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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 20–50 ms. 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-M 2 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 to 255 to 270 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-H 2 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
J . Kriz et al. Brain Research 885 2000 32 –44
35
Fig. 1. Light micrographs of L5 ventral roots from normal A, NF-M2 2 B, NF-L2 2 C, and NF-H 2 2 D mice. The depletion of NF-H 2 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 2 mice C.
Calibration bar550 mm. B Comparison of inner axonal diameters from L5 ventral root axons of control, NF-M 2 2, NF-L 2 2 and NF-H 2 2 mice. Quantitative study of axonal calibers reveals a marked reduction in transgenic sample NF-L2 2 and NF-M 2 2 with unimodal distribution showing the
absence of large myelinated fibers. Axon diameter is preserved in NF-H 2 2 mice 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 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 2 mice, low-threshold stimuli gener-
Table 1
a
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
a
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, P0.05; ANOVA and Tukey’s post hoc test.
J . Kriz et al. Brain Research 885 2000 32 –44
37
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-M 2 2 and NF-H 2 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
was in agreement with the average intra-axonal values see text.
Most of the axons responded to long depolarizing
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Fig. 3. Samples of typical intra-axonally recorded action potentials with corresponding differentiated values dV dt control, NF-M 2 2 and NF-H 2 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 VI 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 VI 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
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-M 2 2 P,0.05, and no correlation for NF-H 2 2 axons. Correlation coefficients are given above the regression lines. Control, n518, NF-M 2 2, n512, and NF-H 2 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 steady R and steady-state resistance, R , the latter identified
p p
ss
state R of the voltage displacement. The V I relation- with the activation of inward rectifier. In axons from NF-H
ss
ship was strongly non-linear, reflecting the activation of 2 2 mice there was a significant increase in R values in
p
more than one voltage-dependent conductance. The input hyperpolarization see Table 1. However, the degree of
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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-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 2 and NF-H 2 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
1
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 were
transgenic animals that will be discussed separately.
ss p
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
p
and at near steady-state point 175 ms later; R . There myelinated nerve fibers [12,46,81,84]. The control of
ss
was no significant difference in R , but the axons of NF-H axonal caliber has a functional significance because diam-
p
2 2 mice showed a significant increase in R resistance
eter is the principal determinant of conduction velocity in
ss
see Table 1. myelinated nerve fibers [30,70,71,78]. The report of axonal
1
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 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
