ISOLATION AND CULTURE OF MOTO-NEURONS FROM EMBRYONIC CHICKEN SPINAL CORDS

A method is described for isolating cholinergic α motoneurons from the spinal cord of chicken embryos at stage 17-18 (Hamburger and Hamilton numbering), at the time when it has shown that motoneurons withdraw from the mitotic cycle but neurons of other types of glia are still mitotic. Fragments of the ventral half of the spinal cord are incubated for 24 hr in the presence of 10 μM 1-β-D-arabinofuranosylcytosine in order to eliminate dividing cells and are subsequently dissociated into a suspension of single cells. The following evidence has been obtained and suggests that these cells are neuronal and appear to be α motoneurons: they are resistant to the lethal effect of arabinofuranosylcytosine, and thus are postmitotic at stage 17-18; when grown in vitro, they exhibit morphological characteristics similar to those of ventral spinal neurons, which include the ability to be stained with silver, Nissl, methylene, blue vital stain at pH 6.5-7.0, and choline acetyltransferase histochemistry; they have high choline acetyltransferase activity; and they are capable of forming functional synapses with muscle.