MATURATION OF NEURITES IN MIXED CULTURES OF SPINAL-CORD NEURONS AND MUSCLE-CELLS FROM XENOPUS-LAEVIS EMBRYOS FOLLOWED WITH ANTIBODIES TO NEUROFILAMENT PROTEINS

Dissociated cell cultures of Xenopus laevis embryonic spinal cord have proved useful for studying the differentiation of neuronal ionic chan nels and membrane properties and for examining the dynamics of microtu bules in developing neurons. To examine their usefulness for studying neurofilaments in developing neurites, we prepared similar cultures fr om stage 22 embryos. Between 3 and 55 h after plating, these cultures were fixed and immunostained with antibodies directed against various epitopes of neurofilament proteins from X. laevis. These antibodies we re specific for nonphosphorylated epitopes of the two low molecular we ight Xenopus neurofilament proteins (Xenopus NF-L and the Xenopus neur onal intermediate filament protein, XNIF), both phosphorylated and non phosphorylated epitopes of the Xenopus middle molecular weight neurofi lament protein (NF-M), and a nonphosphorylated epitope of the Xenopus high molecular weight neurofilament protein (NF-H). The emergence of t hese neurofilament proteins in culture was compared to the time course previously reported for them in Xenopus spinal cord neurons in situ. To facilitate the comparison of times in culture to developmental stag es, the age of cultured neurons was converted to an equivalent Nieuwko op and Faber normal stage using data presented here on the effect of c hanging temperature on developmental rates of X. laevis. With the exce ption of the nonphosphorylated epitope of NF-H, which is indicative of the most mature axons found in situ, the emergence of the other neuro filament protein antibody epitopes closely paralleled that previously reported for these antibodies in situ. Thus, with respect to XNIF, NF- M, and NF-L, the neurites of cultured neurons were typical of young, e mbryonic Xenopus laevis spinal cord axons. This system should prove us eful for studying both the function of these neurofilament proteins du ring the early stages of axonal development and the dynamics of their transport. (C) 1994 John Wiley and Sons, Inc.