INFLUENCE OF ACETYLCHOLINESTERASE ON EMBRYONIC SPINAL RAT MOTONEURONSGROWTH IN CULTURE - A QUANTITATIVE MORPHOMETRIC STUDY

Rat spinal motoneurones sampled at day embryonic 15 were purified usin g a Nycodenz gradient and cultured in defined medium, during 7 days, o n glass coverslips coated with poly-L-lysine and laminine. Purified ac etylcholinesterase (AChE), ecothiopate, BW 284C51 and fasciculin II, i nhibitors of either the catalytic or peripheral site of AChE, were add ed to the defined medium. Morphological changes of spinal motoneurones were measured using a statistical quantitative morphometric method, a llowing the determination of various parameters such as the number of neurites and bifurcations, the length of neurites, the surface and spr eading index. Presence of AChE in the medium (4 units/mL) increases th e surface and the total length of neurites and axons without any chang e in the spreading index. When spinal motoneurones were cultured on AC hE coated substrate, neurones rapidly migrate and form clusters. Addit ion of ecothiopate (10(-6) M) in the medium, which selectively blocks the catalytic site of AChE, leads to a slight increase in the number o f primary neurite and a decrease of the spreading index during the thr ee first days in culture. BW 284C51 (10(-5) M) Which blocks the cataly tic site but also affect the peripheral one, significantly reduces the number of primary neurites and increases the number of bifurcations. Fasciculin II, a potent blocker (10(-9) M) of the AChE peripheral site induces a decrease of both primary neurites and bifurcations with a s ignificant increase of the length and growth velocity of the axon, giv ing a drastic enhancement of the spreading index. These phenomena are discussed in terms of catalytic and non-catalytic function of AChE, in cluding the involvement of the enzyme in adhesive processes, occurring during growth and differentiation of spinal motoneurones.