Purinergic Receptors Mediate Two Distinct Glutamate Release Pathways in Hippocampal Astrocytes

Tommaso Fellin,Tullio Pozzan,Giorgio Carmignoto

doi:10.1074/jbc.m510679200

Tommaso Fellin, Tullio Pozzan + Show 1 more

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https://doi.org/10.1074/jbc.m510679200

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Abstract

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.

Highlights

Synaptic NMDARs2 and triggering episodic, inward currents characterized by slow kinetics (SICs) [10, 11]
Purinergic Receptor Stimulation Evokes Transient and Sustained Activation of Neuronal NMDA Receptors—In hippocampal slices in the absence of extracellular Mg2ϩ and in the presence of 1 ␮M TTX, BzATP (100 ␮M), a potent unselective agonist of P2X7 receptor (P2X7R) [24, 25], evokes a complex response in 20 of 39 (51%) CA1 pyramidal neurons consisting of episodic, transient inward currents and a slowly developing tonic current (Fig. 1, A and A1)
These results clearly indicate that slice perfusion with BzATP triggers the release of glutamate that evokes in CA1 pyramidal neurons both transient and sustained activation of the NMDARs

Summary

Introduction

Synaptic NMDARs2 and triggering episodic, inward currents characterized by slow kinetics (SICs) [10, 11]. In support of the role of the P2X7R, the release of glutamate from murine-cultured astrocytes triggered by purinergic receptor agonists has been described [16] to be (i) larger upon stimulation with BzATP than with ATP (the latter being a weak P2X7R agonist), (ii) greatly potentiated in divalent ion-free medium, i.e. a condition that favors P2X7R openings, and (iii) blocked by P2X7R antagonists These results from cultured astrocytes clearly indicate that under selected experimental conditions the P2X7R allows the efflux of glutamate into the extracellular space, no such evidence exists for astrocytes in situ. Results obtained in patch clamp recordings from neurons and astrocytes of acute hippocampal slices allow us to conclude that P2X7R activation is not involved in the episodic glutamate release that triggers SICs. In contrast, a P2X7-like receptor, possibly expressed in astrocytes and neurons, contributes significantly to increasing the extracellular concentration of glutamate, in particular when extracellular Ca2ϩ decreases to very low levels

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