The central hypothesis of excitotoxicity is that excessive stimulation of neuronal NMDA-sensitive glutamate receptors is harmful to neurons and contributes to a variety of neurological disorders. Glial cells have been proposed to participate in excitotoxic neuronal loss, but their precise role is defined poorly. In this in vivo study, we show that NMDA induces profound nuclear factor B (NF-B) activation in Müller glia but not in retinal neurons. Intriguingly, NMDA-induced death of retinal neurons is effectively blocked by inhibitors of NF-B activity. We demonstrate that tumor necrosis factor ␣ (TNF␣) protein produced in Müller glial cells via an NMDA-induced NF-B-dependent pathway plays a crucial role in excitotoxic loss of retinal neurons. This cell loss occurs mainly through a TNF␣-dependent increase in Ca 2ϩ -permeable AMPA receptors on susceptible neurons. Thus, our data reveal a novel non-cell-autonomous mechanism by which glial cells can profoundly exacerbate neuronal death following excitotoxic injury.

Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs).Tumor necrosis factor ␣ (TNF␣) signaling has been linked to RGC damage, however, the mechanism by which TNF␣ promotes neuronal death remains poorly defined. Using an in vivo rat glaucoma model, we show that TNF␣ is upregulated by Müller cells and microglia/macrophages soon after induction of ocular hypertension. Administration of XPro1595, a selective inhibitor of soluble TNF␣, effectively protects RGC soma and axons. Using cobalt permeability assays, we further demonstrate that endogenous soluble TNF␣ triggers the upregulation of Ca 2ϩ -permeable AMPA receptor (CP-AMPAR) expression in RGCs of glaucomatous eyes. CP-AMPAR activation is not caused by defects in GluA2 subunit mRNA editing, but rather reflects selective downregulation of GluA2 in neurons exposed to elevated eye pressure. Intraocular administration of selective CP-AMPAR blockers promotes robust RGC survival supporting a critical role for non-NMDA glutamate receptors in neuronal death. Our study identifies glia-derived soluble TNF␣ as a major inducer of RGC death through activation of CP-AMPARs, thereby establishing a novel link between neuroinflammation and cell loss in glaucoma.

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Ca 2+ and ⁄ or Zn 2+ entry into neurons and glial cells is often a key step driving the processes of neurodevelopment and disease. As a result, a major pre-occupation of many neuroscientists has been in tracking down when and where nervous tissues express ion channels with appreciable divalent ion permeability. The cobalt (Co 2+ )-staining technique is one of the few techniques that allow a snapshot of the entire neuronal circuit, and selectively labels cells expressing divalent-permeable ion channels with a brown-black precipitate. Despite this, its use has been remarkably limited in the past decade. Reluctance to employ this approach has largely been related to an earlier concern with obtaining a reliable and reproducible means of visualizing transported Co 2+ . Here we show that recent advances have resolved these issues, opening this straightforward and valuable technique to a much larger neuroscience audience.