Activation of Neuronal NMDA Receptors Triggers Transient ATP-Mediated Microglial Process Outgrowth

Abstract

Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron-microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron-microglial communication mediated by ATP.

Effects of the ecto-ATPase apyrase on microglial ramification and surveillance reflect cell depolarization, not ATP depletion

“…[ATP]e, had no effect on microglial surveillance, ramification or membrane potential (5,25,26,33 (36,45). This may involve the generation of ADP from ATP, to activate P2Y12 receptors, but could also involve ecto-ATPase mediated generation of adenosine which acts on microglial A3-type adenosine receptors (41,45,46).…”

“…Any rise of extracellular ATP which might occur following ecto-ATPase block is also expected to induce changes of microglial surveillance and ramification (16,36). To try to detect this, we imaged microglial motility by two-photon microscopy as in Fig.…”

“…This demonstrates that ARL-67156 effectively blocks the hydrolysis of exogenously applied ATP, thus potentiating its action, whereas no detectable turnover of ATP occurs in baseline conditions (Fig. 2b, c) suggesting that endogenous ATP levels are very low.Any rise of extracellular ATP which might occur following ecto-ATPase block is also expected to induce changes of microglial surveillance and ramification (16,36). To try to detect this, we imaged microglial motility by two-photon microscopy as in Fig In summary, chemiluminescence, electrophysiological and imaging experiments exclude the existence of a baseline extracellular ATP concentration high enough to drive microglial purinergic signalling.…”

Receptors, Ion Channels, and Signaling Mechanisms Underlying Microglial Dynamics

“…Expression of P2Y 12 receptor protein on the surface of ramified microglia in vivo (34) is particularly enriched at the tips of the leading processes during chemotaxis (37). Subsequently, as activation of microglia occurs, P2Y 12 receptors are down-regulated, until barely detectable 24 h after the initial damage (29,34).…”

“…Instead of directly communicating to microglia, neurotransmitter signaling may affect microglia motility indirectly by modulating extracellular levels of nucleotides such as ATP, which evoke changes in morphology and process velocity (37,50,75). Indeed, in microglia in situ, only extracellular nucleotides such as ATP, ADP, and UDP evoke inward membrane currents through ionotropic P2X (presumably P2X 4 and P2X 7 ) receptors and outward K ϩ currents via G protein-coupled (P2Y) purinergic receptors (23, 31-33, 35, 50), whereas other neurotransmitters (glutamate, GABA, glycine, acetylcholine, dopamine, and noradrenaline) generate no current (23,32,33,59,60,75).…”