Axons and astrocytes release ATP and glutamate to evoke calcium signals in NG2‐glia

Abstract

NG2-glia are an abundant population of cells in the adult CNS that make up a novel glial cell type. Here, we have examined calcium signals in NG2-glia identified by expression of the fluorescent protein DsRed under the control of the NG2 promoter in the white matter of the mouse optic nerve. We focused on mice aged postnatal day (P)12-16, after the main period of oligodendrocyte generation. Using fluo-4 and fura-2 calcium imaging in isolated intact nerves, we show that glutamate and ATP evoke Ca(2+) signals in NG2-glia in situ, acting on AMPA-type glutamate receptors and P2Y(1) and P2X(7) purine receptors; NMDA evoked a weak Ca(2+) signal in a small proportion of NG2-glia. We show that axonal action potentials and mechanical stimulation of astrocytes effect the release of glutamate and ATP to act on NG2-glia; ATP alone evokes robust Ca(2+) signals, whereas glutamate did not unless AMPA receptor desensitization was blocked with cyclothiazide. We identify the precise contacts that NG2-glia form with axons at nodes of Ranvier, and the intricate bipartite sheaths formed between the processes of NG2-glia and astrocytes. In addition, we provide evidence that NG2-glia express synaptophysin, indicating they have mechanisms for transmitting as well as receiving signals. This study places NG2-glia within a neuron-glial network, and identifies roles for glutamate and ATP in communication with astrocytes as well as axons.

Intertwining extracellular nucleotides and their receptors with Ca2+in determining adult neural stem cell survival, proliferation and final fate

“…The first in vivo evidence that, upon release during axonal action potential, ATP evokes rapid and transient Ca 2þ rises in single OPCs was attested by confocal calcium imaging on optic nerves from a transgenic reporter mouse line in which adult NG2-glia were identified by the expression of the red fluorescent protein DsRed [85]. The predominant mechanism was the activation of metabotropic P2Y 1 R, because response was abolished by the pre-incubation with the P2Y 1 R antagonist MRS2179, but also the P2X7R induced significant calcium increases [85].…”

“…The predominant mechanism was the activation of metabotropic P2Y 1 R, because response was abolished by the pre-incubation with the P2Y 1 R antagonist MRS2179, but also the P2X7R induced significant calcium increases [85]. The Ca 2þ signals increased in both amplitude and duration with increasing stimulus strength, indicating that responses in these cells matched with axonal activity; however, it was clearly demonstrated that ATP is not only released by axons during action potentials, but also comes from astrocytes, in response to axonal activity through vesicles, reverse transport, hemichannels and the P2X7R [86].…”

“…under pathological conditions. Thus, low levels of ATP would activate a transient self-limiting P2Y 1 R-mediated release of Ca 2þ , whereas at higher levels of signalling, Ca 2þ influx through P2X7 would prolong the ATP-evoked Ca 2þ signals for hundreds of seconds [85]. However, this fascinating hypothesis has not been demonstrated yet.…”

Neuroprotective roles of the P2Y2 receptor

“…Extracellular ATP release significantly increases process extension toward an injury site for resting or activated microglia [148]. Astrocytes under pathological conditions also can release ATP to activate P2Rs in neighboring cells [18,188,189], and inflammation in vivo can elevate extracellular ATP levels sufficiently to activate P2 receptors [18].…”

NF‐Y‐dependent regulation of glutamate receptor 4 expression and cell survival in cells of the oligodendrocyte lineage

“…OPC express all four subunits, although GluA1 represents a minor contributor compared with GluA2–4 in vitro (Hossain, Liu, Fragoso, & Almazan, 2014; Itoh et al, 2002), and appears to be entirely absent from OPC in vivo (Kougioumtzidou et al, 2017). Activation of OPC AMPAR provokes an influx of Ca 2+ (Ge et al, 2006; Haberlandt et al, 2011; Hamilton, Vayro, Wigley, & Butt, 2010; Itoh et al, 2002) that can mediate excitotoxic injury in vitro (Alberdi, Sanchez‐Gomez, Marino, & Matute, 2002; Deng, Rosenberg, Volpe, & Jensen, 2003; Li & Stys, 2000; Sanchez‐Gomez & Matute, 1999). These observations suggest that a substantial number of OPC AMPAR lack GluA2 subunits since inclusion of this subunit limits the permeability of AMPAR to Ca 2+ (Geiger et al, 1995; Hollmann, Hartley, & Heinemann, 1991).…”