Astrocyte calcium signaling: from observations to functions and the challenges therein

Abstract

We provide an overview of recent progress on the study of astrocyte intracellular Ca(2+) signaling. We consider the methods that have been used to monitor astrocyte Ca(2+) signals, the various types of Ca(2+) signals that have been discovered (waves, microdomains, and intrinsic fluctuations), the approaches used to broadly trigger and block Ca(2+) signals, and, where possible, the proposed and demonstrated physiological roles for astrocyte Ca(2+) signals within neuronal microcircuits. Although important progress has been made, we suggest that further detailed work is needed to explore the biophysics and molecular mechanisms of Ca(2+) signaling within entire astrocytes, including their fine distal extensions, such as processes that interact spatially with neurons and blood vessels. Improved methods are also needed to mimic and block molecularly defined types of Ca(2+) signals within genetically specified populations of astrocytes. Moreover, it will be essential to study astrocyte Ca(2+) activity in vivo to distinguish between pharmacological and physiological activity, and to study Ca(2+) activity in situ to rigorously explore mechanisms. Once methods to reliably measure, mimic, and block specific astrocyte Ca(2+) signals with high temporal and spatial precision are available, researchers will be able to carefully explore the correlative and causative roles that Ca(2+) signals may play in the functions of astrocytes, blood vessels, neurons, and microcircuits in the healthy and diseased brain.

Figure 1.

Electron and light microscopy analysis of astrocyte processes. (A) Orthogonal slices through a confocal volume of a dye-filled astrocyte. Box counting fractal analysis was performed to determine the local D_F across the astrocyte territory. The vast majority of the territory has a D_F of ∼1.7, extending from the soma to the periphery, as shown by the colored scale bar. This implies that astrocytes are approximately equally complex throughout their territories. (B) Electron microscopic volume of an entire Golgi-impregnated astrocyte. Three perisomatic (yellow) and three peripheral (cyan; one of them is behind a yellow one in the view shown) subvolumes (∼680 µm³ each) have been extracted to determine surface area and volume of astrocyte branchlets. (C) Close-up views of astrocyte processes in perisomatic and peripheral subvolumes demonstrating dense network of fine branchlets in both regions. These analyses reveal that astrocytes have thousands of branches that are the primary sites for interactions with neurons. However, as discussed in the text, Ca²⁺ signals have not been studied in the fine structures. (From Shigetomi et al. 2013a; reproduced, with permission, from the authors as well as by the Creative Commons license for reuse in the public domain.)

Figure 2.

Expression of cytosolic GCaMP3 and Lck-GCaMP3 throughout astrocytes. (A,B) Cartoons of differences between cytosolic and membrane-targeted GECIs. (C) Schematic illustrates the protocol for AAV 2/5 microinjections into the hippocampus. The right-hand image shows the expression of Lck-GCaMP3 throughout the hippocampus, and D shows expression within the stratum radiatum region for Lck-GCaMP3 and cyto-GCaMP3. (E) Representative images showing GFAP and GCaMP3 staining for the stratum radiatum region from control mice that received no AAVs and those that received AAV2/5 Lck-GCaMP3. The image is a zoomed-out view. AAV, adeno-associated virus; gc, genome copy; PM, plasma membrane. (From Shigetomi et al. 2013a; reproduced, with permission, from the authors as well as by the Creative Commons license for reuse under the public domain.)