Cell death of motoneurons in the chick embryo spinal cord. II. A quantitative and qualitative analysis of degenerationin the ventral root, including evidence for axon outgrowth and limb innervation prior to cell death

Development of the ventral roots in the caudal half of the chick lumbar spinal cord (segments 26‐29) was studied by electron microscopy. The ventral root fibers at 4, 5, 6, 7, 8, 9, and 13 days of incubation and at 1, 10 days and 5 weeks post‐hatching were counted either from photomontages or directly in the electron microscope. In the chick, most, if not all, the fibers in the caudal ventral roots studied here probably arise from axons of motoneurons in the lateral and medial motor columns. At four days of incubation, there is an average of 800 axons per segment. The number increases very rapidly reaching a peak of 5,500 axons at day 5.5 In other words, by day 5.5 all the axons of motoneurons hav ealready reached the ventral root region. Between days 6 and 9, the number dreastically declines to 2,200 axons poer segment, a 57% reduction in ventral root fibers. After day 9, there is only aminor and rather slow additional loss of axons, reaching 1, 700 in the 13‐day embryo and 1,500 in the 1‐day post‐hatching chick. In brief, during embryonic development about 71% of the axons are depleted in the ventral roots. Quantitatuve comparisons of Motoneurons in the lateral motor column (LMC) of segments 26‐29 with the axon counts from the same segments have demonstrated: (a) that there is a massive natural cell loss in this region between days 5.5 and 9 amounting to 53%; (b) that axons are lost to the same extent as the motoneurons during this period, Resulting in a close to 1:1 relationship between the two by day 9. When horseradish peroxidase (HRP) was injected into the limb‐buds of 5‐day embryos, prior to the onset of massive cell death, virtually all motoneurons in the LMC were found to contain the HRP reaction product. Since approximately 50% of the cells present on day 5 typically degenerate by day 9, this finding, coupled with the observed close correspondence between axon and cell counts, strongly indicates that all motoneurons, even those destined to die, normally innervate the leg. Ultrastructural changes of motoneuron axons undergoing spontaneous degeneration in the ventral root were also described. The degenerating axons are found in the ventral root as early as the fourth day of incubation, although the number at this time is very low. More massive degeneration occurs between 5.5 and 9 days of incubation. The increrased number of degenerating axons in the ventral root during this period is in agreement with the increased number of degenerating cell bodies in the spinal lateral motor column at these same stages. Between day 4 and day 9, the degenerating axons are characterized by the presence of numerous vesiculated structures, membrane‐bounded autophagic vacuoles, membranous lamellar figures and electron dense bodies in focal, swollen portions of the axon, as well as the disruption of the axolemma. The degeneration process seems to be due to progressive autolysis with the final axonal remnants being phagocytozed by the surrounding Schwann cells and some mononuclear leukocytes. Approximately 60% of the ventral root fibers completely disappear within three to four days, leaving very little evidence of axonal debris. We have found no differences in the details of axonal degeneration of ventral roots from limbbud removal embryos. The spontaneous degeneration of axons continues even after hatching but on a much reduced scale. The post‐hatching degeneration is evidenced by the loosening of myelin sheath, shrinking of th axoplasm, an increase in both multivesicular bodies and lamellated dense bodies, and the disintergration of neurofilaments and neurotubules.