Glutamate receptors on myelinated spinal cord axons: II. AMPA and GluR5 receptors.

OBJECTIVE: Glutamate receptors, which play a major role in the physiology and pathology of central nervous system gray matter, are also involved in the pathophysiology of white matter. However, the cellular and molecular mechanisms responsible for excitotoxic damage to white matter elements are not fully understood. We explored the roles of AMPA and GluR5 kainate receptors in axonal Ca(2+) deregulation. METHODS: Dorsal column axons were loaded with a Ca(2+) indicator and imaged in vitro using confocal microscopy. RESULTS: Both AMPA and a GluR5 kainate receptor agonist increased intraaxonal Ca(2+) in myelinated rat dorsal column fibers. These responses were inhibited by selective antagonists of these receptors. The GluR5-mediated Ca(2+) increase was mediated by both canonical (ie, ionotropic) and noncanonical (metabotropic) signaling, dependent on a pertussis toxin-sensitive G protein/phospholipase C-dependent pathway, promoting Ca(2+) release from inositol triphosphate-dependent stores. In addition, the GluR5 response was reduced by intraaxonal NO scavengers. In contrast, GluR4 AMPA receptors operated via Ca(2+)-induced Ca(2+) release, dependent on ryanodine receptors, and unaffected by NO scavengers. Neither pathway depended on L-type Ca(2+) channels, in contrast with GluR6 kainate receptor action.1 Immunohistochemistry confirmed the presence of GluR4 and GluR5 clustered at the surface of myelinated axons; GluR5 coimmunoprecipitated with nNOS and often colocalized with neuronal nitric oxide synthase clusters on the internodal axon. INTERPRETATION: Central myelinated axons express functional AMPA and GluR5 kainate receptors, and can directly respond to glutamate receptor agonists. These glutamate receptor-dependent signaling pathways promote an increase in intraaxonal Ca(2+) levels potentially contributing to axonal degeneration.

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

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 Ca(2+) deregulation. METHODS: Dorsal column axons were loaded with a Ca(2+) 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 [Ca(2+)]. This Ca(2+) 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 Ca(2+) channels: ryanodine, nimodipine, or nifedipine blocked the agonist-induced Ca(2+) increase. Also, GluR6 stimulation induced intraaxonal production of nitric oxide (NO), which greatly enhanced the Ca(2+) response: quenching of NO with intraaxonal (but not extracellular) scavengers, or inhibition of neuronal NO synthase with intraaxonal Nomega-nitro-L-arginine methyl ester, blocked the Ca(2+) 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 Ca(2+) increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma colocalized with neuronal NO synthase and Ca(v)1.2. 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 Ca(2+) 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.