Spatial properties of astrocyte gap junction coupling in the rat hippocampus

Abstract

Gap junction coupling enables astrocytes to form large networks. Its strength determines how easily a signalling molecule diffuses through the network and how far a locally initiated signal can spread. Changes of coupling strength are well-documented during development and in response to various stimuli. Precise quantification of coupling is needed for studying such modifications and their functional consequences. We therefore explored spatial properties of astrocyte coupling in a model simulating dye loading of single astrocytes. Dye spread into the astrocyte network could be characterized by a coupling length constant and coupling anisotropy. In experiments, the fluorescent marker Alexa Fluor 594 was used to measure these parameters in CA1 and dentate gyrus of the rat hippocampus. Coupling did not differ between regions but showed a temperature-dependence, partially owing to changes of intracellular diffusivity, detected by measuring coupling length constants but not the more variable cell counts of dye-coupled astrocytes. We further found that coupling is anisotropic depending on distance to the pyramidal cell layer, which correlated with regional differences of astrocyte morphology. This demonstrates that applying these new analytical approaches provides useful quantitative information on gap junction coupling and its heterogeneity.

Keywords: astrocytes; dentate gyrus; gap junction coupling; hippocampus; spatial properties; temperature.

Figure 1.

Changes of astrocyte coupling affect the spread of dye injected into a single astrocyte. Diffusion of dye from a simulated patched astrocyte (pipette) in a three-dimensional network was simulated in several conditions (left column, coupling represented by arrows, coupling strength by colour intensity). A single plane of the simulated network containing the patched cell is displayed (right column). Each circle represents a single cell. Dye concentration is colour-coded with bright colours corresponding to high concentrations. (a) Homogeneous coupling between astrocytes results in symmetric dye concentrations around the patched cell. Dye concentrations in gap-junction-coupled astrocytes decrease with increasing distance. (b) Enhancement of coupling between astrocytes increases the spread of injected dye into the network. (c) Increasing coupling along a specific axis, i.e. anisotropic coupling, results in enhanced dye spread in the astrocyte network along this axis (y-axis in this example). (d) Introducing a barrier with limited coupling (e.g. at an anatomical border, dashed line) increases dye spread along and away from that barrier. The shift of the centre of mass (CM, white cross) away from the patched astrocyte reflects the asymmetry of the coupled astrocyte network under these conditions (compared with panel a). (Online version in colour.)

Figure 2.

Visualization and quantification of astrocyte coupling in CA1 stratum radiatum. (a) Astrocytes were held in the whole-cell patch clamp configuration and dialysed with the fluorescent dye Alexa Fluor 594 via the patch pipette (ip, top left panel). X–y–z image stacks were obtained after 20 min and analysed. Right panel: sample slice of an image stack containing the patched astrocyte (dashed lines, patch pipette). (b) Somatic fluorescence intensity of gap-junction-coupled cells was normalized to the patched cell and corrected for depth within the slice (same recording as in (a), 36 cells). The normalized intensity (I) decreases monoexponentially with distance (d) from the patched cell (dashed line, monoexponential fit). The coupling length constant C_λ, I(d) = I₀ exp(−d/C_λ) was used to quantify fluorescent dye spread into the gap-junction-coupled network. (c) Astroglial networks were studied at RT (n = 5) and at 34°C (n = 7). C_λ was temperature-sensitive (unpaired t-test, p = 0.0011) whereas manually determined cell counts were not (p = 0.83, error bars are s.e.m.). (d) Coefficients of variation indicate a lower variability of C_λ analysis (error bars indicate 5–95% confidence intervals obtained by bootstrap analysis). (e) A C_λ similar to CA1 stratum radiatum was observed in the molecular layer of the dentate gyrus (n = 18, p = 0.46, unpaired t-test, dashed line for comparison with CA1). FRAP of EGFP expressed by astrocytes was used to gauge intracellular diffusivity. Bleaching was induced by high power line scanning (f, left panel, dashed line). Fluorescence recovery occurred when unbleached EGFP diffused into the imaged region while the laser shutter was closed (1 s, f, right panel, g, blue bars indicate laser exposure). The bleached fraction of fluorescence ΔF_B and the recovered fraction ΔF_R were measured and used to quantify FRAP = ΔF_R/ΔF_B (g). FRAP was significantly stronger at 34°C than at RT (h_i, n = 10 and 12 for RT and 34°C respectively, unpaired t-test, p = 0.048). (Online version in colour.)

Figure 3.

Astrocyte coupling is anisotropic in CA1 stratum radiatum. (a) Fluorescence intensities (I_i) of gap-junction-coupled cells at positions (x_i, y_i) relative to the patched cell (single experiment, grey values represent I_i, dark is high I_i). The cloud of gap-junction-coupled cells was rotated around the z-axis such that the x-axis is parallel to the stratum pyramidale (str. pyr.). Note the localization of strongly coupled cells (dark) along the y-axis (orthogonal to the str. pyr.). Coupling anisotropy (C_A) was calculated as defined (inset). (b) Coupling anisotropy depends on distance of the patched astrocyte from stratum pyramidale (n = 8, dashed line, linear fit). Arrowhead indicates the data point corresponding to the example shown in (a). Analysis of position-dependent astrocyte morphology was performed on EGFP-expressing astrocytes as illustrated (c, sample astrocyte). Optical cross sections of astrocytes were background-corrected, normalized to somatic fluorescence and subdivided into sectors parallel to the pyramidal cell layer (red, X) and perpendicular (blue, Y). Orientation preferences were quantified by calculating ratios of sector areas (A_Y/A_X) and their average fluorescence intensities (F_Y/F_X). Also see §2 Material and methods. (d) Area ratios showed a directional preference for the y-axis (A_Y/A_X > 1.0), i.e. perpendicular to str. pyr, close to the pyramidal cell layer (n = 19) and in more distal astrocytes (n = 17, p < 0.05, one-population t-tests, p = 0.78, two-population t-test). (e) Average fluorescence intensities ratios showed similar directional preferences only in astrocytes distal to the str. pyr. (F_Y/F_X > 1.0, same n as in d, p < 0.02, one and two-populations t-tests). (Online version in colour.)