Enhanced Astrocytic Ca²⁺Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

Abstract

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 Ca 2ϩ 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 Ca 2ϩ signals for 3 d and that the period of elevated glial Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ chelators selectively into astrocytes after status epilepticus. We demonstrate that the selective attenuation of glial Ca 2ϩ 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.

P2Y1R-Initiated, IP3R-Dependent Stimulation of Astrocyte Mitochondrial Metabolism Reduces and Partially Reverses Ischemic Neuronal Damage in Mouse

“…Presumably because of their proximity to blood vessels, our data indicate that tail-vein loading of TMRM preferentially labels astrocyte mitochondria. We demonstrated that TMRM-stained peri-nuclear mitochondria colocalize with cells that can be labeled with the astrocyte-specific dye sulforhodamine 101 (SR101) 15 as shown in Figures 4D and 4F. To correct for spectral overlap in Figures 4D and 4F, the TMRM image at 6 hours was subtracted from the SR101 plus TMRM image at 7 hours after RB photothrombosis.…”

“…The cortical loading of Fluo4-AM is highly specific for astrocyte labeling in vivo. 15 For mitochondrial membrane potential imaging, potential sensitive dye tetramethyl rhodamine methyl ester (TMRM) was dissolved in DMSO, diluted in sterilized PBS (100 mM) and tail-vein injected. Tetra-methyl rhodamine methyl ester is a lipophilic cation indicator that equilibrates across membranes in a Nernstian fashion.…”

The Role of Astrocytes in Epileptogenesis

“…In general, these observations suggest that widespread intercellular calcium waves are not likely to occur between astrocytes under physiological conditions in situ. However, a recent in vivo study showed that "Ca 2+ signals (…) can propagate through the cell and couple to adjacent astrocytes in the form of a Ca 2+ wave" (Ding et al;. These changes in [Ca 2+ ] i can act as a second messenger in various cellular process, including the release of gliotransmitters or the modification of gene expression patterns.…”

Integrated Brain Circuits: Neuron-Astrocyte Interaction in Sleep-Related Rhythmogenesis

“…Later studies demonstrated that this process can be observed in acute brain slices[71,72,73,74,75,76,77,78,79] and in vivo [80]. Astrocytes were later shown to release a number of chemical transmitters, including ATP[81,82,83,84,85], D-serine[86,87,88,89], TNF-alpha[90,91], and ANP[92], in a process that has recently been termed gliotransmission[93].…”