that bulk loading reflects signaling in relevant astrocyte compartments such as processes. We assessed bulk loading using Sholl analysis (Sholl, 1953) of astrocytes loaded with common calcium indicator dyes and compared these data with Sholl analysis of astrocyte morphology. In the CA1 region of the hippocampus from rats, we found that bulk loading of calcium indicator dyes only reports on calcium signals within the soma and in the most proximal processes, leaving ∼90% of the area of an astrocyte and its extensive processes unsampled. By using morphological reconstructions as "maps" after the imaging session, we present simple procedures that remedy these shortfalls and permit reliable detection of calcium transients in distal astrocyte processes. The data thus reveal limitations in the interpretation of astrocyte calcium imaging data gathered with bulk loading and provide refinements to minimize these shortcomings.

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Figures

Figure 1.

Figure 1.

Differences between calcium signals in astrocyte somata and processes. A, Confocal image of an astrocyte from the stratum radiatum region of a hippocampal slice (P16) loaded with Fluo-4AM. The soma and two processes were visible in this optical plane and regions of interest were selected in the soma (S) and processes (P #1–P #4). Scale bar, 15 μm. B, dF/F traces for the regions shown in A plotted over time. C, Left graph, peak dF/F values for calcium signals measured in the somata and processes of astrocytes. The three distributions were significantly different (Kolmogorov–Smirnov test; bin size = 0.025 dF/F). Right graph, distribution of calcium transient half-widths. Kolmogorov–Smirnov analysis showed that the S and Pi as well as S and Pii transients differed significantly in duration, whereas the Pi and Pii transients did not. D, Scatter plot of peak dF/F values for Pii type transients plotted against their occurrence time relative to the first somatic signal in the cells. E, Flattened 2D image from a 3D reconstruction of a GFAP-labeled astrocyte used to schematically illustrate Sholl analysis. Circles of increasing diameter from the center of the soma were drawn in 1 μm intervals and the number of intersections with the cell measured (red and green dots). F, Exemplar Sholl plot for the astrocyte shown in E.

Figure 2.

Figure 2.

Morphological Sholl analysis of astrocytes. A, Representative maximum projection images from confocal z series for astrocytes loaded with Fluo-4AM, labeled with SR101, or dialyzed with Alexa-488. Scale bars, 15 μm. B, 2D representations of astrocytes loaded with calcium indicator dyes (OGB-AM, Fluo-4AM) and those labeled with SR101 and GFAP, as well as an astrocyte dialyzed with Alexa-488. In the Alexa-488 image, the approximate position of the patch pipette is shown with a red asterisk. C, Average Sholl plots from reconstructions such as those shown in B. D, Average Alexa-488 Sholl plot (orange line and axis) superimposed on the number of calcium transients measured at increasing distances from the soma (black bars) for Fluo-4-loaded astrocytes (Fig. 1; n = 15 astrocytes and 353 transients).

Figure 3.

Figure 3.

Morphological maps improve detection of calcium transients in astrocyte processes. A, Left, Representative image of an astrocyte loaded with Fluo-4AM (acquired in a single confocal plane). Right, Representative image of the same astrocyte dialyzed with Alexa-488 after the calcium imaging session. Scale bars, 15 μm. B, 2D representation of another astrocyte loaded with Fluo-4 (from one confocal plane; green). A 2D representation of the same astrocyte is shown for the z series after loading with Alexa-488 via the patch pipette (orange). C, Top, Average Sholl plots from reconstructions such as those shown in B. Bottom, Number of calcium transients measured at increasing distances from the soma in 10 μm bins. The green bars correspond to transients measured in one confocal plane from the Fluo-4 image. The orange bars represent transients detected using 2D morphological maps of the Alexa-488 z series: the sampled region was larger, as shown by the circles in B. D, Exemplar calcium transient traces for the two different conditions of analysis indicated.

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