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Background: N-Methyl-D-aspartate receptors (NMDARs) play fundamental roles in basic brain functions such as excitatory neurotransmission and learning and memory processes. Their function is largely regulated by factors released by glial cells, including the coagonist d-serine. We investigated whether the activation of microglial CX3CR1 induces the release of factors that modulate NMDAR functions., Methods: We recorded the NMDAR component of the field excitatory postsynaptic potentials (NMDA-fEPSPs) elicited in the CA1 stratum radiatum of mouse hippocampal slices by Shaffer collateral stimulation and evaluated D-serine content in the extracellular medium of glial primary cultures by mass spectrometry analysis., Results: We demonstrated that CX3CL1 increases NMDA-fEPSPs by a mechanism involving the activity of the adenosine receptor type A2 (A2AR) and the release of the NMDAR coagonist D-serine. Specifically (1) the selective A2AR blocker 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) and the genetic ablation of A2AR prevent CX3CL1 action while the A2AR agonist 5-(6-amino-2-(phenethylthio)-9H-purin-9-yl)-N-ethyl-3,4-dihydroxytetrahydrofuran-2-carboxamide (VT7) mimics CX3CL1 effect, and (2) the selective blocking of the NMDAR glycine (and D-serine) site by 5,7-dicholorokynurenic acid (DCKA), the enzymatic degradation of D-serine by D-amino acid oxidase (DAAO) and the saturation of the coagonist site by D-serine, all block the CX3CL1 effect. In addition, mass spectrometry analysis demonstrates that stimulation of microglia and astrocytes with CX3CL1 or VT7 increases D-serine release in the extracellular medium., Conclusions: CX3CL1 transiently potentiates NMDAR function though mechanisms involving A2AR activity and the release of D-serine.

Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-alpha, interleukin-1-beta, and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A(1) receptors (A(1)R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of adenosine from microglia. In this study, we show that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 as reducing adenosine levels in hippocampal cultures, by adenosine deaminase treatment, strongly impairs CX3CL1-mediated neuroprotection. Furthermore, we confirm the predominant role of microglia in mediating the neuronal effects of CX3CL1, because the selective depletion of microglia from hippocampal cultures treated with clodronate-filled liposomes causes the complete loss of effect of CX3CL1. We also show that hippocampal neurons obtained from A(1)R(-/-) mice are not protected by CX3CL1 whereas A(2A)R(-/-) neurons are. The requirement of functional A(1)R for neuroprotection is not unique for CX3CL1 as A(1)R(-/-) hippocampal neurons are not rescued from Glu-induced cell death by other neurotrophins such as brain-derived neurotrophic factor and erythropoietin, which are fully active on wt neurons.

The chemokine fractalkine (CX(3)CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX(3)CR1, CX(3)CL1 exerts both neuroprotection against glutamate (Glu) toxicity and neuromodulation of the glutamatergic synaptic transmission in hippocampal neurons. Using cultured hippocampal neuronal cell preparations, obtained from CX(3)CR1(-/-) (CX(3)CR1(GFP/GFP)) mice, we report that these same effects are mimicked by exposing neurons to a medium conditioned with CX(3)CL1-treated mouse microglial cell line BV2 (BV2-st medium). Furthermore, CX(3)CL1-induced neuroprotection from Glu toxicity is mediated through the adenosine receptor 1 (AR(1)), being blocked by neuronal cell preparations treatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a specific inhibitor of AR(1), and mimicked by both adenosine and the specific AR(1) agonist 2-chloro-N(6)-cyclopentyladenosine. Similarly, experiments from whole-cell patch-clamped hippocampal neurons in culture, obtained from CX(3)CR1(+/+) mice, show that CX(3)CL1-induced depression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA-) type Glu receptor-mediated current (AMPA-current), is associated with AR(1) activity being blocked by DPCPX and mimicked by adenosine. Furthermore, BV2-st medium induced a similar AMPA-current depression in CX(3)CR1(GFP/GFP) hippocampal neurons and this depression was again blocked by DPCPX. We also report that CX(3)CL1 induced a significant release of adenosine from microglial BV2 cells, as measured by HPLC analysis. We demonstrate that (i) CX(3)CL1, along with AR(1), are critical players for counteracting Glu-mediated neurotoxicity in the brain and (ii) AR(1) mediates neuromodulatory action of CX(3)CL1 on hippocampal neurons.