Recovery of Neurofilament Expression Selectively in Regenerating Reticulospinal Neurons

Abstract

During regeneration of lamprey spinal axons, growth cones lack filopodia and lamellipodia, contain little actin, and elongate much more slowly than do typical growth cones of embryonic neurons. Moreover, these regenerating growth cones are densely packed with neurofilaments (NFs). Therefore, after spinal hemisection the time course of changes in NF mRNA expression was correlated with the probability of regeneration for each of 18 identified pairs of reticulospinal neurons and 12 cytoarchitectonic groups of spinal projecting neurons. During the first 4 weeks after operation, NF message levels were reduced dramatically in all axotomized reticulospinal neurons, on the basis of semiquantitative in situ hybridization for the single lamprey NF subunit (NF-180). Thereafter, NF expression returned toward normal in neurons whose axons normally regenerate beyond the transection but remained depressed in poorly regenerating neurons. The recovery of NF expression in good regenerators was independent of axon growth across the lesion, because excision of a segment of spinal cord caudal to the transection site blocked regeneration but did not prevent the return of NF-180 mRNA. The early decrease in NF mRNA expression was not accompanied by a reduction in NF protein content. Thus the axotomy-induced loss of most of the axonal volume resulted in a reduced demand for NF rather than a reduction in volume-specific NF synthesis. We conclude that the secondary upregulation of NF message during axonal regeneration in the lamprey CNS may be part of an intrinsic growth program executed only in neurons with a strong propensity for regeneration.

How the bending kinematics of swimming lampreys build negative pressure fields for suction thrust

“…Prior to surgery, lampreys were anesthetized with Finquel MS-222 (0.1 g l −1 tank water; Argent Chemical Laboratories). Spinal cord transections were performed as detailed previously (Jacobs et al, 1997;Oliphint et al, 2010). Briefly, each lamprey was placed in a Sylgard-lined Petri dish on a paper towel moistened in oxygenated lamprey Ringers (100 NaCl, 2.1 KCl, 2.6 CaCl 2 , 1.8 MgCl 2 , 4 glucose, 0.5 glutamine, 2 HEPES, pH 7.4).…”

Increased expression of multiple neurofilament mRNAs during regeneration of vertebrate central nervous system axons

“…Within a field of view, all cells within the RGC layer were scored as either unlabeled (i.e., labeled below background), weakly (labeled just above background), moderately (visible distinctly and medium blue in color), or strongly labeled (dark blue and labeled over a significant area of the cell body; Jacobs et al, 1997). These numbers were normalized against the total number of cells within the RGC layer for each field of view.…”

“…The behavior of the type IV nIFs in Xenopus regenerating optic nerve resembles that reported in successfully regenerating CNS axons in other systems. For example, in transected spinal cord axons of lamprey, NF-M expression is initially suppressed and then rises in only those axons that successfully regenerate, while remaining suppressed in those that do not (Jacobs et al, 1997). Similarly, in fish optic nerve expression of the xefiltin ortholog, gefiltin, is also initially suppressed, then rises above normal during the peak period of regeneration, and then falls to normal levels after regeneration is complete Asch et al, 1998).…”

Lamprey neurofilaments contain a previously unreported 50‐kDa protein

“…The spinal cord of larval lampreys regenerates morphologically and functionally after transection (Rovainen, 1976;Selzer, 1978;Wood and Cohen, 1981;Jacobs et al, 1997). The growing tips of the regenerating axons have an appearance that is markedly different from the growth cones of embryonic neurons during development or in tissue culture.…”

“…The growing tips of the regenerating axons have an appearance that is markedly different from the growth cones of embryonic neurons during development or in tissue culture. In contrast to that of embryonic neurons, the "growth cones" of regenerating lamprey reticulospinal axons lack filopodia and lamellipodia (Lurie et al, 1994), contain little F-actin (Jacobs et al, 1997), and elongate much more slowly Selzer, 1983, 1984;Lurie and Selzer, 1991). Ultrastructurally the growth cones are densely packed with neurofilaments (NFs) (Lurie et al, 1994), which suggested the hypothesis that assembly and transport of NFs into the growing tip might generate a protrusive force that contributes to regeneration.…”