Early electrophysiological changes after denervation of fast skeletal muscle

EXPERIMENTAL

Early

NEUROLOGY

19,

375-387

(1967)

Electrophysiological

Department

Changes

of

Fast

Skeletal

B.

SALAFSKY

AND

of Pharmacology, Medical Chicago, I&to& Received

August

After

Denervation

Muscle D.

JASINSKI~

College,

University

of Illinois,

60612

13, 1967

Denervation of the anterior tibialis was performed on rabbits; the peroneal nerve being sectioned at the level of the knee and at the level of the thigh. Nerve action potentials monitored in the distal nerve stumps indicated functional failure occurred at 54 and 72 hours for knee and thigh sectioned nerve stumps, respectively. However, data from amplitudes of nerve action potentials and conduction velocities did not indicate that the nerves failed in a centrifugal manner, but rather, failure was abrupt down the entire length of the nerve trunk. Muscle action potentials following indirect stimulation were also recorded. Functional failure in neuromuscular transmission tended to occur prior to neuronal failure but was not complete in some animals when nerve conduction failed. Regardless of the site of denervation junctional failure followed a nearly identical time-course. Muscle tension (tetanic) to indirect stimulation indicated a temporal gradient might exist depending upon the length of the distal nerve stump. Junctional failure assessed by this latter technique revealed loss of function at earlier time periods than did assessment of muscle action potentials following indirect (single shock) stimulation. Introduction Denervation in the periphery produces a loss of conductivity in the distal nerve stump and a failure of neuromuscular transmission in the corresponding muscle. A review of the literature reveals significant discrepancies in: (a) the manner in which conductivity is lost in the distal stump, as assessed by electrophysiological techniques; and (b) the temporal relationship between loss of nerve conductivity in the distal stump and failure of neuromuscular transmission. With regard to (a), Parker (II), Rosenblueth and Dempsey ( 14)) and Causey and Stratmann (3) all reported that degeneration in the distal stump follows a centrifugal course; i.e., it is more marked at a given time in the central than in the peripheral portion of the distal stump. The anatomical basis for this finding was first described by Waller (16). However, Cook and Gerard (4), and Gutmann and Holubar (8) reported that 1 Supported by Illini Foundation and a grant from PHS is a Postdoctoral Fellow, Supported by NIH 2 Tl GM acknowledge the technical assistance of Mr. James Bell. 375

(NINDB 05679). 81-05. We wish

Dr. Jasinski to gratefully

376

SALAFSKY

AND

JASINSKI

they were unable to find any electrophysiological evidence for centrifugal loss of conductivity. They concluded that individual axons fail simultaneously down their entire length, although different groups of fibers may cease to function at slightly different time periods following denervation. Erlanger and Schoepfle (6) also obtained evidence for abrupt conduction failure which they indicated was due to failure at random loci increasing in frequency peripheralwards, or possibly with failure proceeding centripetally. Titeca (15) and Lissak, Dempsey and Rosenblueth (10) stated that transmission at the junction fails before the nerve loses its conductive properties. On the other hand, the finding of Rogers and Parrack (12) and the statements of Cook and Gerard (4) indicate that junctional transmission fails when the nerve action potential ceases. The purpose of this investigation was to assess the rate of functional failure in nerve conduction, and junctional transmission following denervation of a fast muscle. We also wished to determine to what extent the length of the denervated distal stump influenced the time course of failure in nerve and at the junction. Materials

and

Methods

Throughout this study rabbits of the Luenberg breed (male) all weighing approximately 2.0 kg were used. Under pentobarbital anesthesia (40 mg/kg) and aseptic conditions, 2-3 mm of the peroneal nerve was removed. The two sites of neurectomy were at the knee and thigh, i.e., approximately 4 and 8 cm, respectively, proximal to the anterior tibialis muscle (Fig. 1) . Following denervation the severed end of the distal portion of the nerve was sutured to adjacent muscle tissue and each rabbit was given 300,000 units of penicillin (im), and Neosporin R was applied topically to the skin. Control studies were carried out in rabbits in which the muscle was acutely denervated by sections at the sites given above; recordings were made immediately following the section and for several hours thereafter. In a few control experiments the nerve was not cut. The data from these latter experiments did not differ from controls which were denervated and studied at once. Time-course experiments were carried out on rabbits at 6-hour intervals from 6 to 72 hours following nerve section. These animals were again anesthetized with sodium pentobarbital (40 mg/kg) and arranged for recording as shown in Fig. 1. All recordings were carried out in situ. The temperature was maintained at 37 C and a mineral oil pool surrounded the electrodes. Bipolar platinum stimulating electrodes were mounted 3.0 cm (&) cephalad to the site of entry of the peroneal nerve into the anterior tibia1 muscle when denervation was carried out at the level of the knee. However,

FAST

377

MUSCLE

with

denervation at the thigh, stimulating electrodes were positioned both (&) and 8.0 cm (Sp) cephalad from the muscle (Fig. 1). Stimuli were delivered by a Grass S-4 stimulator at a frequency of l/set except under tetanus conditions when the frequency was raised in sequential steps from lO/sec to SO-60/set and to lOO/sec. The duration of the stimulus was set at 0.1 msec and voltage was applied at supramaximal intensity for twitch. The presence of direct excitability of muscle was assessed where indirect excitability testing indicated loss of neuromuscular transmission, Bipolar platinum 3.0

Thigh denrrvation Sciatic

nerve-------kn

si

_a

Knee denervatiow-‘* Distance peroneal

back from insertion of nerve into Tibialis anterior

R1 = 1.0 cm. St = 3.0 cm. S2= 8.0 cm.

Denervations:

To strain gage

a FIG.

lating

1. Schematic and recording

‘snterior muscle

representation electrodes.

of the sites

of denervation,

and

position

of stimu-

electrodes were mounted on a micromanipulator and were used to detect surface action potentials of the muscle during twitch. Bipolar platinum electrodes for nerve recording purposes were mounted 1 cm cephalad to the point at which the peroneal nerve inserts into the muscle. All nerve and muscle action potentials were amplified by a Grass differential a-c preamplifier, displayed on a Tektronix 565 oscilloscope, and photographed with a Polaroid camera, mounted on the oscilloscope. Tension developed by the muscle during single twitch and tetanus was measured by a Grass FT-03 force-displacement transducer and a Grass P-7 polygraph. An initial tension of 50 g was used in all experiments. The leg was fixed with metal pins and the tendon was attached to the transducer with braided silk thread.

378

SALAFSKY

AND

JASINSKI

Results

Nerve. In individual experiments the conducted compound action potential (of the nerve (NAP) and the muscle response to nerve stimulation were recorded. The term “failure” is used in this report to indicate either the absence of a conducted action potential in the nerve, or when applied to the neuromuscular junction, absence of a muscle action potential (MAP) or tension, following indirect stimulation. This design precludes the assessment of neuromuscular transmission in the instance where nerve conduction has ceased. However, a failure of neuromuscular transmission preceding impairment of

I

Control 6

12

I8

24

;6

.42

48

TIME(hrs1

FIG.

where

2. Amplitude I are SE.

of nerve

action

potentials

in all animals

following

knee

denervation,

conduction in the distal nerve stump could be detected as well as concomitant failure of nerve and neuromuscular transmission. Figure 2 represents the amplitudes of the nerve action potentials recorded from S1 at specified time intervals following denervation at the knee. Figure 3 is a similar representation of the nerve action potentials taken at time periods after thigh denervation when the stimulating electrodes were positioned at S1 and Sz. In Figs. 2 and 3 there is a similar trend in the amplitude of the nerve action potential after knee and thigh denervation. In both figures the 6-hour interval after denervation is marked by a significant reduction in NAP amplitude. This is followed by a rise in NAP amplitude over the next three time periods to a peak at the 30-hour period, and then a rapid return in NAP amplitude to essentially control levels. Finally, there

FAST

379

MUSCLE

is a tendency for all NAP amplitudes to fall at the time period just preceding failure, but this response is not equal after knee and thigh denervations. Of considerable interest was the difference in the time intervals at which failure of conduction was observed following knee and thigh denervations. All animals in the knee denervated group tested, failed to exhibit nerve action potentials at the S4-hour interval. On the other hand, all animals in the thigh denervated group failed at the 72-hour interval. This was observed in the latter instance regardlessof stimulation at S1 or Sz. The per-

09 I

FIG.

where

3. Amplitude I are SE.

of nerve

A

action

potentials

A -

Sttmulale

3 Ocm

from

muscle

B ----

Stmwlafe

8 ocm

from

m”xle

in all animals

following

thigh

denervation,

centage of animals exhibiting function failure of NAP at all time intervals is seen in Table 1. In addition, the duration of the compound nerve action potentials were measured in all animals after knee and thigh denervations. This data are shown in Figs. 4 and 5. Figure 4, representing the NAP durations after knee denervation indicates a significant decreasein the first 6-hour interval which is maintained until failure. A similar curve is seenin Fig. 5 following thigh denervation and stimulation at 3.0 cm ( S1). However, stimulation at 8.0 cm (Sz, Fig. 5) shows an initial decreasein NAP duration followed by an increase. This increase is not significant as compared to control NAP durations.

a The

Thigh

Knee

Site of denervation

I

I

of animals

Muscle action potential

Nerve action potential

Muscle action potential

number

1

i

I

Xerve action potential

Parameter measured

TIME

employed

is indicated

3 .O cm cephalad from ant. tib. on peroneal nerve

8.0 cm cephalad from ant. tib. on peroneal nerve

3.0 cm cephalad from ant. tib. on peroneal nerve

3 .O cm cephalad from ant. tib. on peroneal nerve

3.0 cm cephalad from ant. tib. on peroneal

Recording site

FAILURE

TABLE AND

(

).

%

Muscle

Rl

EMG

1 .O cm cephalad from ant. tib. on peroneal nerve =

Rl

40 (5)

(0)

0 (5)

0 (3-5)

0 (5)

0 (3-Q

%

1 .O cm cephalad from ant. tib. on peroneal nerve =

40 (10)

0

0 (7)

(5)

80

0 (5)

0 (5)

(8)

0 (7)

87.5

(7)

(11)

72

48

72

0 (6)

0

42

(6)

36

(3)

30

JUNCTION

(6)

(6)

100

17

0

(6)

(6)

33

0

60

(6)

(91

100

54

of animals exhibiting functional intervals (hours) after denervatior@

NEUROMUSCULAR

Percentage failure at time

AT THE

($7)

6-24

1

Rl Muscle EMG

-

IN NERVE

1 .O cm cephalad from ant. tib. on peroneal nerve =

OF FUNCTIONAL

Stimulus site

COURSE

43 (7)

(7)

57

66

100 (5)

(5)

100

72

6

2

2

FAST

381

MUSCLE

Finally, conduction velocities of the peroneal nerve were measured (Fig. 6) in both control and denervated animals over all the time intervals prior to failure. Generally, there was no significant change in conduction velocities after denervation at either site. The one exception can be seen at the 66-hour interval following denervation at the thigh and stimulation at 3.0 cm (S1)

II

Control

FIG. 4. Duration where I are SE.

L

of nerve

12

action

I6

24

TIME

(hrs.1

potentials

30

of animals

A 8.

060-

36

42

denervated

STIMULATE STIMULATE

48

at the knee

position,

3 0 CM FROM MUSCLE 8.0 CM FROM MUSCLE

T

Control 6

I

12

I8

24

30 TIME

FIG. 5. Duration where I are SE.

of nerve

action

potentials

I

36

42

48

54

60

66

I

72

(hrs)

of animals

denervated

at the thigh

position,

where there is a sharp drop in the conduction velocity as compared to several of the preceding intervals, but not, as compared to control values. Junctional Transmission. The presence of functional transmission across the motor junction was ascertained by recording muscle action potentials (MAP) with gross electrodes following indirect stimulation. Such recordings were made at all the time intervals, as indicated above, following both knee and thigh denervations. In all of these cases the stimulating electrode was

382

SALAFSKY

AND

JASINSKI

placed 3.0 cm (S,) cephalad from the muscle and extreme care was taken to avoid electronically conducted artifacts. Table 1 shows that following knee denervation none of the animals at any time interval up to 36 hours exhibited junctional failure. At 36 hours, however, in 40% of all animals observed at that time period we could no longer detect MAP following indirect stimulation. Likewise, 72% exhibited failure at 42 hours, 87.5% at 48 hours. Functional status of the junction could not be determined at 54 hours in knee denervated animals since at this time neuronal conduction had failed. Of interest in these data is the earlier initial failure at the junction in some

DO0

1

A

Knee Oenerwlion-stlmulote

3 Ocmpxord

LOcm

8

Thigh Denervation-stimulate

3.0cm,record

I Ocm

C

Thigh Oenerwticn-

8-Ocm,record

I.Ocm.

stimulate

> 80.0 h i2 &j 70.0 > zF

60.0

-

3 0 5 ”

50.0

-

L7 Control

6

12

I8

24

30 TIME

FIG. 6. denervation,

36

42

48

54

60

66

,

72

(hrs.)

Conduction velocities of distal nerve stumps in all A, and thigh denervation B, C, where I are SE.

animals

following

knee

animals but a final “apparent” failure of both nerve (NAP) and muscle (MAP) between 48 and 54 hours in all animals. Following thigh denervation (Table 1) an essentially similar time course of junctional failure was observed, particularly with respect to the course of nerve failure. In the first 36 hours there was no impairment of junctional transmission. At the 36-hour time interval we again observed that in 400/O of all animals studied we could not detect MAP, at 42 hours 29% exhibited failure, 80% at 48 hours, and here, we could observe 100% failure at 54 hours. The time course of junctional failure following thigh denervation was not as linear as that seen after knee denervation, but of greater interest, was its approximation to the course of junctional failure seen after knee denervation even though the nerve following thigh denervation survived (NAP) for an additional 18 hours. Muscle. Figure 7 shows the tension produced during isometric contraction

FAST

383

MUSCLE

following indirect stimulation at tetanic frequencies over the specified time intervals both before and after knee and thigh denervation. Essentially no tension could be recorded in animals with knee denervations after the 24-hour time period. Similarly no tension could be recorded in thigh denervated animals after 30 hours. An unexpected result from this data was the significant and unusual rise in tension, following both knee and thigh denervation, which reached a maximum at the 18-hour time interval. Another finding was that 600

500

400

3 E ; .c” c”

300

200

100

L ,

6

FIG. 7. Tension indirect

stimulation

12

18 24 Time in Hours

30

developed in muscles of knee and thigh denervated animals following of respective distal nerve stumps at SO-60/set, where I are SE.

all denervated muscles, regardless of the site of denervation or the time interval at which they were observed, evidenced an increased susceptibility to fatigue. Muscles of control animals would repeatedly give uniform and well-sustainedtetanus recordings. However, all denervated muscles,regardless of the tetanic response to the initial stimulation would, upon subsequent stimulation, yield a substantially reduced tetanic response.In addition, all tetanic responsesin denervated musclewere rather poorly maintained.

384

SALAFSKY

AND

JASINSKI

Throughout, all the denervated muscles in this study responded to direct electrical stimulation. Discussion

Nerve. The time of ultimate failure in the distal stump of a peripheral nerve, following denervation, is influenced by the level at which the neurectomy was performed. In this study where the recording electrode was fixed and the stimulating electrodes variably positioned the animals denervated at the level of the thigh demonstrate NAPS for an additional 18 hours as compared to animals denervated at the knee (Figs. 2, 3). This extended period of survival was observed in nerves of the thigh denervated animals regardless of stimulation at the 3 or 8 cm site. The extended survival of thigh denervated nerves is most apparent when this 3 cm stimulation site is compared to the identical stimulation site for nerves of knee denervated animals. This fact, coupled with simultaneous NAP failure in thigh denervated animals stimulated at the two sites strongly suggests that individual nerve fibers do not undergo a proximodistal pattern of failure but rather that individual fibers fail at the same time down their entire length. These findings are supported by data obtained on conduction velocities in Fig. 6. If individual axons failed in a proximodistal fashion with respect to length, an alteration, probably a reduction, in conduction velocities would be observed under these experimental conditions, We have observed no change in the conduction velocity of the nerves denervated over the period prior to final failure. As would be expected from the data on conduction velocity, the duration of NAP studied over the time intervals after denervation were also relatively unchanged. This is particularly true for the duration of NAP following knee and thigh denervation with stimulation at 3 cm (Figs. 4, 5). Although these experiments show that the absolute rate of failure in two lengths of distal nerve stumps is different, the nature of the NAP during failure is similar. This similarity in the nature of functional failure is exemplified in the striking parallelism seen in curves A and B of Fig. 3 over the entire 72-hour period. Following knee or thigh denervation the amplitude of the NAP fall at the first 6-hour interval, then rise to a peak at the 30-hour interval, subsequently return to control values, and in some cases fall shortly prior to failure. However, in all cases, the failure is seen as an abrupt loss in conduction capabilities as compared to the preceding 6-hour interval. A curious finding in Figs. 2 and 3 is the large increase in the amplitude of the nerve action potential seen 30 hours after knee and thigh denervation, regardless of stimulation site. We have no explanation for this phenomena but suggest that it is possibly related to a critical point in edema formation, particularly to early changes at the nodes of Ranvier. There is histological evidence for these early changes (2). Others have noted similar NAP ampli-

FAST

MUSCLE

385

tude increases after denervation (9, 14). Suggestions have been advanced that edema results in a “shunting” mechanism (8)) but if this is the case one would ordinarily expect a decrease in nerve action potentials. Causey and Stratmann (3) defined the “three schools of thought” which classically represent the findings of conduction failure in degenerating nerve. (i) That following denervation, degeneration occurs uniformly along the nerve distal to the site of section and that conduction fails at a certain time throughout the whole length of the fiber; (ii) that the course of degeneration proceeds centrifugally from the site of the cut; and (iii) that degeneration proceeds at the periphery and proceeds in a central direction. There has been no additional support for the third doctrine for the past 40 years and the current discrepancies appear to center around the first and second theories although the explanations of Erlanger and Schoepile (6) represent a hybrid of schools one and three. The literature that supports centrifugal degeneration is generally based on the findings that following denervation at a single site, stimulation of the distal stump produces compound action potentials of greater amplitude distally and of lesser amplitude closer to the central end of the nerve stump. These differences amount to approximately 10% (14)) and represent insufficient evidence on which to support the concept of centrifugal degeneration. Although our methodology did include multiple sites for stimulation it did not include multiple sites for recording. Nevertheless, data supporting centrifugal failure could have been obtained. If, for example, following denervation at the thigh and stimulation at S1 and S2 the amplitude of the NAP (Fig. 3) had shown an increasing disproportionality with time we would have favored the concept of centrifugal failure. However, the ratio of NAP amplitudes following stimulation at S1 and S2 is remarkably constant. Others (6, 14, 15) also noted that failure occurs at a time when conduction velocities and excitability are essentially normal. We suggest that the observed disproportionality in NAP amplitude following denervation and stimulation recording at multiple sites, as documented by others, is an artifact possibly related to edema effects in these nerve trunks. We, therefore, do not support the concept of centrifugal degeneration. In this regard, our conclusionsare in agreement with Cook and Gerard (4), Gutmann and Holubar (S), and with Erlanger and Schoepfle (6) regarding abrupt faimre, but we can neither clearly support or refute these latter authors in terms of loci of failure increasing in frequency peripheralwards. However, in peripheral nerve trunks there are mixed fiber populations which have varying rates of degeneration (5, 8). To what extent all these fiber types differ in degenerative processesis not known. In a different context, however, we do support the concept of a gradient in degenerative processes:namely, that the longer the distal nerve stump, the greater its functional viability with respect to time. Recently, there has

386

SALAFSKY

AND

JASINSKI

been considerable renewed interest in the “trophic” function of peripheral nerve. It has been generally thought that peripheral nerve directs a trophic influence toward maintenance of skeletal muscles via mechanisms of axoplasmic flow (7, 17, 18). Obviously the nerve utilizes nutritive materials to support itself and quite possibly these materials are found in the axoplasm and at increasing concentrations down the length which may account for the gradient of neuronal failure we have observed. Junctional Transmission. Our data indicates that junctional transmission tends to fail prior to neuronal transmission when the criteria used are either muscle tension or muscle action potentials following indirect stimulation. However, there is a difference in the sensitivity of these two methods to detect failure, the muscle action potentials being recordable some 12-18 hours after tension could no longer be recorded. The findings of earlier junctional failure, as compared to neuronal failure, are in agreement with others (3, 10, 15). A comparison of junctional failure by recording MAP or tension after indirect stimulation yields slightly different data on the nature of neuromuscular degenerative processes. Data from muscle action potentials indicates (Table 1) that regardless of the length of the degenerating nerves, failure follows a similar course. Initially, failure is seen in about 40% of the animals studied at 36 hours after either knee or thigh denervation. All animals observed at 48 hours, regardless of the denervation site exhibited a similar percentage of failure. The course of failure in each of the denervated groups is similar with the exception of the 42-hour period. Here, apparently less of the thigh denervated animals exhibited failure. Tension records more strongly suggest a temporal gradient might exist in that the thigh denervated animals respond over an additional 6-hour period. Such a discrepancy could be accounted for if it were known that degenerative changes of fast and slow muscle fibers were different and if only one type of fiber predominated in the tetanic response 42-30 hours after denervation. Bajusz (1) has shown that following denervation agranular (white) fibers undergo a more rapid atrophy than granular (red) fibers. Since the anterior tibia1 muscle contains a mixed fiber population it is possible that such a hypothesis could account for the difference observed in the nature of junctional failure where the two methods of assessment were employed. Muscle. Our curious finding of increased tetanic tension upon indirect stimulation in degenerating nerves was unexpected. These increased tensions were most marked at 18 hours following either knee or thigh denervation. Gutmann (7) found very early changes in the metabolism of the muscle following denervation, in particular an increase in glycogen and protein. It is possible that these early elevations in tension occurring prior to junctional

FAST

MUSCLE

failure may reflect loss of trophic materials which in turn function late glycogen synthesis or breakdown, or both.

387

to regu-

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

IO.

11. 12. 13. 14.

15. 16.

17.

18.

BAJUSZ, E. 1964. “Red” skeletal muscle fibers: relative independence of neural control. Science 145: 838-839. CAUSEY, G., and E. PALMER. 1952. Early changes in degenerating mammalian nerves. Proc. Roy. Sot. London, Ser. B, 199: 597-609. 1953. The spread of failure of conduction in CAUSEY, G., and C. J. STRATMANN. degenerating mammalian nerve. 3. Physiol. London 121: 215-223. COOK, D. D., and R. W. GERARD. 1931. The effect of stimulation on the degeneration of a severed peripheral nerve. Am. J. Physiol. 97: 412-425. CRAGC, B. G. 1965. Failure of conduction and synaptic transmission in degenerating mammalian C fibers. J. Physiol. London 179: 95-112. ERLANGER, J., and G. M. SCHOEPFLE. 1946. A study of nerve degeneration and regeneration. Am. J. Physiol. 147: 550-581. “The Denervated Muscle.” Publishing House of the CzechoGUTMANN, E. 1962. Slovak Academy of Science, Prague. 1950. The degeneration of peripheral nerve fibers. GUTMANN, E., and J. HOLUBAR. J. Neurol. Neurosurg. Psychiat. 13: 89-105. HEINBECKER, P. 1929. Effect of anoxemia, carbon dioxide, and lactic acid on electrical phenomena of myelinated fibers of the peripheral nervous system. Am. 3. Physiol. 89: 58-83, 1929. LISSAK, E., E. W. DEMPSEY, and A. ROSENBLUETH. 1939. The failure of transmission of motor nerve impulses in the course of Wallerian degeneration. Am. J. Physiol. 298: 45-46. PARKER, G. H. 1933. The progressive degeneration of frog nerve. Am. J. Physiol. 108: 398-403. 1939. Intluence of age on functional survival ROGERS, W. M., and H. 0. PARRACK. of severed mammalian nerves. Am. 1. Physiol. l28: 611-612. 1943. The centrifugal course of Wallerian ROSENBLUETH, .4., and E. C. DEL Pozo. degeneration. Am. I. Physiol. 1%: 247-254. 1939. A study of Wallerian degeneration. ROSENBLUETH, .4., and E. W. DEMPSEY. Am. J. Physiol. 128: 19-30. TITECA, J. 1935. Etude des modifications fonctionelles du nerf au tours da sa degenerescence wallerinne. Arch, Intern. Physiol, 14: 1-56. WALLER, A. 1852. Observations sur les effects de la section des racines spinales et du nerf pheumogastrique au-dessus de son ganglion inferieur chez les mammiferes. Corn@. Rend. 94: 582. WEISS, P. 1961. The concept of perpetual growth and proximo-distal substance convection, pp. 220-242. In “Regional Neurochemistry.” S. S. Kety and J. Elkes leds.1. Pergamon Press, London. WEISS, P. 1963. Self-renewal and proximo-distal convection in nerve fibers, pp. 171-193, In “Symposium on the Effects of Use and Disuse on Neuromuscular Function.” E. Gutmann and P. Hnik [eds.]. Publishing House of the Czechoslovak Academy of Sciences, Prague.