It is well established that peripheral nerves regenerate after injury. Therefore, incomplete functional recovery usually results from
its polysialic acid (PSA) moiety are essential for proper motor axon guidance. In this study we used a well established model of nerve
NCAM (?/?) mice. We found that regenerating axons innervating the muscle pathway and, to a lesser extent, cutaneous axons in the
sensory pathway reexpress high levels of PSA during the time when the cut axons are crossing the lesion site. Second, we found that motor
mice lacking PSA. These results indicate that regenerating motor axons must express polysialylated NCAM, which reduces axon–axon
The degree of functional recovery attained after a peripheral
nerve injury is, to a large extent, dependent on the number of
lesioned axons that have reinnervated their appropriate postsyn-
aptic target(s) (Fu and Gordon, 1997). During development it is
well established that growing axons respond to a combination of
both long- and short-range guidance cues that specifically direct
them to their correct target(s) (Tessier-Lavigne and Goodman,
1996). Moreover, many of the guidance molecules and cellular
mechanisms responsible for precise targeting of axons during
development have been well characterized (Chisholm and
tive targeting of regenerating peripheral axons. One explanation
for this apparent oversight is the general conception that regen-
erating axons do not reinnervate their original targets selectively
are several examples in which selective axon regeneration likely
occurs. For example, physiologically distinct classes of fast and
slow motor neurons have a propensity to reinnervate their ap-
1993; Rafuse and Gordon, 1998; Wang et al., 2002). Similarly,
topographically correct manner as occurs during development
(Laskowski and Sanes, 1988), suggesting that many of the same
guidance cues expressed during embryogenesis are reexpressed
after peripheral nerve injury.
Developmental biologists often have applied simple but ex-
tremely useful model systems to identify specific axon guidance
oral nerve regeneration model that reproducibly demonstrates
that regenerating motor neurons selectively grow down distal
ways (Brushart, 1988, 1993; Al-Majed et al., 2000). This example
of selective motor neuron regeneration is known as preferential
motor reinnervation (PMR).
The molecular mechanisms regulating PMR are not well un-
derstood. However, there are several lines of evidence to suggest
acid (PSA) moiety are involved. PSA and NCAM are both reex-
pressed by regenerating motor axons after peripheral nerve in-
tion of this manuscript and David Fillmore for technical assistance. We also thank Monique Guilderson (Martime
under the auspices of the National Institute of Child Health and Human Development and maintained by the
sie University, Sir Charles Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5.
TheJournalofNeuroscience,February23,2005 • 25(8):2081–2091 • 2081
jury (Zhang et al., 1995; Rutishauser and Landmesser, 1996).
their appropriate muscle target(s) selectively (Tang et al., 1992,
brief electrical stimulation (Al-Majed et al., 2000; Brushart et al.,
2002), which suggests that the guidance molecules responsible
sion of PSA is increased with cell activity (Kiss et al., 1994).
To determine whether PSA and NCAM regulate PMR, we
transection in wild-type, but not NCAM (?/?), mice. PMR was
also absent in wild-type mice when PSA was removed enzymati-
cally from the regenerating nerve. Finally, transgenic mice were
drawal of misprojecting axons more inhibited, in mice lacking
PSA compared with wild-type mice. Together, these results indi-
cate that PSA-dependent growth of motor axons is required for
selective targeting of regenerating motor axons.
Parts of this paper have been published in abstract form
(Franz and Rafuse, 2003).
Mice. Five different strains of mice were used in this study. Wild-type
C57BL/6 mice were obtained from Charles River (Wilmington, MA). In
a C57/B6 background, generated by Cremer et al. (1994), was used. To
control for possible strain differences, we obtained a second strain of
NCAM (?/?) mice from The Jackson Laboratory (Bar Harbor, MA).
at least nine generations (Delling et al., 2002). A transgenic mouse line
(mHb9-Gfp1b), in which enhanced green fluorescent protein (eGFP) is
expressed under the control of the mouse Hb9 promotor (Wichterle et
al., 2002) (a kind gift from Dr. T. M. Jessell, Columbia University, New
York, NY), was used specifically to visualize motor axons in wild-type
mice. Specifically to visualize motor axons in NCAM (?/?) mice, we
bred mHb9-Gfp1b mice with NCAM (?/?) mice to generate NCAM
(?/?) mice that express the mHb9-Gfp1b gene. We designated these
latter mice NCAM (?/?)/Hb9-Gfp. All strains of mice were housed and
bred locally in the animal facilities at Dalhousie University.
Mouse genotyping was performed by PCR. The NCAM mutant allele
was detected as a 336 bp DNA fragment generated by PCR, using a 5?
primer that anneals to the NCAM sequence (5?-GCT CAT GTT CAA
the NCAM sequence (5?-CCT CAG GTA TTA TGG TGT TGG). Ampli-
fication included the following: 95°C for 1 min; 55°C for 30 s; 72°C for 1
min; 30 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s; and, finally,
72°C for 5 min. The eGFP allele was detected as a 650 bp DNA fragment
GC) and a 3? primer (5?-TCC AGC AGG ACC ATG TGA TC). Amplifi-
cation included the following: 94°C for 5 min; 30 cycles of 94°C for 30 s,
60°C for 60 s, 72°C for 60 s; and, finally, 72°C for 5 min.
Nerve transection and repair. All surgeries were performed on young
adult (8–12 weeks) mice under aseptic operating conditions. Mice were
incision was made in the skin to expose the femoral nerve, and one of
three nerve transection and repair surgeries was performed (Fig. 1). (1)
For immunohistochemistry the muscle and cutaneous pathways of the
stumps were joined surgically to the distal stumps with 11-0 nylon su-
(2) For analysis of PMR the femoral nerve was transected and repaired 2
ways. This transection site is distal to the point at which the iliacus nerve
separates from the main femoral nerve, but it is proximal to the diver-
and the proximal and distal nerve stumps were secured together with a
proximal and distal femoral nerve stumps tightly together so that their
cut ends were closely apposed. Because of the very small diameter of the
mouse femoral nerve (?300 ?m), it was not possible to align the proxi-
mal and distal femoral nerve stumps convincingly with the same preci-
al., 2000). Thus no attempt was made to do so. (3) To remove PSA
cutaneous pathways 30 min before the nerve was cut and sutured as
described above. As shown previously, Endo-N injected into the chick
hindlimb effectively removes PSA from developing nerve and muscle
fibers for several days in vivo (Landmesser et al., 1990; Rafuse and Land-
messer, 2000). For pain relief all of the operated mice were administered
ketoprofen (5 mg/kg) after the surgeries.
muscle and cutaneous axons, we harvested the muscle and cutaneous
pathways of the femoral nerve from wild-type C57BL/6 mice at 3, 8, and
15 d after transection and repair (surgery 1, described above). To study
the reexpression of PSA on distal muscle targets in Endo-N- and saline-
injected mice, we dissected out the quadriceps femoris muscle of wild-
and distal stumps. B, PMR was assessed by cutting and repairing the femoral nerve 2 mm
cutaneous branches of the cut and repaired femoral nerve. The cell bodies of motor neurons
Diagrammatic representations of surgeries performed and examples of retro-
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