SHORT EXPOSURE TO METHYLAZOXYMETHANOL CAUSES A LONG-TERM INHIBITION OF AXONAL OUTGROWTH FROM CULTURED EMBRYONIC RAT HIPPOCAMPAL-NEURONS

Methylazoxymethanol (MAM) is an alkylating agent that is used to induc e microencephaly by killing mitotically active neuroblasts. We found t hat at later developmental times, MAM exposure can result in abnormal fiber growth in vivo, However, there have not been any previous studie s on the effects of MAM on differentiating neurons, We examined the ou tcome of short exposure to MAM on postmitotic embryonic hippocampal cu ltures during the establishment of axonal polarity, At 0, 1, or 2 days in vitro (DIV), neurons were treated with 0.1 nM-1 mu M MAM for 3 hr and then transferred to glial conditioned media, At 3 DIV, the cells w ere fixed and analyzed by immunofluorescent staining for neuron viabil ity and differentiation, Control cells initiate several minor processe s; one process elongates rapidly at about 1 DIV eventually becoming an axon, while extensive dendritic growth occurs after 3-4 DIV, Neurons treated with 1 mu M MAM at 0 or 1 DIV showed a marked inhibition of ne urite growth and withdrawal of axons without affecting cell viability, These cells continued to show minimal neurite outgrowth at 7 DIV, eve n when transferred to a glial coculture, In contrast, cells treated in itially with MAM, after neuronal polarity is established at 2 DIV, sho wed no effect on axonal growth, To determine the effects of MAM on the neuronal cytoskeleton, we examined the in vitro assembly of brain mic rotubules in a one cycle assay, Exposure to MAM depleted the soluble p ool of proteins, including microtubule-associated protein 1B (MAP1B) a nd MAP2, which are required for neurite outgrowth, through a nonspecif ic process, Under non-saturating conditions, there were no changes in the total amount of microtubules assembled or the coassembly of MAP1B and MAP2 in the presence of MAM, These results demonstrate that MAM ca n directly affect differentiating neurons, indicating that an early di sruption of axonal outgrowth may have long-term effects. (C) 1996 Wile y-Liss, Inc.