ACETYLCHOLINESTERASE INHIBITOR TREATMENT DELAYS RECOVERY FROM AXOTOMYIN CULTURED DORSAL-ROOT GANGLION NEURONS

We have previously reported that dorsal root ganglion neurons cultured in the presence of the highly specific, reversible acetylcholinestera se inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibr omide (BW284c51), showed significantly reduced neurite outgrowth and c ontained massive perikaryal inclusions of neurofilaments. In the prese nt report we have more closely examined these changes in a time course study over a 21-day culture period using a combined morphological, im munocytochemical and enzymatic approach and additionally, describe, th e effects of acetylcholinesterase inhibitor treatment on the state of neurofilament phosphorylation. Finally, we have examined the effects o f co-administration of N-6,2'-0-dibutyryladenosine 3':5'-cyclic monoph osphate (dbcAMP) with BW284c51. At 1 day in culture, both control and treated cells displayed eccentrically located nuclei, numerous polysom es and perikaryal accumulations of neurofilaments which were immunorea ctive with both phosphorylation- and nonphosphorylation-dependent neur ofilament antibodies. These cytological changes, which are common feat ures of the chromatolytic reaction following axotomy in vivo, rapidly resolved in the control neurons, where by 7 days in culture, the neuro filament accumulations had completely disappeared and neurite outgrowt h was robust. In contrast, inhibitor-treated neurons retained the post -axotomy features up to 21 days and had significantly reduced neurite outgrowth. In addition, we have investigated a possible role of cyclic adenosine monophosphate (cAMP) in the recovery process since it has b een shown to enhance neuritic outgrowth in cultured neurons. Our resul ts demonstrate that the addition of dbcAMP, a membrane permeable analo g of cAMP, significantly enhanced neuritic outgrowth and accelerated t he recovery of BW284c51-treated dorsal root ganglion cells, as gauged by the disappearance of the axotomy-related cytological changes. Treat ment with dbcAMP also increased acetylcholinesterase activity which ha s been positively correlated with neurite outgrowth both in vivo and i n vitro. Together, these observations suggest that acetylcholinesteras e has a non-cholinolytic, neurotrophic role in neuronal regeneration a nd development.