J . Kriz et al. Brain Research 885 2000 32 –44
33
perikaryon and transported down the axon with the slow structure–function relationship for three isoforms of NF
component of axoplasmic transport [33]. Neurofilaments we studied the electrophysiological properties of large
seem not to move along the axon as an organized network, myelinated axons in peripheral nerves of mice with
as originally considered [44,45] but rather as a dynamic targeted disruption of NF genes. We report here that
structure [8,52,53]. There is evidence that subunit ex- changes in expression of NF genes lead to functional
change can occur along the axon [57] and that neurofila- changes in peripheral nerves of transgenic mice. Similar
ment proteins can be transported in an unpolymerized form conclusion stems from our recent studies of mice express-
along axonal microtubules [76]. ing human NF-H [41,42], as well mice expressing neuro-
It is generally considered that one of the functions of filament-beta-galactosidase fusion protein [40]. Prelimin-
neurofilaments is related to the control of radial growth of ary results of the present study have been published in an
large myelinated axons. This notion is largely based on the abstract [43].
correlation between the number of neurofilaments and axonal diameter [22,26,30–32]. Direct support for this
hypothesis has been obtained by studies of three animal
2. Material and methods
models: a mutant quail lacking neurofilaments as a result of a nonsense mutation in the gene coding for the NF-L
2.1. Animals and experimental surgery [56,85,86] a transgenic mouse expressing NF-H b-galacto-
sidase fusion protein that prevents neurofilament transport The experiments were performed on isolated sciatic
into axons [18] and a NF-L knock-out mouse [88]. It has nerves of 3–4-month-old control and transgenic mice NF-
been hypothesized that because the carboxy-terminal do- L2 2, NF-M 2 2and NF-H 2 2. Transgenic animals
main of NF-H and NF-M is associated with NF sidearms were produced and housed in the SPF facility at the
[28,29], increased phosphorylation at their KSP motif may Montreal General Hospital Research Institute [88].
result in increased negative charges associated with these The animals were decapitated under halothane or ether
sidearms and consequently greater electrostatic repulsive anesthesia and the sciatic nerves were quickly dissected
forces between neighboring NFs. The higher level of out proximally at the exit point from the spinal canal, at
phosphorylation of NF-H in myelinated internodal than the lumbo-sacral level and distally above the ankle. The
non-myelinated e.g. nodal regions of axons is associated preparations were pinned to the bottom of the wax-coated
with a 25–50 greater nearest-neighbor spacing of NFs chamber with insect needles and continuously perfused
[17,34,54]. Neurofilaments have also been implicated in with mammalian Ringer saturated with 5 carbon dioxide
regulation of several other cellular functions such as and 95 oxygen pH57.4, temperature 22–258C. The
maturation of regenerating myelinated axons [88] and mammalian Ringer solution contained in mM: 127 NaCl,
regulation of axoplasmic transport [13,51] in which modu- 1.9 KCl, 2.4 CaCl , 1.2 KH PO , 1.3 MgSO , 26 NaHCO
2 2
4 4
3
lation of neurofilament–microtubule interactions and pos- and 10 glucose.
sibly microtubule stabilization [75,87], plays a major role. There is increasing evidence of association between
2.2. Identification of fibers and sampling procedure changes in the normal metabolism of neurofilaments and
disease states [23]. Thus, many human neurodegenerative The intracellular recordings were obtained from 2 to
disorders, such as amyotrophic lateral sclerosis ALS 3-cm segments of sciatic nerves with the impalement zone
[7,11], Parkinson’s disease [27,73] and Alzheimer’s dis- ¯5 mm distal from the spinal roots. The recordings started
ease [73,77] are characterized by NF deposits within after at least 1.5 h incubation of dissected nerves in
neurons so-called spheroids or Lewy bodies. Although it mammalian Ringer solution, a solution that should prompt-
is not known what role these deposits play in pathogenesis ly seal the cut ends of axons [82]. All fibers were impaled
of human diseases, recent studies with transgenic mice blindly in the surface layers of the nerves. On the basis of
clearly demonstrated that disorganized neurofilaments can previous experience with normal animals amphibians and
cause motor neuron degeneration [13,14,47,83]. Additional mammalian, this approach selects the largest axons in the
evidence for neurofilament involvement in human disease population of myelinated axons. This assumption is sup-
originates from the discovery of mutant NF-H alleles in ported by the conduction velocity of axons .30 m s at
some ALS patients [19,39]. Several toxins that disrupt room temperature, control axons and the low probability
neurofilament transport or metabolism result in neuropathy of obtaining stable intra-axonal recordings from axons
[6,25]. ,6–7 mm in diameter. At least two stable recordings were
At present little is known about the NFs relation to the obtained from different axons in each sciatic nerve. Each
specific neuronal elements such as ion channels or trans- experimental group consisted of at least 4–5 experimental
porter and receptors. In this respect, connections between animals. Action potentials were evoked by 0.02 ms current
NFs and other cytoskeletal elements, such as actin net- pulses applied to the proximal trunk of the sciatic nerve via
works, that are known to interact with membrane macro- bipolar platinum electrodes, at frequencies of 0.5–1.0 Hz.
molecules, should be important [23]. In order to establish Axons were considered suitable for experiments when the
34 J
resting membrane potential was more negative than –70 ventral root with the Image-1 software from Universal
mV, with overshooting spikes. The microelectrode tech- Imaging Corp. Pennsylvania [87,88].
nique used in this study allows recording from undissected axons; therefore, it is likely that such recordings better
2.6. Data analysis reflect axonal membrane properties in vivo [65,67,68].
Compound action potentials CAP were measured from Whenever
appropriate results
were expressed
as sciatic nerves using the sucrose-gap method as previously
means6standard error S.E.M.. Statistical significance described [61,62].
was assessed by using one-way analysis of variance ANOVA followed by a Tukey HSD test.
2.3. Intra-axonal microelectrode recording technique Glass microelectrodes were pulled with a Brown-Flam-
3. Results
