Astrocytes as regulators of synaptic function: a quest for the Ca2+ master key

Abstract

The emerging role of astrocytes in neural communication represents a conceptual challenge. In striking contrast to the rapid and highly space- and time-constrained machinery of neuronal spike propagation and synaptic release, astroglia appear slow and imprecise. Although a large body of independent experiments documents active signal exchange between astrocytes and neurons, some genetic models have raised doubts about the major Ca2+ -dependent molecular mechanism routinely associated with release of "gliotransmitters." A limited understanding of astrocytic Ca2+ signaling and the imperfect compatibility between physiology and experimental manipulations seem to have contributed to this conceptual bottleneck. Experimental approaches providing mechanistic insights into the diverse mechanisms of intra-astrocyte Ca2+ signaling on the nanoscale are needed to understand Ca2+ -dependent astrocytic function in vivo. This review highlights limitations and potential advantages of such approaches from the current methodological perspective.

Figure 1

A decrease in external Na⁺ or an increase in internal Na⁺ elevates intracellular Ca²⁺ in Bergmann glia, potentially implicating the sodium-calcium Ca²⁺ exchanger (NCX) mechanism. (A) Fluorescence intensity image (left, fura-2 AM, λ_x = 380 nm) of the cerebellar slice. White circle, region of interest (Bergmann glia cell body); PN, Purkinje cell. (B, C) Intracellular Ca²⁺ concentration reversibly increases in response to application of an Na⁺-free bath solution (B) or to intracellular whole-cell loading of bulk-loaded cells with a high Na⁺ solution (C). Modified from Kirischuk and others (1997).

Figure 2

An example (diffusion computation) illustrating the effect of Ca²⁺ indicators on Ca²⁺ signals inside a small cellular compartment and the relationship between fluorescence recordings and the underlying kinetics of free Ca²⁺. (A) Diagram depicting the modeled Ca²⁺ store (yellow) with Ca²⁺ channels (nine 50-nm channel clusters depicted by dots) facing the cell cytoplasm (~1-μm wide compartment). (B) Simulated Ca²⁺ entry time course; the total entry corresponds to ~10⁴ Ca²⁺ ions (which is approximately twice the spike-evoked presynaptic Ca²⁺ influx in small central synapses [Koester and Sakmann 2000], consistent with the estimate for presynaptic Ca²⁺ store release reported earlier [Scott, Lalic, and others 2008]). (C) Concentration profiles at five time points during Ca²⁺ entry. Top row: free Ca²⁺ landscapes, with no Ca²⁺ buffers present, shown within the 10-nm layer adjacent to the Ca²⁺ store membrane. Middle row: same as top row but in the presence of 0.2 mM Fluo-4 (typical imaging protocol). Bottom row: concentration profile of Ca²⁺-bound Fluo-4 averaged over a 100-nm depth adjacent to the Ca²⁺ store interface, [CaF]_Σ, to reflect recorded fluorescence. False color scale bars as shown. Models adapted and modified from Scott, Lalic, and others (2008) and Henneberger and others (2010) were constructed and run online using Virtual Cell (VCell) version 4.7 at the National Resource for Analysis and Modeling, National Institutes of Health, Connecticut.

Figure 3

Flat ultrathin processes of astrocytes provide favorable conditions for chemical compartmentalization (extended lifetime of concentration hotspots): a Monte Carlo example. (A) A diagram depicting three scatters of 500 small molecules released, from a single-point source, into three representative cellular compartments of an astrocyte, 5 ms postrelease: the soma (represented by a 10-μm-wide sphere, denoted 1), a large primary process (1-μm-wide cylinder, 2), and an ultrathin flat process (50-nm-thick planar slab, 3). In the scatters, individual diffusing molecules are color-coded to report their distance to the release site (as shown by the color scale bar). A dense cluster of molecules near the release site can clearly be seen in the flat process; intracellular diffusion coefficient, D = 0.1 μm²/ms. (B) Time course of the number of molecules (the effective local concentration) remaining within a 200-nm-wide, 50-nm-high “hotspot nanodomain” centered at the release point, for the three loci depicted in A. The life span of the hotspot at the ultrathin flat process appears 5- to 10-fold of that at the other two sites. Modeling environment modified and adapted from Zheng and others 2008.

Figure 4

Fluorescence decay of OGB-1 (O6806; Molecular Probes, Eugene, OR) at various calcium concentrations (calibration kit C3008MP; Molecular Probes), as indicated. Two-photon excitation at ${\lambda }_{\mathrm{x}}^{2\mathrm{p}}=800\mathrm{nm}$ (~100-fs pulses; laser: MaiTai, Spectra Physics, Mountain View, CA), attenuated beam power 9 mW; 20× objective (XLUMPlanFL; Olympus, Tokyo, Japan). Modified from Gersbach and others (2009).