Chick Spinal Somatic Motoneurons in Culture

Abstract

Cultures enriched in spinal somatic motoneurons are useful test systems for delineating the role of trophic factors in motoneuron survival and differentiation (e.g., McManaman et al., 1989, McManaman et al. 1990; Juurlink et al., 1991a; Henderson et al., 1994; Mettling et al., 1995; Kaal et al., 1997). They also form useful test systems for examining the roles of Fas receptor (Raoul et al., 1999) and thrombin receptor (Turgeon and Houenou, 1999) activation in motoneuron survival. Motoneurons in such cultures are obtained from embryonic spinal cord. There are basically two approaches whereby one can obtain cultures highly enriched in spinal motoneurons. One approach takes advantage of the fact that peripheral terminals of motoneurons can take up fluorescent labels and retrogradely transport these labels to the somas. Spinal cords can then be dissociated and labeled motoneurons isolated, using a fluorescence-activated cell sorter (e.g., Calof and Reichardt, 1984; Schaffner et al., 1987); this approach is labor-intensive and yields small numbers of neurons, of which only about 80% are labeled motoneurons. There are also questions concerning the effects of the tracer on the motoneurons (Smith et al., 1986). The second approach takes advantage of the fact that the buoyant density of motoneurons is significantly different from that of the other neural cell populations; here, motoneurons are separated from other cell populations by centrifuging cells through a density gradient (Schnaar and Schaffner, 1981; Dohrmann et al., 1986). This approach has the advantages that, technically, it is much simpler than the fluorescence-activated sorting method, and much larger numbers of motoneurons can be isolated. The cultures established from this latter approach, as described in this chapter, are free of nonneuronal cells, and are comprised of about 95% motoneurons as determined by calcitonin gene-related peptide immunocytochemistry (Juurlink et al., 1990).