Outgrowth of the pyramidal tract in the rat cervical spinal cord: Growth cone ultrastructure and guidance

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Abstract
In order to examine the mode of outgrowth of the pyramidal tract in the rat, the ultrastructure of its pathway in the dorsal funiculus of the spinal cord was analysed. The analysis was performed by means of serial sections of the third cervical segment before and during the arrival of pyramidal tract axons, and focussed on the morphology and microenvironment of the growth cones. Growth cones appear as elongated terminal enlargements without side branches. Two zones could be discerned: the distal, usually lamellipodial fine granular zone, containing no organelles, except for an occasional clear vesicle; and the proximal organelle-rich zone, which contains various organelles, such as agranular reticulum and vesicular structures. In addition, the proximal organelle-rich zone contains round or elliptic structures, limited by two concentric membranes, that enclose reticular and vesicular elements. The electron density of these structures varied from as low as the surrounding growth cone matrix to as dark as lysosomal structures, suggesting their involvement in turnover processes. At embryonic day 20, the most ventral part of the dorsal funiculus, where the first pyramidal tract axons are due to arrive within two days, is populated by axons that are relatively small compared to those in the rest of the dorsal funiculus. At birth, the arrival of the first pyramidal tract axons is marked by the presence of numerous large growth cone profiles in between small axons in the most ventral part of the dorsal funiculus; no circumscript bundle separated from the ascending sensory fiber tracts is present yet. The growth cones descend, club-shaped and 1 to 2 μm in diameter, without lamellipodia or filopodia. Within the same area a second growth cone type is present, which contains dense-core vesicles and has spread-out lamellipodia. Most of these growth cones are ascending and they probably belong to primary afferent or propriospinal fibers. At postnatal day 2, the pyramidal tract can be readily delineated from the adjacent fasciculus cuneatus where myelination has already started, but no glial boundary is present. The abundant growth cones are 1–2 μm wide and extend single unbranched lamellipodia, up to 15 μm long, which often enfold parallel axons or other growth cones. At postnatal day 4, growth cones are scarce in the tract. They measure 1 μm or less in diameter and each extends a single, straight lamellipodium or filopodium over 1 to 7 μm in the caudal direction. At all ages examined, most of the surface of the growth cones is in apposition to longitudinally oriented axons and growth cones. The caudally outgrowing growth cones also make contact with glial elements. These are predominantly oriented perpendicularly to the longitudinal axis and are not joined together into continuous arrays. Furthermore, growth cones form synaptic contacts with dendrites that extend into the dorsal funiculus from the adjacent spinal grey. These observations indicate that the morphology of growth cones changes during the formation of the pyramidal tract in the cervical spinal cord. It is proposed that the outgrowing axons are probably guided into their pathway by selective fasciculation on their precursors, and by local differences in oligodendrocyte and axon maturation within the dorsal funiculus. Furthermore, it is suggested that the leading growth cones of the pyramidal tract grow into the most immature region of the ascending sensory fiber tracts without actively fasciculating on the axons of these tracts.