Structure of reticulospinal axon growth cones and their cellular environment during regeneration in the lamprey spinal cord

Abstract

The large larval sea lamprey is a primitive vertebrate that recovers coordinated swimming following complete spinal transection. An ultrastructural study was performed in order to determine whether morphologic features of regenerating axons and their cellular environment would provide clues to their successful regeneration compared to their mammalian counterparts. Three larval sea lampreys were studied at 3, 4 and 11 weeks following complete spinal transection and compared with an untransected control. Müller and Mauthner cells or their giant reticulospinal axons (GRAs) were impaled and injected with horseradish peroxidase (HRP). Alternating thick and thin sections were collected for light and electron microscopy. A total of 9 neurites were examined. At all times, growth cones of GRAs differed from those of cultured mammalian neurons in being packed with neurofilaments and in lacking long filopodia, suggesting possible differences in the mechanisms of axon outgrowth. Morphometric analysis suggested that GRA growth cones contact glial fibers disproportionately compared to the representation of glial surface membranes in the immediate environment of these growth cones. No differences were found between glial cells in regenerating spinal cords and those of untransected control animals with regard to the size of the cell body and nucleus and the packing density of their intermediate filaments. Glial fibers in control animals and glial fibers located far from a transection were oriented transversely. Glial cells adjacent to the transection site sent thickened, longitudinally oriented processes into the blood clot at the transection site. These longitudinal glial processes preceded the regenerating axons. Desmosomes were observed on glia adjacent to the lesion but were scarce in the lesion during the first four weeks post-transection. These findings suggest that longitudinally oriented glial fibers may serve as a bridge along which axons can regenerate across the lesion. The presence of desmosomes might prevent migration of astrocytes near the transection, thus stabilizing the glial bridge.

Microglial-neuronal interactions in synaptic damage and recovery

“…It is also important to consider that microglial activation promotes the proliferation of astrocytes, and the presence of an astrocytic scar following brain injury has been thought to be an a priori impassable barrier for axons. However, pronounced astrocytic scars are readily crossed by regenerating axons in lower vertebrates (Lurie et al, 1994;Reier, 1979), and astrocytes also provide potent support for neurite outgrowth in vitro (Hatten et al, 1984;Noble et al, 1984). Taken together, these observations allude to an intricate balance in the interactions of neurons, synapses, microglia, astrocytes, and oligodendrocytes following injury to the CNS.…”

Recovery of Neurofilament Expression Selectively in Regenerating Reticulospinal Neurons

“…Growth cones of these regenerating axons lack filopodia and lamellipodia (Lurie et al, 1994). They have few microfilaments, are densely packed with phosphorylated neurofilaments (NFs) (Hall et al, 1991;Lurie et al, 1994;Hall and Lee, 1995;McHale et al, 1995;Pijak et al, 1996), elongate slowly (Ͻ120 m /d) Selzer, 1983, 1984;Lurie and Selzer, 1991a;Davis and McClelland, 1994a), and contain very little actin (G. F. Hall, J. Yao, K. S. Kosik, and M. E. Selzer, unpublished observations).…”

“…Growth cones of these regenerating axons lack filopodia and lamellipodia (Lurie et al, 1994). They have few microfilaments, are densely packed with phosphorylated neurofilaments (NFs) (Hall et al, 1991;Lurie et al, 1994;Hall and Lee, 1995;McHale et al, 1995;Pijak et al, 1996), elongate slowly (Ͻ120 m /d) Selzer, 1983, 1984;Lurie and Selzer, 1991a;Davis and McClelland, 1994a), and contain very little actin (G. F. Hall, J. Yao, K. S. Kosik, and M. E. Selzer, unpublished observations). Thus the mechanism of growth cone motility in these regenerating central axons must be different from that of embryonic neuronal growth cones in vitro (Letourneau, 1981;Gordon-Weeks, 1989) or in situ (Ho and Goodman, 1982;Keshishian and Bentley, 1983;Tosney and Landmesser, 1985;Bovalenta and Mason, 1987;Nordlander and Singer, 1987;Bovalenta and Dodd, 1990;Yaginuma et al, 1991), which contain no NFs and grow 1-3 mm /d in a process involving complex interactions between actin microfilaments, myosin, and microtubules (Lin and Forscher, 1995;.…”

“…Differences in growth cone morphology between regenerating and developing axons suggest differences in the mechanisms of growth Unlike embryonic growth cones, growth cones of regenerating lamprey axons lack filopodia and lamellipodia, have few microfilaments, and are filled with NFs (Lurie et al, 1994;McHale et al, 1995;Pijak et al, 1996), similar to growth cones of regenerating goldfish optic nerve (Lanners and Grafstein, 1980). Simple growth cones without lamellipodia or filopodia have also been described in regenerating optic nerves of frog (Scalia and Matsumoto, 1985) and myelin-deficient rats (Gocht and Löhler, 1993), and in the late-developing corticospinal tract of the rat (Gorgels, 1991).…”

“…Thus it is not yet clear whether NFs are involved in the mechanism of embryonic axon elongation. In regenerating lamprey growth cones, NFs are present in swirls rather than parallel longitudinal arrays (Lurie et al, 1994;McHale et al, 1995;Pijak et al, 1996) and despite being highly phosphorylated (Hall et al, 1991;Pijak et al, 1996) are more densely packed than they are in the axon (Pijak et al, 1996). Thus they may be under increased pressure, suggesting that transport of NFs into the injured axon tip may provide an internal propulsive force driving the growth cone forward.…”