ArticleDifferent receptors mediate motor neuron death induced by short and long exposures to excitotoxicity
Introduction
The excitatory neurotransmitter glutamate can induce neuronal death through activation of two classes of ionotropic receptors, N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate (KA) receptors. Brief, intense periods of glutamate receptor activation are damaging to neurons and may contribute to brain and spinal cord injury after acute insults, including stroke and trauma 7, 18. This acute excitotoxicity seems to be mediated mostly by Ca2+ entry into the cells via the NMDA receptors [14], although non-NMDA receptors may also play an important role [13]. Long-term exposures to a relatively low concentration of glutamate may also cause cell death and this excitotoxic mechanism seems to play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS) [19]. ALS is a neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. Although the cause of the selective motor neuron degeneration is unknown, a substantial amount of circumstantial evidence indicates that excitotoxicity is involved both in vivo 9, 10 and in organotypic and dissociated spinal cord cultures 2, 5, 20. This excitotoxicity during ALS seems to be mediated by Ca2+ entry via Ca2+-permeable AMPA receptors.
The Ca2+-permeability of the AMPA receptor is determined by the edited GluR2 [11]. If GluR2 is present in the receptor complex, the channel’s permeability for divalent ions is drastically reduced. We have shown that the high Ca2+-permeability of the AMPA receptors in motor neurons compared to the ones in dorsal horn neurons is not due to a difference in the editing of the GluR2 mRNA [24] and that it is not caused by the absence or lower relative level of the GluR2 subunit in motor neurons 22, 24. At this moment it is not clear which factor(s) determine the higher Ca2+ permeability of the AMPA receptors present in the cultured motor neurons.
To contribute to the understanding of the pathogenesis of ALS, we investigated which glutamate receptors are involved in excitotoxic motor neuron death in culture. Neuronal excitotoxic death can be induced by short exposure to high concentrations of neurotransmitter, or by exposure to a lower concentration of this neurotransmitter during prolonged periods of time. These two paradigms may be mediated through different receptors, as mentioned above. Therefore, both the effect of the concentration of the excitotoxin and of the time of exposure were investigated.
Section snippets
Cell cultures
Motor neurons and glial cells were prepared as described before 23, 25. Briefly, spinal cords were dissected from 14-day-old Wistar rat embryos in Hanks’ Balanced Salt Solution (HBSS) and stripped of meninges and dorsal root ganglia (E0 being the day of sperm-positivity). The ventral cord was cut in pieces of about 1 mm and digested for 15 min in 0.05% trypsin in HBSS at 37°C, as described by Camu and Henderson [4]. After treatment with DNase, the tissue was further dissociated by trituration.
Results
To compare glutamate-induced cell death after short and long exposures, cultured motor neurons were incubated for 20 min (Fig. 1A) or 24 h (Fig. 1B) in the presence of glutamate. Motor neuron death was evaluated 24 h after the start of the treatment and was in both cases dose-dependent (Fig. 1, insets). After a short exposure, motor neuron death leveled off at a concentration of 100 μM glutamate, while exposing motor neurons for 24 h to increasing amounts of glutamate ultimately resulted in
Discussion
Exposing motor neurons for a short or a long period to glutamate made no difference in terms of the glutamate receptor involved in the induction of motor neuron death. Inhibition of the NMDA receptor was in both cases sufficient to inhibit motor neuron death. This resembles cell death found in cerebellar granule cells [1], cortical neurons [6] and mixed spinal cord neurons [17] as selective NMDA-receptor blockade also prevented cell death induced by brief intense exposure of these cells to
Acknowledgements
We thank H. Klaassen and E. Van Houtte for technical assistance. This work was supported by a grant from the Research Council of the University of Leuven and from the Fund for Scientific Research-Flanders (FWO). L.V.D.B. is a Postdoctoral Fellow and W.R. is a Clinical Investigator of the FWO.
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