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Status epilepticus (SE), an unremitting seizure, is known to cause a variety of traumatic responses including delayed neuronal death and later cognitive decline. Although excitotoxicity has been implicated in this delayed process, the cellular mechanisms are unclear. Because our previous brain slice studies have shown that chemically induced epileptiform activity can lead to elevated astrocytic Ca2+ signaling and because these signals are able to induce the release of the excitotoxic transmitter glutamate from these glia, we asked whether astrocytes are activated during status epilepticus and whether they contribute to delayed neuronal death in vivo. Using two-photon microscopy in vivo, we show that status epilepticus enhances astrocytic Ca2+ signals for 3 d and that the period of elevated glial Ca2+ signaling is correlated with the period of delayed neuronal death. To ask whether astrocytes contribute to delayed neuronal death, we first administered antagonists which inhibit gliotransmission: MPEP [2-methyl-6-(phenylethynyl)pyridine], a metabotropic glutamate receptor 5 antagonist that blocks astrocytic Ca2+ signals in vivo, and ifenprodil, an NMDA receptor antagonist that reduces the actions of glial-derived glutamate. Administration of these antagonists after SE provided significant neuronal protection raising the potential for a glial contribution to neuronal death. To test this glial hypothesis directly, we loaded Ca2+ chelators selectively into astrocytes after status epilepticus. We demonstrate that the selective attenuation of glial Ca2+ signals leads to neuronal protection. These observations support neurotoxic roles for astrocytic gliotransmission in pathological conditions and identify this process as a novel therapeutic target.

Although almost all GFAP(+) cells in primary astrocyte cultures show functional beta-adrenergic (beta-AR) and metabotropic purinergic (P2Y) receptors, the fewer studies on astrocytes in situ have shown that a much smaller proportion express these same receptor-mediated activities. Here we show, by multiplex single cell RT-PCR, that 44% of freshly isolated, GFAP(+) astrocytes (FIAs) from the CA1 of P8-12 rat hippocampus always co-express beta-adrenergic receptor mRNA subtypes with metabotropic ATP receptor mRNA subtypes (P2Y1, P2Y2 or P2Y4). We also found that beta2 mRNA was the dominant beta-AR subtype expressed. P2Y1 mRNA always co-expresses with either one or two subtypes of P2U-like receptor (P2Y2 or P2Y4) mRNAs. Immunocytochemical studies showed a similar percentage of all FIAs expressed beta-AR and P2Y1 protein (54% and 52%, respectively), as for the mRNAs (46% and 65%, respectively). The staining of hippocampal sections for beta-AR or P2Y1 receptor plus GFAP shows that there are quite numerous, scattered star-shaped GFAP(+) astrocytes in the CA1 region of P9-10 rat hippocampus that stained positive for either of these receptors. These data show that astrocytes in situ express, and to a large extent likely co-express, beta-AR and P2Y receptors.