Survival and death of mature avian motoneurons in organotypic slice culture: trophic requirements for survival and different types of degeneration.
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.
Brunet
Núria
N
Unitat de Neurobiologia Cel.lular, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida and IRB Lleida, 25008 Lleida, Catalonia, Spain.
Tarabal
Olga
O
Portero-Otín
Manel
M
Oppenheim
Ronald W
RW
Esquerda
Josep E
JE
Calderó
Jordi
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002642:Chick Embryo; D000242:Cyclic AMP / Q000378:metabolism; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism / Q000494:pharmacology; D005911:Gliosis / Q000188:drug therapy / Q000378:metabolism / Q000503:physiopathology; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D046529:Microscopy, Electron, Transmission; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009410:Nerve Degeneration / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D009414:Nerve Growth Factors / Q000378:metabolism* / Q000494:pharmacology; D009924:Organ Culture Techniques; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D013116:Spinal Cord / Q000196:embryology* / Q000378:metabolism* / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000494:pharmacology
D061945:Caspase Inhibitors; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D042461:Vascular Endothelial Growth Factor A; D000242:Cyclic AMP; D053148:Caspase 3; D011188:Potassium