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.

Complex interplay between glutamate receptors and intracellular Ca2+ stores during ischaemia in rat spinal cord white matter.

Electrophysiological recordings of propagated compound action potentials (CAPs) and axonal Ca(2+) measurements using confocal microscopy were used to study the interplay between AMPA receptors and intracellullar Ca(2+) stores in rat spinal dorsal columns subjected to in vitro combined oxygen and glucose deprivation (OGD). Removal of Ca(2+) or Na(+) from the perfusate was protective after 30 but not 60 min of OGD. TTX was ineffective with either exposure, consistent with its modest effect on ischaemic depolarization. In contrast, AMPA antagonists were very protective, even after 60 min of OGD where 0Ca(2+) + EGTA perfusate was ineffective. Similarly, blocking ryanodine receptor-mediated Ca(2+) mobilization from internal stores (0Ca(2+) + nimodipine or 0Ca(2+) + ryanodine), or inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release (block of group 1 metabotropic glutamate receptors with 1-aminoindan-1,5-dicarboxylic acid, inhibition of phospholipase C with U73122 or IP(3) receptor block with 2APB; each in 0Ca(2+)) were each very protective, with the combination resulting in virtually complete functional recovery after 1 h OGD (97 +/- 32% CAP recovery versus 4 +/- 6% in artificial cerebrospinal fluid). AMPA induced a rise in Ca(2+) concentration in normoxic axons, which was greatly reduced by blocking ryanodine receptors. Our data therefore suggest a novel and surprisingly complex interplay between AMPA receptors and Ca(2+) mobilization from intracellular Ca(2+) stores. We propose that AMPA receptors may not only allow Ca(2+) influx from the extracellular space, but may also significantly influence Ca(2+) release from intra-axonal Ca(2+) stores. In dorsal column axons, AMPA receptor-dependent mechanisms appear to exert a greater influence than voltage-gated Na(+) channels on functional outcome following OGD.

Spatiotemporal distribution of spectrin breakdown products induced by anoxia in adult rat optic nerve in vitro.

Hypoxic/ischemic and traumatic injury to central nervous system myelinated axons is heavily dependent on accumulation of Ca ions in the axoplasm, itself promoted by Na influx from the extracellular space. Given the high density of nodal Na channels, we hypothesized that nodes of Ranvier might be particularly vulnerable to Ca overload and subsequent damage, as this is the expected locus of maximal Na influx. Adult rat optic nerves were exposed to in vitro anoxia and analyzed immunohistochemically for the presence of spectrin breakdown. Cleavage of spectrin became detectable between 15 and 30 mins of anoxia, and increased homogeneously along the lengths of fibers; localized breakdown was not observed at nodes of Ranvier at any time point analyzed. Spectrin breakdown was also found in glial processes surrounding axons. Confocal imaging of axoplasmic Ca also revealed a gradual and nonlocalized increase as anoxia progressed, without evidence of Ca 'hot-spots' anywhere along the axons at any time between 0 and 30 mins of anoxic exposure in vitro. Calculations of Ca diffusion rates indicated that even if Ca entered or was released focally in axons, this ion would diffuse rapidly into the internodes and likely produce diffuse injury by activating Ca-dependent proteases. Western blot analysis for voltage-gated Na channel protein revealed that key functional proteins such as these are also degraded by anoxia/ischemia. Thus, proteolysis of structural and functional proteins will conspire to irreversibly injure central axons and render them nonfunctional, eventually leading to transection, degradation, and Wallerian degeneration.

Depolarization-induced Ca2+ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors.

The mechanisms of Ca(2+) release from intracellular stores in CNS white matter remain undefined. In rat dorsal columns, electrophysiological recordings showed that in vitro ischemia caused severe injury, which persisted after removal of extracellular Ca(2+); Ca(2+) imaging confirmed that an axoplasmic Ca(2+) rise persisted in Ca(2+)-free perfusate. However, depletion of Ca(2+) stores or reduction of ischemic depolarization (low Na(+), TTX) were protective, but only in Ca(2+)-free bath. Ryanodine or blockers of L-type Ca(2+) channel voltage sensors (nimodipine, diltiazem, but not Cd(2+)) were also protective in zero Ca(2+), but their effects were not additive with ryanodine. Immunoprecipitation revealed an association between L-type Ca(2+) channels and RyRs, and immunohistochemistry confirmed colocalization of Ca(2+) channels and RyR clusters on axons. Similar to "excitation-contraction coupling" in skeletal muscle, these results indicate a functional coupling whereby depolarization sensed by L-type Ca(2+) channels activates RyRs, thus releasing damaging amounts of Ca(2+) under pathological conditions in white matter.

Aberrant chloride transport contributes to anoxic/ischemic white matter injury.

Rundown of ionic gradients is a central feature of white matter anoxic injury; however, little is known about the contribution of anions such as Cl-. We used the in vitro rat optic nerve to study the role of aberrant Cl- transport in anoxia/ischemia. After 30 min of anoxia (NaN3, 2 mm), axonal membrane potential (V(m)) decreased to 42 +/- 11% of control and to 73 +/- 11% in the presence of tetrodotoxin (TTX) (1 microm). TTX + 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid disodium salt (500 microm), a broad spectrum anion transport blocker, abolished anoxic depolarization (95 +/- 8%). Inhibition of the K-Cl cotransporter (KCC) (furosemide 100 microm) together with TTX was also more effective than TTX alone (84 +/- 14%). The compound action potential (CAP) area recovered to 26 +/- 6% of control after 1 hr anoxia. KCC blockade (10 microm furosemide) improved outcome (40 +/- 4%), and TTX (100 nm) was even more effective (74 +/- 12%). In contrast, the Cl- channel blocker niflumic acid (50 microm) worsened injury (6 +/- 1%). Coapplication of TTX (100 nm) + furosemide (10 microm) was more effective than either agent alone (91 +/- 9%). Furosemide was also very effective at normalizing the shape of the CAPs. The KCC3a isoform was localized to astrocytes. KCC3 and weaker KCC3a was detected in myelin of larger axons. KCC2 was seen in oligodendrocytes and within axon cylinders. Cl- gradients contribute to resting optic nerve membrane potential, and transporter and channel-mediated Cl- fluxes during anoxia contribute to injury, possibly because of cellular volume changes and disruption of axo-glial integrity, leading to propagation failure and distortion of fiber conduction velocities.

Active and passive membrane properties of rat sympathetic preganglionic neurones innervating the adrenal medulla.

The intravascular release of adrenal catecholamines is a fundamental homeostatic process mediated via thoracolumbar spinal sympathetic preganglionic neurones (AD-SPN). To understand mechanisms regulating their excitability, whole-cell patch-clamp recordings were obtained from 54 retrogradely labelled neonatal rat AD-SPN. Passive membrane properties included a mean resting membrane potential, input resistance and time constant of -62 +/- 6 mV, 410 +/- 241 MOmega and 104 +/- 53 ms, respectively. AD-SPN were homogeneous with respect to their active membrane properties. These active conductances included transient outward rectification, observed as a delayed return to rest at the offset of the membrane response to hyperpolarising current pulses, with two components: a fast 4-AP-sensitive component (A-type conductance), contributing to the after-hyperpolarisation (AHP) and spike repolarisation; a slower prolonged Ba(2+)-sensitive component (D-like conductance). All AD-SPN expressed a Ba(2+)-sensitive instantaneous inwardly rectifying conductance activated at membrane potentials more negative than around -80 mV. A potassium-mediated, voltage-dependent sustained outward rectification activated at membrane potentials between -35 and -15 mV featured an atypical pharmacology with a component blocked by quinine, reduced by low extracellular pH and arachidonic acid, but lacking sensitivity to Ba(2+), TEA and intracellular Cs(+). This quinine-sensitive outward rectification contributes to spike repolarisation. Following block of potassium conductances by Cs(+) loading, AD-SPN revealed the capability for autorhythmicity and burst firing, mediated by a T-type Ca(2+) conductance. These data suggest the output capability is dynamic and diverse, and that the range of intrinsic membrane conductances expressed endow AD-SPN with the ability to generate differential and complex patterns of activity. The diversity of intrinsic membrane properties expressed by AD-SPN may be key determinants of neurotransmitter release from SPN innervating the adrenal medulla. However, factors other than active membrane conductances of AD-SPN must ultimately regulate the differential ratio of noradrenaline (NA) versus adrenaline (A) release secreted in response to various physiological and environmental demands.