Morphological features and responses to AMPA receptor-mediated excitotoxicity of mouse motor neurons: comparison in purified, mixed anterior horn or motor neuron/glia cocultures

Abstract

Primary motor neuron cultures are widely used as in vitro model to study the early mechanisms involved in the aetiology of amyotrophic lateral sclerosis. In this study, we directly compared the morphological features and the responses to AMPA receptor (AMPAR) activation of mouse spinal cord motor neurons under different culture conditions (OptiPrep-purified, mixed anterior horn or motor neuron/glia cocultures). Motor neurons cocultured with a confluent glial layer had significant improvements in axonal length and in somata perimeter and area, compared both to mixed anterior horn cultures and to purified cultures, suggesting that the presence of more “mature” glial cells was determinant to obtain healthier motor neurons. By immuno-cytochemical assays we found that both in mixed anterior horn cultures and in cocultures, lower AMPA (0.3 μM) or kainate (5 μM) concentrations, but not the higher (1 or 15 μM, respectively), induced classical apoptotic events such as the nuclear fragmentation, the membrane externalization of phosphatidylserine residues and the activation of caspases-9 and -3. The morphological features and the different degenerative pathways induced by AMPAR agonist concentrations suggest that the experimental conditions used for in vitro studies are key factors that should be deeply considered to obtain more valid and reproducible results.