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Research report

Electrophysiological properties of axons in mice lacking neurofilament

subunit genes: disparity between conduction velocity and axon

diameter in absence of NF-H

a ,1 b b a ,

_*

ˇ

Jasna Kriz

, Qinzang Zhu , Jean-Pierre Julien , Ante L. Padjen

Department of Pharmacology and Therapeutics, the Montreal General Hospital Research Institute, McGill University,

3655 Sir William Osler Promenade (ex-Drummond Street), Montreal, QC H3G 1Y6, Canada

Centre for Research in Neuroscience, the Montreal General Hospital Research Institute, McGill University,

3655 Sir William Osler Promenade (ex-Drummond Street), Montreal, QC H3G 1Y6, Canada Accepted 22 August 2000

Abstract

Neurofilament proteins (NFs) are made by co-polymerization of three intermediate filament proteins, NF-L, NF-M and NF-H and constitute the most abundant cytoskeletal element in large myelinated axons. NFs have a well-established role as intrinsic determinants of axon caliber with all the functional implications, but the role of each individual NF subunit is much less clear. The aim of our study was to examine functional properties of large myelinated axons with altered morphology from mice bearing a targeted disruption of each NF genes (NF-L2/2, NF-M2/2and NF-H 2/2mice). Membrane properties, action potentials and single axon refractory period were measured in isolated sciatic nerves in vitro, using intra-axonal microelectrode recording in conjunction with current–clamp technique. Some results were obtained from whole nerves by sucrose-gap recording. The NF-knockout mice showed several deficits in physiological properties of low-threshold fibers. In keeping with smaller axon diameter, the conduction velocity was significantly decreased in NF-L

2/2 and NF-M 2/2 transgenic animals (control, 39.961.8 m / s, NF-M 2/2; 23.561.4 m / s, and NF-L2/2; 12.060.7 m / s, mean6S.E.M.; intra-axonal recording; similar ratios obtained by sucrose-gap recording; 22–268C). However, in spite of their preserved caliber, large myelinated axons in NF-H 2/2 mice also showed a significant decrease in conduction velocity (22.861.0 m / s, mean6S.E.M.). Although action potential amplitudes, duration and shape did not differ between control axons and transgenic animals, the refractory period was prolonged in NF-H 2/2 and NF-M 2/2 animals. Intracellular injections of 200 ms depolarizing and hyperpolarizing currents revealed outward and inward rectification in all animal groups. In comparison to control animals, NF-H2/2

mice expressed a significant decrease in outward rectification. Potassium channel blockers (4AP and TEA) and cesium ions were able to block outward and inward rectification in all myelinated axons in qualitatively the same manner. These results suggest that NF-H may have a specific role in modulating ion channel functions in large myelinated fibers.  2000 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Genetic models

Keywords: Neurofilament; Transgenic mouse; Myelinated axon; Intra-axonal recording; Conduction velocity

1. Introduction ponent in large myelinated axons of the peripheral nervous

system (PNS). The three neurofilaments subunits (NF-L, Neurofilaments represent the major cytoskeletal com- 68 kDa; NF-M, 134 kDa and NF-H, 200 kDa [33,50]) share a conserved domain, common with other inter-mediate filaments (IF), involved in the formation of

*Corresponding author. Tel.: 11-514-398-3603; fax: 11-514-398- _{coiled–coil structure [23,24,74]. Neurofilaments form} het-4449.

eropolymers, which in mice require NF-L with either E-mail address: [email protected] (A.L. Padjen).

1 _{NF-M or NF-H proteins [48,49].}

Present address: Department of Pharmacology, Faculty of Medicine,

University of Rijeka, Rijeka, Croatia. The three neurofilaments are synthesized in the

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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 NaHCO2 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 pathogenesprompt-is 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]. ,6–7mm 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

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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

ing puller (Sutter Instruments Co., San Francisco, CA)

filled with 3 M KCl and had a direct current resistance 3.1. Distribution of axon calibers in peripheral nerves of 60–80 MV. They were selected for their low noise and normal and transgenic mice

their ability to pass #2 nA without rectification. The

microelectrodes were connected to an amplifier with fast To examine the relationship between neurofilament rise time (Axoclamp 2B, Axon Instruments, Foster City, content and the radial growth and functional properties of CA). A bridge circuit allowed simultaneous current in- the peripheral axons, axonal diameters were measured in jection and recording through the same electrode (‘cur- the L5 ventral roots of 3-month-old animals (Fig. 1A). The rent–clamp’ technique). After capacity compensation, the ventral roots of wild-type mice expressed a bimodal rise time of 50 mV voltage calibration pulse was 20–50ms. distribution of axon calibers representing the populations This bandwidth allowed for appropriate bridge balance. of small and large myelinated axons. A bimodal dis-When possible, bridge balance was checked independently tribution with similar axonal diameters was preserved in by ensuring that the action potential overshoot remained NF-H 2/2 mice. However, peripheral nerves of NF-L constant [20]. After differential amplification at 20 kHz, 2/2 and NF-M 2/2 expressed a unimodal distribution following the low pass 8-pole Bessel filter set at 10 kHz, of small axon calibers with the large myelinated axons and a 16 bit A / D converter (ITC 16 Instrutech Corp., NY) shifted to the small category (Fig. 1B). The average caliber with appropriate software (TIDA for Windows) records of myelinated axons in L5 ventral roots of NF-H 2/2 were stored on a computer for further analysis. Sucrose- mice (4.6 mm) did not differ from controls (4.8 mm), gap recording was done with use of a differential amplifier unlike lower average values in NF-M2/2 (3.2 mm) and

[61]. NF-L (2.6 mm).

2.4. Application of potassium channel blockers 3.2. Resting and action potentials of axons in isolated

sciatic nerves

Potassium channel blockers were applied by diffusion

from 3 M KCl recording microelectrodes containing 50 Electrophysiological results described in this study were mM 4-AP and 20 mM TEA (DC resistance ,80 MV). obtained from over 60 stable recordings from large myeli-Previous studies in this laboratory indicate that their effects nated axons in 25–30-mm segments of sciatic nerve. In appear after 10–15 min of recording, reaching approxi- standard medium, intracellular placement of the microelec-mately 80% of maximal values 20–30 min after impale- trode was indicated by a sharp drop in the recorded ment [67]. Cesium ions (CsCl, 3 mM) were added to the potential (usually to255 to270 mV) and the appearance

perfusate. of brief action potentials (,1.5 ms duration and

overshoot-ing). After penetration, it usually required about 10–15

2.5. Histological methods min for the resting potential to stabilize. There was no

significant difference in average resting membrane po-Mice were killed by an overdose of pentobarbital; their tential (RMP) in axons from different groups of animals heart were perfused with 0.9% NaCl and then with fixative (control axons: 275.560.5 mV; NF-M2/2: 274.261 (2.5% glutaraldehyde, 0.5% paraformaldehyde in 0.1 M mV; NF-H 2/2: 276.160.7 mV; mean6S.E., n524–32 sodium phosphate buffer), and postfixed in 1% phosphate- in each group). Similarly, there was no significant differ-buffered osmium tetroxide. After three washes with phos- ence in average amplitude of action potentials between the phate buffer, each sample was dehydrated in a graded animal groups (see Table 1; control: 85.762.9 mV; NF-M series of ethanol solutions and embedded in Epon. Thin 2/2: 7963.9 mV; NF-H2/2: 85.663.0 mV; n516–19). sections were stained with Toluidine blue and examined Presumably due to smaller axon diameter in NF-L 2/2 under a Polyvar microscope. Axons were counted and mice, it was very difficult to obtain stable RMP values for axonal diameter and myelin thickness measured in the L5 more than 5–10 min, so that intra-axonal recordings in

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Fig. 1. Light micrographs of L5 ventral roots from normal (A), NF-M2/2(B), NF-L2/2(C), and NF-H2/2(D) mice. The depletion of NF-H2/2

(D) had the least effects on axonal calibers followed by the depletion of NF-M2/2(B). Note the absence of large motor axons in NF-L2/2mice (C). Calibration bar550mm. (B) Comparison of inner axonal diameters from L5 ventral root axons of control, NF-M2/2, NF-L2/2and NF-H2/2mice. Quantitative study of axonal calibers reveals a marked reduction in transgenic sample (NF-L2/2and NF-M2/2) with unimodal distribution showing the absence of large myelinated fibers. Axon diameter is preserved in NF-H2/2mice with apparent maintenance of bimodal distribution as compared to the control. The histogram represents the distribution of inner axonal diameter of axons in L5 ventral roots from 3 to 5 animals per each animal group.

these animals were suitable only for measurements of ms (n525). Two groups of transgenic animals showed a

conduction velocity (CV). significant decrease in CV of their axons (NF-M 2/2:

23.5461.41 m / s n525; NF-L 2/2: 12.0560.69 m / s,

3.3. Conduction velocity n522), as expected from their smaller diameters.

Un-expectedly, CV in axons from NF-H 2/2 mice was also Conduction velocity was measured at supra-maximal significantly decreased (22.861.0 ms, n524; Fig. 3), stimulation of the proximal trunk of the isolated sciatic though in these animals the distribution of axon diameters nerve segments. It was calculated by comparing the latency was similar to that in wild-type mice (See Fig. 1). between the stimulus artifact and either the rising phase of In several animals from each group the values of CVs action potential, in case of the intra-axonal recordings, or recorded in single axons were compared with the measure-the peak compound action potentials (CAP), in case of measure-the ments on whole sciatic nerves using sucrose-gap recording sucrose-gap recordings, and the distance between stimulat- of compound action potentials (CAPs). This approach also ing electrode and the site of recording. allowed determination of the spike threshold for population At room temperature (238C), the average CV from of axons. In wild-type mice, stimulation of the low-thres-single myelinated axons of control animals was 39.961.8 hold fibers (pulses of 0.02 ms, 6–9 V) generated CAP with

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Fig. 1. (continued )

one peak. Fig. 2 shows samples of typical CAPs obtained higher room temperature (268C), which explains their from sciatic nerves of different transgenic animals. The CV faster CV compared to single axons recordings, but the in all transgenic animal groups was significantly slower relative values are essentially identical to those obtained by than controls (control: 48 m / s; NF-H 2/2: 33 m / s; intra-axonal recording. In sciatic nerves from NF-L2/2, NF-M: 28 m / s; NF-L 2/2: 18 m / s for the fastest fiber unlike the CAPs in nerves from wild-type controls, NF-population). These measurements were done at somewhat H2/2 and NF-M2/2mice, low-threshold stimuli

gener-Table 1

Functional properties of large myelinated axons of mice with different neurofilament expression

RMP (mV) AP (mV) AP/ 2 (ms) Hyperpolarization Depolarization

Mean6SE n Mean6SE n Mean6SE n Rp (MV) Rss (MV) Rp (MV) Rss (MV) Mean6SE n Mean6SE n Mean6SE n Mean6SE n

Control 275.560.5 31 85.763.0 19 0.6560.02 19 33.562.4 28 25.863.1 28 14.261.0 28 12.161.0 28 NF-M2/2 274.261.0 24 79.063.9 16 0.6860.02 16 31.263.0 11 27.262.7 11 15.561.2 11 12.961.2 11 NF-H2/2 276.160.7 25 85.663.0 16 0.7160.02 16 42.663.9* 14 34.463.4 14 16.861.0 14 15.561.0* 14

Results obtained by intra-axonal recording. RMP, resting membrane potential; AP, action potential amplitude; AP/ 2, action potential duration at half-amplitude; R and R , peak and steady state input resistance (measured 15–20 ms after pulse initiation and 190–195 ms after pulse initiation,p ss

respectively) during hyperpolarization or depolarization, as indicated. Values represent means6SEM and n. Values in bold* characters significantly different from control, P#0.05; ANOVA and Tukey’s post hoc test.

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ated CAPs with several peaks, suggesting the presence of populations of fibers with similar thresholds but with different CVs. Multiple action potentials have never been observed in intra-axonal recordings from NF-L 2/2 axons, arguing against the alternative possibility that the multiple peaks were due to repetitive activity in these low-threshold fibers.

3.4. Action potential shape and conduction velocity There was no significant difference in AP amplitude or AP duration between axons from transgenic and control animals, although values recorded from axons of NF-H 2/2 mice showed a tendency for longer APs. Thus, the average AP duration at half-amplitude (AP/ 2) in axons from various mice did not differ (see Table 1; see also samples in Fig. 3). At membrane potentials close to the RMP, action potentials were only occasionally followed by slow depolarizing afterpotentials (DAP, as originally de-scribed in amphibian axons [2] and mammalian axons [16]. In those cases, the DAPs showed voltage dependence by increasing in amplitude with membrane hyperpolariza-tion.

An analysis of the time course of AP (dV / dt of rising and falling phase) revealed no significant differences between the nerves from control and transgenic animals (rising phase: 475634 V/ s in controls, 438629 V/ s in NF-M2/2; 457617 V/ s in NF-H2/2 mice; corre-sponding values of falling phase: 213466 V/ s in control, 2134610 V/ s in NF-M2/2; 213767 in NF-H 2/2 mice, mean6S.E., n513–19).

However, examination of the correlation between the rates of rising and falling phases of AP and CV and / or AP/ 2 revealed significant differences among the tested groups. In control animals the rate of AP rise vs. CV (Fig. 4A), and the rate of AP decline vs. AP duration (Fig. 4B) showed significant positive correlation (P,0.05). In axons from transgenic mice (Fig. 4A,B) only NF-M2/2 mice expressed a significant (positive) correlation between the rate of AP decline and AP duration (Fig. 4B).

3.5. Recovery period of AP generation

Recovery of AP generation (refractory or recovery period) was obtained by double stimulation using supra-maximal stimuli and varying the inter-pulse interval. Recovery of AP generation in axons from NF-M2/2and NF-H2/2 mice was significantly slower than in control animals. In the first 3–5 ms, recovery reached 75–80% of

Fig. 2. Compound action potentials (CAPs) were simultaneously

re-the test AP amplitude compared to wild-type mice where

corded from 20 to 25-mm long segments of sciatic nerves isolated from

control and transgenic mice (as labeled). Note that the difference in these values were close to 90% of the initial spike height

delays do not reflect the true difference in CV since the nerve pairs were _{(Fig. 5).} not of the same length (see text for details). Samples of CAPs induced by

supra-maximal stimulation for low-threshold fibers show multiple peaks

3.6. Accommodation

in nerves from NF-L 2/2 mice as compared with control and other knockouts. The conduction velocity obtained from sucrose-gap recordings

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Fig. 3. Samples of typical intra-axonally recorded action potentials with corresponding differentiated values (dV / dt) (control, NF-M2/2and NF-H2/2

mice).

current pulses with only a few action potentials, indicating hyperpolarizing current steps were used to test the ex-the presence of a strong accommodation. This is similar to citability of axons and construct V/I relationships. In all previous observations in other axonal preparations animal groups the voltage responses were characterized by [1,64,67]. Although some of the fibers in this study a fast rise to depolarizing pulses and a noticeably slower showed differences in accommodation no clear pattern response to the hyperpolarizing pulses. The

hyperpolariz-could be established. ing voltage responses featured a ‘sag’, typically observed

100–150 ms after the initiation of current pulse (not 3.7. Membrane responses and V/I relationship shown), and interpreted as evidence of inward rectifier. The ‘sag’ reached the steady state within 150–200 ms, at Intracellular injections of 200 ms depolarizing and membrane potentials 10–40 mV below RMP. There was

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Fig. 4. (A) Correlation between dV / dt of rising phase of an action potential and its CV. Note significant positive correlation (P,0.05) for controls, but not for axons from transgenic animal. (B) Correlation between dV / dt of falling phase of action potential and its duration at half-amplitude (AP/ 2). Note significant positive correlation for controls and NF-M2/2(P,0.05), and no correlation for NF-H2/2axons. Correlation coefficients are given above the regression lines. (Control, n518, NF-M2/2, n512, and NF-H2/2, n513).

considerable variety in the expression of inward rectifica- resistance ranged from 1 to 3 MV in responses to tion in the population of axons, but no consistent pattern depolarizing pulses, to 60–70 MV at the peak of

hy-could be established. perpolarizing pulses. There were two regions of linear V /I

To obtain voltage–current (V /I ) curves the membrane relationship below 2100 mV (defined as peak resistance, potential was measured at the peak (R ) and the steadyp R ) and steady-state resistance, R ), the latter identifiedp ss

state (R ) of the voltage displacement. The V /I relation-ss with the activation of inward rectifier. In axons from NF-H ship was strongly non-linear, reflecting the activation of 2/2 mice there was a significant increase in R values inp

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subunit genes in order to establish their role in functional properties of axons. As expected, the conduction velocity of axons appeared to be related to their diameter (see Section 3), the smaller average diameters of axons in NF-L 2/2 and NF-M 2/2 being associated with slower conduction in records from whole sciatic nerves. Similar results were obtained at the single axon level using microelectrode recording from large myelinated nerve fibers of NF-L2/2 and NF-M2/2mice. Unexpectedly, this relationship did not hold for the NF-H 2/2 mice where the conduction velocity measured in whole nerves or single axons was significantly decreased in spite of a normal distribution of diameters.

Intra-axonal studies revealed several differences in physiological parameters of peripheral nerves in transgenic mice: (1) the refractory period in NF-M2/2 and NF-H 2/2 mice was prolonged; (2) in contrast to axons from

Fig. 5. Refractory period of action potential (AP) generation is increased

in single sciatic axons of transgenic mice. In intra-axonal recordings wild-type and NF-M2/2, in NF-H2/2 mice there was

samples were obtained by varying delay to second identical stimulus in _{no correlation between the rate of rise and decay of action} dual stimulation protocol (see Section 2). Recovery of AP generation was

potential and conduction velocity (as well as AP duration

measured 3, 5 and 7 ms after first stimulating pulse. Note almost

at half amplitude); (3) changes in rectification in axons of

complete recovery of AP amplitude after delay of 5 ms in axons of

NF-H 2/2 mice (increase in input resistance during

control animals. Recovery of AP in axons of NF-M2/2and NF-H2/2

mice is decreased in first 3–5 ms reaching the control values by 7 ms. hyperpolarization and in the input resistance in the steady

Values represent mean6S.E.; *significantly different from control, P# _{state period during depolarization). These findings suggest}

0.05 (n53).

changes in the function of axonal ion channels (K and 1

Na ) and / or possible morphological changes of axons in inward rectification (measured as a ratio of R /R ) weress p transgenic animals that will be discussed separately. similar in all animal groups (0.77, 0.87, 0.81, for control,

NF-M2/2 and NF-H 2/2, respectively)

The shape of the V /I relationship obtained by injection 4.1. Neurofilaments, axonal caliber and conduction of depolarizing currents was highly non-linear, indicating velocity

strong outward rectification of axonal membrane (cf. Refs.

[59,67]). Input resistance was measured at two points; at The present understanding of the role of NFs is that they the voltage displacement 15–20 ms after pulse onset (R )p are major intrinsic determinants of axon caliber in large and at near steady-state point (175 ms later; R ). Theress myelinated nerve fibers [12,46,81,84]. The control of was no significant difference in R , but the axons of NF-Hp axonal caliber has a functional significance because diam-2/2 mice showed a significant increase in Rss resistance eter is the principal determinant of conduction velocity in

(see Table 1). myelinated nerve fibers [30,70,71,78]. The report of axonal

The effect of K channel blockade was examined using hypotrophy and reduced conduction velocity in Japanese microelectrodes containing 50 mM 4-AP and 20 mM TEA quail mutant lacking NFs as a result of nonsense mutation (see Section 2). Twenty minutes after impalement there in the NF-L gene [56,72] is particularly strong evidence in was a marked decrease in outward rectification as expected favor of this notion. Reduced axonal calibers accompanied from previous studies [1,67] (data not shown). Approxi- by decreased conduction velocity have also been observed mately 10 min after addition of CsCl (3 mM) to the in transgenic mice expressing NF-H /b-galactosidase fu-perfusate, the impaled axons of control animals lost the sion protein that interferes with neurofilament transport characteristic ‘sag’ in hyperpolarizing potentials. Cesium into axon [18,40]. Reduced axonal diameter (with neurode-did not affect outward rectification in the region positive to generation) has been also observed in various transgenic and just negative to the RMP, suggesting that extracellular mice over-expressing any single neurofilament subunit cesium ions did not affect any conductances activated in [13,14,41,83]. The positive correlation between fiber diam-the region around RMP. TEA (20 mM) depolarized diam-the eter and conduction velocity or between fiber diameter and

fibers 5–10 mV. neurofilament gene expression seems to hold even for

changes following nerve injury or during neuronal disease [30,38,39]. As expected, our study provides evidence in

4. Discussion support of a positive correlation between reduction in the

diameter and conduction velocity in large myelinated fibers We have examined the electrophysiological properties of of sciatic nerve, but only for the NF-L and NF-M knockout large myelinated axons in mice lacking neurofilament mice.

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A plausible view of the mechanism by which NF 4.2. Differences in ion channel functions maintain axon caliber involves the highly phosphorylated

carboxy-tail domain of NF-H that orders axoplasmic The results presented here, and in our recent study of structure through interaction (cross-bridging) between mice expressing human NF-H [41], indicate that mem-adjacent neurofilaments [28,29]. The phosphorylated NF-H brane voltage responses and action potentials recorded in tail may contribute to axonal caliber through a three- axons from normal mice are similar to those obtained by dimensional filament scaffold and / or through interactions intra-axonal recordings from other vertebrate myelinated with other axonal components, such as microtubules, axons. Thus, responses to hyperpolarizing current in cortical actin arrays, and potentially many other com- normal mice axons demonstrate a long charging time ponents. The preserved diameter of large myelinated axons constant, similar in duration to that in amphibian [2,58] in NF-H 2/2 mice could probably be explained by and rat axons [1,5] as well as outward and inward hyperphosphorylation of the NF-H tails that could in- rectifications. In agreement with the findings in rats [1], fluence the nearest inter-filament distance and / or twofold outward rectification is suppressed by 4-AP and TEA, increase in microtubules [69,81,87]. indicating the involvement of at least two

voltage-depen-1

Why the myelinated axons in NF-H 2/2 mice do not dent K internodal conductances [1,9,10,16,70,79,80]; follow the predicted structure–function (i.e., diameter and while the inward rectification is sensitive to cesium, as in CV) relationship? In these mice, the nerve fiber diameter other peripheral axons [1,66,67] and other cells [63]. has the typical bimodal distribution. The fact that the CV Action potentials in mice axons are only occasionally measured by intra-axonal sampling agrees with the CV of followed by a voltage-dependent DAP (increased by the compound action potentials argues against the unrepre- hyperpolarization), as observed in rats [4,5,16], unlike their sentative sampling by the microelectrodes (in any case, common appearance in amphibian axons [2,64]. These intra-axonal recordings appear to preferentially sample the differences in DAPs expression are considered to result

largest axons, see Section 2). Preliminary morphological from enhanced activation of K conductances in mam-examination of large myelinated axon from NF-H 2/2 malian axons [16] rather than any difference in the high show that both the G ratio (ratio of axonal diameter vs. conductance pathway through myelin sheets (Barrett–Bar-total myelinated axon diameter [21]; ca. 0.6–0.7) and rett conductance [2,4,70]), as mentioned above.

internodal distances (ca. 1 mm) are similar to those in The upstroke (dV / dt) of the AP in transgenic mice was control mice. Thus, the fibers in NF-H 2/2 mice would not significantly different from the control values, sug-belong to Rushton’s category of ‘dimensionally similar’ gesting that there was no alteration in sodium conductance fibers [71], i.e. one would expect CV to be proportional to activation. However, the refractory period (action potential the axonal diameter. Yet, there is approximately 20% recovery) in transgenic animals was significantly delayed reduction in conduction velocity in axons from NF-H2/2 in the first 3–5 ms after a test AP, suggesting a delay in

mice as compared to control fibers suggesting that mem- recovery from Na conductance inactivation. Since several 1

brane characteristics of axons have changed properties. lines of evidence link Na channel phosphorylation and One may also take the differences in correlation between the inactivation process [15,36,55], one may speculate action potential properties and CV (see Section 3) as whether a change in phosphorylation may be involved in another indication of differences in membrane properties in the observed results.

transgenic animals, although for the moment we do not What still remains to be explained are the differences in have a plausible explanation of this phenomenon. correlation between the AP time course on the one hand Axonal diameter has been established as the primary and with the other, conduction velocity and duration of AP. determinant of CV in myelinated axons. Previous theoret- Theoretical studies have identified the rise time of the ical evaluations indicate that CV should be relatively action potential as having an important bearing on the insensitive to nodal parameters, moderately sensitive to conduction velocity in myelinated axons [37]. Thus, at axoplasmic conductance (as well as diameter) and quite physiological temperature the relationship for large myeli-insensitive to internodal parameters [37,70,78]. This early nated axons is found to be approximately 6 m / s per work may not be entirely relevant because it does not take micron of overall fiber diameter [35]. Any deviation from into consideration the recent finding that the internodal this relationship, as was observed in axons from transgenic

membrane, with its spectrum of voltage dependent K mice (NF-H 2/2), would suggest a difference in the conductances [79] does participate in the conduction of membrane properties. Preliminary morphological studies impulses via the so-called Barrett–Barrett pathway [2– indicate that the internodal distance in transgenic animals 4,59,70]. Additional evidence in favor of this model of falls within the normal range, suggesting that this mor-axons has recently been obtained in simulation experi- phological parameter cannot be involved. One remaining ments (‘leaky myelin’ model [60]). The present results unchecked parameter that could change (decrease) the CV

demonstrate a decrease in internodal conductance (i.e. K is an increase in axoplasmic resistance. At present it is not conductances, see below) in axons of NF-H 2/2 mice. known whether disturbance of the intracellular architecture This decrease may be relevant for the decrease in CV in the transgenic animals may affect the axial resistance.

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sclerosis, in: R.A. Smith (Ed.), Handbook of Amyotrophic Lateral

regulatory mechanism(s) linking changes in NF and ion

Sclerosis, Marcel Dekker, New York, 1992, pp. 133–181.

channel functions. However, the existence of a complex

[12] D.W. Cleveland, M.J. Monteiro, P.C. Wong, S.R. Gill, J.D. Gearhart,

and yet unidentified link between NF expression and ion _{P.N. Hoffman, Involvement of neurofilaments in the radial growth of} channel function in myelinated axons is corroborated by axons, J. Cell Sci. Suppl. 15 (1991) 85–95.

our recent study of mice expressing human NF-H [41,42]. [13] J.F. Collard, F. Cote, J.P. Julien, Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis (see

In summary, this study describes some of the basic

comments), Nature 375 (1995) 61–64.

electrophysiological properties of large myelinated fibers

[14] F. Cote, J.F. Collard, J.P. Julien, Progressive neuronopathy in

in mice with different neurofilament subunit expression. _{transgenic mice expressing the human neurofilament heavy gene: a} Our results indicate that myelinated axons of NF-H 2/2 _{mouse model of amyotrophic lateral sclerosis, Cell 73 (1993)} mice, in comparison with control and NF-M2/2 mice, 35–46.

[15] S. Cukierman, Regulation of voltage-dependent sodium channels

express different functional properties than expected from

(Review), J. Membr. Biol. 151 (1996) 203–214.

morphological changes alone (decreased CV without a

[16] G. David, B. Modney, K.A. Scappaticci, J.N. Barrett, E.F. Barrett,

change in axon size), suggesting that NF-H itself or

Electrical and morphological factors influencing the depolarizing

through its interaction with other axonal components _{after-potential in rat and lizard myelinated axons, J. Physiol. 489} (microtubules, actin or perhaps other cytoskeletal mole- (1995) 141–157.

[17] S.M. De Waegh, V.M.Y. Lee, S.T. Brady, Local modulation of

cules) may have a role in modifying the functional

neurofilament phosphorylation, axonal caliber, and slow axonal

properties of large myelinated fibers. In addition, the

transport by myelinating Schwann cells, Cell 68 (1992) 451–463.

electrical properties defined in this study should help to

[18] J. Eyer, A. Peterson, Neurofilament-deficient axons and perikaryal

generate a computational model that would further com- _{aggregates in viable transgenic mice expressing a} neurofilament-plete our understanding of these complex neural structures. beta-galactosidase fusion protein, Neuron 12 (1994) 389–405.

[19] D.A. Figlewicz, A. Krizus, M.G. Martinoli, V. Meininger, M. Dib, G.A. Rouleau, J.P. Julien, Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral

Acknowledgements _{sclerosis, Hum. Mol. Genet. 3 (1994) 1757–1761.}

[20] K. Frank, M.G.F. Fuortes, Stimulation of spinal motoneurons with intracellular electrodes, J. Physiol. (Lond.) 134 (1956) 451–470.

This work has been supported in part by the Canadian

[21] R.L. Friede, R. Bischhausen, How are sheath dimensions affected by

ALS Association, Canadian Diabetes Association and

axon caliber and internode length?, Brain Res. 235 (1982) 335–350.

MRC of Canada. We thank Dr K. Krnjevic for comments. _{[22] R.L. Friede, T. Samorajski, Axon caliber related to neurofilaments}

and microtubules in sciatic nerve fibers of rats and mice, Anat. Rec. 167 (1970) 379–387.

[23] E. Fuchs, D.W. Cleveland, A structural scaffolding of intermediate

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J. Kriz et al. / Brain Research 885 (2000) 32 –44 39

Fig. 4. (A) Correlation between dV / dt of rising phase of an action potential and its CV. Note significant positive correlation (P,0.05) for controls, but not for axons from transgenic animal. (B) Correlation between dV / dt of falling phase of action potential and its duration at half-amplitude (AP/ 2). Note significant positive correlation for controls and NF-M2/2(P,0.05), and no correlation for NF-H2/2axons. Correlation coefficients are given above the regression lines. (Control, n518, NF-M2/2, n512, and NF-H2/2, n513).

considerable variety in the expression of inward rectifica-

resistance ranged from 1 to 3 MV

in responses to

tion in the population of axons, but no consistent pattern

depolarizing pulses, to 60–70 M

V

at the peak of

hy-could be established.

perpolarizing pulses. There were two regions of linear V /I

To obtain voltage–current (V /I ) curves the membrane

relationship below

2100 mV (defined as peak resistance,

potential was measured at the peak (R ) and the steady

R ) and steady-state resistance, R ), the latter identified

p ss

state (R ) of the voltage displacement. The V /I relation-

with the activation of inward rectifier. In axons from NF-H

ship was strongly non-linear, reflecting the activation of

2 /2

mice there was a significant increase in R values in

(2)

subunit genes in order to establish their role in functional

properties of axons. As expected, the conduction velocity

of axons appeared to be related to their diameter (see

Section 3), the smaller average diameters of axons in NF-L

2 /2

and NF-M

2 /

2 being associated with slower

conduction in records from whole sciatic nerves. Similar

results were obtained at the single axon level using

microelectrode recording from large myelinated nerve

fibers of NF-L

2 /2

and NF-M

2 /

2 mice. Unexpectedly,

this relationship did not hold for the NF-H

2 /

2 mice

where the conduction velocity measured in whole nerves

or single axons was significantly decreased in spite of a

normal distribution of diameters.

Intra-axonal studies revealed several differences in

physiological parameters of peripheral nerves in transgenic

mice: (1) the refractory period in NF-M2

/

2 and NF-H

2 /2

mice was prolonged; (2) in contrast to axons from

Fig. 5. Refractory period of action potential (AP) generation is increased

in single sciatic axons of transgenic mice. In intra-axonal recordings

wild-type and NF-M2

/

2, in NF-H2

/

2 mice there was

samples were obtained by varying delay to second identical stimulus in

_{no correlation between the rate of rise and decay of action}

dual stimulation protocol (see Section 2). Recovery of AP generation was

potential and conduction velocity (as well as AP duration

measured 3, 5 and 7 ms after first stimulating pulse. Note almost

at half amplitude); (3) changes in rectification in axons of

complete recovery of AP amplitude after delay of 5 ms in axons of

NF-H

2 /

2 mice (increase in input resistance during

control animals. Recovery of AP in axons of NF-M2/2and NF-H2/2

mice is decreased in first 3–5 ms reaching the control values by 7 ms.

hyperpolarization and in the input resistance in the steady

Values represent mean6S.E.; *significantly different from control, P#

_{state period during depolarization). These findings suggest}

0.05 (n53).

changes in the function of axonal ion channels (K

and

Na ) and / or possible morphological changes of axons in

inward rectification (measured as a ratio of R /R ) were

ss p

transgenic animals that will be discussed separately.

similar in all animal groups (0.77, 0.87, 0.81, for control,

NF-M

2 /

2 and NF-H

2 /

2, respectively)

The shape of the V /I relationship obtained by injection

4.1. Neurofilaments, axonal caliber and conduction

of depolarizing currents was highly non-linear, indicating

velocity

strong outward rectification of axonal membrane (cf. Refs.

[59,67]). Input resistance was measured at two points; at

The present understanding of the role of NFs is that they

the voltage displacement 15–20 ms after pulse onset (R )

are major intrinsic determinants of axon caliber in large

and at near steady-state point (175 ms later; R ). There

myelinated nerve fibers [12,46,81,84]. The control of

was no significant difference in R , but the axons of NF-H

axonal caliber has a functional significance because

diam-2

/2

mice showed a significant increase in R

resistance

eter is the principal determinant of conduction velocity in

(see Table 1).

myelinated nerve fibers [30,70,71,78]. The report of axonal

The effect of K

channel blockade was examined using

hypotrophy and reduced conduction velocity in Japanese

microelectrodes containing 50 mM 4-AP and 20 mM TEA

quail mutant lacking NFs as a result of nonsense mutation

(see Section 2). Twenty minutes after impalement there

in the NF-L gene [56,72] is particularly strong evidence in

was a marked decrease in outward rectification as expected

favor of this notion. Reduced axonal calibers accompanied

from previous studies [1,67] (data not shown). Approxi-

by decreased conduction velocity have also been observed

mately 10 min after addition of CsCl (3 mM) to the

in transgenic mice expressing NF-H /b-galactosidase

fu-perfusate, the impaled axons of control animals lost the

sion protein that interferes with neurofilament transport

characteristic ‘sag’ in hyperpolarizing potentials. Cesium

into axon [18,40]. Reduced axonal diameter (with

neurode-did not affect outward rectification in the region positive to

generation) has been also observed in various transgenic

and just negative to the RMP, suggesting that extracellular

mice over-expressing any single neurofilament subunit

cesium ions did not affect any conductances activated in

[13,14,41,83]. The positive correlation between fiber

diam-the region around RMP. TEA (20 mM) depolarized diam-the

eter and conduction velocity or between fiber diameter and

fibers 5–10 mV.

neurofilament gene expression seems to hold even for

changes following nerve injury or during neuronal disease

[30,38,39]. As expected, our study provides evidence in

4. Discussion

support of a positive correlation between reduction in the

diameter and conduction velocity in large myelinated fibers

We have examined the electrophysiological properties of

of sciatic nerve, but only for the NF-L and NF-M knockout

large myelinated axons in mice lacking neurofilament

mice.

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J. Kriz et al. / Brain Research 885 (2000) 32 –44 41

A plausible view of the mechanism by which NF

4.2. Differences in ion channel functions

maintain axon caliber involves the highly phosphorylated

carboxy-tail domain of NF-H that orders axoplasmic

The results presented here, and in our recent study of

structure through interaction (cross-bridging) between

mice expressing human NF-H [41], indicate that

mem-adjacent neurofilaments [28,29]. The phosphorylated NF-H

brane voltage responses and action potentials recorded in

tail may contribute to axonal caliber through a three-

axons from normal mice are similar to those obtained by

dimensional filament scaffold and / or through interactions

intra-axonal recordings from other vertebrate myelinated

with other axonal components, such as microtubules,

axons. Thus, responses to hyperpolarizing current in

cortical actin arrays, and potentially many other com-

normal mice axons demonstrate a long charging time

ponents. The preserved diameter of large myelinated axons

constant, similar in duration to that in amphibian [2,58]

in NF-H

2 /

2 mice could probably be explained by

and rat axons [1,5] as well as outward and inward

hyperphosphorylation of the NF-H tails that could in-

rectifications. In agreement with the findings in rats [1],

fluence the nearest inter-filament distance and / or twofold

outward rectification is suppressed by 4-AP and TEA,

increase in microtubules [69,81,87].

indicating the involvement of at least two

voltage-depen-1

Why the myelinated axons in NF-H

2 /

2 mice do not

dent K

internodal conductances [1,9,10,16,70,79,80];

follow the predicted structure–function (i.e., diameter and

while the inward rectification is sensitive to cesium, as in

CV) relationship? In these mice, the nerve fiber diameter

other peripheral axons [1,66,67] and other cells [63].

has the typical bimodal distribution. The fact that the CV

Action potentials in mice axons are only occasionally

measured by intra-axonal sampling agrees with the CV of

followed by a voltage-dependent DAP (increased by

the compound action potentials argues against the unrepre-

hyperpolarization), as observed in rats [4,5,16], unlike their

sentative sampling by the microelectrodes (in any case,

common appearance in amphibian axons [2,64]. These

intra-axonal recordings appear to preferentially sample the

differences in DAPs expression are considered to result

largest axons, see Section 2). Preliminary morphological

from enhanced activation of K

conductances in

mam-examination of large myelinated axon from NF-H

2 /

2 malian axons [16] rather than any difference in the high

show that both the G ratio (ratio of axonal diameter vs.

conductance pathway through myelin sheets

(Barrett–Bar-total myelinated axon diameter [21]; ca. 0.6–0.7) and

rett conductance [2,4,70]), as mentioned above.

internodal distances (ca. 1 mm) are similar to those in

The upstroke (dV / dt) of the AP in transgenic mice was

control mice. Thus, the fibers in NF-H

2 /2

mice would

not significantly different from the control values,

sug-belong to Rushton’s category of ‘dimensionally similar’

gesting that there was no alteration in sodium conductance

fibers [71], i.e. one would expect CV to be proportional to

activation. However, the refractory period (action potential

the axonal diameter. Yet, there is approximately 20%

recovery) in transgenic animals was significantly delayed

reduction in conduction velocity in axons from NF-H

2 /

2 in the first 3–5 ms after a test AP, suggesting a delay in

mice as compared to control fibers suggesting that mem-

recovery from Na

conductance inactivation. Since several

brane characteristics of axons have changed properties.

lines of evidence link Na

channel phosphorylation and

One may also take the differences in correlation between

the inactivation process [15,36,55], one may speculate

action potential properties and CV (see Section 3) as

whether a change in phosphorylation may be involved in

another indication of differences in membrane properties in

the observed results.

transgenic animals, although for the moment we do not

What still remains to be explained are the differences in

have a plausible explanation of this phenomenon.

correlation between the AP time course on the one hand

Axonal diameter has been established as the primary

and with the other, conduction velocity and duration of AP.

determinant of CV in myelinated axons. Previous theoret-

Theoretical studies have identified the rise time of the

ical evaluations indicate that CV should be relatively

action potential as having an important bearing on the

insensitive to nodal parameters, moderately sensitive to

conduction velocity in myelinated axons [37]. Thus, at

axoplasmic conductance (as well as diameter) and quite

physiological temperature the relationship for large

myeli-insensitive to internodal parameters [37,70,78]. This early

nated axons is found to be approximately 6 m / s per

work may not be entirely relevant because it does not take

micron of overall fiber diameter [35]. Any deviation from

into consideration the recent finding that the internodal

this relationship, as was observed in axons from transgenic

membrane, with its spectrum of voltage dependent K

mice (NF-H

2 /

2), would suggest a difference in the

conductances [79] does participate in the conduction of

membrane properties. Preliminary morphological studies

impulses via the so-called Barrett–Barrett pathway [2–

indicate that the internodal distance in transgenic animals

4,59,70]. Additional evidence in favor of this model of

falls within the normal range, suggesting that this

mor-axons has recently been obtained in simulation experi-

phological parameter cannot be involved. One remaining

ments (‘leaky myelin’ model [60]). The present results

unchecked parameter that could change (decrease) the CV

demonstrate a decrease in internodal conductance (i.e. K

is an increase in axoplasmic resistance. At present it is not

conductances, see below) in axons of NF-H

2 /

2 mice.

known whether disturbance of the intracellular architecture

This decrease may be relevant for the decrease in CV

in the transgenic animals may affect the axial resistance.

observed in these NF-H

2 /

2 mice.

For the moment we can only speculate about possible

(4)

sclerosis, in: R.A. Smith (Ed.), Handbook of Amyotrophic Lateral

regulatory mechanism(s) linking changes in NF and ion

Sclerosis, Marcel Dekker, New York, 1992, pp. 133–181.

channel functions. However, the existence of a complex

[12] D.W. Cleveland, M.J. Monteiro, P.C. Wong, S.R. Gill, J.D. Gearhart,

and yet unidentified link between NF expression and ion

_{P.N. Hoffman, Involvement of neurofilaments in the radial growth of}

channel function in myelinated axons is corroborated by

axons, J. Cell Sci. Suppl. 15 (1991) 85–95.

our recent study of mice expressing human NF-H [41,42].

[13] J.F. Collard, F. Cote, J.P. Julien, Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis (see

In summary, this study describes some of the basic

comments), Nature 375 (1995) 61–64.

electrophysiological properties of large myelinated fibers

[14] F. Cote, J.F. Collard, J.P. Julien, Progressive neuronopathy in

in mice with different neurofilament subunit expression.

_{transgenic mice expressing the human neurofilament heavy gene: a}

Our results indicate that myelinated axons of NF-H

2 /

2

_{mouse model of amyotrophic lateral sclerosis, Cell 73 (1993)}

mice, in comparison with control and NF-M2

/

2 mice,

35–46.

[15] S. Cukierman, Regulation of voltage-dependent sodium channels

express different functional properties than expected from

(Review), J. Membr. Biol. 151 (1996) 203–214.

morphological changes alone (decreased CV without a

[16] G. David, B. Modney, K.A. Scappaticci, J.N. Barrett, E.F. Barrett,

change in axon size), suggesting that NF-H itself or

Electrical and morphological factors influencing the depolarizing

through its interaction with other axonal components

_{after-potential in rat and lizard myelinated axons, J. Physiol. 489}

(microtubules, actin or perhaps other cytoskeletal mole-

(1995) 141–157.

[17] S.M. De Waegh, V.M.Y. Lee, S.T. Brady, Local modulation of

cules) may have a role in modifying the functional

neurofilament phosphorylation, axonal caliber, and slow axonal

properties of large myelinated fibers. In addition, the

transport by myelinating Schwann cells, Cell 68 (1992) 451–463.

electrical properties defined in this study should help to

[18] J. Eyer, A. Peterson, Neurofilament-deficient axons and perikaryal

generate a computational model that would further com-

_{aggregates in viable transgenic mice expressing a}

neurofilament-plete our understanding of these complex neural structures.

beta-galactosidase fusion protein, Neuron 12 (1994) 389–405.

[19] D.A. Figlewicz, A. Krizus, M.G. Martinoli, V. Meininger, M. Dib, G.A. Rouleau, J.P. Julien, Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral

Acknowledgements

_{sclerosis, Hum. Mol. Genet. 3 (1994) 1757–1761.}

[20] K. Frank, M.G.F. Fuortes, Stimulation of spinal motoneurons with intracellular electrodes, J. Physiol. (Lond.) 134 (1956) 451–470.

This work has been supported in part by the Canadian

[21] R.L. Friede, R. Bischhausen, How are sheath dimensions affected by

ALS Association, Canadian Diabetes Association and

axon caliber and internode length?, Brain Res. 235 (1982) 335–350.

MRC of Canada. We thank Dr K. Krnjevic for comments.

_{[22] R.L. Friede, T. Samorajski, Axon caliber related to neurofilaments} and microtubules in sciatic nerve fibers of rats and mice, Anat. Rec. 167 (1970) 379–387.

[23] E. Fuchs, D.W. Cleveland, A structural scaffolding of intermediate

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