Separation of cell types from embryonic chicken and rat spinal cord: characterization of motoneuron-enriched fractions

Abstract

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.

Motor Neurons Are Selectively Vulnerable to AMPA/Kainate Receptor-Mediated InjuryIn Vitro

“…4, 5), further substantiating the critical role of Ca 2ϩ entry in rapidly triggered injury to large SMI-32(ϩ) neurons. In addition, because the motor neuron identity of large SMI-32(ϩ) neurons has not been proven, we repeated Ca 2ϩ -dependence experiments with two other indices of the motor neuron population: peripherin immunocytochemistry (Parysek and Goldman, 1988;Escurat et al, 1990) and ChAT enzymatic activity (Schnaar and Schaffner, 1981;Schaffner et al, 1987;Martinou et al, 1989b;Rothstein et al, 1993). As with large SMI-32(ϩ) neurons, both peripherin(ϩ) neurons and ChAT activity were lost preferentially after brief kainate exposures.…”

“…Motor neuron enrichment techniques such as density gradients (sorting by size: Schnaar and Schaffner, 1981;Dohrmann et al, 1986;Martinou et al, 1989a), retrograde labeling (Calof and Reichardt, 1984;O'Brian and Fischbach, 1986a;Schaffner et al, 1987), and immunopanning (Camu and Henderson, 1992;Mettling et al, 1995) have been used often, but generally give limited yield or lack complete specificity for motor neurons. In addition, enriching for motor neurons does not in itself provide the morphological detail that can be revealed with direct labeling, and removal of other spinal cord neurons with which motor neurons may interact may markedly alter their survival or phenotype (O'Brian and Fischbach, 1986b).…”

Trophic Support of Motoneurons: Physiological, Pathophysiological, and Therapeutic Implications

“…Experimental achievements were confined to the demonstration that skeletal muscle extract or conditioned medium of primary cultures of skeletal muscle my ob lasts trophically supported motoneurons in vivo (75) and in vitro (11,21,24,25,57,73,89,99).…”

Advantages and Pitfalls in Experimental Models Of ALS

“…Many authors have developed protocols to isolate MNs from newborn or embryonic murine spinal cord (Berg & Fischbach, 1978;Gingras et al, 2007;Schnaar & Schaffner, 1981), allowing to identify MNs for their size and their Choline Acetyl Transferase (ChAT) activity. However, more recently, Wiese and coll.…”