ABSTRACT

The nonphosphorylated neurofilament marker SMI-32 stains motor neurons in spinal cord slices and stains a subset of cultured spinal neurons neurons"], which have a morphology consistent with motor neurons identified in vitro: large cell body, long axon, and extensive dendritic arborization. They are found preferentially in ventral spinal cord cultures, providing further evidence that large SMI-32(ϩ) neurons are indeed motor neurons, and SMI-32 staining often colocalizes with established motor neuron markers (including acetylcholine, calcitonin gene-related peptide, and peripherin). Additionally, choline acetyltransferase activity (a frequently used index of the motor neuron population) and peripherin(ϩ) neurons share with large SMI-32(ϩ) neurons an unusual vulnerability to AMPA /kainate receptor-mediated injury. Kainateinduced loss of these motor neuron markers is Ca 2ϩ -dependent, which supports a critical role of Ca 2ϩ ions in this injury. Raising extracellular Ca 2ϩ exacerbates injury, whereas removal of extracellular Ca 2ϩ is protective. A basis for this vulnerability is provided by the observation that most peripherin(ϩ) neurons, like large SMI-32(ϩ) neurons, are subject to kainate-stimulated Co 2ϩ uptake, a histochemical stain that identifies neurons possessing Ca 2ϩ -permeable AMPA /kainate receptor-gated channels. Finally, of possibly greater relevance to the slow motor neuronal degeneration in diseases, both large SMI-32(ϩ) neurons and peripherin(ϩ) neurons are selectively damaged by prolonged (24 hr) low-level exposures to kainate (10 M) or to the glutamate reuptake blocker L-transpyrrolidine-2,4-dicarboxylic acid (100 M). During these lowlevel kainate exposures, large SMI-32(ϩ) neurons showed higher intracellular Ca 2ϩ concentrations than most spinal neurons, suggesting that Ca 2ϩ ions are also important in this more slowly evolving injury.

The reason for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is primarily unknown. A possible factor is the expression by motor neurons of Ca 2ϩ -permeable AMPA/kainate channels, which may permit rapid Ca 2ϩ influx in response to synaptic receptor activation. However, other subpopulations of central neurons, most notably forebrain GABAergic interneurons, consistently express large numbers of these channels but do not degenerate in ALS. Indeed, when subjected to identical excitotoxic exposures, motor neurons were more susceptible than GABAergic neurons to AMPA/kainate receptor-mediated neurotoxicity. Microfluorimetric studies were performed to examine the basis for the difference in vulnerability. First, AMPA or kainate exposures appeared to trigger substantial mitochondrial Ca 2ϩ loading in motor neurons, as indicated by a sharp increase in intracellular Ca 2ϩ after addition of the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl hydrazone (FCCP) after the agonist exposure. The same exposures caused little mitochondrial Ca 2ϩ accumulation in GABAergic cortical neurons. Subsequent experiments examined other measures of mitochondrial function to compare sequelae of AMPA/kainate receptor activation between these populations. Brief exposure to either AMPA or kainate caused mitochondrial depolarization, assessed using tetramethylrhodamine ethylester, and reactive oxygen species (ROS) generation, assessed using hydroethidine, in motor neurons. However, these effects were only seen in the GABAergic neurons after exposure to the nondesensitizing AMPA receptor agonist kainate. Finally, addition of either antioxidants or toxins (FCCP or CN Ϫ ) that block mitochondrial Ca 2ϩ uptake attenuated AMPA/kainate receptor-mediated motor neuron injury, suggesting that the mitochondrial Ca 2ϩ uptake and consequent ROS generation are central to the injury process.

The widespread neuronal injury that results after brief activation of highly Ca2+-permeable NMDA channels may, in large part, reflect mitochondrial Ca2+ overload and the consequent production of injurious oxygen radicals. In contrast, AMPA/kainate receptor activation generally causes slower toxicity, and most studies have not found evidence of comparable oxygen radical production. Subsets of central neurons, composed mainly of GABAergic inhibitory interneurons, express AMPA/kainate channels that are directly permeable to Ca2+ ions. Microfluorometric techniques were performed by using the oxidation-sensitive dye hydroethidine (HEt) to determine whether the relatively rapid Ca2+ flux through AMPA/kainate channels expressed on GABAergic neurons results in oxygen radical production comparable to that triggered by NMDA. Consistent with previous studies, NMDA exposures triggered increases in fluorescence in most cultured cortical neurons, whereas high K+ (50 mM) exposures (causing depolarization-induced Ca2+ influx through voltage-sensitive Ca2+ channels) caused little fluorescence change. In contrast, kainate exposure caused fluorescence increases in a distinct subpopulation of neurons; immunostaining for glutamate decarboxylase revealed the responding neurons to constitute mainly the GABAergic population. The effect of NMDA, kainate, and high K+ exposures on oxygen radical production paralleled the effect of these exposures on intracellular Ca2+ levels when they were monitored with the low-affinity Ca2+-sensitive dye fura-2FF, but not with the high-affinity dye fura-2. Inhibition of mitochondrial electron transport with CN- or rotenone almost completely blocked kainate-triggered oxygen radical production. Furthermore, antioxidants attenuated neuronal injury resulting from brief exposures of NMDA or kainate. Thus, as with NMDA receptor activation, rapid Ca2+ influx through Ca2+-permeable AMPA/kainate channels also may result in mitochondrial Ca2+ overload and consequent injurious oxygen radical production.

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