Glutamate receptors on myelinated spinal cord axons: I. GluR6 kainate receptors

Abstract

Objective The deleterious effects of glutamate excitotoxicity are well described for central nervous system gray matter. Although overactivation of glutamate receptors also contributes to axonal injury, the mechanisms are poorly understood. Our goal was to elucidate the mechanisms of kainate receptor–dependent axonal Ca²⁺ deregulation. Methods Dorsal column axons were loaded with a Ca²⁺ indicator and imaged in vitro using confocal laser‐scanning microscopy. Results Activation of glutamate receptor 6 (GluR6) kainate receptors promoted a substantial increase in axonal [Ca²⁺]. This Ca²⁺ accumulation was due not only to influx from the extracellular space, but a significant component originated from ryanodine‐dependent intracellular stores, which, in turn, depended on activation of L‐type Ca²⁺ channels: ryanodine, nimodipine, or nifedipine blocked the agonist‐induced Ca²⁺ increase. Also, GluR6 stimulation induced intraaxonal production of nitric oxide (NO), which greatly enhanced the Ca²⁺ response: quenching of NO with intraaxonal (but not extracellular) scavengers, or inhibition of neuronal NO synthase with intraaxonal Nω‐nitro‐L‐arginine methyl ester, blocked the Ca²⁺ increase. Loading axons with a peptide that mimics the C‐terminal PDZ binding sequence of GluR6, thus interfering with the coupling of GluR6 to downstream effectors, greatly reduced the agonist‐induced axonal Ca²⁺ increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma colocalized with neuronal NO synthase and Ca_v1.2. Interpretation Myelinated spinal axons express functional GluR6‐containing kainate receptors, forming part of novel signaling complexes reminiscent of postsynaptic membranes of glutamatergic synapses. The ability of such axonal “nanocomplexes” to release toxic amounts of Ca²⁺ may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur. Ann Neurol 2009