Astrocytes, but Not Neurons, Exhibit Constitutive Activation of P2X7 Receptors in Mouse Acute Cortical Slices under Non-stimulated Resting Conditions

Abstract

We previously demonstrated that the P2X7 receptor (P2X7R), a purinergic receptor, expressed by mouse cultured cortical astrocytes is constitutively activated without any exogenous stimulus, differing from the case of neurons. It is well known that astrocytic morphology differs between in vitro and in vivo situations, implying different functionalities. Brain acute slices are widely accepted as an in vitro experimental system that reflects in vivo cell conditions better than in vitro cell culture ones. We examined whether astrocytic P2X7Rs exhibited constitutive activation in mouse cortical slices. In acute cortical slices, P2X7R-immunoreactivity was detected in both glial fibrillary acidic protein-immunopositive astrocytes and microtubuleassociated protein 2-immunopositive neurons. Astrocytic, but not neuronal, spontaneous uptake of propidium iodide, an indicator of P2X7R channel/pore activity, was inhibited by representative antagonists of P2X7R, but they had no effect on the uptake by astrocytes in membrane-permeabilized fixed slices. These findings indicate that astrocytes, but not neurons, in acute cortical slices exhibit constitutive activation of P2X7Rs under non-stimulated resting conditions as in the case of cell culture systems.Key words P2X7 receptor; astrocyte; mouse cortical slice; neuron Among purine receptors, in particular, P2X7 receptors (P2X7Rs) have unique characteristics, and are activated by high concentrations (ca. mM) of ATP, resulting in the formation of a non-selective cationic channel/pore, through which large molecules of up to 900 Da can pass. Through activation of P2X7Rs in neurons, cell death is induced via calcium influx, followed by related cellular events, while activation of P2X7Rs expressed by microglia, brain-resident immune cells, causes generation and/or release of reactive oxygen species (ROS) and pro-inflammatory cytokines. These responses help keep the brain environment clean, while under severe pathological conditions, neuronal injury is exacerbated.

Detection and Functional Evaluation of the P2X7 Receptor in hiPSC Derived Neurons and Microglia-Like Cells

“…Interestingly, a number of articles reported the presence of P2X7R in astrocytes ( Kamatsuka et al, 2014 ; Gao et al, 2017 ; Khan et al, 2019 ), including a work on iPSC-derived cells ( Kesavan et al, 2021 ). Some of these reports suggest that the astrocytic P2X7R indirectly regulates neuronal activity ( Khan et al, 2019 ).…”

Circadian ATP Release in Organotypic Cultures of the Rat Suprachiasmatic Nucleus Is Dependent on P2X7 and P2Y Receptors

“…Gap junctional networks of astrocytes are more common and generally more extensive, whereas little evidence is available for gap junctions among SCN neurons (Welsh and Reppert, 1996 ; Colwell, 2000 ). The P2X7R protein is also well-documented to be expressed on astrocytes (Narcisse et al, 2005 ; Sperlágh et al, 2006 ; Hamilton et al, 2008 ; Kamatsuka et al, 2014 ; Zhao et al, 2016 ), whereas its expression in neurons is still questionable (Illes et al, 2017 ). Our present study also shows the expression of the P2X7R protein in SCN astrocytes.…”

Spontaneous and multifaceted ATP release from astrocytes at the scale of hundreds of synapses

“…We found transient increases in extracellular ATP within restricted subcellular regions of astrocytes (Fig. 2C-E; area: 124.16 (37.28, 381.6) µm 2 ; full width at half maximum (FWHM): 6 (5,14) sec; peak ∆F/F0: 51.4% (41.7%, 65.8%); median (interquartile ranges [IQRs]) in 12 events from 30 cells in 6 mice). Such transient and localized ATP events were also found in acute brain slices without stimulation (Fig.…”

“…Not only is ATP released from astrocytes in response to neuronal inputs, but Koizumi et al found tetrodotoxin-insensitive ATP release in neuron-glia co-culture (12). Tonic ATP release was reported in brain slice preparations (13)(14)(15), but it was unclear whether astrocytes release ATP spontaneously or in response to the basal neuronal firing. Investigating basal ATP release activity will provide a further perspective on how astrocytes are involved in the neuron network function.…”