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.
Highlights
A distinct feature of astrocytes is their extensive gap junction coupling [1,2,3]
We further found that coupling is anisotropic depending on distance to the pyramidal cell layer, which correlated with regional differences of astrocyte morphology
Coupling length constants were similar in CA1 stratum radiatum and the molecular layer of the dentate gyrus and along its dorsal –ventral axis
Summary
A distinct feature of astrocytes is their extensive gap junction coupling [1,2,3]. It is thought to be essential for neuronal circuit function in many brain regions including the hippocampus. An inverse experimental approach is to load single astrocytes with a gap-junction-permeable dye, or other inert tracer, and visualize its spread into the astrocyte network after a defined time. Astrocyte networks of substantial size have been visualized by taking advantage of fast-diffusing tracers and/or ionotophoretic loading and allowing enough time for diffusion [4,9,20] In these networks, hundreds of cells are gap-junction-coupled to a single astrocyte in CA1 stratum radiatum [4,9,20]. Dye spread in astrocyte networks was first simulated to identify suitable parameters These were tested on dye coupling data obtained from the hippocampal CA1 stratum radiatum and the molecular layer of the dentate gyrus, where fluorescent dye was loaded into astrocytes via whole-cell patch clamp pipettes and later visualized by two-photon excitation fluorescence microscopy
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