Astrocyte calcium waves propagate proximally by gap junction and distally by extracellular diffusion of ATP released from volume-regulated anion channels

Yuki Fujii,Shohei Maekawa,Mitsuhiro Morita

doi:10.1038/s41598-017-13243-0

Abstract

Wave-like propagation of [Ca2+]i increases is a remarkable intercellular communication characteristic in astrocyte networks, intercalating neural circuits and vasculature. Mechanically-induced [Ca2+]i increases and their subsequent propagation to neighboring astrocytes in culture is a classical model of astrocyte calcium wave and is known to be mediated by gap junction and extracellular ATP, but the role of each pathway remains unclear. Pharmacologic analysis of time-dependent distribution of [Ca2+]i revealed three distinct [Ca2+]i increases, the largest being in stimulated cells independent of extracellular Ca2+ and inositol 1,4,5-trisphosphate-induced Ca2+ release. In addition, persistent [Ca2+]i increases were found to propagate rapidly via gap junctions in the proximal region, and transient [Ca2+]i increases were found to propagate slowly via extracellular ATP in the distal region. Simultaneous imaging of astrocyte [Ca2+]i and extracellular ATP, the latter of which was measured by an ATP sniffing cell, revealed that ATP was released within the proximal region by volume-regulated anion channel in a [Ca2+]i independent manner. This detailed analysis of a classical model is the first to address the different contributions of two major pathways of calcium waves, gap junctions and extracellular ATP.

Highlights

Deeper understanding of the mechanisms underlying the spatio-temporal diversity and complexity of [Ca2+]i increases in astrocytes is crucial for exploring the physiological and pathological functions of this glial cell population
Transient [Ca2+]i increases, which are characteristic of astrocyte [Ca2+]i increases in the distal region, were induced in HEK293 + GCaMP2 cells by astrocyte calcium waves and ATP, as well as in astrocytes by ATP (Fig. 5c), indicating that the temporal dynamics of GCaMP2-expressing HEK293 cells is comparable to that of Fura2-loaded astrocytes
The mean distances between stimulated astrocytes and HEK293 + GCaMP2 cells showing 25–50% increases in [Ca2+]i and the amplitudes and area under the curve (AUC) of their [Ca2+]i increases were not affected by the presence of astrocytes (Fig. 6e and f). These results indicate that the GCaMP2 responses do not decline in the distal region, and are not affected by the presence of astrocytes. These findings suggest that the transient [Ca2+]i increases in the distal region is attributed to the diffusion of ATP released in the proximal region

Summary

Introduction

Deeper understanding of the mechanisms underlying the spatio-temporal diversity and complexity of [Ca2+]i increases in astrocytes is crucial for exploring the physiological and pathological functions of this glial cell population. Various pharmacologic and physical stimuli have been found to induce [Ca2+]i increases propagating between astrocytes in cell cultures[1,2], in brain slices[3,4], and in other in vivo preparations[5,6] These calcium waves are regarded as transmitting physiologic and pathologic signals within the brain, because they influence the activities of adjacent neurons[7,8], microglia[9], and endothelial cells[10]. The present analysis of this classical model pharmacologically and by using an ATP sniffing cell revealed distinct [Ca2+]i increases during calcium waves. Study was designed to assess the distinct contributions of gap junction and extracellular ATP and the ATP release mechanism in calcium waves, revealing novel aspects of the diverse and complicated dynamics of astrocyte [Ca2+]i

Methods

Results

Conclusion