- Email: [email protected]
Axonal excitability in the forearm: Normal data and differences along the median nerve
Clinical Neurophysiology 120 (2009) 167–173
Contents lists available at ScienceDirect
Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Axonal excitability in the forearm: Normal data and differences along the median nerve S.K. Jankelowitz *, David Burke Institute of Clinical Neurosciences, Royal Prince Alfred Hospital and University of Sydney, Medical Foundation Building K-25, Sydney, NSW 2006, Australia
See Editorial, pages 1–2
a r t i c l e
i n f o
Article history: Accepted 10 August 2008 Available online 22 November 2008 Keywords: Axonal excitability Median nerve Forearm
a b s t r a c t Objective: This study was designed to validate excitability studies of motor axons in the median nerve at the elbow, innervating forearm muscles (flexor carpi radialis, FCR) and collect normal data for this stimulation site. The differences in measures of excitability due to different sites of stimulation or a different test muscle group was also determined. Methods: The median nerve was stimulated at the elbow and excitability studies were recorded from FCR and abductor policis brevis, APB. Further studies stimulated the median nerve at the wrist and recorded from APB. The study was performed using the TRONDCM protocol of QTRACS. Results: Stimulus-response curves for FCR were significantly less steep than those for APB. There was ‘‘fanning in” of threshold electrotonus and less accommodation to hyperpolarising pulses for FCR axons. FCR studies showed significantly less supernormality. Conclusion: There are differences in axonal excitability along a single nerve in the human upper limb, even when the nerve is stimulated at one site and recordings are made from different muscles. Significance: Normal values for one muscle group cannot be assumed to be the same for a different muscle group. The findings for FCR axons will be of value in studies on patients with central nervous system disorders. Ó 2008 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Nerve excitability studies can allow inferences to be drawn about the activity of ion channels within the peripheral axon in vivo (Bostock et al., 1998; Kiernan et al., 2000; Burke et al., 2001). They are therefore able to provide insight into the pathophysiology of peripheral nerve disease (e.g., Krishnan and Kiernan, 2005; Lin et al., 2006). Excitability studies are relatively easy to perform and can constitute a useful addition to the armamentarium of neurophysiological investigations. To date, most upper limb motor studies have stimulated the median nerve at the wrist and recorded from the thenar eminence (over abductor pollicis brevis, APB; e.g., Cappelen-Smith et al., 2002; Lin et al., 2006; Jankelowitz et al., 2007a,b). However, upper limb pathology may involve more proximal limb muscles. To this end, we have validated the reproducibility of excitability studies on motor axons in the median nerve at the elbow, innervating forearm muscles (flexor carpi radi-
* Corresponding author. Tel.: +612 9036 3091; fax: +612 9036 3092 E-mail address: [email protected] (S.K. Jankelowitz).
alis, FCR), and have collected normal data for stimulation at this site. To determine whether differences in the measures of excitability were due to a different site of stimulation or a different test muscle group, comparisons were made between the recordings to median nerve stimulation at the wrist (recording from APB) and at the elbow (recording from FCR and APB).
2. Methods Nerve excitability studies were performed on 15 subjects (seven male, mean age 44.2 years, range 28–63), none of whom had a history of neurological disease or were on drugs that affect voltagedependent ion channels. The study was approved by the Human Research Ethics Committee of the University of Sydney and all the subjects gave written, informed consent prior to the commencement of the study. For all experiments, skin temperature was monitored close to the stimulation site and maintained at 32 degrees Celsius using blankets. The median nerve was stimulated at the elbow, medial to the brachial artery, just proximal to the elbow crease using a 1-cm
1388-2457/$34.00 Ó 2008 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2008.08.017
168
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
diameter Ag–AgCl electrode strapped to the arm as the cathode. The stimulus was generated by a custom-made isolated linear bipolar constant current stimulator (maximal output ± 50 mA) of the type used by Kiernan et al. (2000). The anode was a non-polarisable surface electrode (Maersk Medical, Stonehouse, UK), placed 10 cm proximally over the belly of the biceps brachii muscle. Recordings of compound muscle action potentials (CMAPs) were made using disposable surface Ag–AgCl electrodes (Maersk Medical, Stonehouse, UK). The active electrode was placed over the body of FCR at the site recommended for needle EMG (Lees and DeLisa, 2000). The reference recording electrode was placed over the tendon in the midline at the wrist. This electrode configuration ensured that the recorded CMAP had a relatively simple waveform (Fig. 1A), even though a number of forearm muscles could have been activated. The earth electrode was affixed to the olecranon. In five subjects, further studies were performed recording from abductor policis brevis (APB) stimulating the median nerve at the elbow. In another five subjects, additional recordings were obtained by stimulating the median nerve at the wrist and recording from APB. All studies were performed with the arm slightly flexed at the elbow, resting on a bench in the neutral position, with fixation applied to the hand when necessary to control stimulus-induced movement. The position of the arm was kept constant throughout the study to avoid movement of the surface EMG electrodes relative to the underlying muscle. EMG signals were sampled at a rate of 10 kHz, amplified, filtered (3 Hz to 3 kHz) and digitized. Stimulus waveforms were generated by computer and converted to current by an isolated linear bipolar constant-current source (maximal output ± 50 mA). Studies were performed using the TRONDCM protocol of QTRAC (Ó Professor Hugh Bostock, Institute of Neurology, London). This protocol studies motor nerve excitability and cycles through five subroutines sequentially; these are the stimulus-response curve, determination of the strength-duration time constant, threshold electrotonus, the current-threshold relationship and the recovery cycle. A stimulus-response curve was recorded first using stimuli of 1ms duration, and the morphology of the CMAP was monitored to ensure that it did not change as stimulus intensity increased (Fig. 1A and B). When the CMAP was bipeaked (Fig. 1A), the first negative peak of the CMAP was used for ‘‘tracking” the potential. The target potential was set to be approximately 40% of the maximal CMAP, on the fast rising phase of the stimulus-response curve, and the current required to produce a CMAP of this size is referred to as the ‘‘threshold” for the CMAP. Except for the measurement of strength-duration properties, the test stimuli were of 1-ms duration. Stimuli were delivered regularly at 0.8-ms intervals.
The protocol then cycled automatically through subroutines 2– 5. The strength-duration curve of motor axons in the median nerve was measured by assessing the change in stimulus current required to produce the test CMAP (40% of maximum) using five different stimulus durations from 0.2 to 1.0 ms. The strengthduration time constant (SDTC) and rheobase were estimated from plots of stimulus charge against stimulus duration (Fig. 2A), as in Mogyoros et al. (1996). In the third and fourth subroutines, prolonged subthreshold currents were used to alter the potential difference across the nodal and internodal membrane. The changes in threshold associated with electrotonic changes in membrane potential are termed threshold electrotonus. Subthreshold polarising currents of 100ms duration were set at +40 and +20% (depolarising) and 40 and 20% (hyperpolarising) of the control threshold current (i.e., the current required to produce the unconditioned target CMAP). Threshold was tested at different time points during and after the polarising 100-ms current (Fig. 2B). Threshold was then measured with 1-ms stimuli delivered 200 ms after the onset of a long-lasting (220-ms) subthreshold polarising current. The strength of the conditioning current was changed in 10% steps from +50% (depolarising) to 100% (hyperpolarising) of the control threshold. This produced a current-threshold (IV) relationship that depends on the rectifying properties of the axonal membrane and is analogous to the current-voltage relationship (Fig. 2C). Greater rectification, inward or outward, would result in a lesser threshold change for the injected current, and would produce a steeper slope for the response to hyperpolarising or depolarising currents, respectively. The final subroutine measured the recovery cycle, i.e., the changes in axonal excitability following a supramaximal conditioning stimulus of 1-ms duration. The recovery cycle documents the changes in excitability as axons pass through the relative refractory, supernormal (or superexcitable) and late subnormal (or subexcitable) periods. The changes in current necessary to produce the test CMAP were recorded at 18 conditioning-test intervals between 2 and 200 ms (Fig. 2D). The CMAP produced by the supramaximal conditioning stimulus was subtracted at each conditioning-test interval to define the response to the test stimulus uncontaminated by the maximal CMAP due to the conditioning stimulus. The measures given in Table 1 are SDTC (i.e., strength duration time constant); stimulus-response slope (i.e., the maximal slope of the curve of increasing stimulus plotted against increasing CMAP amplitude); TEd 10–20 (i.e., the mean threshold change 10–20 ms after the onset of the depolarising current); TEd 90–100 (i.e., the mean threshold change 90–100 ms after the onset of the depolaris-
Fig. 1. Growth of the CMAP recorded over FCR as the stimulus strength increases. (A) and (B), data from one subject. There is no change in CMAP morphology (A). The stimulus-response curve for the recording is shown in (B). Panel (C) shows the mean stimulus-response curves for FCR (n = 15, circles, 1 ms stimulus) and for APB (n = 29, lines, 1 ms and 0.2 ms stimuli), with the amplitude of the maximal CMAP normalised to 100%. Mean ± SEM.
169
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
A
B 100
5
APB
Threshold reduction (%)
Threshold charge (mA.ms)
FCR
0
0
-100
-200 -1
0 Stimulus width (ms)
1
C
0
100 Delay (ms)
200
D Threshold change (%)
Current (% threshold)
100
0
0
-100 -500
0 Threshold reduction (%)
10 100 Interstimulus interval (ms)
Fig. 2. Excitability of median motor axons innervating FCR and APB. Comparison between control data for studies stimulating the median nerve at the wrist and recording from APB (n = 29, open circles) and data from this study, i.e., stimulating the median nerve at the elbow and recording from FCR (n = 15, filled circles). (A) strength-duration properties shown in a plot of threshold charge vs stimulus duration, (B) threshold electrotonus, (C) current-threshold relationship, and (D) recovery cycle. Data are mean ± SEM. In A, SDTC (given by the intercept with the X-axis) is similar for both axonal populations, but rheobase (given by the slope of the relationship) is significantly greater for FCR axons. In B, there is ‘‘fanning in” of threshold electrotonus for the FCR recordings (with lesser threshold changes to both depolarisation and hyperpolarisation) with the corresponding decreases in undershoot and overshoot when the polarising stimulus is switched off. In C, the current-threshold relationships are not different in the depolarising direction, but there is a lesser slope in the hyperpolarising direction for FCR studies. In the recovery cycles of D, there is greater refractoriness and less supernormality for FCR axons.
ing pulse); TEh 10–20 (i.e., the mean change in threshold 10–20 ms after the onset of the hyperpolarising pulse); S2 accommodation (i.e,. the slow accommodative change in threshold in the depolarising direction in the threshold electrotonus curve); hyperpolarising IV slope (i.e., the slope of the IV curve beyond 40% in the hyperpolarising direction); resting IV slope (i.e., the slope of the IV curve from zero to 40%). The data for the threshold for the target CMAP, strength-duration time constant (SDTC), threshold electrotonus, current-threshold relationship and recovery cycle were averaged across subjects. The overall data for FCR recordings were compared to the control data for APB from Kiernan et al. (2000) included in the QTRAC software (n = 29; mean age 39.4 years, range 24–56). Some of those studies were performed in Sydney using a similar stimulator (custom-made ‘‘HiDC”), and a similar recording setup as in these studies. Further comparisons were made between the data for FCR and APB to the stimulation of the median nerve at the elbow (FCR vs APBe, n = 5) and for APB to the stimulation of the median nerve at the wrist (FCR vs APBw, n = 5). Statistical analyses were performed using Student’s t-test with Bonferroni corrections where appropriate. Significance was taken at P < 0.05. Data are given as mean ± SEM. 3. Results The mean data for studies on FCR motor axons to stimulation of the median nerve at the elbow are presented in Fig. 2 and Table 1,
where they are contrasted with the control data for APB. The maximal CMAP was 9.6 ± 1.1 mV. The mean stimulus required to produce the unconditioned test CMAP (40% of maximum) of FCR was 10.1 ± 1.1 mA. This value represents ‘‘threshold” for the CMAP. 3.1. Comparison of data for FCR with control data for APB The growth of the stimulus-response curve for FCR was smooth (Fig. 1B and C), but its slope (3.0 ± 1.0) was less than the control for APB with stimulation at the wrist (4.9 ± 1.0, P < 0.001). The mean strength-duration time constant was 0.43 ± 0.01 ms and rheobase was 6.9 ± 1.1 mA. The former is the same as for the control data for APB but the latter was significantly greater (0.43 ± 0.02 ms and 3.1 ± 1.1 mA). However, when comparing different sites, this means little because all threshold values, including rheobase, depend on the accessibility of the nerve. In the studies of threshold electrotonus, the maximal threshold decrease 10–20 ms after the onset of the +40% depolarising conditioning stimulus (i.e., TEd 10–20) was less in the FCR recordings than in the APB recordings (P < 0.001). This implies a lesser depolarising change in membrane potential for the equivalent stimulus, and accordingly there was also less S2 accommodation for FCR recordings (P < 0.001), and the TEd 40–60 threshold reduction for FCR was also significantly less (P < 0.001). As would be expected from the lesser S2 accommodation, the undershoot of threshold following the end of the 100 ms depolarisation was significantly less at the elbow than the wrist (P < 0.001). Similar differences between axons
170
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
Table 1 Measures from excitability studies on median motor axons Elbow to FCR n = 15 Mean stimulus (mA) SDTC (ms) Rheobase (mA) Stimulus-Response Slope Maximal CMAP (mV) TEd (10–20) (%) TEd (40–60) (%) TEd (90–100) (%) TEd Undershoot (%) TEd Peak (%) S2 Accommodation (%) TEh (90–100) (%) TEh (10–20) (%) TEh (20–40) (%) TEh Overshoot (%) TEh slope 101-140 Resting IV Slope Minimal IV Slope Hyperpolarising IV slope Supernormality (%) Subnormality (%) Relative Refractory Period (ms) Refractoriness at 2.5 ms (%) Refractoriness at 2 ms (%)
10.1 ± 1.1 0.43 ± 0.01 6.9 ± 1.1 3.0 ± 1.1 9.6 ± 1.1 55.9 ± 1.3 45.1 ± 0.9 41.7 ± 0.6 11.4 ± 1.0 56.2 ± 1.3 14.6 ± 1.1 117.3 ± 3.9 64.6 ± 2.0 81.7 ± 2.3 7.2 ± 0.5 1.7 ± 0.5 0.64 ± 0.0 0.20 ± 0.0 0.3 ± 0.0 9.1 ± 1.5 14.7 ± 2.2 3.7 ± 1.0 47.0 ± 4.2 89.0 ± 5.5
Wrist to APB n = 29 4.5 ± 1.1 0.43 ± 0.02 3.1 ± 1.1 4.9 ± 1.0 8.4 ± 1.1 69.3 ± 0.9 53.0 ± 0.9 45.7 ± 0.8 19.3 ± 0.8 69.6 ± 0.9 24.0 ± 0.6 120.8 ± 2.8 73.6 ± 0.9 92.3 ± 1.4 16.6 ± 0.7 2.1 ± 0.1 0.6 ± 0.0 0.3 ± 8.1 0.4 ± 0.0 25.4 ± 1.1 14.5 ± 0.8 3.1 ± 1.0 31.5 ± 3.5 90.1 ± 6.3
P-value <0.001 0.936 <0.001 <0.001 0.276 <0.001 <0.001 0.002 <0.001 <0.001 <0.001 0.482 <0.001 <0.001 <0.001 <0.001 0.436 <0.001 0.001 <0.001 0.943 <0.001 0.009 0.874
Values for studies stimulating the median nerve at the elbow and recording from FCR (n = 15) and control data for stimulating the median nerve at the wrist and recording from APB (n = 29). All values are given as mean ± SEM. Probabilities are corrected for multiple comparisons.
innervating FCR and APB were seen in the hyperpolarising direction, with less change in threshold (TEh 10–20 and TEh 20–40) and less overshoot following the end of the hyperpolarising current (P < 0.001). In other words, threshold electrotonus for motor axons innervating FCR was ‘‘fanned in” compared to the control data for APB (Fig. 2B). It is notable that TEh 90–100 differed little for FCR and APB despite the significant differences at the earlier intervals (TEh 10–20 and TEh 20–40). This suggests less accommodation to the hyperpolarising current for FCR axons, even when the ‘‘fanning in” is taken into account, and this interpretation is supported by the currentthreshold data. The slope of the current-threshold relationship in the hyperpolarising direction was significantly less steep for FCR. This implies that inward rectification, presumably due to activation of the hyperpolarisation-activated conductance (Ih), is less for FCR axons. However, in the depolarising direction, the current-threshold relationships were similar for median motor axons at the elbow and wrist (Fig. 2C), suggesting that outward rectification due to K+ currents was similar for the two axonal populations. In the recovery cycle the relative refractory period was longer, and refractoriness at 2.5-ms was greater for FCR axons (P < 0.001 for both, Fig. 2D). There was significantly less supernormality for FCR than in control data for APB. For FCR, mean supernormality was 9.1 ± 1.5% and in two (male) subjects there was no measurable decrease in threshold, whereas for APB, mean supernormality was 25.4%, and it was present in all subjects (P < 0.001). Mean subnormality was 14.7 ± 2.2%, much as in the control APB data. 3.2. Comparison of different stimulation and recording sites within subjects To exclude the possibility that inter-individual differences might have been responsible for the differences described above, FCR recordings to stimulation at the elbow and APB recordings to stimulation at the wrist were made in five subjects (Fig. 3, right panel). There were similar differences to those described above, indicating that the differences reported above were not due to the choice of the subject (or to differences between investigators).
In a further five subjects, recordings were obtained from FCR and APB to stimulation of the median nerve at the elbow. Again there were similar differences to those reported above between FCR and APB to stimulation at the wrist. There was less supernormality (P = 0.007) for FCR studies (Fig. 3, left panels). In threshold electrotonus there was a smaller threshold reduction with depolarisation (P = 0.017) and less S2 accommodation (P = 0.01), but threshold change to hyperpolarisation was greater for FCR than in the APB studies (P = 0.024). These findings suggest that the differences between motor axons innervating FCR and APB largely result from the innervated muscle rather than the site of stimulation. 3.3. Reproducibility The reproducibility of data across subjects and for different sites is detailed above. The data appear quite reproducible, as evidenced by the low SEMs, which are generally similar to those for the conventional test site at the wrist (Table 1). The test-retest reproducibility of studies on axons innervating the forearm muscles was demonstrated by the small variation in the data for ten separate studies performed on a single subject over four months (Fig. 4). Specific measures were threshold 6.1 ± 1.1 mA, maximal CMAP 8.3 ± 1.2 mV, SDTC 0.47 ± 0.08 ms, TEd 10–20 34.5 ± 0.4%, S2 accommodation 21.8 ± 0.7%, TEh 90–100 113.9 ± 3.2%, refractoriness at 2.5 ms 21.6 ± 3.5%, supernormality 16.7 ± 1.4,% and late subnormality 15.9 ± 2.9%.
4. Discussion This study has demonstrated the feasibility of excitability studies on axons innervating forearm muscles, and that the results are reproducible between and within subjects. The study has also revealed differences in excitability parameters when recordings are made from proximal and distal muscles innervated by the same nerve, even when the nerve is stimulated at the same site, and this emphasises that control data are needed for different muscle groups as well as different stimulation sites. When stimulating the median nerve at the elbow and recording from FCR, the threshold changes produced by subthreshold currents in threshold electrotonus studies were reduced, and there was reduced supernormality and increased refractoriness. Supernormality is determined by the depolarising afterpotential and occurs as a result of the passive discharge along a low-resistance pathway through the myelin of the current stored on the internodal membrane (Barrett and Barrett, 1982; David et al., 1995). A longer relative refractory period, ‘‘fanning in” of the threshold electrotonus and reduced supernormality could occur if there was membrane depolarisation (Bostock et al., 1998). While a greater stimulus threshold would not be expected in the presence of a depolarised membrane, this may have been due to lesser accessibility of motor axons in the median nerve at the elbow. Krishnan et al. (2004) studied lower limb nerves at two different sites and noted that with proximal stimulation there was a reduction in threshold, rheobase and supernormality and an increase in subnormality and the relative refractory period. Threshold electrotonus showed a ‘‘fanning in” with proximal stimulation. They concluded that axonal depolarisation was the most likely cause for the findings, and that this could explain the greater propensity for ectopic activity in proximal axons. Kuwabara et al. (2000) compared the median nerve at the wrist with the peroneal nerve at the ankle and fibular head. Median motor axons at the wrist had greater supernormality and late subnormality, greater threshold changes in threshold electrotonus and greater accommodation to prolonged depolarising current. Comparisons
171
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
D
100
Threshold reduction (%)
Threshold reduction (%)
A
0
-100
-200
100
0
-100
-200 0
100
0
200
100
E
B
100 Threshold reduction (%)
Threshold reduction (%)
100
0
0
10 100 Interstimulus interval (ms)
C
10 100 Interstimulus interval (ms)
F Current (% threshold)
Current (% threshold)
200
Delay (ms)
Delay (ms)
0
-100
0
-100 -500
0 Threshold reduction (%)
-500
0 Threshold reduction (%)
Fig. 3. Effects of the stimulation site and of the innervated muscle on axonal excitability. (Panels A, B and C) Comparison of data for studies of median motor axons at the elbow innervating FCR (filled circles) and APB (open circles) in the same subjects (n = 5). (Panels D, E and F) The data for FCR (filled circles) and APB at the wrist (open circles, n = 5). For FCR axons, there is fanning in of threshold-electrotonus, less supernormality in the recovery cycle and a less steep slope of the current-threshold relationship in the hyperpolarising direction than for APB axons whether the APB data came from stimulation at the elbow (left panels) or wrist (right panels).
between the two sites along the peroneal nerve showed significantly greater supernormality and a trend towards greater subnormality at the knee. Threshold electrotonus at the ankle was ‘‘fanned-in” relative to the knee. There were, however, no significant differences in strength-duration properties, stimulus-response curves and current-threshold relationships. These findings were thought to suggest differences in internodal rather than nodal properties. The authors speculated that the differences could reflect the (passive) properties of the axolemma and the myelin sheath or the functioning of ion channels, or both. Axon size was also considered a possible factor, but not temperature, which was controlled. While this study has found similar changes for median motor axons, it is notable that they are the opposite for proximal/distal sites. This suggests that morphological factors such as distal tapering of axons are not the sole factor. Given that the stimulus reaches axons through the skin, the anatomical arrange-
ment of the nerve at the elbow may be a factor. However, the nerve was stimulated where it is superficial (and palpable), proximal to its branching. It is also possible that the differences in excitability may reflect the different properties and functions of the innervated motor units, e.g., discharge parameters, fibre type, and functional use of the muscle. There is a linear relationship between conduction velocity and the cross-sectional area of myelinated fibres (Hursh, 1939), and McIntyre et al. (2002) showed that smaller fibres generated a greater passive depolarising afterpotential and thereby greater supernormality. There is also a greater subnormal period in smaller fibres because the greater depolarising afterpotential prolongs the activation of the slow K+ conductance. Some of the differences in excitability observed at different sites along the same nerve could be a function of nerve fibre diameter, but one would then expect similar trends along different nerves (e.g., median and peroneal).
172
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
B Threshold charge (mA.ms)
Current (% threshold)
A 0
-100
5
0 -500 0 Threshold reduction (%)
D
100
Threshold change (%)
Threshold reduction (%)
C
-1
0
-100
0 Stimulus width (ms)
1
100
0
-200 0
100 Delay (ms)
10 100 Interstimulus interval (ms)
200
Fig. 4. Intrasubject reproducibility. Data for 10 studies performed on one subject over 3 months. The figures show the individual recordings for each study with the mean (± SEM) superimposed as filled circles.
It is relevant that Caruso et al. (1992) found that nerve fibre diameter varies little between the wrist, finger and elbow. Internodal length is, however, significantly longer at the elbow than more distally. It has been suggested that short internodes have greater Na+/K+ pump activity because there is greater Na+ influx over the same length of nerve (Moldovan and Krarup, 2007). The Na+/K+ pump is electrogenic, with three Na+ ions pumped out for every two K+ ions pumped into the axon (Rakowski et al., 1989). This results in a net deficit of positive charge on the inner aspect of the axonal membrane. When the activity of the Na+/K+ pump is less, as could occur with relatively long internodes, there would be more accumulation of extracellular K+ ions, and this could lead to a more depolarised membrane potential (Ritchie and Straub, 1959; Kaji and Sumner, 1989). Therefore internodal length could play a role in some of the differences observed in axonal excitability at different sites along the nerve. The opposite findings for the peroneal nerve might then be explained by pathologically shorter internodes at the fibular head due to recurrent minor trauma. However, this would not explain the differences at the elbow between axons innervating FCR and APB. This study shows a significant difference in the current-threshold relationships in the hyperpolarising direction for proximal and distal stimulations. This alteration implies a reduction in inward rectification at the more proximal site. Such a change has been observed on distal stimulation in stroke subjects, and the preferred explanation, supported by computer modelling, was reduced hyperpolarisation-activated current (Ih) in the nerve (Jankelowitz et al., 2007a). This study has confirmed that there are differences in axonal excitability along a single nerve in the human upper limb, even when the nerve is stimulated at one site and recordings are made from different muscles. However, the extent to which changes in channel currents or differences in the morphological properties of axons account for the differences in the measures of excitability
is conjectural. This study provides normal data for motor axons innervating forearm muscles for a site that may well be more appropriate for in-vivo studies of pathophysiology in neuromuscular diseases. Whichever site is used, it would be prudent to keep in mind that ion channel types, densities and membrane dynamics of the mammalian node of Ranvier and internode are not completely characterised. References Barrett EF, Barrett JN. Intracellular recording from vertebrate myelinated axons: mechanism of the depolarizing afterpotential. J Physiol 1982;323:117–44. Bostock H, Cikurel K, Burke D. Threshold tracking techniques in the study of human peripheral nerve. Muscle Nerve 1998;21:137–58. Burke D, Kiernan MC, Bostock H. Excitability of human axons. Clin Neurophysiol 2001;112:1575–85. Cappelen-Smith C, Lin CS-Y, Kuwabara S, Burke D. Conduction block during and after ischaemia in chronic inflammatory demyelinating neuropathy. Brain 2002;125:1850–8. Caruso G, Massini R, Crisci C, Nilsson J, Catalano A, Santoro L, et al. The relationship between electrophysiological findings, upper limb growth and histological features of median and ulnar nerves in man. Brain 1992;115:1925–45. David G, Modney B, Scappaticci KA, Barrett JN, Barrett EF. Electrical and morphological factors influencing the depolarizing after-potential in rat and lizard myelinated axons. J Physiol 1995;489:141–57. Hursh JB. Conduction velocity and diameter of nerve fibres. J Physiol 1939;127:131–9. Jankelowitz SK, Howells J, Burke D. Plasticity of inwardly rectifying conductances following a corticospinal lesion in human subjects. J Physiol 2007a;581:927–40. Jankelowitz SK, McNulty PA, Burke D. Changes in measures of motor axon excitability with age. Clin Neurophysiol 2007b;118:1397–404. Kaji R, Sumner AJ. Ouabain reverses conduction disturbances in single demyelinated nerve fibres. Neurology 1989;39:1364–8. Kiernan MC, Burke D, Andersen KV, Bostock H. Multiple measures of axonal excitability: a new approach in clinical testing. Muscle Nerve 2000;23:399–409. Krishnan AV, Lin CS, Kiernan MC. Nerve excitability properties in lower limb axons: evidence for a length dependent gradient. Muscle Nerve 2004;29:645–55. Krishnan AV, Kiernan MC. Altered nerve excitability properties in established diabetic neuropathy. Brain 2005;128:1178–87. Kuwabara S, Cappelen-Smith C, Lin CS, Mogyoros I, Bostock H, Burke D. Excitability properties of median and peroneal motor axons. Muscle Nerve 2000;23:1365–73.
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173 Lees HJ, DeLisa JA. Surface anatomy for clinical needle electromyography. New York: Demos Medical Publishing Inc.; 2000. Lin CS-Y, Kiernan MC, Burke D, Bostock H. Assessment of nerve excitability properties in peripheral nerve disease. In: Kimura J, Daube JR, Mauguière F, editors. Handbook of clinical neurophysiology. peripheral nerve diseases 2006;vol. 7. Amsterdam: Elsevier; 2006. p. 381–403. McIntyre CC, Richardson AG, Grill WM. Modelling the excitability of mammalian nerve fibres: influence of afterpotentials on the recovery cycle. J Neurophysiol 2002;87:995–1006.
173
Mogyoros I, Kiernan MC, Burke D. Strength-duration properties of human peripheral nerve. Brain 1996;119:439–47. Moldovan M, Krarup C. Internodal function in normal and regenerated mammalian axons. Acta Physiol 2007;189:191–200. Rakowski RF, Gadsby DC, De Weer P. Stoichiometry and voltage dependence of the sodium pump in voltage clamped, internally dialyzed squid giant axon. J Gen Physiol 1989;93:903–41. Ritchie JM, Straub RW. The hyperpolarization which follows activity in mammalian non-medullated fibers. J Physiol 1959;136:80–97.
Contents lists available at ScienceDirect
Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Axonal excitability in the forearm: Normal data and differences along the median nerve S.K. Jankelowitz *, David Burke Institute of Clinical Neurosciences, Royal Prince Alfred Hospital and University of Sydney, Medical Foundation Building K-25, Sydney, NSW 2006, Australia
See Editorial, pages 1–2
a r t i c l e
i n f o
Article history: Accepted 10 August 2008 Available online 22 November 2008 Keywords: Axonal excitability Median nerve Forearm
a b s t r a c t Objective: This study was designed to validate excitability studies of motor axons in the median nerve at the elbow, innervating forearm muscles (flexor carpi radialis, FCR) and collect normal data for this stimulation site. The differences in measures of excitability due to different sites of stimulation or a different test muscle group was also determined. Methods: The median nerve was stimulated at the elbow and excitability studies were recorded from FCR and abductor policis brevis, APB. Further studies stimulated the median nerve at the wrist and recorded from APB. The study was performed using the TRONDCM protocol of QTRACS. Results: Stimulus-response curves for FCR were significantly less steep than those for APB. There was ‘‘fanning in” of threshold electrotonus and less accommodation to hyperpolarising pulses for FCR axons. FCR studies showed significantly less supernormality. Conclusion: There are differences in axonal excitability along a single nerve in the human upper limb, even when the nerve is stimulated at one site and recordings are made from different muscles. Significance: Normal values for one muscle group cannot be assumed to be the same for a different muscle group. The findings for FCR axons will be of value in studies on patients with central nervous system disorders. Ó 2008 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Nerve excitability studies can allow inferences to be drawn about the activity of ion channels within the peripheral axon in vivo (Bostock et al., 1998; Kiernan et al., 2000; Burke et al., 2001). They are therefore able to provide insight into the pathophysiology of peripheral nerve disease (e.g., Krishnan and Kiernan, 2005; Lin et al., 2006). Excitability studies are relatively easy to perform and can constitute a useful addition to the armamentarium of neurophysiological investigations. To date, most upper limb motor studies have stimulated the median nerve at the wrist and recorded from the thenar eminence (over abductor pollicis brevis, APB; e.g., Cappelen-Smith et al., 2002; Lin et al., 2006; Jankelowitz et al., 2007a,b). However, upper limb pathology may involve more proximal limb muscles. To this end, we have validated the reproducibility of excitability studies on motor axons in the median nerve at the elbow, innervating forearm muscles (flexor carpi radi-
* Corresponding author. Tel.: +612 9036 3091; fax: +612 9036 3092 E-mail address: [email protected] (S.K. Jankelowitz).
alis, FCR), and have collected normal data for stimulation at this site. To determine whether differences in the measures of excitability were due to a different site of stimulation or a different test muscle group, comparisons were made between the recordings to median nerve stimulation at the wrist (recording from APB) and at the elbow (recording from FCR and APB).
2. Methods Nerve excitability studies were performed on 15 subjects (seven male, mean age 44.2 years, range 28–63), none of whom had a history of neurological disease or were on drugs that affect voltagedependent ion channels. The study was approved by the Human Research Ethics Committee of the University of Sydney and all the subjects gave written, informed consent prior to the commencement of the study. For all experiments, skin temperature was monitored close to the stimulation site and maintained at 32 degrees Celsius using blankets. The median nerve was stimulated at the elbow, medial to the brachial artery, just proximal to the elbow crease using a 1-cm
1388-2457/$34.00 Ó 2008 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2008.08.017
168
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
diameter Ag–AgCl electrode strapped to the arm as the cathode. The stimulus was generated by a custom-made isolated linear bipolar constant current stimulator (maximal output ± 50 mA) of the type used by Kiernan et al. (2000). The anode was a non-polarisable surface electrode (Maersk Medical, Stonehouse, UK), placed 10 cm proximally over the belly of the biceps brachii muscle. Recordings of compound muscle action potentials (CMAPs) were made using disposable surface Ag–AgCl electrodes (Maersk Medical, Stonehouse, UK). The active electrode was placed over the body of FCR at the site recommended for needle EMG (Lees and DeLisa, 2000). The reference recording electrode was placed over the tendon in the midline at the wrist. This electrode configuration ensured that the recorded CMAP had a relatively simple waveform (Fig. 1A), even though a number of forearm muscles could have been activated. The earth electrode was affixed to the olecranon. In five subjects, further studies were performed recording from abductor policis brevis (APB) stimulating the median nerve at the elbow. In another five subjects, additional recordings were obtained by stimulating the median nerve at the wrist and recording from APB. All studies were performed with the arm slightly flexed at the elbow, resting on a bench in the neutral position, with fixation applied to the hand when necessary to control stimulus-induced movement. The position of the arm was kept constant throughout the study to avoid movement of the surface EMG electrodes relative to the underlying muscle. EMG signals were sampled at a rate of 10 kHz, amplified, filtered (3 Hz to 3 kHz) and digitized. Stimulus waveforms were generated by computer and converted to current by an isolated linear bipolar constant-current source (maximal output ± 50 mA). Studies were performed using the TRONDCM protocol of QTRAC (Ó Professor Hugh Bostock, Institute of Neurology, London). This protocol studies motor nerve excitability and cycles through five subroutines sequentially; these are the stimulus-response curve, determination of the strength-duration time constant, threshold electrotonus, the current-threshold relationship and the recovery cycle. A stimulus-response curve was recorded first using stimuli of 1ms duration, and the morphology of the CMAP was monitored to ensure that it did not change as stimulus intensity increased (Fig. 1A and B). When the CMAP was bipeaked (Fig. 1A), the first negative peak of the CMAP was used for ‘‘tracking” the potential. The target potential was set to be approximately 40% of the maximal CMAP, on the fast rising phase of the stimulus-response curve, and the current required to produce a CMAP of this size is referred to as the ‘‘threshold” for the CMAP. Except for the measurement of strength-duration properties, the test stimuli were of 1-ms duration. Stimuli were delivered regularly at 0.8-ms intervals.
The protocol then cycled automatically through subroutines 2– 5. The strength-duration curve of motor axons in the median nerve was measured by assessing the change in stimulus current required to produce the test CMAP (40% of maximum) using five different stimulus durations from 0.2 to 1.0 ms. The strengthduration time constant (SDTC) and rheobase were estimated from plots of stimulus charge against stimulus duration (Fig. 2A), as in Mogyoros et al. (1996). In the third and fourth subroutines, prolonged subthreshold currents were used to alter the potential difference across the nodal and internodal membrane. The changes in threshold associated with electrotonic changes in membrane potential are termed threshold electrotonus. Subthreshold polarising currents of 100ms duration were set at +40 and +20% (depolarising) and 40 and 20% (hyperpolarising) of the control threshold current (i.e., the current required to produce the unconditioned target CMAP). Threshold was tested at different time points during and after the polarising 100-ms current (Fig. 2B). Threshold was then measured with 1-ms stimuli delivered 200 ms after the onset of a long-lasting (220-ms) subthreshold polarising current. The strength of the conditioning current was changed in 10% steps from +50% (depolarising) to 100% (hyperpolarising) of the control threshold. This produced a current-threshold (IV) relationship that depends on the rectifying properties of the axonal membrane and is analogous to the current-voltage relationship (Fig. 2C). Greater rectification, inward or outward, would result in a lesser threshold change for the injected current, and would produce a steeper slope for the response to hyperpolarising or depolarising currents, respectively. The final subroutine measured the recovery cycle, i.e., the changes in axonal excitability following a supramaximal conditioning stimulus of 1-ms duration. The recovery cycle documents the changes in excitability as axons pass through the relative refractory, supernormal (or superexcitable) and late subnormal (or subexcitable) periods. The changes in current necessary to produce the test CMAP were recorded at 18 conditioning-test intervals between 2 and 200 ms (Fig. 2D). The CMAP produced by the supramaximal conditioning stimulus was subtracted at each conditioning-test interval to define the response to the test stimulus uncontaminated by the maximal CMAP due to the conditioning stimulus. The measures given in Table 1 are SDTC (i.e., strength duration time constant); stimulus-response slope (i.e., the maximal slope of the curve of increasing stimulus plotted against increasing CMAP amplitude); TEd 10–20 (i.e., the mean threshold change 10–20 ms after the onset of the depolarising current); TEd 90–100 (i.e., the mean threshold change 90–100 ms after the onset of the depolaris-
Fig. 1. Growth of the CMAP recorded over FCR as the stimulus strength increases. (A) and (B), data from one subject. There is no change in CMAP morphology (A). The stimulus-response curve for the recording is shown in (B). Panel (C) shows the mean stimulus-response curves for FCR (n = 15, circles, 1 ms stimulus) and for APB (n = 29, lines, 1 ms and 0.2 ms stimuli), with the amplitude of the maximal CMAP normalised to 100%. Mean ± SEM.
169
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
A
B 100
5
APB
Threshold reduction (%)
Threshold charge (mA.ms)
FCR
0
0
-100
-200 -1
0 Stimulus width (ms)
1
C
0
100 Delay (ms)
200
D Threshold change (%)
Current (% threshold)
100
0
0
-100 -500
0 Threshold reduction (%)
10 100 Interstimulus interval (ms)
Fig. 2. Excitability of median motor axons innervating FCR and APB. Comparison between control data for studies stimulating the median nerve at the wrist and recording from APB (n = 29, open circles) and data from this study, i.e., stimulating the median nerve at the elbow and recording from FCR (n = 15, filled circles). (A) strength-duration properties shown in a plot of threshold charge vs stimulus duration, (B) threshold electrotonus, (C) current-threshold relationship, and (D) recovery cycle. Data are mean ± SEM. In A, SDTC (given by the intercept with the X-axis) is similar for both axonal populations, but rheobase (given by the slope of the relationship) is significantly greater for FCR axons. In B, there is ‘‘fanning in” of threshold electrotonus for the FCR recordings (with lesser threshold changes to both depolarisation and hyperpolarisation) with the corresponding decreases in undershoot and overshoot when the polarising stimulus is switched off. In C, the current-threshold relationships are not different in the depolarising direction, but there is a lesser slope in the hyperpolarising direction for FCR studies. In the recovery cycles of D, there is greater refractoriness and less supernormality for FCR axons.
ing pulse); TEh 10–20 (i.e., the mean change in threshold 10–20 ms after the onset of the hyperpolarising pulse); S2 accommodation (i.e,. the slow accommodative change in threshold in the depolarising direction in the threshold electrotonus curve); hyperpolarising IV slope (i.e., the slope of the IV curve beyond 40% in the hyperpolarising direction); resting IV slope (i.e., the slope of the IV curve from zero to 40%). The data for the threshold for the target CMAP, strength-duration time constant (SDTC), threshold electrotonus, current-threshold relationship and recovery cycle were averaged across subjects. The overall data for FCR recordings were compared to the control data for APB from Kiernan et al. (2000) included in the QTRAC software (n = 29; mean age 39.4 years, range 24–56). Some of those studies were performed in Sydney using a similar stimulator (custom-made ‘‘HiDC”), and a similar recording setup as in these studies. Further comparisons were made between the data for FCR and APB to the stimulation of the median nerve at the elbow (FCR vs APBe, n = 5) and for APB to the stimulation of the median nerve at the wrist (FCR vs APBw, n = 5). Statistical analyses were performed using Student’s t-test with Bonferroni corrections where appropriate. Significance was taken at P < 0.05. Data are given as mean ± SEM. 3. Results The mean data for studies on FCR motor axons to stimulation of the median nerve at the elbow are presented in Fig. 2 and Table 1,
where they are contrasted with the control data for APB. The maximal CMAP was 9.6 ± 1.1 mV. The mean stimulus required to produce the unconditioned test CMAP (40% of maximum) of FCR was 10.1 ± 1.1 mA. This value represents ‘‘threshold” for the CMAP. 3.1. Comparison of data for FCR with control data for APB The growth of the stimulus-response curve for FCR was smooth (Fig. 1B and C), but its slope (3.0 ± 1.0) was less than the control for APB with stimulation at the wrist (4.9 ± 1.0, P < 0.001). The mean strength-duration time constant was 0.43 ± 0.01 ms and rheobase was 6.9 ± 1.1 mA. The former is the same as for the control data for APB but the latter was significantly greater (0.43 ± 0.02 ms and 3.1 ± 1.1 mA). However, when comparing different sites, this means little because all threshold values, including rheobase, depend on the accessibility of the nerve. In the studies of threshold electrotonus, the maximal threshold decrease 10–20 ms after the onset of the +40% depolarising conditioning stimulus (i.e., TEd 10–20) was less in the FCR recordings than in the APB recordings (P < 0.001). This implies a lesser depolarising change in membrane potential for the equivalent stimulus, and accordingly there was also less S2 accommodation for FCR recordings (P < 0.001), and the TEd 40–60 threshold reduction for FCR was also significantly less (P < 0.001). As would be expected from the lesser S2 accommodation, the undershoot of threshold following the end of the 100 ms depolarisation was significantly less at the elbow than the wrist (P < 0.001). Similar differences between axons
170
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
Table 1 Measures from excitability studies on median motor axons Elbow to FCR n = 15 Mean stimulus (mA) SDTC (ms) Rheobase (mA) Stimulus-Response Slope Maximal CMAP (mV) TEd (10–20) (%) TEd (40–60) (%) TEd (90–100) (%) TEd Undershoot (%) TEd Peak (%) S2 Accommodation (%) TEh (90–100) (%) TEh (10–20) (%) TEh (20–40) (%) TEh Overshoot (%) TEh slope 101-140 Resting IV Slope Minimal IV Slope Hyperpolarising IV slope Supernormality (%) Subnormality (%) Relative Refractory Period (ms) Refractoriness at 2.5 ms (%) Refractoriness at 2 ms (%)
10.1 ± 1.1 0.43 ± 0.01 6.9 ± 1.1 3.0 ± 1.1 9.6 ± 1.1 55.9 ± 1.3 45.1 ± 0.9 41.7 ± 0.6 11.4 ± 1.0 56.2 ± 1.3 14.6 ± 1.1 117.3 ± 3.9 64.6 ± 2.0 81.7 ± 2.3 7.2 ± 0.5 1.7 ± 0.5 0.64 ± 0.0 0.20 ± 0.0 0.3 ± 0.0 9.1 ± 1.5 14.7 ± 2.2 3.7 ± 1.0 47.0 ± 4.2 89.0 ± 5.5
Wrist to APB n = 29 4.5 ± 1.1 0.43 ± 0.02 3.1 ± 1.1 4.9 ± 1.0 8.4 ± 1.1 69.3 ± 0.9 53.0 ± 0.9 45.7 ± 0.8 19.3 ± 0.8 69.6 ± 0.9 24.0 ± 0.6 120.8 ± 2.8 73.6 ± 0.9 92.3 ± 1.4 16.6 ± 0.7 2.1 ± 0.1 0.6 ± 0.0 0.3 ± 8.1 0.4 ± 0.0 25.4 ± 1.1 14.5 ± 0.8 3.1 ± 1.0 31.5 ± 3.5 90.1 ± 6.3
P-value <0.001 0.936 <0.001 <0.001 0.276 <0.001 <0.001 0.002 <0.001 <0.001 <0.001 0.482 <0.001 <0.001 <0.001 <0.001 0.436 <0.001 0.001 <0.001 0.943 <0.001 0.009 0.874
Values for studies stimulating the median nerve at the elbow and recording from FCR (n = 15) and control data for stimulating the median nerve at the wrist and recording from APB (n = 29). All values are given as mean ± SEM. Probabilities are corrected for multiple comparisons.
innervating FCR and APB were seen in the hyperpolarising direction, with less change in threshold (TEh 10–20 and TEh 20–40) and less overshoot following the end of the hyperpolarising current (P < 0.001). In other words, threshold electrotonus for motor axons innervating FCR was ‘‘fanned in” compared to the control data for APB (Fig. 2B). It is notable that TEh 90–100 differed little for FCR and APB despite the significant differences at the earlier intervals (TEh 10–20 and TEh 20–40). This suggests less accommodation to the hyperpolarising current for FCR axons, even when the ‘‘fanning in” is taken into account, and this interpretation is supported by the currentthreshold data. The slope of the current-threshold relationship in the hyperpolarising direction was significantly less steep for FCR. This implies that inward rectification, presumably due to activation of the hyperpolarisation-activated conductance (Ih), is less for FCR axons. However, in the depolarising direction, the current-threshold relationships were similar for median motor axons at the elbow and wrist (Fig. 2C), suggesting that outward rectification due to K+ currents was similar for the two axonal populations. In the recovery cycle the relative refractory period was longer, and refractoriness at 2.5-ms was greater for FCR axons (P < 0.001 for both, Fig. 2D). There was significantly less supernormality for FCR than in control data for APB. For FCR, mean supernormality was 9.1 ± 1.5% and in two (male) subjects there was no measurable decrease in threshold, whereas for APB, mean supernormality was 25.4%, and it was present in all subjects (P < 0.001). Mean subnormality was 14.7 ± 2.2%, much as in the control APB data. 3.2. Comparison of different stimulation and recording sites within subjects To exclude the possibility that inter-individual differences might have been responsible for the differences described above, FCR recordings to stimulation at the elbow and APB recordings to stimulation at the wrist were made in five subjects (Fig. 3, right panel). There were similar differences to those described above, indicating that the differences reported above were not due to the choice of the subject (or to differences between investigators).
In a further five subjects, recordings were obtained from FCR and APB to stimulation of the median nerve at the elbow. Again there were similar differences to those reported above between FCR and APB to stimulation at the wrist. There was less supernormality (P = 0.007) for FCR studies (Fig. 3, left panels). In threshold electrotonus there was a smaller threshold reduction with depolarisation (P = 0.017) and less S2 accommodation (P = 0.01), but threshold change to hyperpolarisation was greater for FCR than in the APB studies (P = 0.024). These findings suggest that the differences between motor axons innervating FCR and APB largely result from the innervated muscle rather than the site of stimulation. 3.3. Reproducibility The reproducibility of data across subjects and for different sites is detailed above. The data appear quite reproducible, as evidenced by the low SEMs, which are generally similar to those for the conventional test site at the wrist (Table 1). The test-retest reproducibility of studies on axons innervating the forearm muscles was demonstrated by the small variation in the data for ten separate studies performed on a single subject over four months (Fig. 4). Specific measures were threshold 6.1 ± 1.1 mA, maximal CMAP 8.3 ± 1.2 mV, SDTC 0.47 ± 0.08 ms, TEd 10–20 34.5 ± 0.4%, S2 accommodation 21.8 ± 0.7%, TEh 90–100 113.9 ± 3.2%, refractoriness at 2.5 ms 21.6 ± 3.5%, supernormality 16.7 ± 1.4,% and late subnormality 15.9 ± 2.9%.
4. Discussion This study has demonstrated the feasibility of excitability studies on axons innervating forearm muscles, and that the results are reproducible between and within subjects. The study has also revealed differences in excitability parameters when recordings are made from proximal and distal muscles innervated by the same nerve, even when the nerve is stimulated at the same site, and this emphasises that control data are needed for different muscle groups as well as different stimulation sites. When stimulating the median nerve at the elbow and recording from FCR, the threshold changes produced by subthreshold currents in threshold electrotonus studies were reduced, and there was reduced supernormality and increased refractoriness. Supernormality is determined by the depolarising afterpotential and occurs as a result of the passive discharge along a low-resistance pathway through the myelin of the current stored on the internodal membrane (Barrett and Barrett, 1982; David et al., 1995). A longer relative refractory period, ‘‘fanning in” of the threshold electrotonus and reduced supernormality could occur if there was membrane depolarisation (Bostock et al., 1998). While a greater stimulus threshold would not be expected in the presence of a depolarised membrane, this may have been due to lesser accessibility of motor axons in the median nerve at the elbow. Krishnan et al. (2004) studied lower limb nerves at two different sites and noted that with proximal stimulation there was a reduction in threshold, rheobase and supernormality and an increase in subnormality and the relative refractory period. Threshold electrotonus showed a ‘‘fanning in” with proximal stimulation. They concluded that axonal depolarisation was the most likely cause for the findings, and that this could explain the greater propensity for ectopic activity in proximal axons. Kuwabara et al. (2000) compared the median nerve at the wrist with the peroneal nerve at the ankle and fibular head. Median motor axons at the wrist had greater supernormality and late subnormality, greater threshold changes in threshold electrotonus and greater accommodation to prolonged depolarising current. Comparisons
171
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
D
100
Threshold reduction (%)
Threshold reduction (%)
A
0
-100
-200
100
0
-100
-200 0
100
0
200
100
E
B
100 Threshold reduction (%)
Threshold reduction (%)
100
0
0
10 100 Interstimulus interval (ms)
C
10 100 Interstimulus interval (ms)
F Current (% threshold)
Current (% threshold)
200
Delay (ms)
Delay (ms)
0
-100
0
-100 -500
0 Threshold reduction (%)
-500
0 Threshold reduction (%)
Fig. 3. Effects of the stimulation site and of the innervated muscle on axonal excitability. (Panels A, B and C) Comparison of data for studies of median motor axons at the elbow innervating FCR (filled circles) and APB (open circles) in the same subjects (n = 5). (Panels D, E and F) The data for FCR (filled circles) and APB at the wrist (open circles, n = 5). For FCR axons, there is fanning in of threshold-electrotonus, less supernormality in the recovery cycle and a less steep slope of the current-threshold relationship in the hyperpolarising direction than for APB axons whether the APB data came from stimulation at the elbow (left panels) or wrist (right panels).
between the two sites along the peroneal nerve showed significantly greater supernormality and a trend towards greater subnormality at the knee. Threshold electrotonus at the ankle was ‘‘fanned-in” relative to the knee. There were, however, no significant differences in strength-duration properties, stimulus-response curves and current-threshold relationships. These findings were thought to suggest differences in internodal rather than nodal properties. The authors speculated that the differences could reflect the (passive) properties of the axolemma and the myelin sheath or the functioning of ion channels, or both. Axon size was also considered a possible factor, but not temperature, which was controlled. While this study has found similar changes for median motor axons, it is notable that they are the opposite for proximal/distal sites. This suggests that morphological factors such as distal tapering of axons are not the sole factor. Given that the stimulus reaches axons through the skin, the anatomical arrange-
ment of the nerve at the elbow may be a factor. However, the nerve was stimulated where it is superficial (and palpable), proximal to its branching. It is also possible that the differences in excitability may reflect the different properties and functions of the innervated motor units, e.g., discharge parameters, fibre type, and functional use of the muscle. There is a linear relationship between conduction velocity and the cross-sectional area of myelinated fibres (Hursh, 1939), and McIntyre et al. (2002) showed that smaller fibres generated a greater passive depolarising afterpotential and thereby greater supernormality. There is also a greater subnormal period in smaller fibres because the greater depolarising afterpotential prolongs the activation of the slow K+ conductance. Some of the differences in excitability observed at different sites along the same nerve could be a function of nerve fibre diameter, but one would then expect similar trends along different nerves (e.g., median and peroneal).
172
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173
B Threshold charge (mA.ms)
Current (% threshold)
A 0
-100
5
0 -500 0 Threshold reduction (%)
D
100
Threshold change (%)
Threshold reduction (%)
C
-1
0
-100
0 Stimulus width (ms)
1
100
0
-200 0
100 Delay (ms)
10 100 Interstimulus interval (ms)
200
Fig. 4. Intrasubject reproducibility. Data for 10 studies performed on one subject over 3 months. The figures show the individual recordings for each study with the mean (± SEM) superimposed as filled circles.
It is relevant that Caruso et al. (1992) found that nerve fibre diameter varies little between the wrist, finger and elbow. Internodal length is, however, significantly longer at the elbow than more distally. It has been suggested that short internodes have greater Na+/K+ pump activity because there is greater Na+ influx over the same length of nerve (Moldovan and Krarup, 2007). The Na+/K+ pump is electrogenic, with three Na+ ions pumped out for every two K+ ions pumped into the axon (Rakowski et al., 1989). This results in a net deficit of positive charge on the inner aspect of the axonal membrane. When the activity of the Na+/K+ pump is less, as could occur with relatively long internodes, there would be more accumulation of extracellular K+ ions, and this could lead to a more depolarised membrane potential (Ritchie and Straub, 1959; Kaji and Sumner, 1989). Therefore internodal length could play a role in some of the differences observed in axonal excitability at different sites along the nerve. The opposite findings for the peroneal nerve might then be explained by pathologically shorter internodes at the fibular head due to recurrent minor trauma. However, this would not explain the differences at the elbow between axons innervating FCR and APB. This study shows a significant difference in the current-threshold relationships in the hyperpolarising direction for proximal and distal stimulations. This alteration implies a reduction in inward rectification at the more proximal site. Such a change has been observed on distal stimulation in stroke subjects, and the preferred explanation, supported by computer modelling, was reduced hyperpolarisation-activated current (Ih) in the nerve (Jankelowitz et al., 2007a). This study has confirmed that there are differences in axonal excitability along a single nerve in the human upper limb, even when the nerve is stimulated at one site and recordings are made from different muscles. However, the extent to which changes in channel currents or differences in the morphological properties of axons account for the differences in the measures of excitability
is conjectural. This study provides normal data for motor axons innervating forearm muscles for a site that may well be more appropriate for in-vivo studies of pathophysiology in neuromuscular diseases. Whichever site is used, it would be prudent to keep in mind that ion channel types, densities and membrane dynamics of the mammalian node of Ranvier and internode are not completely characterised. References Barrett EF, Barrett JN. Intracellular recording from vertebrate myelinated axons: mechanism of the depolarizing afterpotential. J Physiol 1982;323:117–44. Bostock H, Cikurel K, Burke D. Threshold tracking techniques in the study of human peripheral nerve. Muscle Nerve 1998;21:137–58. Burke D, Kiernan MC, Bostock H. Excitability of human axons. Clin Neurophysiol 2001;112:1575–85. Cappelen-Smith C, Lin CS-Y, Kuwabara S, Burke D. Conduction block during and after ischaemia in chronic inflammatory demyelinating neuropathy. Brain 2002;125:1850–8. Caruso G, Massini R, Crisci C, Nilsson J, Catalano A, Santoro L, et al. The relationship between electrophysiological findings, upper limb growth and histological features of median and ulnar nerves in man. Brain 1992;115:1925–45. David G, Modney B, Scappaticci KA, Barrett JN, Barrett EF. Electrical and morphological factors influencing the depolarizing after-potential in rat and lizard myelinated axons. J Physiol 1995;489:141–57. Hursh JB. Conduction velocity and diameter of nerve fibres. J Physiol 1939;127:131–9. Jankelowitz SK, Howells J, Burke D. Plasticity of inwardly rectifying conductances following a corticospinal lesion in human subjects. J Physiol 2007a;581:927–40. Jankelowitz SK, McNulty PA, Burke D. Changes in measures of motor axon excitability with age. Clin Neurophysiol 2007b;118:1397–404. Kaji R, Sumner AJ. Ouabain reverses conduction disturbances in single demyelinated nerve fibres. Neurology 1989;39:1364–8. Kiernan MC, Burke D, Andersen KV, Bostock H. Multiple measures of axonal excitability: a new approach in clinical testing. Muscle Nerve 2000;23:399–409. Krishnan AV, Lin CS, Kiernan MC. Nerve excitability properties in lower limb axons: evidence for a length dependent gradient. Muscle Nerve 2004;29:645–55. Krishnan AV, Kiernan MC. Altered nerve excitability properties in established diabetic neuropathy. Brain 2005;128:1178–87. Kuwabara S, Cappelen-Smith C, Lin CS, Mogyoros I, Bostock H, Burke D. Excitability properties of median and peroneal motor axons. Muscle Nerve 2000;23:1365–73.
S.K. Jankelowitz, D. Burke / Clinical Neurophysiology 120 (2009) 167–173 Lees HJ, DeLisa JA. Surface anatomy for clinical needle electromyography. New York: Demos Medical Publishing Inc.; 2000. Lin CS-Y, Kiernan MC, Burke D, Bostock H. Assessment of nerve excitability properties in peripheral nerve disease. In: Kimura J, Daube JR, Mauguière F, editors. Handbook of clinical neurophysiology. peripheral nerve diseases 2006;vol. 7. Amsterdam: Elsevier; 2006. p. 381–403. McIntyre CC, Richardson AG, Grill WM. Modelling the excitability of mammalian nerve fibres: influence of afterpotentials on the recovery cycle. J Neurophysiol 2002;87:995–1006.
173
Mogyoros I, Kiernan MC, Burke D. Strength-duration properties of human peripheral nerve. Brain 1996;119:439–47. Moldovan M, Krarup C. Internodal function in normal and regenerated mammalian axons. Acta Physiol 2007;189:191–200. Rakowski RF, Gadsby DC, De Weer P. Stoichiometry and voltage dependence of the sodium pump in voltage clamped, internally dialyzed squid giant axon. J Gen Physiol 1989;93:903–41. Ritchie JM, Straub RW. The hyperpolarization which follows activity in mammalian non-medullated fibers. J Physiol 1959;136:80–97.