Neurite outgrowth from embryonic chicken spinal neurons is promoted by media conditioned by muscle cells.

Abstract

The effect of media conditioned by muscle cells on the development in vitro ofchicken spinal neurons was studied. Neural tube cells of 4.5-day chicken embryos were dissociated after trypsinization and cultured in serum-free minimum essential medium conditioned for 4 days over cultures of fused chicken myotubes. After 20 hr in conditioned medium (protein concentration, 10-50 ftg/ml), about 50% of surviving cells had extended neurites, whereas in cultures in nonconditioned medium this value was about 10%. The active factor(s) in conditioned medium is macromolecular and its activity was completely destroyed by incubation with trypsin. Concentrated samples ofconditioned medium were analyzed by gel filtration on columns of Sepharose CL-6B. The activity was recovered in peaks with apparent molecular weights of 40,000 and 500,000 and at the exclusion volume of the column. Media conditioned by chicken liver and skin cells also contained neurite-promoting activity but at lower levels. No activity was detected in Nerve Growth Factor, insulin, fetal calf serum, or horse serum or in media conditioned by chicken lung, chicken heart, or C6 glioma cells.During the phase of embryogenesis leading up to the formation of a given synapse, a complex set of interactions between the nerve cell and its potential target is brought into play (1, 2). These interactions may be qualified as "anterograde," such as for example the effect of the nerve on the distribution and metabolism of receptors in the target cell membrane, or, conversely, as "retrograde" (3). It has been postulated for many systems (3-5) that the target cell liberates soluble retrograde factors upon which the neuron depends for its survival and morphological and biochemical differentiation.The best candidate for such a role is the nerve growth factor (NGF) (6), but there is indirect evidence that, in the embryonic spinal cord, motoneurons destined to innervate skeletal muscle may be affected by comparable retrograde factors liberated by their target tissues. Results obtained in vivo, such as the enhancement of naturally occurring cell death by limb extirpation in the chicken embryo (7) and the rescue of some motoneurons by the graft of a supernumerary limb (8), suggested that these factors might be necessary for cell survival. More recent experiments. in vitro have pointed to roles for muscle-derived factors in enhancing neurite extension (9), transmitter synthesis (10, 11), and cell survival (12) of spinal neurons.In this paper, we report the effects of media conditioned by skeletal muscle cells upon the development of dissociated cell cultures ofembryonic chicken spinal cord. We have established a rapid quantitative assay for factors affecting one crucial aspect of the development of spinal neurons: neurite extension. Investigations using this assay have provided a preliminary characterization of the substance(s) responsible for the growth-promoting effect of muscle-conditioned medium. MATERIALS AND METHODSSpinal Cord Cultures. Neural tubes were dissected from 4....

Potentiation of miniature endplate potential frequency by ATP in Xenopus tadpoles

“…Release of trophic factors from the presynaptic nerve terminals may regulate the distribution and the ion-conducting properties of transmitter receptors in the postsynaptic membrane (Usdin & Fishbach, 1986;Nitkin et al, 1987;Laufer & Changeux, 1987;Mulle et al, 1988). Factors secreted by the postsynaptic cell may regulate the growth and survival of presynaptic neurones (Henderson et al, 1981;Gurney, 1984;Oppenheim & Haverkamp, 1988). It is now widely recognized that many mature neurones contain more than one neurotransmitter (Hokfelt et al, 1984), and in at least some instances, both substances have physiological effects on the postsynaptic cell (see, for example, Jan et al, 1983).…”

Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture

“…For example it has been shown that cells dissociated from early embryonie chick, rat, or mouse spinal cords can be maintained in culture, and that media taken from cultured muscle cells or soluble extracts of muscle tissue increased the Ievels of cell survival, neurite outgrowth, and/or transmitter synthesis (see, e.g., Giller et aL, 1977;Bennett et aL, 1980;Brookes et aL, 1980;Godfrey et aL, 1980;Henderson et al, 1981;Longo et aL, 1982;Tanaka and Obata, 1982;Tanaka and Obata, 1983;Smith and Appel, 1983;Kaufman et al, 1985;Smith et aL, 1985). The fact, however, that motor neurons were not separated from other spinal cells makes the interpretation of these results ambiguous because it is not possible to decide whether the observed effects result from a direct action on the neurons in question or an indirect effect via other neuronal or nonneuronal cells (see Fukada, 1980).…”

Motoneurons purified by cell sorting respond to two distinct activities in myotube-conditioned medium

“…The presence of muscle cells, or medium conditioned by muscle cells, has been shown to affect several aspects of the development of cultured spinal cord cells. These include survival (Bennett et al, 1980), neurite outgrowth (Dribin and Barrett, 1980;Henderson et al, 1981), and neurotransmitter synthesis and metabolism (Giller et ab, 1977;Godfrey et al, 1980;Brookes et ak, 1980). However, in studies using whole spinal cord cultures, it is impossible to distinguish effects on motoneurons from effects on other classes of spinal cord cells.…”