J. Physiol. (1977), 270, pp. 165-180 With 7 text-ftgureM Printed in Great Britain
165
MEMBRANE PROPERTIES AND CONDUCTION VELOCITY IN SENSORY NEURONES FOLLOWING CENTRAL OR PERIPHERAL AXOTOMY
BY G. CZRH, N. KUDO AND M. KUNO From the Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514, U.S.A.
(Received 20 December 1976) SUMMARY
1. The properties of dorsal root ganglion cells in the lumbosacral segments were examined with intracellular electrodes about 3 weeks after section of the central (dorsal roots) or peripheral processes in the cat. 2. Chronic section of the peripheral nerve in the hind limb resulted in a reduction in conduction velocity of both the central and peripheral processes of sensory neurones. 3. Sensory neurones arising from the triceps surae and plantaris muscles were subject to 'disuse' conditions for about 3 weeks by section of the ventral roots combined with severance of the Achilles tendon. Under such conditions, the central and peripheral conduction velocities of these sensory neurones tended to decrease, but the decrease was significantly less than that following peripheral nerve section. 4. Chronic section of the dorsal roots produced no significant changes in conduction velocity of the central processes of muscle sensory neurones but caused a significant increase in the peripheral conduction velocity. 5. The only electrophysiological property of dorsal root ganglion cells which altered following axotomy was the time-dependent membrane rectification in response to hyperpolarizing current pulses. The rectification characteristics were modified by chronic section of the peripheral nerve but not by chronic section of the dorsal root. 6. It is concluded that injuries in nerve fibres per se do not necessarily result in a decrease of their conduction velocity and that a decrease in their conduction velocity is associated with changes in the properties of the cell bodies. 7. It is suggested that a decrease in conduction velocity following nerve section may require the participation of changes in the neurone cell body.
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1. CZEH, N. KUDO AND M. KUNO INTRODUCTION
Injuries in peripheral nerve fibres result in a reduction of conduction velocity proximal to the lesion (Acheson, Lee & Morison, 1942; Gutmann & Holubar, 1951; Eccles & McIntyre, 1953; Eccles, Krnjevic & Miledi, 1959; Kiraly & Krnjevic, 1959; Cragg & Thomas, 1961; Aitken & Thomas, 1962; Bagust & Lewis, 1974; Kuno, Miyata & Munioz-Martinez, 1974; Mendell, Munson & Scott, 1976; Schmidt & Stefani, 1976). The reduced conduction velocity appears to be associated with a decrease in axon diameter (Greenman, 1913; Gutmann & Sanders, 1943; Gutmann & Holuba6, 1951; Cragg & Thomas, 1961; Aitken & Thomas, 1962). Mendell et al. (1976) have attempted to determine if decreased conduction velocity proceeds centrally from the cut end toward the neurone cell body or in the reverse direction. However, the results failed to show in which direction the changes in conduction velocity might spread. Thus, it remains uncertain whether a reduction in conduction velocity following nerve section is a primary change in the axon induced by the injury or whether it is a reaction of the axon secondary to chromatolytic changes in the neurone cell body. In sensory neurones it is generally agreed that the cell body shows a chromatolytic reaction to section of the peripheral process (peripheral axotomy), whereas section of the central process (central axotomy) induces no morphological changes in the cell body (Anderson, 1902; Ranson, 1914; Hare & Hinsey, 1940; Scott, Gutmann & Horsky, 1968; Carmel & Stein, 1969). This unique behaviour may provide a favourable condition to test the question as to whether the change in the cell body is a necessary correlate of a reduction in conduction velocity following nerve section. In the present study, the conduction velocity of dorsal root ganglion cells was measured following chronic section of the central (dorsal roots) or peripheral processes. The results show that changes in electrophysiological properties of the cell body as well as a reduction in the axonal conduction velocity occur within about 3 weeks after section of the peripheral processes, whereas these changes are absent following section of the central processes. It is suggested that a decrease in conduction velocity following nerve section may require the participation of changes in the neurone cell body. METHODS Adult cats, 1-7-3.2 kg in body weight, were anaesthetized by an I[.P. injection of sodium pentobarbitone (35-40 mglkg). In the first group of animals (twelve cats), several peripheral nerves (see below) in the left hind leg were sectioned, and the central and distal cut ends were ligated. In the second group of animals (thirteen cats), the seventh lumbar (L 7) and first sacral (S 1) dorsal roots on the left side
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were cut intradurally near the entry into the cord, and the cut ends were ligated. The third group of animals (seven cats) was prepared to examine the possible 'disuse' effects on sensory fibres arising from the triceps surae and plantaris muscles. For this purpose, the L 7 and S 1 ventral roots on the left side were sectioned intra durally, and the Achilles tendon was severed (tenotomy) in the left hind leg. To impede reconnexion, the proximal end of the cut tendon was pulled into the space between the skin and the surface of the triceps surae muscle and anchored to the skin with a thread. All chronic operations were performed under aseptic conditions. The results obtained from operated animals were compared with those observed in twelve control, unoperated cats. The statistical analysis of the results was made by two-tailed t tests with significance limit of 2P < 0 05. After a post-operative period ranging from 20 to 24 days, the animal was anaesthetized with sodium pentobarbitone (35-40 mg/kg; i.P.). The bilateral common carotid and vertebral arteries were permanently occluded. The brain was thus ischaemically impaired, as evidenced by cessation of respiration and by dilatation of the pupils. The spinal cord was then transacted at the first cervical level, and the brain rostral to the transaction was pithed. Thereafter, the animal was maintained on artificial respiration and immobilized by i.v. injections of gallamine triethiodide throughout the experiment. Dorsal root ganglia of the L 7 and S 1 segments on the left side were exposed by lumbosacral laminectomy. One of these ganglia was supported on a Lucite platform (Bessou, Burgess, Perl & Taylor, 1971) and prepared for intracellular recording with glass micro-electrodes filled with 2*5 M-KCl. The resistance of the electrodes was between 8 and 30 Mn. Insertion of the electrode was facilitated by incision of the connective tissue over the ganglion. Action potentials in each ganglion cell were generated successively by electrical stimuli applied to an array of bipolar electrodes placed on the dorsal root and on the peripheral nerve in the left hind leg (Fig. 1 A). Usually, four pairs of stimulating electrodes were placed on the dorsal root, and seven pairs on the peripheral nerve. Every pair of electrodes was arranged so that the cathode pole was located on the side proximal to the ganglion. The pulse duration of electrical stimuli was approximately 0 12 msec. The latency of action potentials evoked in the ganglion cell was measured at an intensity twice the threshold of the sensory fibre under study at each site of stimulation (Fig. 1 B, C). At the end of the experiment, the cathode sites of all stimulating electrodes were marked, and the peripheral nerve with attached dorsal root was excised. The conduction distance was measured from the sites of stimulation to the centre of the ganglion. The dorsal root ganglion was 3-5 mm in size. Therefore, the measured conduction distance might include an error of about 2 mm because of variability of the actually recorded site relative to the centre of the ganglion (Loeb, 1976). The conduction velocity of each ganglion cell was calculated separately for its central (dorsal root fibres; dotted lines) and peripheral (continuous) processes from the slope of the relation between the conduction time (latency) and the conduction distance (Fig. 2). The best fitting line intersecting the origin (site of the ganglion) was judged by eye (Fig. 2). Often, some points showed a considerable deviation from the line. Such a deviation occurred at random with respect to site of stimulation and was found only in some sensory neurones recorded from the same animal. No obvious explanation was found for the scatter of the points, but selection of the results was made based on two arbitrary criteria: (1) when a point deviated vertically from the line by more than 2 mm in the case of central processes and by more than 5 mm in the peripheral processes (Fig. 2, points indicated by arrows), the point was considered to be false, and (2) the results for the central processes were discarded when points more than one out of the four points were false, whereas the results for the peripheral processes were rejected
G. CZJH, N. KUDO AND M. KUNO
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when false points exceeded 35 % (one or two points) of the total points (four to seven) used for the measurement. About 86 % of the observed ganglion cells satisfied these criteria for measurement of the peripheral conduction velocity, but only 61 % of the cells could be accepted for the analyses of the central conduction velocity. The ratio of acceptance of the data based on these criteria did not differ between A
Dorsal
B
root
DRG
Sciatic
2
Sural
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Fig. 1. A, schematic diagram of the experiment for intracellular recordings from dorsal root ganglion (DRG) cells in response to stimulation of the dorsal root and the peripheral nerves in the hind leg. TS + P1, triceps surae and plantaris muscle nerves. B, C, examples of action potentials recorded from a ganglion cell in response to stimulation of the dorsal root (B) and the peripheral nerve (C). Downward arrows indicate the onset of action potentials. Upward arrow, onset of stimuli. Stimulus artifacts retouched except for the third record from top. operated and unoperated animals. The above procedure of selection of the results might have caused some bias of the samplings. However, since the source of the observed false points was inexplicable, the direction of the possible bias was not clear; also, no adequate correction was possible for uncertainty of the conduction velocity observed in those units with several false points. Intracellular recordings were made largely from sensory neurones arising from the triceps surae and plantaris muscles (Fig. 1 A, TS + P1), but in a few preparations the observations were made on cutaneous sensory neurones identified by stimulation of the sural nerve (Fig. 1 A) or on unidentified sensory neurones responding to
AXOTOMIZED SENSORY NEURONES
169 stimulation of the tibial or common peroneal nerve (mixed nerve). Similarly, in most cases, chronic peripheral section was made on the triceps surae and plantaris nerves about 10 mm from the muscles, except for some animals in which the sural nerve and the tibial or common peroneal nerve were sectioned in the popliteal fossa. The distance between site of section and the dorsal root ganglion was 26-34 mm for the central process (dorsal root) and 103-146 mm for the peripheral process. When the micro-electrode is inserted into the dorsal root ganglion, intracellular potentials can be recorded from neurone cell bodies or from their axons. The two instances could be distinguished on the basis of potential configuration described in
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Fig. 2. Relations between conduction distance and conduction time (latency) of two sensory fibres (open and filled circles) arising from the triceps surae and plantaris muscles recorded from the same animal. The origin corresponds to site of the dorsal root ganglion. Dotted lines, for the central processes (dorsal root fibres, DR). Continuous lines, for the peripheral processes. Arrows indicate false points (see text).
previous reports (Sato & Austin, 1961; Letbetter & Willis, 1969). Measurements of axonal conduction velocity were made whether intracellular potentials were recorded from cell bodies or from axons, if the action potential was large enough (usually > 40 mV) to reveal its onset clearly from the base line. Electrophysiological properties of the cell body were measured only in those units with an action potential in
CZJ9H, N. KUDO
G.
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AND M. KUNO
excess of 80 mV. In order to minimize a bias of sampling from particular animals, the maximum number of cells observed from one animal was limited to twenty. All exposed tissues were covered with pools of paraffin oil, and external heat aided in keeping the rectal temperature between 36 and 380 C. The pool temperature ranged from 31 to 340 C. The pool temperature around the dorsal root ganglion was consistently higher than that in the hind leg, but the difference was less than 1° C. 0
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I- . . M M N 10 50 100 10 50 100 Peripheral velocity (m/sec) Fig. 3. Relations between central (dorsal root fibres) and peripheral conduction velocities. Open circles, muscle sensory fibres. Filled circles, cutaneous sensory fibres. A, from control, unoperated cats. B, 20-23 days after section of the peripheral nerves. Regression lines (continuous lines) are y = 0-91x-6-9 for A and y = 0-73x+3-8 for B. The correlation coefficients are 0-85 for A and 0-86 for B. Dotted lines, relations expected for *0g
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RESULTS
Conduction velocity in control animals. In agreement with previous observations (Loeb, 1976), the majority of sensory neurones showed a faster conduction velocity in their peripheral processes than in the central processes (Fig. 2). Fig. 3A illustrates the relation between the central and peripheral conduction velocities of cutaneous (filled circles) and muscle (open circles) sensory neurones observed in control, unoperated cats. On the average, the central conduction velocity was about 82 % of the velocity in the peripheral process. Conduction velocity following peripheral nerve section. Fig. 3B shows a similar relationship between the central and peripheral conduction velocities of sensory neurones recorded 20-23 days after section of the peripheral nerves. Compared with those in control animals (Fig. 3A), the points show a general shift toward the lower left (Fig. 3B). However, the slope
171 AXOTOMIZED SENSORY NEURONES of the regression line between the central and peripheral conduction velocities following section of the peripheral nerves (0.73; Fig. 3B) did not significantly (0.10 > 2P > 0.05) differ from that in control animals (0.91; Fig. 3A). This suggests that a reduction in conduction velocity of sensory neurones occurs proportionately in both the central and peripheral processes after section of the peripheral nerves (see below). A
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50
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40 k B 30
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50 100 Conduction velocity (misec) Fig. 4. Histograms of central (A) and peripheral (B) conduction velocities of sensory neurones arising from the triceps surae and plantaris muscles. Stippled histograms, from control, unoperated cats. Heavy line histograms, 20-23 days after section of the muscle nerves. Single arrows, average conduction velocities for control animals. Double arrows, average conduction velocities for the animals with peripheral axotomy. 0
Intracellular recordings from cutaneous sensory ganglion cells were more difficult than from muscle sensory neurones with fast conduction velocities presumably because of the small cell size (Bessou et al. 1971). Therefore, changes in conduction velocity following section of the central or peripheral processes were systematically examined only in sensory neurones arising from the muscle. Muscle sensory fibres can be classified into distinct groups, each of which is composed of fibres with a relatively narrow range
172 G. CZJ9H, N. KUDO AND M. KUNO of conduction velocity (Hunt, 1954). This situation was favourable for detection of the change in conduction velocity under different conditions because of a shift of the modal peak in the histograms (see below). Fig. 4 shows histograms of the frequency of occurrence of sensory fibres arising from the triceps surae and plantaris muscles as a function of conduction velocities of the central processes (A) and of the peripheral processes (B) following chronic section of the muscle nerves (heavy lines). Similar histograms for control, unoperated animals are illustrated in the same Figure for comparison (thin lines, stippled). The results include the cases in which only central or peripheral conduction velocity could be measured. Judging from the peripheral conduction velocities, the majority of sensory neurones examined in the present study represented group I fibres (velocity > 72 m/sec; Hunt, 1954). A small proportion of the sample included group II fibres (velocity = 24-72 m/sec; Hunt, 1954), but group III or IV fibres were practically absent in our sample. In the preparations with peripheral axotomy, the average central conduction velocity decreased from 75 m/sec (Fig. 4A, single arrow for control animals) to 58 m/sec (double arrow). Similarly, the average peripheral conduction velocity (Fig. 4B) decreased from 86 m/sec (single arrow for control animals) to 75 m/sec (double arrow). Both changes were statistically significant (2P < 0 001). The distribution of conduction velocities appears to show a greater skewness toward slow speed (< 50 m/sec) in the animals with peripheral axotomy than in control cats (Fig. 4A, B). However, the differences in the average central and peripheral conduction velocities between operated and unoperated animals were still highly significant (2P < 0.001) when the values were calculated after exclusion of those units with conduction velocity slower than 50 m/sec. Therefore, there was no indication that a reduction in conduction velocity following section of the peripheral processes might be attributed to a bias in sampling of different groups of sensory neurones. Conduction velocity following dorsal root section. Fig. 5 shows histograms of conduction velocities for the central (A) and peripheral (B) processes of sensory neurones arising from the triceps surae and plantaris muscles 20-24 days after section of the dorsal roots (heavy lines). In comparison with the results obtained from control animals (Fig. 5, thin lines, stippled), the average central conduction velocity showed no significant (0-20 > 2P > 0.10) difference (70 and 75 m/sec; single and double arrows in Fig. 5A). Unexpectedly, however, the average peripheral conduction velocity following central axotomy (94 m/sec; double arrow in Fig. 5B) showed a significant (2P < 0*001) increase compared with that in control animals (86 m/sec; single arrow in Fig. 5B). It could be argued that section of the dorsal roots might result in some shrinkage of the ganglion cells, so that
173 AXOTOMIZED SENSORY NEURONES the sampling may have been biased in favour of cells with large diameters. However, morphologically no evidence has been found for cell shrinkage or cell death in the dorsal root ganglion following section of the dorsal root (Anderson, 1902; Ranson, 1914; Hare & Hinsey, 1940; Carmel & Stein, 1969). Furthermore, the distribution of the central conduction velocities A
n
20 H 10 M
0
50
100
40k B 30 20
10
0
50 100 Conduction velocity (msec) Fig. 5. Histograms of central (A) and peripheral (B) conduction velocities of sensory neurones arising from the triceps surae and plantaris muscles. Stippled histograms, from control, unoperated cats. Heavy line histograms, 20-24 days after section of the dorsal roots. Single arrows, average conduction velocities for control animals. Double arrows, average conduction velocities for the animals with central axotomy.
following central axotomy was strikingly similar to that in control animals (Fig. 5A). Therefore, an increase in peripheral conduction velocity associated with central axotomy could not adequately be accounted for on the basis of a bias in sampling. Effects of 'disuse'. When the peripheral nerve is sectioned, impulse activity would no longer be present in the sectioned sensory fibres. It is then possible that decreased conduction velocity in the central and peripheral processes of sensory neurones following section of the peripheral
174 1. CZ.9H, N. KUDO AND M. KUNO nerve (Fig. 4) may be due to 'disuse' rather than to interruption of axonal continuity (Gutmann & Holubar, 1951). To test this possibility, alpha and gamma motor innervation to the triceps surae and plantaris muscles was interrupted by section of the ventral roots, and the muscle length as well as its tension was reduced by section of the Achilles tendon (see Methods). Under such conditions, activity of sensory fibres arising from spindles (group Ia and group II) and tendon organs (group Ib) in these muscles may be assumed to be virtually, if not completely, abolished (Hnik, 1970; Hnik & Lessler, 1971). The average central conduction velocity of the muscle sensory fibres measured in a post-operative period of 20-23 days was 69 m/sec (n = 52), and the average peripheral conduction velocity was 82 m/sec (n = 96). Both the values were not significantly different from the values obtained from unoperated, control animals (75 m/sec and 86 m/sec) when examined by two-tailed t tests (0.10 > 2P > 0.05). However, when one-tailed t tests were applied, both the values observed in 'disuse' conditions were significantly (0.05 > P > 0 025) slower than the control values. On the other hand, the central and peripheral conduction velocities following section of the peripheral nerves (58 and 75 m/sec) were significantly (2P < 0-001) slower than those obtained under 'disuse' conditions. Thus, while sensory fibres tend to decrease their conduction velocity under 'disuse' conditions, decreased conduction velocity of sensory fibres following section of the peripheral nerves does not seem to be accounted for entirely on the basis of a lack of impulse activity. Reaction of cell bodies to axotomy. Electrophysiological properties (see below) of dorsal root ganglion cell bodies were indistinguishable between cutaneous and muscle sensory neurones. Therefore, measurements of the properties of ganglion cell bodies included both cutaneous and muscle sensory neurones as well as unidentified sensory neurones responding to stimulation of the mixed nerves. However, sensory neurones with a peripheral conduction velocity slower than 40 m/sec showed a longer duration of after-hyperpolarization (28-100 msec) than those with a velocity faster than 40 m/sec (5-42 msec). Those units with a peripheral conduction velocity slower than 40 m/sec were excluded from the analyses. Dorsal root ganglion cells recorded from control cats showed an average resting membrane potential of 63 mV (n = 49) and an average action potential of 87 mV (Fig. 6A; cf. Sato & Austin, 1961; Downes & Franz, 1971). Each action potential was followed by after-hyperpolarization (Fig. 6B), the average duration of whir' was 13 msec (n = 46; cf. Sato & Austin, 1961). The input membrane resistance was calculated from the linear portion of the current-voltage relationship with relatively weak (< 2 nA) depolarizing and hyperpolarizing current pulses applied through the intracellular electrode (Fig. 6C). The average input resistance was
175 AXOTOMIZED SENSORY NEUR ONES 34 MQ (n = 28; cf. Downes & Franz, 1971). None of these parameters showed significant changes about 3 weeks after section of the central or peripheral processes of sensory neurones. The only property of ganglion cell bodies which was detected to be altered by axotomy was the rectification characteristics of the cell membrane. As described by Ito (1957), hyperpolarizing responses of the ganglion cell show an initial peak subsequently followed by a decrement to a steady level when the applied C
A
D
B-
10-40 mVl 1-4 msec
10 mV to 5 nAl 10 msec7V I
Fig. 6. Intracellular potentials recorded from an unidentified sensory neurone responding to stimulation of the tibial nerve of a control, unoperated cat. A, resting and action potentials. B, after-hyperpolarization following the action potential evoked by intracellular stimulation. C, potential changes (lower traces) produced by the application of current pulses (upper traces) through the intracellular electrode. Upward deflexion, depolarization, and downward deflexion, hyperpolarization. D, similar to C, but two short hyperpolarizing current pulses were applied at the resting level and during hyperpolarization. 40 mV, 1 msec calibration for A. 10 mV, 4 msec calibration for B.
current pulses are increased beyond a certain intensity (Fig. 6C). The ionic mechanisms underlying this rectification are not clear (see small print below), and this phenomenon is referred to below as the 'timedependent hyperpolarizing (inward-going) rectification'. All ganglion cells (twenty-eight) examined in control animals showed the rectification (Fig. 6C). The average hyperpolarizing current intensity at which the time-dependent rectification began to appear was 3-6 + 1-8 (S.D.) nA. Similarly, all ganglion cells (twenty-six) tested about 3 weeks after section of the dorsal roots showed the time-dependent hyperpolarizing rectification (Fig. 7 A). The average 'threshold' for the rectification in these animals was 3-7 + 2-6 (S.D.) nA. In contrast, in the animals with peripheral axotomy, seven out of 15 ganglion cells showed no sign of the timedependent rectification in response to strong hyperpolarizing current
176 G. CZEH, N. KUDO AND M. KUNO pulses up to 16 nA (Fig. 7B). The rest of ganglion cells (eight) revealed the rectification, but the average 'threshold' was 6-8+3.6 (S.D.) nA which was significantly (2P < 0-01) higher than the control value. Whatever the mechanisms responsible for the time-dependent hyperpolarizing rectification, the ganglion cell bodies apparently undergo a change in the properties in response to peripheral axotomy, whereas such changes are lacking in response to central axotomy. Phenomenologically, these results coincide with morphological observations that chromatolytic changes in sensory neurones can be induced by peripheral axotomy but not by central axotomy (see Introduction). A
B
m
10 msOmV to 5 nA,
Fig. 7. Potential changes (lower traces) produced by the application of current pulses (upper traces) through the intracellular electrode. Upward defiexion, depolarization, and downward deflexion, hyperpolarization. A, a cutaneous neurone recorded 22 days after section of the dorsal root. B, a muscle sensory neurone recorded 22 days after section of the muscle nerve.
The rectification property was examined only in four ganglion cells under 'disuse' conditions. The 'threshold' for rectification in these cells was similar to the control value, being in the range of 1-9-5*7 nA (average, 3.5 nA). The time-dependent inward-going (anomalous) rectification recently analysed in the egg cell membrane of a tunicate (Miyazaki, Takahashi, Tsuda & Yoshii, 1974) and a starfish (Hagiwara & Takahashi, 1974; Hagiwara, Miyazaki & Rosenthal, 1976) has been attributed to an increase in the potassium conductance which is initially high and subsequently declines to a steady level during hyperpolarization. However, the same mechanism may not apply to the time-dependent rectification described in the present study for two reasons: (1) the peak hyperpolarization can reach over -I100 mV, exceeding the reversal potential of after-hyperpolarization which may reflect the potassium equilibrium potential, and (2) there is an indication of a decrease, rather than an increase, in the membrane conductance at the peak
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of hyperpolarization when constant current pulses are superimposed on the hyperpolarizing response (Fig. 6D). Ito (1957) has suggested a delayed increase in the chloride conductance during hyperpolarization for the time-dependent rectification in dorsal root ganglion cells of the toad. This possibility is also unlikely since the membrane conductance is lower at the late steady level of hyperpolarization than at the resting level as shown by superimposed current pulses (Fig. 6D). The membrane conductance was slightly (about 3 %) higher at the steady level of hyperpolarization than at the peak when measured by superimposed current pulses (Fig. 6D). However, this slight difference in the membrane resistance was not sufficient to account for the potential difference (> 40 %) between the peak and the late steady level of hyperpolarization (Fig. 6D). It is possible that the ganglion cell has the outward-going (delayed) rectification and that the sodium and/or calcium conductance may slightly increase with a certain delay after the onset of hyperpolarizing response presumably by removal of partial inactivation existing under the resting conditions. DISCUSSION
The present results show that dorsal root fibres do not change their conduction velocity when the dorsal roots are sectioned, at least in muscle sensory fibres with relatively large diameters. This implies that injuries in nerve fibres per se do not necessarily result in a decrease in their conduction velocity. On the other hand, chronic section of the peripheral processes of sensory neurones caused a decrease in conduction velocity for both the central and peripheral processes, and these changes were associated with changes in the properties of the neurone cell body. This behaviour is in contrast with that following section of the dorsal roots which does not produce detectable changes in the cell body properties. Thus, it is suggested that a decrease in conduction velocity following nerve section may require the participation of changes in the neurone cell body. The simplest explanation would be that peripheral section of sensory neurones may induce some alterations in the cell body and that the signal originating from the altered cell body may exert the influence on both the central and peripheral processes, thereby reducing their conduction velocities. However, a possibility still exists that the change in the neurone cell body and a decrease in the axonal conduction velocity may be controlled by a common factor following section of the peripheral nerve (see below). A puzzling finding was that central axotomy caused an increase in conduction velocity of the peripheral processes (Fig. 5B) without changes in the central conduction velocity (Fig. 5A) or in the properties of the cell body (Fig. 7A). In the Aplysia cerebral neurone, section of one branch of the axon has been shown to cause an increase in amounts and rates of axonal transport in the remaining intact branch (Goldberg, Goldman & Schwartz, 1976). If an increase in the peripheral conduction velocity following central axotomy is assumed to be related to some changes in the
178 1. CZtH, N. KUDO AND M. KUNO axonal transport, one may speculate that the transport material originally destined to the central process may be diverted to the peripheral processes because of occlusion at the central cut end. Thus, the peripheral axonal transport may be increased in a manner similar to the process suggested by Goldberg et al. (1976). A similar explanation may apply to the difference in conduction velocity between the central and peripheral processes of the dorsal root ganglion cell. In mammalian myelinated sensory fibres, there is as yet no evidence that the central processes are smaller in diameter than the peripheral processes (Dale, 1900; Rexed & sourander, 1949; Ha, 1970). Nevertheless, the present study confirmed previous observations that the majority of sensory neurones have a slower conduction velocity in their central processes than in the peripheral processes (Loeb, 1976; also, cf. Fu & Schomburg, 1974; Willis, Nufiez & Rudomin, 1976). This difference might be related to the amount of axoplasmic transport which is smaller in the central processes of the dorsal root ganglion cell than in the peripheral processes (Lasek, 1968; Ochs, 1972; also, cf. Smith, 1973). The absence of chromatolytic or electrophysiological reaction in the cell body to central axotomy of sensory neurones is another puzzling problem. Both the central and peripheral processes of sensory neurones appear to possess the retrograde transport system as evidenced by injection of horseradish peroxidase into the spinal cord (Maynard, Leonard, Coulter, Coggeshall & Willis, 1975) and into the muscle (Burke, Strick, Kanda, Kim & Walmsley, 1977; also, cf. Furstman, Saporta & Kruger, 1975). In sympathetic ganglion cells, both chromatolytic (West & Bunge, 1976) and electrophysiological (Purves & Nja, 1976) reactions following section of the postganglionic axon can be prevented by the application of nerve growth factor. Similarly, nerve growth factor prevents the biochemical changes that follow axotomy of the sympathetic ganglion (Hendry, 1975). Thus, it has been suggested that the normal properties of sympathetic neurones are maintained by nerve growth factor transported from the effector organ (Hendry & Iversen, 1973). If one assumes a similar mechanism fot sensory neurones, such a putative material that maintains their normal properties would be present only in the peripheral organs and be lacking in the central nervous system. The authors wish to thank Miss Cynthia V. Taylor for technical assistance. This work was supported by project (NS 11132) and research (NS 10319) grants from the U.S. Public Health Service. REFERENCES
ACHESON, G. H., LEE, E. S. & MORISON, R. S. (1942). A deficiency in the phrenic respiratory discharges parallel to retrograde degeneration. J. Neurophy8iol. 5, 269-273.
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AITKEN, J. T. & THOMAS, P. K. (1962). Retrograde changes in fibre size following nerve section. J. Anat. 96, 121-129. ANDERSON, H. K. (1902). The nature of the lesions which hinder the development of nerve cells and their processes. J. Physiol. 28, 499-513. BAGUST, J. & LEWIS, D. M. (1974). Isometric contractions of motor units in selfreinnervated fast and slow twitch muscles of the cat. J. Phy8iol. 237, 91-102. BESSOU, P., BURGESS, P. R., PERL, E. R. & TAYLOR, C. B. (1971). Dynamic properties of mechanoreceptors with unmyelinated (C) fibers. J. Neurophysiol. 34, 116-131. BuRK1E, R. E., STRICK, P. L., KANDA, K., KIM, C. C. & WALMSLEY, B. (1977). The anatomy of the medial gastroecnemius and soleus motor nuclei in the cat spinal cord. J. Neurophy8iol. 40 (in the Press). CARMEL, P. W. & STEIN, B. M. (1969). Cell changes in sensory ganglia following proximal and distal nerve section in the monkey. J. comp. Neurol. 135, 145-166. CRAGG, B. G. & THOMAS, P. K. (1961). Changes in conduction velocity and fibre size proximal to peripheral nerve lesion. J. Phy8iol. 157, 315-327. DALE, H. H. (1900). On some numerical comparison of the centripetal and centrifugal medullated nerve-fibres arising in the spinal ganglia of the mammal. J. Physiol. 25, 196-206. DOwNES, H. & FRANZ, D. N. (1971). Effects of a convulsant barbiturate on dorsal root ganglion cells and dorsal root discharges. J. Pharmac. exp. Ther. 179, 660-670. EccuEs, J. C., KRNJEVI6, K. & MILEDI, R. (1959). Delayed effects of peripheral severance of afferent nerve fibres on the efficacy of their central synapses. J. Physiol. 145, 204-220. ECCLES, J. C. & MCINTYRE, A. K. (1953). The effects of disuse and of activity on mammalian spinal reflexes. J. Physiol. 121, 492-516. Fu, T. C. & SCHOMBURG, E. D. (1974). Electrophysiological investigation of the projection of secondary muscle spindle afferents in the cat spinal cord. Acta phy8iol. 8cand. 91, 314-329. FURSTMAN, L., SAPORTA, S. & KRUGER, L. (1975). Retrograde axonal transport of horseradish peroxidase in sensory nerves and ganglion cells of the rat. Brain Re8. 84, 320-324. GOLDBERG, D. J., GOLDMAN, J. E. & SCHWARTZ, J. H. (1976). Alterations in amounts and rates of serotonin transported in an axon of the giant cerebral neurone of Aply8ia californica. J. Phyaiol. 259, 473-490. GREENMAN, M. J. (1913). Studies on the regeneration of the peroneal nerve of the albino rat: number and sectional areas of fibers: area relation of axis to sheath. J. comp. Neurol. 23, 479-513. GUTMANN, E. & HOLUBAI%, J. (1951). Atrophy of nerve fibres in the central stump following nerve section and the possibilities of its prevention. Arch8 int. Stud. Neurol. 1, 1-11. GUTMANN, E. & SANDERS, F. K. (1943). Recovery of fibre numbers and diameters in the regeneration of peripheral nerves. J. Physiol. 101, 489-518. HA, H. (1970). Axonal bifurcation in the dorsal root ganglion of the cat: a light and electron microscopic study. J. comp. Neurol. 140, 227-240. HAGIWARA, S., MIYAZAKI, S. & ROSENTHAL, N. P. (1976). Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish. J. gen. Phygiol. 67, 621-638. HAGIWARA, S. & TAKAHASHI, K. (1974). The anomalous rectification and cation selectivity of the membrane of a starfish egg cell. J. Membrane Biol. 18, 61-80. HARE, W. K. & HINSEY, J. C. (1940). Reaction of dorsal root ganglion cells to section of peripheral and central processes. J. comp. Neurol. 73, 489-502.
8. CZEH, N. KUDO AND M. KUNO 180 HENDRY, I. A. (1975). The response of adrenergic neurones to axotomy and nerve growth factor. Brain Re-s. 94, 87-97. HENDRY, I. A. & IVERSEN, L. L. (1973). Reduction in the concentration of nerve growth factor in mice after sialectomy and castration. Nature, Lond. 243, 500-504. HNiK, P. (1970). The increased response of chronically de-efferented rat muscle spindles to stretch. Brain Re8. 21, 448-451. iHNix, P. & LESSLER, M. J. (1971). The enhanced spindle response to stretch of tenotomized gastrocnemius muscle of the rat. Brain Re8. 33, 237-240. HUNT, C. C. (1954). Relation of function to diameter in afferent fibers of muscle nerves. J. gen. Phy8iol. 38, 117-131. ITO, M. (1957). The electrical activity of spinal ganglion cells investigated with intracellular microelectrodes. Jap. J. Phy8iol. 7, 297-323. KIRALY, J. K. & KRNJEVI6, K. (1959). Some retrograde changes in function of nerves after peripheral section. Q. JL exp. Phyaiol. 44, 244-257. KUNO, M., MIYATA, Y. & MUNOZ-MARTINEZ, E. J. (1974). Differential reaction of fast and slow a-motoneurones to axotomy. J. Physiol. 240, 725-739. LASEK, R. (1968). Axoplasmic transport in cat dorsal root ganglion cells: as studied with [3H]-L-leucine. Brain Re8. 7, 360-377. LETBETTER, W. D. & WILLIS, W. D. (1969). Electrophysiological characteristics of cat dorsal root ganglion cells. Phy8iologi8t, Wa8h. 12, 283. LOEB, G. E. (1976). Decreased conduction velocity in the proximal projections of myelinated dorsal root ganglion cells in the cat. Brain Re8. 103, 381-385. MAYNARD, C. W., LEONARD, R. B., COULTER, J. D., COGGESHALL, R. E. & WILLIS, W. D. (1975). Cells of origin of ventral root afferents. Annl8 Soc. Neuro8ci. 5, 141. MENDELL, L. M., MUNSON, J. B. & SCOTT, J. G. (1976). Alterations of synapses on axotomized motoneurones. J. Physiol. 255, 67-79. MIYAZAKI, S., TAKAHASHI, K., TSUDA, K. & YOSHII, M. (1974). Analysis of nonlinearity observed in the current-voltage relation of the tunicate embryo. J. Phy8iOl. 238, 55-77. OCHS, S. (1972). Rate of fast axoplasmic transport in mammalian nerve fibres. J. Physiol. 227, 627-645. PURvES, D. & NJA, A. (1976). Effect of nerve growth factor on synaptic depression after axotomy. Nature, Lond. 260, 535-536. RANSON, S. W. (1914). Transplantation of the spinal ganglion with observations on the significance of the complex types of spinal ganglion cells. J. comp. Neurol. 24, 547-558.
REXED, B. & SOURANDER, P. (1949). The caliber of central and peripheral neurites of spinal ganglion cells and variations in fiber size at different levels of dorsal spinal root. J. comp. Neurol. 91, 297-306. SATO, M. & AUSTIN, G. (1961). Intracellular potentials of mammalian dorsal root ganglion cells. J. Neurophy8iol. 24, 569-582. SCHMIDT, H. & STEFANI, E. (1976). Re-innervation of twitch and slow muscle fibres of the frog after crushing the motor nerves. J. Phy8iol. 258, 99-123. & HORSKY, P. (1968). Regeneration in spinal neurons: proteosynthesis following nerve growth factor administration. Science, N.Y. 152, 787-788. SMITH, R. S. (1973). Microtuble and neurofilament densities in amphibian spinal root nerve fibers: relationship to axoplasmic transport. Can. J. Phy8iol. Pharmacol. 51, 798-806. WEST, N. R. & BUNGE, R. P. (1976). Prevention of the chromatolytic response in rat superior ganglion neurons by nerve growth factor. Ann. Soc. Neuro8ci. 6, 1038. WILLIS, W. D., NYJEZ, R. & RUDOMIN, P. (1976). Excitability changes of terminal arborization of single Ia and Ib afferent fibers produced by muscle and cutaneouis conditioning volleys. J. Neurophy.9iol. 39, 1150-1159.
SCOTT, D., GUTMANN, E.
165
MEMBRANE PROPERTIES AND CONDUCTION VELOCITY IN SENSORY NEURONES FOLLOWING CENTRAL OR PERIPHERAL AXOTOMY
BY G. CZRH, N. KUDO AND M. KUNO From the Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514, U.S.A.
(Received 20 December 1976) SUMMARY
1. The properties of dorsal root ganglion cells in the lumbosacral segments were examined with intracellular electrodes about 3 weeks after section of the central (dorsal roots) or peripheral processes in the cat. 2. Chronic section of the peripheral nerve in the hind limb resulted in a reduction in conduction velocity of both the central and peripheral processes of sensory neurones. 3. Sensory neurones arising from the triceps surae and plantaris muscles were subject to 'disuse' conditions for about 3 weeks by section of the ventral roots combined with severance of the Achilles tendon. Under such conditions, the central and peripheral conduction velocities of these sensory neurones tended to decrease, but the decrease was significantly less than that following peripheral nerve section. 4. Chronic section of the dorsal roots produced no significant changes in conduction velocity of the central processes of muscle sensory neurones but caused a significant increase in the peripheral conduction velocity. 5. The only electrophysiological property of dorsal root ganglion cells which altered following axotomy was the time-dependent membrane rectification in response to hyperpolarizing current pulses. The rectification characteristics were modified by chronic section of the peripheral nerve but not by chronic section of the dorsal root. 6. It is concluded that injuries in nerve fibres per se do not necessarily result in a decrease of their conduction velocity and that a decrease in their conduction velocity is associated with changes in the properties of the cell bodies. 7. It is suggested that a decrease in conduction velocity following nerve section may require the participation of changes in the neurone cell body.
166
1. CZEH, N. KUDO AND M. KUNO INTRODUCTION
Injuries in peripheral nerve fibres result in a reduction of conduction velocity proximal to the lesion (Acheson, Lee & Morison, 1942; Gutmann & Holubar, 1951; Eccles & McIntyre, 1953; Eccles, Krnjevic & Miledi, 1959; Kiraly & Krnjevic, 1959; Cragg & Thomas, 1961; Aitken & Thomas, 1962; Bagust & Lewis, 1974; Kuno, Miyata & Munioz-Martinez, 1974; Mendell, Munson & Scott, 1976; Schmidt & Stefani, 1976). The reduced conduction velocity appears to be associated with a decrease in axon diameter (Greenman, 1913; Gutmann & Sanders, 1943; Gutmann & Holuba6, 1951; Cragg & Thomas, 1961; Aitken & Thomas, 1962). Mendell et al. (1976) have attempted to determine if decreased conduction velocity proceeds centrally from the cut end toward the neurone cell body or in the reverse direction. However, the results failed to show in which direction the changes in conduction velocity might spread. Thus, it remains uncertain whether a reduction in conduction velocity following nerve section is a primary change in the axon induced by the injury or whether it is a reaction of the axon secondary to chromatolytic changes in the neurone cell body. In sensory neurones it is generally agreed that the cell body shows a chromatolytic reaction to section of the peripheral process (peripheral axotomy), whereas section of the central process (central axotomy) induces no morphological changes in the cell body (Anderson, 1902; Ranson, 1914; Hare & Hinsey, 1940; Scott, Gutmann & Horsky, 1968; Carmel & Stein, 1969). This unique behaviour may provide a favourable condition to test the question as to whether the change in the cell body is a necessary correlate of a reduction in conduction velocity following nerve section. In the present study, the conduction velocity of dorsal root ganglion cells was measured following chronic section of the central (dorsal roots) or peripheral processes. The results show that changes in electrophysiological properties of the cell body as well as a reduction in the axonal conduction velocity occur within about 3 weeks after section of the peripheral processes, whereas these changes are absent following section of the central processes. It is suggested that a decrease in conduction velocity following nerve section may require the participation of changes in the neurone cell body. METHODS Adult cats, 1-7-3.2 kg in body weight, were anaesthetized by an I[.P. injection of sodium pentobarbitone (35-40 mglkg). In the first group of animals (twelve cats), several peripheral nerves (see below) in the left hind leg were sectioned, and the central and distal cut ends were ligated. In the second group of animals (thirteen cats), the seventh lumbar (L 7) and first sacral (S 1) dorsal roots on the left side
AXOTOMIZED SENSORY NEURONES
167
were cut intradurally near the entry into the cord, and the cut ends were ligated. The third group of animals (seven cats) was prepared to examine the possible 'disuse' effects on sensory fibres arising from the triceps surae and plantaris muscles. For this purpose, the L 7 and S 1 ventral roots on the left side were sectioned intra durally, and the Achilles tendon was severed (tenotomy) in the left hind leg. To impede reconnexion, the proximal end of the cut tendon was pulled into the space between the skin and the surface of the triceps surae muscle and anchored to the skin with a thread. All chronic operations were performed under aseptic conditions. The results obtained from operated animals were compared with those observed in twelve control, unoperated cats. The statistical analysis of the results was made by two-tailed t tests with significance limit of 2P < 0 05. After a post-operative period ranging from 20 to 24 days, the animal was anaesthetized with sodium pentobarbitone (35-40 mg/kg; i.P.). The bilateral common carotid and vertebral arteries were permanently occluded. The brain was thus ischaemically impaired, as evidenced by cessation of respiration and by dilatation of the pupils. The spinal cord was then transacted at the first cervical level, and the brain rostral to the transaction was pithed. Thereafter, the animal was maintained on artificial respiration and immobilized by i.v. injections of gallamine triethiodide throughout the experiment. Dorsal root ganglia of the L 7 and S 1 segments on the left side were exposed by lumbosacral laminectomy. One of these ganglia was supported on a Lucite platform (Bessou, Burgess, Perl & Taylor, 1971) and prepared for intracellular recording with glass micro-electrodes filled with 2*5 M-KCl. The resistance of the electrodes was between 8 and 30 Mn. Insertion of the electrode was facilitated by incision of the connective tissue over the ganglion. Action potentials in each ganglion cell were generated successively by electrical stimuli applied to an array of bipolar electrodes placed on the dorsal root and on the peripheral nerve in the left hind leg (Fig. 1 A). Usually, four pairs of stimulating electrodes were placed on the dorsal root, and seven pairs on the peripheral nerve. Every pair of electrodes was arranged so that the cathode pole was located on the side proximal to the ganglion. The pulse duration of electrical stimuli was approximately 0 12 msec. The latency of action potentials evoked in the ganglion cell was measured at an intensity twice the threshold of the sensory fibre under study at each site of stimulation (Fig. 1 B, C). At the end of the experiment, the cathode sites of all stimulating electrodes were marked, and the peripheral nerve with attached dorsal root was excised. The conduction distance was measured from the sites of stimulation to the centre of the ganglion. The dorsal root ganglion was 3-5 mm in size. Therefore, the measured conduction distance might include an error of about 2 mm because of variability of the actually recorded site relative to the centre of the ganglion (Loeb, 1976). The conduction velocity of each ganglion cell was calculated separately for its central (dorsal root fibres; dotted lines) and peripheral (continuous) processes from the slope of the relation between the conduction time (latency) and the conduction distance (Fig. 2). The best fitting line intersecting the origin (site of the ganglion) was judged by eye (Fig. 2). Often, some points showed a considerable deviation from the line. Such a deviation occurred at random with respect to site of stimulation and was found only in some sensory neurones recorded from the same animal. No obvious explanation was found for the scatter of the points, but selection of the results was made based on two arbitrary criteria: (1) when a point deviated vertically from the line by more than 2 mm in the case of central processes and by more than 5 mm in the peripheral processes (Fig. 2, points indicated by arrows), the point was considered to be false, and (2) the results for the central processes were discarded when points more than one out of the four points were false, whereas the results for the peripheral processes were rejected
G. CZJH, N. KUDO AND M. KUNO
168
when false points exceeded 35 % (one or two points) of the total points (four to seven) used for the measurement. About 86 % of the observed ganglion cells satisfied these criteria for measurement of the peripheral conduction velocity, but only 61 % of the cells could be accepted for the analyses of the central conduction velocity. The ratio of acceptance of the data based on these criteria did not differ between A
Dorsal
B
root
DRG
Sciatic
2
Sural
TS+Pl
0-5 msec ~ii
Fig. 1. A, schematic diagram of the experiment for intracellular recordings from dorsal root ganglion (DRG) cells in response to stimulation of the dorsal root and the peripheral nerves in the hind leg. TS + P1, triceps surae and plantaris muscle nerves. B, C, examples of action potentials recorded from a ganglion cell in response to stimulation of the dorsal root (B) and the peripheral nerve (C). Downward arrows indicate the onset of action potentials. Upward arrow, onset of stimuli. Stimulus artifacts retouched except for the third record from top. operated and unoperated animals. The above procedure of selection of the results might have caused some bias of the samplings. However, since the source of the observed false points was inexplicable, the direction of the possible bias was not clear; also, no adequate correction was possible for uncertainty of the conduction velocity observed in those units with several false points. Intracellular recordings were made largely from sensory neurones arising from the triceps surae and plantaris muscles (Fig. 1 A, TS + P1), but in a few preparations the observations were made on cutaneous sensory neurones identified by stimulation of the sural nerve (Fig. 1 A) or on unidentified sensory neurones responding to
AXOTOMIZED SENSORY NEURONES
169 stimulation of the tibial or common peroneal nerve (mixed nerve). Similarly, in most cases, chronic peripheral section was made on the triceps surae and plantaris nerves about 10 mm from the muscles, except for some animals in which the sural nerve and the tibial or common peroneal nerve were sectioned in the popliteal fossa. The distance between site of section and the dorsal root ganglion was 26-34 mm for the central process (dorsal root) and 103-146 mm for the peripheral process. When the micro-electrode is inserted into the dorsal root ganglion, intracellular potentials can be recorded from neurone cell bodies or from their axons. The two instances could be distinguished on the basis of potential configuration described in
ob
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Fig. 2. Relations between conduction distance and conduction time (latency) of two sensory fibres (open and filled circles) arising from the triceps surae and plantaris muscles recorded from the same animal. The origin corresponds to site of the dorsal root ganglion. Dotted lines, for the central processes (dorsal root fibres, DR). Continuous lines, for the peripheral processes. Arrows indicate false points (see text).
previous reports (Sato & Austin, 1961; Letbetter & Willis, 1969). Measurements of axonal conduction velocity were made whether intracellular potentials were recorded from cell bodies or from axons, if the action potential was large enough (usually > 40 mV) to reveal its onset clearly from the base line. Electrophysiological properties of the cell body were measured only in those units with an action potential in
CZJ9H, N. KUDO
G.
170
AND M. KUNO
excess of 80 mV. In order to minimize a bias of sampling from particular animals, the maximum number of cells observed from one animal was limited to twenty. All exposed tissues were covered with pools of paraffin oil, and external heat aided in keeping the rectal temperature between 36 and 380 C. The pool temperature ranged from 31 to 340 C. The pool temperature around the dorsal root ganglion was consistently higher than that in the hind leg, but the difference was less than 1° C. 0
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I- . . M M N 10 50 100 10 50 100 Peripheral velocity (m/sec) Fig. 3. Relations between central (dorsal root fibres) and peripheral conduction velocities. Open circles, muscle sensory fibres. Filled circles, cutaneous sensory fibres. A, from control, unoperated cats. B, 20-23 days after section of the peripheral nerves. Regression lines (continuous lines) are y = 0-91x-6-9 for A and y = 0-73x+3-8 for B. The correlation coefficients are 0-85 for A and 0-86 for B. Dotted lines, relations expected for *0g
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RESULTS
Conduction velocity in control animals. In agreement with previous observations (Loeb, 1976), the majority of sensory neurones showed a faster conduction velocity in their peripheral processes than in the central processes (Fig. 2). Fig. 3A illustrates the relation between the central and peripheral conduction velocities of cutaneous (filled circles) and muscle (open circles) sensory neurones observed in control, unoperated cats. On the average, the central conduction velocity was about 82 % of the velocity in the peripheral process. Conduction velocity following peripheral nerve section. Fig. 3B shows a similar relationship between the central and peripheral conduction velocities of sensory neurones recorded 20-23 days after section of the peripheral nerves. Compared with those in control animals (Fig. 3A), the points show a general shift toward the lower left (Fig. 3B). However, the slope
171 AXOTOMIZED SENSORY NEURONES of the regression line between the central and peripheral conduction velocities following section of the peripheral nerves (0.73; Fig. 3B) did not significantly (0.10 > 2P > 0.05) differ from that in control animals (0.91; Fig. 3A). This suggests that a reduction in conduction velocity of sensory neurones occurs proportionately in both the central and peripheral processes after section of the peripheral nerves (see below). A
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40 k B 30
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50 100 Conduction velocity (misec) Fig. 4. Histograms of central (A) and peripheral (B) conduction velocities of sensory neurones arising from the triceps surae and plantaris muscles. Stippled histograms, from control, unoperated cats. Heavy line histograms, 20-23 days after section of the muscle nerves. Single arrows, average conduction velocities for control animals. Double arrows, average conduction velocities for the animals with peripheral axotomy. 0
Intracellular recordings from cutaneous sensory ganglion cells were more difficult than from muscle sensory neurones with fast conduction velocities presumably because of the small cell size (Bessou et al. 1971). Therefore, changes in conduction velocity following section of the central or peripheral processes were systematically examined only in sensory neurones arising from the muscle. Muscle sensory fibres can be classified into distinct groups, each of which is composed of fibres with a relatively narrow range
172 G. CZJ9H, N. KUDO AND M. KUNO of conduction velocity (Hunt, 1954). This situation was favourable for detection of the change in conduction velocity under different conditions because of a shift of the modal peak in the histograms (see below). Fig. 4 shows histograms of the frequency of occurrence of sensory fibres arising from the triceps surae and plantaris muscles as a function of conduction velocities of the central processes (A) and of the peripheral processes (B) following chronic section of the muscle nerves (heavy lines). Similar histograms for control, unoperated animals are illustrated in the same Figure for comparison (thin lines, stippled). The results include the cases in which only central or peripheral conduction velocity could be measured. Judging from the peripheral conduction velocities, the majority of sensory neurones examined in the present study represented group I fibres (velocity > 72 m/sec; Hunt, 1954). A small proportion of the sample included group II fibres (velocity = 24-72 m/sec; Hunt, 1954), but group III or IV fibres were practically absent in our sample. In the preparations with peripheral axotomy, the average central conduction velocity decreased from 75 m/sec (Fig. 4A, single arrow for control animals) to 58 m/sec (double arrow). Similarly, the average peripheral conduction velocity (Fig. 4B) decreased from 86 m/sec (single arrow for control animals) to 75 m/sec (double arrow). Both changes were statistically significant (2P < 0 001). The distribution of conduction velocities appears to show a greater skewness toward slow speed (< 50 m/sec) in the animals with peripheral axotomy than in control cats (Fig. 4A, B). However, the differences in the average central and peripheral conduction velocities between operated and unoperated animals were still highly significant (2P < 0.001) when the values were calculated after exclusion of those units with conduction velocity slower than 50 m/sec. Therefore, there was no indication that a reduction in conduction velocity following section of the peripheral processes might be attributed to a bias in sampling of different groups of sensory neurones. Conduction velocity following dorsal root section. Fig. 5 shows histograms of conduction velocities for the central (A) and peripheral (B) processes of sensory neurones arising from the triceps surae and plantaris muscles 20-24 days after section of the dorsal roots (heavy lines). In comparison with the results obtained from control animals (Fig. 5, thin lines, stippled), the average central conduction velocity showed no significant (0-20 > 2P > 0.10) difference (70 and 75 m/sec; single and double arrows in Fig. 5A). Unexpectedly, however, the average peripheral conduction velocity following central axotomy (94 m/sec; double arrow in Fig. 5B) showed a significant (2P < 0*001) increase compared with that in control animals (86 m/sec; single arrow in Fig. 5B). It could be argued that section of the dorsal roots might result in some shrinkage of the ganglion cells, so that
173 AXOTOMIZED SENSORY NEURONES the sampling may have been biased in favour of cells with large diameters. However, morphologically no evidence has been found for cell shrinkage or cell death in the dorsal root ganglion following section of the dorsal root (Anderson, 1902; Ranson, 1914; Hare & Hinsey, 1940; Carmel & Stein, 1969). Furthermore, the distribution of the central conduction velocities A
n
20 H 10 M
0
50
100
40k B 30 20
10
0
50 100 Conduction velocity (msec) Fig. 5. Histograms of central (A) and peripheral (B) conduction velocities of sensory neurones arising from the triceps surae and plantaris muscles. Stippled histograms, from control, unoperated cats. Heavy line histograms, 20-24 days after section of the dorsal roots. Single arrows, average conduction velocities for control animals. Double arrows, average conduction velocities for the animals with central axotomy.
following central axotomy was strikingly similar to that in control animals (Fig. 5A). Therefore, an increase in peripheral conduction velocity associated with central axotomy could not adequately be accounted for on the basis of a bias in sampling. Effects of 'disuse'. When the peripheral nerve is sectioned, impulse activity would no longer be present in the sectioned sensory fibres. It is then possible that decreased conduction velocity in the central and peripheral processes of sensory neurones following section of the peripheral
174 1. CZ.9H, N. KUDO AND M. KUNO nerve (Fig. 4) may be due to 'disuse' rather than to interruption of axonal continuity (Gutmann & Holubar, 1951). To test this possibility, alpha and gamma motor innervation to the triceps surae and plantaris muscles was interrupted by section of the ventral roots, and the muscle length as well as its tension was reduced by section of the Achilles tendon (see Methods). Under such conditions, activity of sensory fibres arising from spindles (group Ia and group II) and tendon organs (group Ib) in these muscles may be assumed to be virtually, if not completely, abolished (Hnik, 1970; Hnik & Lessler, 1971). The average central conduction velocity of the muscle sensory fibres measured in a post-operative period of 20-23 days was 69 m/sec (n = 52), and the average peripheral conduction velocity was 82 m/sec (n = 96). Both the values were not significantly different from the values obtained from unoperated, control animals (75 m/sec and 86 m/sec) when examined by two-tailed t tests (0.10 > 2P > 0.05). However, when one-tailed t tests were applied, both the values observed in 'disuse' conditions were significantly (0.05 > P > 0 025) slower than the control values. On the other hand, the central and peripheral conduction velocities following section of the peripheral nerves (58 and 75 m/sec) were significantly (2P < 0-001) slower than those obtained under 'disuse' conditions. Thus, while sensory fibres tend to decrease their conduction velocity under 'disuse' conditions, decreased conduction velocity of sensory fibres following section of the peripheral nerves does not seem to be accounted for entirely on the basis of a lack of impulse activity. Reaction of cell bodies to axotomy. Electrophysiological properties (see below) of dorsal root ganglion cell bodies were indistinguishable between cutaneous and muscle sensory neurones. Therefore, measurements of the properties of ganglion cell bodies included both cutaneous and muscle sensory neurones as well as unidentified sensory neurones responding to stimulation of the mixed nerves. However, sensory neurones with a peripheral conduction velocity slower than 40 m/sec showed a longer duration of after-hyperpolarization (28-100 msec) than those with a velocity faster than 40 m/sec (5-42 msec). Those units with a peripheral conduction velocity slower than 40 m/sec were excluded from the analyses. Dorsal root ganglion cells recorded from control cats showed an average resting membrane potential of 63 mV (n = 49) and an average action potential of 87 mV (Fig. 6A; cf. Sato & Austin, 1961; Downes & Franz, 1971). Each action potential was followed by after-hyperpolarization (Fig. 6B), the average duration of whir' was 13 msec (n = 46; cf. Sato & Austin, 1961). The input membrane resistance was calculated from the linear portion of the current-voltage relationship with relatively weak (< 2 nA) depolarizing and hyperpolarizing current pulses applied through the intracellular electrode (Fig. 6C). The average input resistance was
175 AXOTOMIZED SENSORY NEUR ONES 34 MQ (n = 28; cf. Downes & Franz, 1971). None of these parameters showed significant changes about 3 weeks after section of the central or peripheral processes of sensory neurones. The only property of ganglion cell bodies which was detected to be altered by axotomy was the rectification characteristics of the cell membrane. As described by Ito (1957), hyperpolarizing responses of the ganglion cell show an initial peak subsequently followed by a decrement to a steady level when the applied C
A
D
B-
10-40 mVl 1-4 msec
10 mV to 5 nAl 10 msec7V I
Fig. 6. Intracellular potentials recorded from an unidentified sensory neurone responding to stimulation of the tibial nerve of a control, unoperated cat. A, resting and action potentials. B, after-hyperpolarization following the action potential evoked by intracellular stimulation. C, potential changes (lower traces) produced by the application of current pulses (upper traces) through the intracellular electrode. Upward deflexion, depolarization, and downward deflexion, hyperpolarization. D, similar to C, but two short hyperpolarizing current pulses were applied at the resting level and during hyperpolarization. 40 mV, 1 msec calibration for A. 10 mV, 4 msec calibration for B.
current pulses are increased beyond a certain intensity (Fig. 6C). The ionic mechanisms underlying this rectification are not clear (see small print below), and this phenomenon is referred to below as the 'timedependent hyperpolarizing (inward-going) rectification'. All ganglion cells (twenty-eight) examined in control animals showed the rectification (Fig. 6C). The average hyperpolarizing current intensity at which the time-dependent rectification began to appear was 3-6 + 1-8 (S.D.) nA. Similarly, all ganglion cells (twenty-six) tested about 3 weeks after section of the dorsal roots showed the time-dependent hyperpolarizing rectification (Fig. 7 A). The average 'threshold' for the rectification in these animals was 3-7 + 2-6 (S.D.) nA. In contrast, in the animals with peripheral axotomy, seven out of 15 ganglion cells showed no sign of the timedependent rectification in response to strong hyperpolarizing current
176 G. CZEH, N. KUDO AND M. KUNO pulses up to 16 nA (Fig. 7B). The rest of ganglion cells (eight) revealed the rectification, but the average 'threshold' was 6-8+3.6 (S.D.) nA which was significantly (2P < 0-01) higher than the control value. Whatever the mechanisms responsible for the time-dependent hyperpolarizing rectification, the ganglion cell bodies apparently undergo a change in the properties in response to peripheral axotomy, whereas such changes are lacking in response to central axotomy. Phenomenologically, these results coincide with morphological observations that chromatolytic changes in sensory neurones can be induced by peripheral axotomy but not by central axotomy (see Introduction). A
B
m
10 msOmV to 5 nA,
Fig. 7. Potential changes (lower traces) produced by the application of current pulses (upper traces) through the intracellular electrode. Upward defiexion, depolarization, and downward deflexion, hyperpolarization. A, a cutaneous neurone recorded 22 days after section of the dorsal root. B, a muscle sensory neurone recorded 22 days after section of the muscle nerve.
The rectification property was examined only in four ganglion cells under 'disuse' conditions. The 'threshold' for rectification in these cells was similar to the control value, being in the range of 1-9-5*7 nA (average, 3.5 nA). The time-dependent inward-going (anomalous) rectification recently analysed in the egg cell membrane of a tunicate (Miyazaki, Takahashi, Tsuda & Yoshii, 1974) and a starfish (Hagiwara & Takahashi, 1974; Hagiwara, Miyazaki & Rosenthal, 1976) has been attributed to an increase in the potassium conductance which is initially high and subsequently declines to a steady level during hyperpolarization. However, the same mechanism may not apply to the time-dependent rectification described in the present study for two reasons: (1) the peak hyperpolarization can reach over -I100 mV, exceeding the reversal potential of after-hyperpolarization which may reflect the potassium equilibrium potential, and (2) there is an indication of a decrease, rather than an increase, in the membrane conductance at the peak
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of hyperpolarization when constant current pulses are superimposed on the hyperpolarizing response (Fig. 6D). Ito (1957) has suggested a delayed increase in the chloride conductance during hyperpolarization for the time-dependent rectification in dorsal root ganglion cells of the toad. This possibility is also unlikely since the membrane conductance is lower at the late steady level of hyperpolarization than at the resting level as shown by superimposed current pulses (Fig. 6D). The membrane conductance was slightly (about 3 %) higher at the steady level of hyperpolarization than at the peak when measured by superimposed current pulses (Fig. 6D). However, this slight difference in the membrane resistance was not sufficient to account for the potential difference (> 40 %) between the peak and the late steady level of hyperpolarization (Fig. 6D). It is possible that the ganglion cell has the outward-going (delayed) rectification and that the sodium and/or calcium conductance may slightly increase with a certain delay after the onset of hyperpolarizing response presumably by removal of partial inactivation existing under the resting conditions. DISCUSSION
The present results show that dorsal root fibres do not change their conduction velocity when the dorsal roots are sectioned, at least in muscle sensory fibres with relatively large diameters. This implies that injuries in nerve fibres per se do not necessarily result in a decrease in their conduction velocity. On the other hand, chronic section of the peripheral processes of sensory neurones caused a decrease in conduction velocity for both the central and peripheral processes, and these changes were associated with changes in the properties of the neurone cell body. This behaviour is in contrast with that following section of the dorsal roots which does not produce detectable changes in the cell body properties. Thus, it is suggested that a decrease in conduction velocity following nerve section may require the participation of changes in the neurone cell body. The simplest explanation would be that peripheral section of sensory neurones may induce some alterations in the cell body and that the signal originating from the altered cell body may exert the influence on both the central and peripheral processes, thereby reducing their conduction velocities. However, a possibility still exists that the change in the neurone cell body and a decrease in the axonal conduction velocity may be controlled by a common factor following section of the peripheral nerve (see below). A puzzling finding was that central axotomy caused an increase in conduction velocity of the peripheral processes (Fig. 5B) without changes in the central conduction velocity (Fig. 5A) or in the properties of the cell body (Fig. 7A). In the Aplysia cerebral neurone, section of one branch of the axon has been shown to cause an increase in amounts and rates of axonal transport in the remaining intact branch (Goldberg, Goldman & Schwartz, 1976). If an increase in the peripheral conduction velocity following central axotomy is assumed to be related to some changes in the
178 1. CZtH, N. KUDO AND M. KUNO axonal transport, one may speculate that the transport material originally destined to the central process may be diverted to the peripheral processes because of occlusion at the central cut end. Thus, the peripheral axonal transport may be increased in a manner similar to the process suggested by Goldberg et al. (1976). A similar explanation may apply to the difference in conduction velocity between the central and peripheral processes of the dorsal root ganglion cell. In mammalian myelinated sensory fibres, there is as yet no evidence that the central processes are smaller in diameter than the peripheral processes (Dale, 1900; Rexed & sourander, 1949; Ha, 1970). Nevertheless, the present study confirmed previous observations that the majority of sensory neurones have a slower conduction velocity in their central processes than in the peripheral processes (Loeb, 1976; also, cf. Fu & Schomburg, 1974; Willis, Nufiez & Rudomin, 1976). This difference might be related to the amount of axoplasmic transport which is smaller in the central processes of the dorsal root ganglion cell than in the peripheral processes (Lasek, 1968; Ochs, 1972; also, cf. Smith, 1973). The absence of chromatolytic or electrophysiological reaction in the cell body to central axotomy of sensory neurones is another puzzling problem. Both the central and peripheral processes of sensory neurones appear to possess the retrograde transport system as evidenced by injection of horseradish peroxidase into the spinal cord (Maynard, Leonard, Coulter, Coggeshall & Willis, 1975) and into the muscle (Burke, Strick, Kanda, Kim & Walmsley, 1977; also, cf. Furstman, Saporta & Kruger, 1975). In sympathetic ganglion cells, both chromatolytic (West & Bunge, 1976) and electrophysiological (Purves & Nja, 1976) reactions following section of the postganglionic axon can be prevented by the application of nerve growth factor. Similarly, nerve growth factor prevents the biochemical changes that follow axotomy of the sympathetic ganglion (Hendry, 1975). Thus, it has been suggested that the normal properties of sympathetic neurones are maintained by nerve growth factor transported from the effector organ (Hendry & Iversen, 1973). If one assumes a similar mechanism fot sensory neurones, such a putative material that maintains their normal properties would be present only in the peripheral organs and be lacking in the central nervous system. The authors wish to thank Miss Cynthia V. Taylor for technical assistance. This work was supported by project (NS 11132) and research (NS 10319) grants from the U.S. Public Health Service. REFERENCES
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