During CNS development, neuroblasts proliferate within germinal zones of the neuroepithelium, and then migrate to their final positions. Although many neurons are thought to migrate along processes of radial glial fibers, increasing evidence suggests environmental factors also influence nerve cell movement. Extracellular matrix molecules are thought to be involved in guiding neuronal migration, and molecules such as NGF and GABA exert trophic effects on immature neurons. The nature of the signals that initiate and direct neuroblast migration, however, is unknown. In vitro, NGF and GABA promote neurite outgrowth from cultured cells, and NGF induces axonal chemotaxis (directed migration along a chemical gradient). At earlier developmental stages, these molecules could influence neuroblast movement. Therefore, we investigated whether these molecules induce embryonic neuronal migration. Using an in vitro microchemotaxis assay, we show that rat embryonic spinal cord neurons migrate toward picomolar NGF and femtomolar GABA beginning at embryonic day 13 (E13). Cells exhibit chemotactic responses to NGF while GABA stimulates chemokinesis (increased random movement). GABA effects are mimicked by muscimol and inhibited by bicuculline and picrotoxin, suggesting GABA motility signals are mediated by GABA receptor proteins. Expression of GABA receptors by embryonic cord cells has been previously reported (Mandler et al., 1990; Walton et al., 1993). We used polymerase chain reaction analysis to demonstrate the presence of NGF and trk mRNA in E13 and E14 cord cells, indicating the cells express message for both NGF and high-affinity NGF receptors. Immunohistochemistry of E13 spinal cord sections indicates that NGF and GABA colocalize in fibers close to the target destinations of migrating neurons, suggesting diffusible gradients of these molecules provide chemoattractant signals to migratory cells. Thus, in vitro, neuroblast migration is induced by specific signaling molecules that are present in the developing spinal cord, and may stimulate migration of embryonic neurons prior to synaptogenesis.

It is not well understood when during embryonic development the elements of a cell's responsiveness first appear, nor the factors controlling their appearance. A strategy to approach this issue is to determine which aspects of neuronal development are highly stereotyped in presence, timing, or pattern across a variety of cell types, and which are more diversified by cell type, region, or other parameters. We have used a fluorescent potentiometric oxonol dye in conjunction with a digital video imaging system to record the emergence and distribution of specific forms of excitability in dissociated embryonic rat spinal cord cells. We studied the expression of responses to veratridine, a sodium channel activator; muscimol, a GABAA receptor agonist; and kainic acid, an agonist at a class of glutamate receptors. Responses were consistently detectable in a percentage of cells dissociated from the earliest age examined, embryonic day 13, and increased progressively in later ages. Cells were examined from four regions, with cervical-lumbosacral and ventrodorsal distinctions. In the population of cells from each region, functional sodium channels appeared prior to GABAA receptors, which in turn emerged prior to kainate-activated glutamate receptors. This pattern was common to all spinal cord regions and revealed ventrodorsal and rostrocaudal gradients reflecting the known pattern of spinal cord neurogenesis. Analysis of the individual cell responses indicated that the stereotypical pattern of sequential channel development occurs individually on most cells in each region.

Single cell suspensions prepared from embryonic chick or rat spinal cords were separated into morphologically and functionally distinct subpopulation based on their buoyant densities The lightest fraction (F-1) was highly enriched for cells containing the enzyme choline acetyltransferase (CAT), a marker for developing motoneurons. The morphology biochemistry, and in vitro development of this and other spinal cord cell fractions isolated by the outlined procedure were investigated. Spinal cords, dissected from 6-day chick or 12-day rat embryos, were dissociated with trypsin and applied to iso-osmotic metrizamide density gradients. After brief centrifugation, biochemical analysis revealed that cholinergic cells migrated to lower densities than other spinal cord cells. The use of discontinuous density gradients allowed rapid and simple isolation of three fractions of viable cells (designated F-1 to F-3, lowest to highest density). Characterization of chicken and rat embryo cell fractions gave similar results. The cells in Fraction 1 were large with prominent nuclei and nucleoli, while those in F-2 and F-3 were distinctly smaller. Fraction 1 was highly enriched for cholinergic cells. The CAT specific activity (CAT/cell) was increased 400% in Fraction 1 compared to unfractionated cells, while CAT specific activity in F-2 and F-3 was reduced to 25% and less than 4% that of unfractionated cells, respectively. The recovery of cholinergic cells using this procedure was much better than with other published procedures; greater than half the spinal cord CAT activity was routinely recovered in the enriched fraction. The cholinergic-enriched cells (F-1) were unique in their in vitro growth characteristics. All fractions had neuronal cells, while non-neuronal cells were distributed primarily in F-3, fewer in F-2, and were essentially absent from F-1. Neurons in F-2 and F-3 remained viable under a variety of conditions, most of which were not supportive of F-1 cell survival. The cholinergic-enriched F-1 cells survived and developed only in the presence of muscle cells or in muscle-conditioned medium on highly adhesive substrata. Large, multipolar neurons predominated under these conditions. The method described provides a means of characterizing the factors involved in the development of distinct populations of cells from the embryonic spinal cord.