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Glutamate receptor-mediated excitotoxicity and mitochondrial dysfunction appear to play an important role in motoneuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). In the present study we used an organotypic slice culture of chick embryo spinal cord to explore the responsiveness of mature MNs to different excitotoxic stimuli and mitrochondrial inhibition. We found that, in this system, MNs are highly vulnerable to excitotoxins such as glutamate, N-methyl-D-aspartate (NMDA), and kainate (KA), and that the neuroprotective drug riluzole rescues MNs from KA-mediated excitotoxic death. MNs are also sensitive to chronic mitochondrial inhibition induced by malonate and 3-nitropropionic acid (3-NP) in a dose-dependent manner. MN degeneration induced by treatment with mitochondrial toxins displays structural changes similar to those seen following excitotoxicity and can be prevented by applying either the antiexcitotoxic drug 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) or riluzole. Excitotoxicity results in an increased frequency of normal spontaneous Ca2+ oscillations in MNs, which is followed by a sustained deregulation of intracellular Ca2+. Tolerance to excitotoxic MN death resulting from chronic exposure to excitotoxins correlates with a reduced excitotoxin-induced increase in intracellular Ca2+ and increased thapsigargin-sensitive Ca2+ stores.

We have developed an organotypic culture technique that uses slices of chick embryo spinal cord, in which trophic requirements for long-term survival of mature motoneurons (MNs) were studied. Slices were obtained from E16 chick embryos and maintained for up to 28 days in vitro (DIV) in a basal medium. Under these conditions, most MNs died. To promote MN survival, 14 different trophic factors were assayed. Among these 14, glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor were the most effective. GDNF was able to promote MN survival for at least 28 DIV. K(+) depolarization or caspase inhibition prevented MN death but also induced degenerative-like changes in rescued MNs. Agents that elevate cAMP levels promoted the survival of a proportion of MNs for at least 7 DIV. Examination of dying MNs revealed that, in addition to cells exhibiting a caspase-3-dependent apoptotic pattern, some MNs died by a caspase-3-independent mechanism and displayed autophagic vacuoles, an extremely convoluted nucleus, and a close association with microglia. This organotypic spinal cord slice culture may provide a convenient model for testing conditions that promote survival of mature-like MNs that are affected in late-onset MN disease such as amyotrophic lateral sclerosis.

To investigate the presumptive role of excitatory amino acids (EAAs) in the regulation of normally occurring motoneuron (MN) death, chick embryos were treated with the glutamate receptor antagonists dizocilpine maleate and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium. Both failed to alter the number of surviving MNs at the end of the critical period of programmed cell death. However, treatment with 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, a competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, was able to rescue a significant number of MNs from death. Treatment with several EAA agonists induced extensive excitotoxic lesions in the spinal cord. MN degeneration induced by excitotoxins exhibited changes characteristic of necrosis rather than apoptosis. However, when either 0.5 or 1 mg of NMDA was applied acutely on embryonic day (E) 7, about 50% of treated embryos failed to exhibit NMDA-induced excitoxicity but rather showed a clear reduction in the number of pyknotic MNs. This apparent neuroprotective effect of NMDA was also observed in a subset of embryos chronically treated with NMDA, in which an excessive number of MNs was detected when examined on E9. Surprisingly, in the same experiment other embryos showed either normal or highly reduced MN numbers. Embryos with motoneuronal depletion induced by NMDA also showed a delayed impairment of later neuromuscular development with the appearance of degenerative changes in surviving MNs and apoptosis of skeletal muscle cells. Because some of the alterations reported here are similar to those described in MN diseases, our experimental model may be useful for gaining insights into the mechanisms that control both developmentally regulated and pathological MN death.