Journal of Neuroscience, Vol 6, 3265-3274, Copyright © 1986 by Society for Neuroscience
Isolation of embryonic chick motoneurons and their survival in vitro
RJ O'Brien and GD Fischbach
This is the first of a series of 4 papers in which we describe the
regulation of excitatory amino acid receptors on embryonic chick
motoneurons dissociated from the lateral motor column and maintained in
cell culture. Techniques are described for labeling embryonic chick
motoneurons in vivo with Lucifer Yellow or fluorescein isothiocyanate
conjugates of wheat germ agglutinin (Fl-WGA). We estimate that 65-95% of
the motoneurons in the lateral motor column survive tissue dissociation and
settle on an appropriate culture surface. The number of fluorescent
motoneurons observed in heterogeneous spinal cord cell cultures decreases
with a half-life of 2 d. The decline is due to fading of the fluorescent
tracer rather than to loss of cells. Techniques are also described for
separating motoneurons from other spinal cord cells with a
fluorescence-activated cell sorter. Approximately 24% of the motoneurons in
the lateral motor column can be isolated, and motoneurons comprise more
than 90% of the population in cultures seeded with sorted cells. The
survival of sorted and unsorted motoneurons in vitro is enhanced in the
presence of skeletal myotubes or muscle conditioned medium, but the
survival of non-motoneurons is not influenced by muscle. Electrophysiologic
properties of sorted and unsorted motoneurons determined with patch-clamp
techniques are similar. Both differ from mature motoneurons in their lower
resting membrane potential (-50 mV), larger input resistance (450 M omega),
and longer time constant (39 msec). Also they do not exhibit anomalous
rectification or a calcium-activated potassium after hyperpolarization.
Motoneurons grown in the absence of interneurons differ from motoneurons in
heterogeneous spinal cord cell cultures in that their neurites (dendrites)
are shorter and they branch less often.
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