Abstract
‘Calcium signalling’ is the ubiquitous response of glial cells to multiple extracellular stimuli. The primary mechanism of glial calcium signalling is by release of calcium from intracellular stores of the endoplasmic reticulum (ER). Replenishment of ER Ca2+ stores relies on store-operated calcium entry (SOCE). However, despite the importance of calcium signalling in glial cells, little is known about their mechanisms of SOCE. Here, we investigated SOCE in glia of the mouse optic nerve, a typical CNS white matter tract that comprises bundles of myelinated axons and the oligodendrocytes and astrocytes that support them. Using quantitative RT-PCR, we identified Orai1 channels, both Stim1 and Stim2, and the transient receptor potential M3 channel (TRPM3) as the primary channels for SOCE in the optic nerve, and their expression in both astrocytes and oligodendrocytes was demonstrated by immunolabelling of optic nerve sections and cultures. The functional importance of SOCE was demonstrated by fluo-4 calcium imaging on isolated intact optic nerves and optic nerve cultures. Removal of extracellular calcium ([Ca2+]o) resulted in a marked depletion of glial cytosolic calcium ([Ca2+]i), which recovered rapidly on restoration of [Ca2+]o via SOCE. 2-aminoethoxydiphenylborane (2APB) significantly decreased SOCE and severely attenuated ATP-mediated calcium signalling. The results provide evidence that Orai/Stim and TRPM3 are important components of the ‘calcium toolkit’ that underpins SOCE and the sustainability of calcium signalling in white matter glia.
Highlights
Glial cells respond to a wide range of extracellular stimuli, including neurotransmitters, by a rise in cytosolic calcium ([Ca2+]i) termed ‘Ca2+ signalling’, which is the basis of ‘glial excitability’ (Khakh and McCarthy 2015)
The results indicate that IP3 receptor 2 (IP3R2) is the primary endoplasmic reticulum (ER) Ca2+ release channel in optic nerve glia, with significantly lower expression of IP3R1 (p < 0.01, unpaired t test), whilst ryanodine receptor 3 (RyR3) was barely detectable; RyR3 is the main subtype expressed in the brain and RyR1 and RyR2 were not included in the Mouse Neuronal Ion Channels RT2 ProfilerTM assay
The most prominent expression of Orai1 in optic nerve sections was in rows of oligodendroglial cell somata identified by expression of the proteolipid protein (PLP)-DsRed reporter (Fig. 2ai–iii), which was confirmed by the generation of a colocalization channel that identifies the individual voxels in which the red (PLP-DsRed) and green (Orai1 immunostaining) channels overlap with the same intensity (Fig. 2aiv), as described previously (Hawkins and Butt 2013)
Summary
Glial cells respond to a wide range of extracellular stimuli, including neurotransmitters, by a rise in cytosolic calcium ([Ca2+]i) termed ‘Ca2+ signalling’, which is the basis of ‘glial excitability’ (Khakh and McCarthy 2015). Glial calcium signalling is mainly dependent on inositol 1,4,5-triphosphate (IP3)-mediated release of C a2+ from the endoplasmic reticulum (ER), which depends on the replenishment of intracellular stores through store-operated calcium entry (SOCE) (Verkhratsky and Parpura 2014). The two main plasmalemmal channels mediating SOCE are transient receptor potential (TRP) channels, comprising six subfamilies, and calcium release-activated calcium channels (CRAC), which are formed of Orai, the plasmalemma spanning channels and STIM (Stromal Interaction Molecule), which function as ER C a2+ sensors (Verkhratsky and Parpura 2014). Depletion of ER Ca2+ triggers STIM to interact with plasmalemmal SOCE channels, both Orai and TRP (Huang et al 2006; Mercer et al 2006), resulting in influx of Ca2+ from the extracellular milieu into. We show that astrocytes and oligodendrocytes express both Orai/Stim and TRP channels in the mouse optic nerve, a typical CNS white matter tract
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