Motor neurons are selectively vulnerable to AMPA/kainate receptor-mediated injury in vitro

Abstract

The nonphosphorylated neurofilament marker SMI-32 stains motor neurons in spinal cord slices and stains a subset of cultured spinal neurons ["large SMI-32(+) 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(+) 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(+) neurons share with large SMI-32(+) neurons an unusual vulnerability to AMPA/kainate receptor-mediated injury. Kainate-induced loss of these motor neuron markers is Ca2+-dependent, which supports a critical role of Ca2+ ions in this injury. Raising extracellular Ca2+ exacerbates injury, whereas removal of extracellular Ca2+ is protective. A basis for this vulnerability is provided by the observation that most peripherin(+) neurons, like large SMI-32(+) neurons, are subject to kainate-stimulated Co2+ uptake, a histochemical stain that identifies neurons possessing Ca2+-permeable AMPA/kainate receptor-gated channels. Finally, of possibly greater relevance to the slow motor neuronal degeneration in diseases, both large SMI-32(+) neurons and peripherin(+) neurons are selectively damaged by prolonged (24 hr) low-level exposures to kainate (10 microM) or to the glutamate reuptake blocker L-trans-pyrrolidine-2,4-dicarboxylic acid (100 microM). During these low-level kainate exposures, large SMI-32(+) neurons showed higher intracellular Ca2+ concentrations than most spinal neurons, suggesting that Ca2+ ions are also important in this more slowly evolving injury.

Fig. 1.

Motor neuron markers label large ventral horn neurons in spinal cord slices. Photomicrographs show slices of adult spinal cord stained (as described) with antibody to SMI-32 (A), peripherin (B), CGRP (C), ACh (D), or ChAT (E). For each marker, thebottom panel shows high-power (400×) detail of neurons indicated by arrowhead in top panel. Scale bar, 300 μm.

Fig. 2.

Motor neuron markers label neurons in spinal cord cultures. Photomicrographs show dissociated spinal cord cultures under phase-contrast microscopy (A, arrows show the typical appearance of putative motor neurons before staining) or after immunostaining (as described) with antibody to SMI-32 (B,arrow shows axonal branch), peripherin (C), CGRP (D), ACh (E), or ChAT (F). Note the extensive morphological detail provided by SMI-32 and peripherin staining (generally showing an extensive dendritic arborization as well as a single axon-like process that often extends for several millimeters). In comparison, relatively little morphological detail is provided by the other stains (D–F, arrowheadsindicate representative stained neurons). Scale bar, 100 μm.

Fig. 3.

Top left. Most peripherin(+) neurons are also SMI-32(+). Photomicrographs show a culture double-stained for peripherin and SMI-32 under visible light (A, peripherin staining) and under fluorescence microscopy (B, SMI-32 labeling). A majority of peripherin(+) neurons were found to express SMI-32 immunoreactivity. Other double-labeling studies showed most ACh- or CGRP-immunoreactive neurons to be SMI-32(+) (see Results). Scale bar, 50 μm.

Fig. 4.

Kainate injury to peripherin(+) and large SMI-32(+) neurons is Ca²⁺-dependent: morphological appearance. Spinal cord cultures were exposed to kainate (100 μm for 10 min), in either the presence of 1.8 mm Ca²⁺ (A, C) or the absence of Ca²⁺ (B, D), and were stained 24 hr later for either peripherin (A, B) or SMI-32 (C, D). Although these submaximal exposures in the presence of Ca²⁺ caused severe damage to many (but not all) labeled neurons, removal of Ca²⁺during the exposure resulted in good preservation of most neurons. Scale bar, 100 μm.

Fig. 5.

Kainate injury to the spinal motor neuronal population is Ca²⁺-dependent. A, Kainate injury to large SMI-32(+) and peripherin(+) neurons is Ca²⁺-dependent. Cultures were exposed to kainate (100 μm for 10 min) in the presence of the indicated Ca²⁺ concentration. Overall neuronal loss and loss of large SMI-32(+) or peripherin(+) neurons were evaluated 20–24 hr later (as described in Materials and Methods). Values represent mean ± SEM compiled from four experiments;n = 9–15 cultures per condition. & indicates labeled neuronal loss significantly different from labeled neuronal loss seen in the 1.8 mmCa²⁺ condition (p < 0.05 by two-tailed t test). # indicates labeled neuronal loss significantly different from total neuronal loss after the same exposure (p < 0.01 by two-tailedt test). B, Kainate-induced loss of spinal cord ChAT activity is Ca²⁺-dependent. Cultures were exposed to kainate (100 μm for 15 min) in the presence of the indicated Ca²⁺ concentration. Overall neuronal loss and loss of ChAT activity were assessed 20–24 hr later (as described). Values represent mean ± SEM compiled from 10 experiments; n = 27–36 cultures for each condition.& indicates ChAT activity loss significantly different from that seen in the 10 mm Ca²⁺condition (p < 0.01 by two-tailed ttest). # indicates ChAT activity loss significantly different from overall neuronal injury after the same exposure (p < 0.01 by two-tailed t test).

Fig. 7.

Motor neurons are selectively vulnerable to slow, excitotoxic injury. A, Large SMI-32(+) neurons and peripherin(+) neurons are selectively damaged by chronic kainate exposures. Cultures were exposed to the indicated kainate concentration for 20–24 hr, followed by evaluation of injury to the overall neuronal population and to the labeled neuronal population. Values represent mean ± SEM compiled from three to four representative experiments;n = 10–12 cultures per condition. # indicates labeled neuronal loss significantly different from total neuronal loss after the same exposure (p < 0.01 by two-tailedt test). B, Large SMI-32(+) neurons are selectively damaged by chronic exposure to the glutamate reuptake blocker PDC. Cultures were exposed for 24 hr to PDC (100 μm) alone or with the addition of glutamate receptor antagonists as indicated (each at 10 μm), followed by evaluation of damage to the overall neuronal population and to large SMI-32(+) neurons. Values represent mean ± SEM compiled from three to four representative experiments; n = 9–12 cultures per condition.# indicates large SMI-32(+) neuronal loss significantly different from total neuronal loss after the same exposure (p < 0.01 by two-tailed t test).& indicates large SMI-32(+) neuronal loss significantly different from that obtained in the 100 μm PDC condition (p < 0.01 by two-tailed ttest).

Fig. 9.

Large SMI-32(+) neurons show substantial [Ca²⁺]_i elevations during low-level kainate exposures. A, Distribution of [Ca²⁺]_i values in spinal neurons (528 neurons from eight experiments) 8–10 min after the addition of kainate (10 μm). All large SMI-32(+) neurons have higher than average [Ca²⁺]_i values.B, Time course of [Ca²⁺]_i changes. [Ca²⁺]_i levels are plotted in individual neurons before and for 9 min after addition of kainate (10 μm). Of the 44 neurons shown, the 2 neurons represented by solid lines are large SMI-32(+) neurons.