- Email: [email protected]
The effect of peripheral nerve injury on dorsal root potentials and on transmission of afferent signals into the spinal cord
Brain Research, 209 (1981) 95-111
95
© Elsevier/North-Holland Biomedical Press
T H E E F F E C T OF P E R I P H E R A L N E R V E I N J U R Y O N D O R S A L R O O T P O T E N T I A L S A N D O N T R A N S M I S S I O N OF A F F E R E N T S I G N A L S I N T O THE SPINAL CORD
PATRICK D. WALL and MARSHALL DEVOR Cerebral Functions Research Group, Department of Anatomy, University College, London WC1E 6BT (U.K.) and Institute of Life Sciences, The Hebrew University, Jerusalem (Israel)
(Accepted August 28th, 1980) Key words: peripheral nerve - - spinal cord - - dorsal root potentials - - sensory afferents - - spinal
cord tracts
SUMMARY The sciatic nerve of adult rats was either cut and ligated or was crushed on one side. The response of the spinal cord to stimulation of the proximal part of the injured nerve was examined at various times after the lesion and compared to the effects of stimulating the intact nerve on the other side. During the first 10 days after nerve section the following measures were not affected: (i) the size of the input volley (compound action potential, CAP, measured on a dorsal root that carried sciatic nerve afferents (L5); (ii) the volley running in the dorsal columns; (iii) the dorsal root potential (DRP) evoked on neighbouring dorsal roots which do not contain sciatic afferents (L2 and L3); (iv) the post-synaptic volleys ascending in the spinal cord. However, by the fourth day after nerve section, there was a decrease of the D R P evoked on the ipsilateral L5 dorsal root by stimulation of the cut nerve. By 10 days this D R P had decreased by 50 ~ . There was also a decrease in the D R P on the L5 root evoked by stimulation of the contralateral intact nerve. Crush lesions of the sciatic nerve did not produce D R P change. Beginning 10-20 days after nerve cut, there was a decrease in the amplitude of the afferent CAP and of all the measures of central response to the afferent volley. We discuss the possibility that the loss of the D R P may be associated with a disinhibition which results in novel receptive fields which we observe in cord cells deafferented by the peripheral nerve section. The decrease of D R P and the appearance of novel receptive fields do not occur if the peripheral nerve is crushed rather than cut.
96 INTRODUCTION It has become apparent recently that there are important alterations in the dorsal horn following peripheral nerve injury. Subtle ultrastructural changes in afferent terminals in lamina 2 have been seen to begin by 6 days after sciatic nerve section in the rat 2. These early anatomical changes are not thought to signal degeneration 2. Furthermore we have been unable to detect substantial degeneration by the FinkHeimer technique in rat or cat cord examined up to 100 days after sciatic section (Devor, in preparation). While morphological changes in the first 10 days may be small, there is no doubt that there are rapid and striking biochemical changes. The enzyme fluoride-resistant acid phosphatase, which is generated in small dorsal root ganglion cells and transported to central terminals in laminae 1 and 2, disappears from these terminals within 4 days after peripheral nerve section or crush2,4, 23. Similarly, substance P exists in afferent terminals in laminae 1 and 2 and decreases after dorsal root or peripheral nerve section tS. Barbut et al. 1 have examined the time course of this effect and find that substance P disappears in 5-9 days after sciatic section in rats. This change is small and delayed if the nerve is crushed rather than cut. The central effect of peripheral nerve section also includes the appearance of non-neural 'reactive cells'. These begin to accumulate in dorsal horn by 3 days after sciatic section or crush 11. Finally, Devor and Wall s showed that dorsal horn ceils in cat which had lost their normal afferent input as a result of peripheral denervation adopted new receptive fields supplied by the nearest intact nerves. We have recently repeated these experiments in rats and find that the switching of receptive fields after the nerves have been cut begins by 4 days. The switch does not occur after nerve crush. The present experiments were aimed at finding an explanation for this rapid synaptic reorganization. The anatomical and biochemical changes that occur in the dorsal horn are concentrated in the region of the substantia gelatinosa. One of us has given evidence that this region has important gating functions at least partially involving its ability to generate primary afferent depolarization (PAD) and its peripheral manifestation, the dorsal root potential (DRP) 25-2s. It was therefore natural that we should investigate the ability of the parent fibres proximal to a nerve cut or crush to generate a DRP. There was one incidental observation in the literature to encourage this search. Horch 13 cut the sural nerve in cats and noticed that, during the period of regeneration, there was a disappearance of the dorsal root reflex (DRR) even though the peripheral axons and their extensions into dorsal columns were still functional. The D R R consists of antidromic impulses generated during the rapidly rising onset of PAD 19. Therefore, since the D R R and DRP are coupled, it seemed possible that both might be changed by peripheral nerve injury. Horch and Lisney have repeated and extended their observations 14. Here we examined the ability of the central part of cut and ligated, or of crushed nerves to generate DRPs. It was obviously necessary to assure ourselves that the afferent volley was in fact arriving in spinal cord and therefore we had to study the dorsal root volley generated by normal and cut nerves. It was also of interest to examine the ability of the afferent volley to excite post-synaptic structures in the cord,
97 and we therefore recorded the massed volleys ascending in cord. We did not record ventral root reflexes since motor axons had been cut and we would not have been able to differentiate between the well known effects of axotomy on motoneuron excitability from the effects of the afferent volley which interested us is,z2. Although rats were examined up to 136 days after the lesion, the experiments concentrated on the first 10 days since the synaptic reorganization on second order sensory neurons in the rat dorsal horn was complete by this time. METHODS Initial experiments were carried out on hooded rats but the results reported here were on adult male albino rats of the Sabra strain weighing 250--400 g at the time of operation. The number of animals used for each experimental group is noted with the results.
Preliminary operation Section and ligation. Rats were anaesthetized with ether and the sciatic nerve was exposed in the popliteal fossa. The nerve was dissected free locally and inspected to ensure that the sural nerve was included with the tibial and peroneal nerves. A 4-0 or 50 silk suture was passed around the entire nerve and firmly tied. The nerve was then cut 1-2 mm distal to the ligature. Muscle and skin were closed in layers. Topical antibiotic powder and a single injection of 30,000 U penicillin (i.m.) was given and all animals recovered uneventfully. Crush. Here the nerve was firmly clamped for 30 sec with a hemostat whose blades (2.5 mm across) had been ground fiat. The blades were covered with carbon so that the site of crush was marked black. This crush procedure was previously shown to produce conduction block followed by rapid degeneration in all myelinated and unmyelinated fibres in the distal nerve stumpS, 7. In other respects the operation was identical to that used for section and ligation.
Eleetrophysiological preparation The experiment. (Fig. 1). On the experimental day the animal was anaesthetized with intraperitoneal Nembutal (50 mg/kg) and additional doses of 30 mg/kg were given every 45-60 min. Both sciatic nerves were exposed and covered with warmed (37 °C) mineral oil. An extensive laminectomy exposed the spinal cord from the cauda equina to the T8 segment. The cord was covered with mineral oil (37 °C) and the dura opened. Temperature, heart rate and tidal pCO2 and arterial (carotid) blood pressure were monitored. Fluids and drugs were given as required through a carotid cannula. Ventilation was maintained artificially during gallamine paralysis. Stimulation. The lesioned sciatic nerve was dissected free of connective tissue in a region 10-15 mm proximal to the lesion and placed on an insulating sheet of parafilm. A pair of silver-silver chloride stimulating electrodes was placed in contact on either side of the nerve. The intact contralateral nerve was prepared similarly. The stimulus was a 1-2 Hz, 0.1 msec square wave delivered through a constant current photoelectri-
98 cally isolated source. T h e current was m o n i t o r e d a n d raised to a level which generated a response o f all m y e l i n a t e d (A) afferents. W e shall r e p o r t elsewhere on the effect o f C volleys. A t the end o f the experiment, the a r e a o f the lesion was dissected free to ensure t h a t the entire nerve was included in the lesion. The full length o f the l u m b o s a c r a l t r u n k was then exposed to confirm the identity o f the roots which h a d been used for r e c o r d i n g and, b y laying a t h r e a d a l o n g the nerve, to determine the c o n d u c t i o n distance.
Recording (1} The posterior biceps nerve (R5 in Fig. 1). Since it was o u r intention to stimulate only lesioned axons, we checked that the stimulus d i d n o t spread to m o r e p r o x i m a l intact nerves. The p o s t e r i o r biceps nerve which leaves the l u m b o s a c r a l t r u n k 10-15 m m p r o x i m a l to the stimulus site is the intact nerve closest to the stimulating site. Therefore it was dissected free, placed on r e c o r d i n g h o o k s a n d checked to ensure t h a t n o n e o f its axons were being stimulated. (2} The distal part of the cut dorsal root L5 ( R4 in Fig. 1}. Sciatic nerve afferents enter the spinal c o r d along dorsal r o o t s ( D R ' s ) L4, L5 a n d L6 (ref. 16 a n d D e v o r unpublished). The L5 r o o t was cut bilaterally a n d m o u n t e d o n r e c o r d i n g electrodes to m e a s u r e the c o m p o u n d action potential ( C A P ) arriving f r o m the sciatic nerve (Fig. 2). In all r e c o r d i n g situations, p a i r e d recordings were m a d e on the lesion side a n d on the intact side o f the cord. I n each case the same r e c o r d i n g electrode pair was used for the two sides to ensure identical electrode separation. (3} The DRP on the L5 dorsal root (R3 in Fig. 1). The L5 D R was cut a n d carefully dissected free u p to the d o r s a l r o o t entry zone. One r e c o r d i n g electrode was placed u n d e r the r o o t as close as possible to the c o r d w i t h o u t t o u c h i n g it. T h e other
T8
~
R1
Fig. 1. Diagram of the stimulus, S, and recording sites, R. All pairs of electrodes were placed in symmetrical locations on the lesion side and on the intact side. S is the stimulus point on the sciatic nerve 15 mm proximal to the lesion on one side and at a similar point on the intact nerve. The recording points are: RI, electrodes on the transected spinal cord at T8; R2, on the dorsal roots of L2 and/or L3 with the proximal electrode close to the cord and the distal electrode on the cut end; R3, similar to R2 but placed on the cut L5 dorsal root; R4, on the distal part of the cut dorsal root to record the input volley; R5, on the cut posterior biceps nerve to monitor stimulus spread from the main part of the sciatic nerve.
99 was placed on the cut end of the root. A fixed interelectrode distance o f 5 mm was used (Fig. 6). (4) The DRP on the L2 and L3 dorsal roots (R2 in Fig. 1). These roots were cut peripherally near the dorsal root ganglion and dissected free up to the cord. Electrodes were mounted on the roots as for L5 and the DRPs evoked by sciatic stimulation were recorded. (5) The tract volleys in the thoracic spinal cord (R1 in Fig. 1). The spinal cord was transected at T8. The dorsal and ventral roots of T9 were cut bilaterally and the entire cord was gently elevated and placed on an insulating sheet of parafilm. A single pair of recording electrodes was placed on the cord. One electrode rested against the cut end and the other was placed either 5 mm or 10--15 mm caudally on the surface of the dorsal columns in the midline. Stimuli were delivered to the intact or severed sciatic nerve and in some experiments, to the central branch of dorsal roots. Examples of spinal tract recording are shown in Fig. 4. The CAP, DRP and tract volley recordings were led through a high impedance head stage to conventional amplifiers with filters set to pass 0.1 Hz to 10 kHz. In general, 16 or 32 responses were averaged and written out on a chart recorder. The heights and areas of responses to be reported were measured from these records; heights from peak to baseline and area from the initial inflexion until the trace crossed the baseline once again. In order to ensure that each recording was made with a minimal interruption of the input, measurements were made in the following order. First the spinal cord tract volley on R1 was measured with all roots intact. Then dorsal roots L2 and L3 which do not contain sciatic afferents were cut and their DRPs measured, R2. Finally dorsal root L5, which contains many sciatic afferents was cut and the L5 DRP and the input CAP were measured, R3 and R4. In some experiments a terminal check of the input CAP on dorsal root L4 was made. All statistical evaluations were based on two-tailed Student's t-tests. RESULTS
The first 10 days During the first 10 days after operation the input CAP and the tract volleys were relatively unaffected while striking changes occurred in the DRPs. We will discuss the effects of section and ligation of the sciatic nerve and then contrast this with the effect of crushing the sciatic nerve. (1) The input as measured on dorsal root L5 (Figs. 2 and 3) The lesioned and intact sciatic nerves were stimulated and recordings were made on the cut L5 dorsal root (R4 in Fig. 1). The pairs of stimulating and recording electrodes were placed in as symmetrical locations as possible on the two sides. Examples of the recordings obtained are shown in Fig. 2 from an animal 5 days after sciatic section. The maximal volley, the top line, shows the CAP, which includes all types of A fibres. The middle line is the CAP produced when the stimulus current was
100
m '
Fig. 2. The input compound action potentials, CAPs. The upper three records came from stimulation of the intact sciatic nerve recorded on the ipsilateral L5 dorsal root, R4 in Fig. 1. The largest volley is generated by a maximal stimulus for myelinated afferents. The middle sweep shows the shape of the CAP when the stimulus was reduced to reduce the CAP height by 50 % and the smallest CAP has 25 of the maximal height. The lower three records are the same as the upper three except that the stimuli were applied to the sciatic whichhad been cut and ligated 5 days previouslyand recorded on that nerve's ipsilateral L5 dorsal root. Time, 0.5 msec. Height of maximal volley upper records, 8 mV.
reduced to give a reduction of 50 ~ of the maximal CAP amplitude (about 30 ~ of maximal CAP area), and the lower line was produced by a stimulus sufficient to produce 25 ~ of the maximal CAP amplitude (about 15 ~ of maximal CAP area). As expected, the reduced stimuli produced little change of the initial latency but the later parts of the CAP disappeared since this is produced by the slower fibres with higher thresholds. The best measure of the total number of nerve impulses in the volley is the area under the curve 10,12. In the upper graph of Fig. 3 we show the ratio of the area of the CAP recorded on L5 after stimulating the lesioned nerve over the CAP area from the intact side. This ratio is somewhat variable around the 1.0 line for the first 10 days (mean ratio 0.95 4- 0.13) and then there is a decline. There are two reasons for this variability in the first 10 days. The first and most important is that the contribution of the sciatic nerve to the L5 dorsal root varies somewhat between animals and between sides in the same animal. In Sabra rats, the great bulk of the sciatic afferents enter the spinal cord over dorsal roots L4 and 5 with minor contributions in L6. However the relative fraction shared between L4 and 5 varies and this fraction need not be precisely the same on the two sides (Devor, in preparation). We had to leave the major input L4 intact since our main interest was to study the central effects of the arriving volley. A second smaller source of variation is that the exact position and the contact resistance of the recording electrodes may vary between the two sides 12.
101 The latency o f the beginning o f the C A P and the conduction distance were used to calculate conduction velocity o f the fastest fibres in the nerve. This measure is not subject to the sources o f variability o f the C A P area. Again, as in all subsequent graphs, we plot the ratio o f the measure from the lesioned nerve over that f r o m the intact nerve. The conduction velocity o f the fastest fibres in the lesioned nerve began to decline by 5 days after nerve section and ligation. Crushed rat nerves behaved similarlyL It is noteworthy that the decline in C A P area begins a b o u t 5 days after that o f C A P conduction velocity. We suspect that the retrograde reduction in axon diameter, which partly accounts for C A P deceleration, moves centrally at this pace. Only when axon diameter at the recording site falls does the c o m p o u n d extracellular field potential, the C A P area, do so.
1.0 0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
_z
,1, .60
\
~,
0 ILl
.40 0
t
1..00
@
'
i
I
I
I
I
I
I
" ~ ° . % - ° % . . . . . . -I. . . . . . . . . . . . . . .
\ ' .~ ~
.80
CAP
•
VELOCITY
L..
0
0
.60
~
.40
.20
0
' 2
' ,
6
POST
' 810
20
,
i O
OPE R A T I V E
6
I0
i. 80
,
DAYS
Fig. 3. The ratio of the input on the L5 dorsal root from the lesioned sciatic nerve over that from the intact nerve. Each data point represents results from a single rat, and the solid line joins the mean ratio for each survival interval checked. Upper graph, the area under the CAP from the first inflexion until the curve returned to baseline was measured on each side from 16 averaged responses. Lower graph, the velocity of the beginning of the CAP was compared between the lesion and intact side. In this and subsequent graphs a vertical line marks the 10th postoperative day and the scale is expanded for the first 10 days.
102
m
Fig. 4. The ascending volleys recorded on the transected spinal cord at T8 (RI, Fig. 1). The wave shows three components. Wave 1 disappeared when the dorsal columns were cut. Waves 2 and 3 were signs of volleys ascending in the axons of post-synaptic spinal cord cells excited by the sciatic afferents. The upper trace was evoked by maximal stimulation of the intact sciatic while the lower trace was produced by a maximal stimulus to the sciatic nerve which had been cut and ligated 5 days previously. Each is an original record and not a product of averaging. Time, 2 msec; wave 3 peak voltage, 200/~V.
(2) The spinal tract volleys at T8 (Figs. 4 and 5) When the sciatic nerve is maximally stimulated and the entire cut spinal cord at T8 is mounted on recording electrodes, a characteristic wave is observed (Fig. 4). It has three major components which we have labelled waves 1, 2 and 3. The first wave is produced by action potentials travelling in the dorsal columns, especially the ascending collaterals of the entering primary afferent fibers. The evidence for this is: (i) the height of wave 1 was unaffected when the stimulus frequency was increased to over 200 Hz; and (ii) the wave disappeared ifa complete lesion was made of dorsal columns by a jewellers forceps crush at L1 or L2 leaving other white matter intact. Waves 2 and 3 were not affected by dorsal column transection and decreased their amplitude if the frequency of stimulation was raised to 20 Hz or more. We propose that waves 2 and 3 represent the massed activity in various post synaptic ascending systems which originate in cord cells excited by the arriving sciatic nerve volley20. We use them as a general quantitative measure of the amount of post synaptic firing in certain systems without attempting to dissect their origin in detail. It is worth noting that wave 3 can first be detected (with averaging) with threshold input volleys and that it continues to increase in area until the input CAP is fully saturated for its A component. The height of wave 1 was measured in all animals in which it was discernible and the ratio of the two sides is shown day by day in the upper graph of Fig. 5. There was no significant change of the ratio in the first 10 days (days 0-4 versus 5-10, P > 0.2). The ratio of the height of wave 2 varied between rats (range -1- 20 ~o to ---14 ~ ) but here, too, there was no significant trend over the first 10 days (days 0--4 versus 5-10, P :> 0.2). Finally, the ratio of the area under the entire evoked potential, reflecting in particular the size of wave 3, was measured. In spite of its variation (range -t-20 ~ to --12 ~ ) there was no significant change during the first 10 days (days 0-4 versus 5-10, P ~- 0.2). In 8 rats, the central portion of DR's L2, L3 or L5 was stimulated. L5 contains sciatic fibers, L2 and L3 do not. Root stimulation resulted in spinal tract volleys of similar form to that shown in Fig. 4 for stimulation of the sciatic nerve but with a
103 shorter latency due to the shorter conduction distance. There was no significant difference (P > 0.2) between the waves evoked by stimulation o f the L2 and L3 dorsal roots on either side during the first 10 days after unilateral nerve section (mean ratio of ligated over intact was 1.02, range 0.92-1.26 in 4 rats), or even, during the following 58 days (mean ratio 1.07, range 0.94-1.18 in 4 rats). Waves evoked by stimulation o f the L5 dorsal root were also the same (P > 0.2) on the two sides for first 10 days (mean ratio 1.04, range 0.88-1.20 in 3 rats). These data add to the evidence already shown that the response o f dorsal column afferents and relay cells to a single stimulus is not affected during the early period. 1.20
1.00
-
WAVE
I
.80
I-- .60 U 1.20
i
i
i
i
i
i
i
i
!
i
!
z 1.00 •
ILl
>
0 .80~ a
~.60
0 ~
.40
~ 1.20 r
I
WAVE
3
.80
.60
.40
.20
•
•
•
i
~.
4
6
8
,
I0
POST
20
,
,
40
60
OPERATIVE
i
80
100
i
i
120 130
DAYS
Fig. 5. Ratio of the three ascending volleys from the lesioned nerve. Each data point represents results from a single rat. All three waves appear to be unaffected by the peripheral lesion until 10-20 days after the nerve has been cut. For waves 1 and 2 we measured the peak amplitude. For wave 3 we measured the area under the entire curve from the first inflexion until the curve returned to baseline from averaged records of 16 or 32 responses.
104
L_ Fig. 6. The ipsilateral dorsal root potential (DRP) recorded on the cut dorsal root L5, R3 in Fig. 1. The upper trace shows the DRP evoked by stimulation of the intact nerve. It has two obvious components: first a brief positive (downward) deflection,DRP IV19,next a prolonged negative (upward) wave, DRP V14. It was the area under this long negativeDRP V averaged from 16 or 32 stimuli, which was used in the subsequent graphs for comparison of the two sides. On the rising phase of the negative DRP from the intact side (upper trace) a ragged dischargeis visible whichis the dorsal root reflex, DRR. The lower trace is the ipsilateral DRP evoked from the sciatic nerve which had been cut and ligated 5 days previously. The DRR has disappeared and the area of the negative wave is smaller than that produced by the intact nerve. Time, 20 sec; voltage 200 #V.
(3) Dorsal root potentials (Figs. 6 and 7) The shape of passive D R P has been described by Lloyd 19. The first three components, D R P I, II and III, are not easily distinguishable when peripheral nerve is stimulated since the arriving volley is not sufficiently synchronized. The first obvious wave (Fig. 6) is Lloyd's D R P IV where the proximal electrode goes positive. This is rapidly followed by a prolonged negative wave, D R P V, which peaks at about 15 msec and then slowly declines to the original baseline. The late positive D R P VI of Lloyd is not present in animals anesthetized with barbiturate. On the rising phase of the negative D R P V in Fig. 6 top, a brief antidromic volley of action potentials, the dorsal root reflex (DRR) is recorded. (a) DRPs evoked on dorsal roots L2 or L3 - - top graph, Fig. 7. The two sciatic nerves were stimulated maximally and DRPs on the ipsilateral dorsal roots L2 or L3 were recorded and averaged. In some animals both roots were inspected one after the other. L2 and L3 roots were selected as the closest dorsal roots rostral to the entry point of sciatic afferents which contain no significant number of sciatic axons (ref. 16 and Devor unpublished). The area under the negative wave D R P V was measured and the ratio of the lesioned side over the intact side was calculated (Fig. 7, top). No significant difference between the two sides was observed over the first 10 days (mean ratio ----- 1.0 q- 0.1, days 0--4 versus 5-10, P > 0.2).
(b) DRPs on the L5 dorsal root evoked by a sciatic volley--mid and bottom graphs, Fig. 7. The L5 dorsal root contains about half of the total of sciatic afferents (ref. 16 and Devor in preparation). The afferent volley on these fibers was recorded on the peripheral part of the cut dorsal root and used to construct Figs. 2 and 3. The evoked D R P was recorded on the central part of the same root. There was no change in the D R P for the first 3 days after the sciatic nerve had been sectioned, but by day 4 the cut nerve evoked a 25 ~ smaller ipsilateral D R P than that evoked by the intact
105 nerve. This fall continued so that by day 8 the ipsilateral L5 D R P from the cut nerve
50 % of that from the normal side. A s the D R P fell, the D R R (Fig. 6). A D R R was seen o n the experimental side in only 1 o f the 15 rats
was only
disappeared studied 4 or
more days after nerve section.
1.20
1.00
'•"••IX DRP
.80
.60
on
L2
or L 3
~ r
a
.4O
z uJ >
I
I
I
|
a
,
1.00
,80 MAX
DRP
on
L5
0 t'~ uJ t-
0m
~)
.60
.40 .30
*
i
|
,
I.O0[ . . . . . . . . .
~ . . . . . . . . . . . . . . . . . . . . . . . . . .
0
1/4
~_ .80
DRP
on
L5
tv
.60
/
o i tj
.20
0
2
4
6
8
I0
POST
• I
.40
610
OPERATIVE
800
I
IO0
I
120
140
DAYS
Fig. 7. The ratio of DRPs produced by the lesioned nerve over that produced by the intact nerve. In the upper graph, the ratio is shown for the DRPs recorded on dorsal roots L2 or L3, R2 in Fig. 1. The comparison is made using maximal sciatic volleys and it is evident that a striking decline begins only after 22 days. In the middle graph, the results are shown for the DRPs on dorsal root L5 evoked by maximal sciatic volleys, R3 in Fig. 1. The DRP produced by cut nerves declines by 4 days after the lesion. DRPs produced after nerve crush are shown by the open circles. These do not change from the normal range. The lower graph shows the ratio of DRPs on L5 produced by a sciatic input volley reduced to 25 % of maximal height. The changes here are more marked than those produced by the maximal input.
106 u.l
O 1.20
CONTRALATERAL
DRPs
0 1.oo
~
.80
Z u~. .60
O O
~-..40
I 2
POST
i 4
i 6
OPERATIVE
i 8
i I0
112
, 14
DAYS
Fig. 8. DRPs were recorded from the L5 dorsal root contralateral to the stimulated nerve. The area of the contralateral DRP evoked by stimulation of the intact nerve was divided by the area of the contralateral DRP evoked by stimulation of the injured nerve. The results show that by 4 days the intact nerve produces a smaller contralateral DRP than that produced by the cut nerve. After recording the L5 D R P to a maximal input volley, stimulus strength was lowered until the height of the CAP recorded on the peripheral part of the cut L5 dorsal root was a quarter of its maximum (see Fig. 2). The decline of the D R P on the operated side was more dramatic, but had the same time course as when maximal input volleys were used. (c) Contralateral DRPs evoked on dorsal root L5 - - Fig. 8. It is evident that a sectioned nerve was unable to evoke the normal D R P on a root which contained many axons from the sectioned nerve, L5, and yet was still able to evoke normal D R P s on nearby roots which contained few if any sectioned axons, L2 and L3. It was of interest, therefore, to observe DRPs evoked by the two nerves on contralateral dorsal roots. The contralateral passive D R P in rats has a single negative component which begins some 5 msec later than the ipsilateral negative D R P V, and is smaller in area than the ipsilateral DRP. By 4 days after unilateral sciatic nerve transection the intact sciatic nerve evoked a smaller D R P on the opposite (experimental) L5 dorsal root than the lesioned nerve evoked on its opposite (intac0 L5 dorsal root. Put another way, the mechanism for depolarising the terminals of primary afferent fibres that have been cut in the periphery is defective whether the afferent volley is carried by peripherally cut afferents or by intact afferents. (d) DRPs evoked by stimulation o f a nerve which has been crushed. In 6 animals, we crushed the sciatic nerve rather than cutting and ligating it. Sensory testing in the first 10 days after operation showed that the sciatic area of the foot was completely anestehticS,L Signs of reinnervation of the foot from the sciatic nerve did not begin for 18-20 days. As after cut and ligation, the area of the afferent CAP recorded on the L5 dorsal roots following maximal sciatic stinmlation did not change within the first 10 days. The initial conduction velocity of the CAP, however, fell to an average of 83 ~ (range 81-86 ~o) of normal by the 10th day (see also ref. 5). The size of the three waves on the cut cord representing the tract volleys also did not differ from normal (P > 0.1) within the first 10 days: wave 1 averaged 1 0 2 ~ of normal (range
107 100-104~o); wave 2, 98~o (range 96-107~o) and wave 3, 97~o (range 91-103~o). In striking contrast to cut and ligated nerves, however, the crushed nerves were capable of producing normal DRPs throughout the first 10 postoperative days and beyond (Fig. 7 middle). The average size of the DRP generated by crushed nerves was 101 ~ of normal (range 98-102~) in the period 5-10 days after crush. In contrast, the DRP generated by cut nerves in the same period was 62 ~ of normal (range 46--86 ~o).
The period after 10 days After the 10th postoperative day, the size of the arriving volley from the cut nerve began to decline and reached 40 ~ of normal by the 80th day (Fig. 3, top). The conduction velocity of the CAP continued its decline which began on day 5 and reached 40 ~oof normal by day 80 (Fig. 3 bottom). These data are consistent with earlier reports of retrograde conduction change after axotomy especially in sensory axonslL It is therefore of no great surprise that the various central effects of the afferent volley also declined. The details of these later developments can be seen to the right of the vertical line in Figs. 5 and 7. DISCUSSION We conclude that following peripheral nerve section there is an early failure of the mechanism which produces dorsal root potentials (DRPs) and depolarization of the terminals of primary afferents (PAD) in the dorsal horn. This occurs at a time when the afferent volley carried centrally by the peripherally cut axons, and many postsynaptic responses to this volley are unchanged. This finding is interesting because it coincides with changes in the chemistry of afferent terminalsl,Z, 2a and with changes of synaptic connectivitys. The DRP has previously been shown to be a widespread and stable phenomenon19,25-27. We have made two direct measures of the size of the afferent volley from the cut nerve. The first is the area of the CAP recorded on the L5 dorsal root. The second, a direct measure of a special fraction of the afferent volley, is the first wave of the cord tract volley which is generated by action potentials in branches of peripheral axons which ascend in dorsal columns. We observed no consistent changes in either of these measures during the first 10 days after sciatic nerve cut and ligation. Yet during that time the DRP fell to 50 ~o of normal (Fig. 7). It is possible that there was a small drop in the afferent volley that we failed to discriminate, and that this drop accounts for the observed decrease of the DRP. We obviously need to know how sensitive the DRP is to the size of the afferent volley. We obtained the necessary data by recording from the L5 dorsal root on the intact side of operated animals and comparing the area of the DRP evoked by a maximal input volley, a half amplitude volley and a quarter amplitude volley (Fig. 2). The half maximal volley (about 30~ of maximal area) produced a DRP 69 ~o of maximal (n -- 14, range 38-98 ~o). The quarter maximal volley (about 15~ of maximal area) produced a DRP 62~ of maximal (n -- 14, range 28-95 ~). It is apparent that the size of the DRP is not extremely sensitive to the
108 size of the input volley. In order to produce the 50 ~ drop of DRP observed 10 days after nerve section, it would be necessary to reduce the input volley to below 25 ~ of its maximum amplitude and/or to below 1 5 ~ of its maximal area. In spite of the variability of the input measure, it is quite clear that we would have detected such a massive shift. The one change in the afferent volley which coincides with the change in the DRP is the early reduction in conduction velocity (Fig. 3 bottom, also see ref. 4). We must obviously ask if this could be the cause of the decreased DRP, perhaps by desynchronizing the afferent volley. We can dismiss this possibility for two reasons. If an animal is cooled from 38 °C to 30 °C, there is a larger decrease of afferent conduction velocity than observed here but there is a marked increase of both the D R R and the DRP 19. A more powerful argument comes from our own results on crushed nerve. At least for the first 10 days the conduction velocity of the crushed afferents falls in exactly the same manner as after cut and ligation 5. Yet, as we have shown, there was no change in the ability of the central part of a crushed nerve to produce a DRP (Fig. 7, middle). Other evidence that the failure of the DRP generating mechanism is not simply due to failure of input comes from the fact that the contralateral DRP generated by intact nerve also failed in the region of terminals of peripherally cut axons (Fig. 8). The failure of the intact nerve to produce a contralateral DRP begins at the same time, 4 days, as the cut nerve's failure to produce a normal ipsilateral DRP. At the time of the DRP failure in the region of the terminals of cut afferents, the cut nerve produced normal sized DRPs on the adjacent intact ipsilateral roots, L2 and L3 (top line, Fig. 7). Furthermore, the cut nerve produced a much larger crossed D R P than did the intact nerve. This suggests that the failure is related to elements other than the afferents themselves. Another indicator of successful synaptic transmission of primary afferent information into the spinal cord is provided by the size of waves 2 and 3 (Fig. 5). These waves, which remained intact during the first 10 days, represent the firing of structures in the lumbar cord which send projecting axons as far as the lower thoracic cord. The simplest interpretation of the preservation of the spinal tract volleys is that the cut nerve has retained its normal postsynaptic excitatory power on the cells from which these axons originate. Alternatively, it could be that the excitatory effect of the input volley decreased, but that this was exactly counteracted by a rise of postsynaptic excitability. This possibility was checked by measuring the size of waves 2 and 3 evoked by stimulation of dorsal roots L2 and L3. Since many of the axons in roots L2 and L3 synapse on the same cells that receive sciatic afferents6, postsynaptic denervation supersensitivity would be expected to increase the postsynaptic response to this convergent input. No such increase was observed at least for the first 68 days after nerve section (P > 0.1). Since there are changes in the proximal part of the cut fibers as judged by their reduced conduction velocity, a possible explanation for the collapse of the DRP is a decrease in the space constant of the central terminal arborization. If this occurred, the normal depolarization of the terminals would no longer be conducted as far as previously out onto the dorsal roots. Czeh et al. 3 made intracellular recordings from
109 primary sensory neuron somata after peripheral axotomy and failed to find a change in the passive properties of the membranes. We can also dismiss this possibility on the basis of experiments we have done 29 in which we stimulated the terminals of the cut nerves with local microelectrodes. This technique25 generates an antidromic volley recorded on the peripheral nerve. Changes in the size of this antidromic volley provide a measure of excitability change at the terminals. The results of this test show that by 8 days after nerve section, the afferent volley from the cut nerve produces no signs at all of excitability change (i.e. PAD) in its own axon terminals. The same volley, however, produces the full expected increase in the excitability (PAD) in nearby axon terminals whose parent axons have not been cut in the periphery. Similar results are reported by Horch and Lisney14 for cat. This result fits well in another way with the results reported here. We show by comparing the area of the CAP on the L4 and L5 dorsal roots as evoked by maximal stimulation of the dorsal roots themselves with the CAP evoked by stimulation of the sciatic nerve, that approximately 50 ~ of the fibres in these roots are of sciatic origin (Devor, in preparation). Therefore if a volley from a cut sciatic nerve produced no DRP in its own afferents in the L5 dorsal root and a normal DRP in intact L5 dorsal root afferents one would expect the observed 50 decrease of the overall DRP. We conclude that peripheral nerve section and ligation leads to a failure of the DRP generating mechanism around the central terminals of the cut fibers. Nerve crush does not do so. Let us consider other central correlates of these two peripheral operations. We will concentrate on the first 10 days after injury since the DRP changes in this time while the afferent volley remains normal. The following three changes appear identical in crushed and sectioned nerves: (i) decrease of conduction velocity5; (ii) disappearance of fluoride-resistant acid phosphatase from the primary afferent terminals2,4,23; (iii) appearance of reactive cells in the dorsal horn 11. Since these alterations occur with both operations we can tentatively eliminate them as being causally related to the DRP change which occurs only after nerve section. We have recently found that ligation and section produces a marked loss of substance P in the central terminals of sectioned axons whereas we have been unable to detect any loss during the first ten days after crush 1. Finally, the two operations have different effects in the periphery. For example, ligation and crush differ in the degrees to which they disrupt axoplasmic transport 17, the blood-nerve barrier 21 and, of course, the outgrowth of regenerating sprouts 24. What significance might a failure of the DRP or PAD have on other aspects of spinal cord physiology? PAD has been associated with presynaptic surround inhibition or blockg, 27. Its failure might therefore be associated with disinhibition and an amplification of otherwise weak afferent signals. In fact, with respect to peripheral nerve injury, the disappearance of PAD might represent part of a homeostatic process whose net result is to increase the effect of whatever afferent inputs might have survived the injury. We have recently reported on the synaptic reorganization of a second order sensory neurons whose major primary afferent input had been cut by peripheral nerve sections. Specifically within 21 days following cut and ligation of foot nerves in cats and by 4 days in rats, cells formerly responding exclusively to foot stimulation began
110 to r e s p o n d to stimulation o f n o r m a l l y innervated thigh skin. The change o f receptive fields was n o t observed if the nerves were crushed rather t h a n cut. These d a t a suggested to us either that new connections from thigh afferents s p r o u t within the spinal cord, or that previously existing b u t ineffective thigh afferents become effective. W e have so far thiled to find evidence o f central s p r o u t i n g 4. O u r present observations o f D R P changes lend s u p p o r t to the alternative hypothesis o f disinhibition. The possible linkage between the D R P change a n d the receptive field change is s u p p o r t e d by the fact t h a t they b o t h occur with the same time course after nerve section and b o t h changes fail to occur after crush injuries. It is quite clear that the physiological changes within the spinal cord that follow peripheral nerve injury are n o t limited to the terminals o f the axons which have been cut. ACKNOWLEDGE MENTS W e w o u l d like to t h a n k Dr. M a r i a F i t z g e r a l d for her help with the early experiments a n d D i n a h Schonfeld a n d R u t h G o v r i n - L i p p m a n n for technical assistance. The w o r k was s u p p o r t e d in L o n d o n by the M e d i c a l Research Council a n d the N a t i o n a l Institutes o f H e a l t h a n d in J e r u s a l e m b y the Thyssen F o u n d a t i o n a n d the l s r a e l - U . S . Binational Science F o u n d a t i o n .
REFERENCES 1 Barbut, D.,Polak, J. M. andWalI, P.D.,SubstancePinspinalcorddorsalhorndecreasesfollowing peripheral nerve injury, Brain Research, in press. 2 Csillik, B. and Knyihar, E., Biodynamic plasticity in the Rolando substance, Progr. NeurobioL, 10 (1978) 203-230. 3 Czeh, G., Kudo, N. and Kuno, M. Membrane properties and conduction velocity in sensory neurons following central or peripheral axotomy, J. Physiol. (Lond.), 270 (1977) 165-180. 4 Devor, M. and Claman, D., Mapping and plasticity of acid phosphatase afferents in the rat dorsal horn, Brain Research, 190 (1980) 17-28. 5 Devor, M. and Govrin-Lippmann, R., Selective regeneration of sensory fibers following nerve crush injury, Exp. Neurol., 64 (1979) 260-270. 6 Devor, M., Merrill, E. G. and Wall, P. D., Dorsal horn cells that respond to stimulation of distant dorsal roots, J. PhysioL (Lond.), 270 (1977) 519-531. 7 Devor, M., Schonfeld, D., Seltzer, Z. and Wall, P. D., Two modes of cutaneous reinnervation following peripheral nerve injury, J. comp. Neurol., 185 (1979) 211-220. 8 Devor, M. and Wall, P. D., Reorganisation of spinal cord sensory map after peripheral nerve injury, Nature (Lond.), 276 (1978) 75-76. 9 Eccles, J. C., Presynaptic inhibition in the spinal cord. In J. C. Eccles and J. P. Schad6 (Eds.), Physiology of Spinal Neurons, Progr. Brain Res., Vol. 12, Elsevier, Amsterdam, 1964. 10 Gasser, H. S. and Grundfest, H., Axon diameters in relation to the spike dimensions and conduction velocity in mammalian A fibers, Amer. J. Physiol., 127 (1939) 393414. 11 Gilmore, S. A. and Skinner, R. D., Intraspinal non-neuronal cellular responses to peripheral nerve injury, Anat. Rec., 194 (1979) 369-388. 12 Hoffer, J. A., Stein, R. B. and Gordon, T., Differential atrophy of sensory and motor fibers following section of cat peripheral nerves, Brain Research, 178 (1979) 347-361. 13 Horch, K. W., Ascending collaterals of cutaneous neurons in the fasciculus gracilis of the cat during peripheral nerve regeneration, Brain Research, 117 (1976) 19-32. 14 Horch, K. W. and Lisney, S. J. W., Changes in primary afferent depolarisation of myelinated cutaneous sensory neurones during nerve regeneration in the cat, J. PhysioL (Lond.), in press.
111 15 Jessell, T., Tsundo, A., Kanazawa, I. and Otsuka, M., Substance P depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurones, Brain Research, 168 (1979) 247-259. 16 Kaizawa, J. and Takahashi, I., Fiber analysis of the lumbar spinal roots and their sciatic branches in rats, Tohoku J. exp. Med., 100 (1970) 61-74. 17 Karlstron, L. and Dahistr6m, A., The effect of different types of axonal trauma on the synthesis and transport of amine storage granules in rat sciatic nerves, J. NeurobioL, 4 (1973) 191-200. 18 Lieberman, A. R., The axon reaction, Int. Rev. NeurobioL, 14 (1971) 49-124. 19 Lloyd, D. P. C., Electrotonus in dorsal roots, ColdSpr. Harb. Symp. quant. BioL, 17 (1952) 203-219. 20 Lundberg, A., Ascending spinal hindlimb pathways in the cat. In J. C. Eccles and J. P. Schad~ (Eds.), Physiology of Spinal Neurons, Progr. Brain Res., I/oL 12, Elsevier, Amsterdam, 1964. 21 Mellick, R. and Cavanagh, J. B., Longitudinal movement of radioiodinated albumin within extravascular spaces of peripheral nerves following three systems of experimental trauma, J. NeuroL Neurosurg. Psychiat., 30 (1967) 458. 22 Mendell, L. M., Manson, J. B. and Scott, J. G., Connectivity changes in la afferents on axotomized motoneurons, Brain Research, 73 (1974) 338-342. 23 Schoenen, J., Budo, C. and Poncelet, G., Effet de la section du sciatique sur l'activit6 de l'isoenzyme fluororesistant de la phosphatase acide dans la moelle epiniere du rat, C.R. Soc. BioL, 162 (1968) 2035-2037. 24 Sunderland, S., Nerves and Nerve Injuries, 2nd edn., Churchill Livingstone, Edinburgh, 1978. 25 Wall, P. D., Excitability changes in afferent fiber terminations and their relation to slow potentials, J. Physiol. (Lond.), 142 (1958) 1-21. 26 Wall, P. D., The origin of a spinal cord slow potential., J. PhysioL (Lond.), 164 (1962) 508-526. 27 Wall, P. D., Presynaptic control of impulses at the first central synapse. In J. C. Eccles and J. P. Schad6 (Eds.), Physiology of Spinal Neurones, Progr. Brain Res., VoL 12, Elsevier, Amsterdam, 1964. 28 Wall, P. D., The role of substantia gelatinosa as a gate control, Res. PubL Ass. nerv. rnent. Dis., 58 (1980) 205-231. 29 Wall, P. D., Changes in primary afferent depolarisation following peripheral nerve injury, submitted.
95
© Elsevier/North-Holland Biomedical Press
T H E E F F E C T OF P E R I P H E R A L N E R V E I N J U R Y O N D O R S A L R O O T P O T E N T I A L S A N D O N T R A N S M I S S I O N OF A F F E R E N T S I G N A L S I N T O THE SPINAL CORD
PATRICK D. WALL and MARSHALL DEVOR Cerebral Functions Research Group, Department of Anatomy, University College, London WC1E 6BT (U.K.) and Institute of Life Sciences, The Hebrew University, Jerusalem (Israel)
(Accepted August 28th, 1980) Key words: peripheral nerve - - spinal cord - - dorsal root potentials - - sensory afferents - - spinal
cord tracts
SUMMARY The sciatic nerve of adult rats was either cut and ligated or was crushed on one side. The response of the spinal cord to stimulation of the proximal part of the injured nerve was examined at various times after the lesion and compared to the effects of stimulating the intact nerve on the other side. During the first 10 days after nerve section the following measures were not affected: (i) the size of the input volley (compound action potential, CAP, measured on a dorsal root that carried sciatic nerve afferents (L5); (ii) the volley running in the dorsal columns; (iii) the dorsal root potential (DRP) evoked on neighbouring dorsal roots which do not contain sciatic afferents (L2 and L3); (iv) the post-synaptic volleys ascending in the spinal cord. However, by the fourth day after nerve section, there was a decrease of the D R P evoked on the ipsilateral L5 dorsal root by stimulation of the cut nerve. By 10 days this D R P had decreased by 50 ~ . There was also a decrease in the D R P on the L5 root evoked by stimulation of the contralateral intact nerve. Crush lesions of the sciatic nerve did not produce D R P change. Beginning 10-20 days after nerve cut, there was a decrease in the amplitude of the afferent CAP and of all the measures of central response to the afferent volley. We discuss the possibility that the loss of the D R P may be associated with a disinhibition which results in novel receptive fields which we observe in cord cells deafferented by the peripheral nerve section. The decrease of D R P and the appearance of novel receptive fields do not occur if the peripheral nerve is crushed rather than cut.
96 INTRODUCTION It has become apparent recently that there are important alterations in the dorsal horn following peripheral nerve injury. Subtle ultrastructural changes in afferent terminals in lamina 2 have been seen to begin by 6 days after sciatic nerve section in the rat 2. These early anatomical changes are not thought to signal degeneration 2. Furthermore we have been unable to detect substantial degeneration by the FinkHeimer technique in rat or cat cord examined up to 100 days after sciatic section (Devor, in preparation). While morphological changes in the first 10 days may be small, there is no doubt that there are rapid and striking biochemical changes. The enzyme fluoride-resistant acid phosphatase, which is generated in small dorsal root ganglion cells and transported to central terminals in laminae 1 and 2, disappears from these terminals within 4 days after peripheral nerve section or crush2,4, 23. Similarly, substance P exists in afferent terminals in laminae 1 and 2 and decreases after dorsal root or peripheral nerve section tS. Barbut et al. 1 have examined the time course of this effect and find that substance P disappears in 5-9 days after sciatic section in rats. This change is small and delayed if the nerve is crushed rather than cut. The central effect of peripheral nerve section also includes the appearance of non-neural 'reactive cells'. These begin to accumulate in dorsal horn by 3 days after sciatic section or crush 11. Finally, Devor and Wall s showed that dorsal horn ceils in cat which had lost their normal afferent input as a result of peripheral denervation adopted new receptive fields supplied by the nearest intact nerves. We have recently repeated these experiments in rats and find that the switching of receptive fields after the nerves have been cut begins by 4 days. The switch does not occur after nerve crush. The present experiments were aimed at finding an explanation for this rapid synaptic reorganization. The anatomical and biochemical changes that occur in the dorsal horn are concentrated in the region of the substantia gelatinosa. One of us has given evidence that this region has important gating functions at least partially involving its ability to generate primary afferent depolarization (PAD) and its peripheral manifestation, the dorsal root potential (DRP) 25-2s. It was therefore natural that we should investigate the ability of the parent fibres proximal to a nerve cut or crush to generate a DRP. There was one incidental observation in the literature to encourage this search. Horch 13 cut the sural nerve in cats and noticed that, during the period of regeneration, there was a disappearance of the dorsal root reflex (DRR) even though the peripheral axons and their extensions into dorsal columns were still functional. The D R R consists of antidromic impulses generated during the rapidly rising onset of PAD 19. Therefore, since the D R R and DRP are coupled, it seemed possible that both might be changed by peripheral nerve injury. Horch and Lisney have repeated and extended their observations 14. Here we examined the ability of the central part of cut and ligated, or of crushed nerves to generate DRPs. It was obviously necessary to assure ourselves that the afferent volley was in fact arriving in spinal cord and therefore we had to study the dorsal root volley generated by normal and cut nerves. It was also of interest to examine the ability of the afferent volley to excite post-synaptic structures in the cord,
97 and we therefore recorded the massed volleys ascending in cord. We did not record ventral root reflexes since motor axons had been cut and we would not have been able to differentiate between the well known effects of axotomy on motoneuron excitability from the effects of the afferent volley which interested us is,z2. Although rats were examined up to 136 days after the lesion, the experiments concentrated on the first 10 days since the synaptic reorganization on second order sensory neurons in the rat dorsal horn was complete by this time. METHODS Initial experiments were carried out on hooded rats but the results reported here were on adult male albino rats of the Sabra strain weighing 250--400 g at the time of operation. The number of animals used for each experimental group is noted with the results.
Preliminary operation Section and ligation. Rats were anaesthetized with ether and the sciatic nerve was exposed in the popliteal fossa. The nerve was dissected free locally and inspected to ensure that the sural nerve was included with the tibial and peroneal nerves. A 4-0 or 50 silk suture was passed around the entire nerve and firmly tied. The nerve was then cut 1-2 mm distal to the ligature. Muscle and skin were closed in layers. Topical antibiotic powder and a single injection of 30,000 U penicillin (i.m.) was given and all animals recovered uneventfully. Crush. Here the nerve was firmly clamped for 30 sec with a hemostat whose blades (2.5 mm across) had been ground fiat. The blades were covered with carbon so that the site of crush was marked black. This crush procedure was previously shown to produce conduction block followed by rapid degeneration in all myelinated and unmyelinated fibres in the distal nerve stumpS, 7. In other respects the operation was identical to that used for section and ligation.
Eleetrophysiological preparation The experiment. (Fig. 1). On the experimental day the animal was anaesthetized with intraperitoneal Nembutal (50 mg/kg) and additional doses of 30 mg/kg were given every 45-60 min. Both sciatic nerves were exposed and covered with warmed (37 °C) mineral oil. An extensive laminectomy exposed the spinal cord from the cauda equina to the T8 segment. The cord was covered with mineral oil (37 °C) and the dura opened. Temperature, heart rate and tidal pCO2 and arterial (carotid) blood pressure were monitored. Fluids and drugs were given as required through a carotid cannula. Ventilation was maintained artificially during gallamine paralysis. Stimulation. The lesioned sciatic nerve was dissected free of connective tissue in a region 10-15 mm proximal to the lesion and placed on an insulating sheet of parafilm. A pair of silver-silver chloride stimulating electrodes was placed in contact on either side of the nerve. The intact contralateral nerve was prepared similarly. The stimulus was a 1-2 Hz, 0.1 msec square wave delivered through a constant current photoelectri-
98 cally isolated source. T h e current was m o n i t o r e d a n d raised to a level which generated a response o f all m y e l i n a t e d (A) afferents. W e shall r e p o r t elsewhere on the effect o f C volleys. A t the end o f the experiment, the a r e a o f the lesion was dissected free to ensure t h a t the entire nerve was included in the lesion. The full length o f the l u m b o s a c r a l t r u n k was then exposed to confirm the identity o f the roots which h a d been used for r e c o r d i n g and, b y laying a t h r e a d a l o n g the nerve, to determine the c o n d u c t i o n distance.
Recording (1} The posterior biceps nerve (R5 in Fig. 1). Since it was o u r intention to stimulate only lesioned axons, we checked that the stimulus d i d n o t spread to m o r e p r o x i m a l intact nerves. The p o s t e r i o r biceps nerve which leaves the l u m b o s a c r a l t r u n k 10-15 m m p r o x i m a l to the stimulus site is the intact nerve closest to the stimulating site. Therefore it was dissected free, placed on r e c o r d i n g h o o k s a n d checked to ensure t h a t n o n e o f its axons were being stimulated. (2} The distal part of the cut dorsal root L5 ( R4 in Fig. 1}. Sciatic nerve afferents enter the spinal c o r d along dorsal r o o t s ( D R ' s ) L4, L5 a n d L6 (ref. 16 a n d D e v o r unpublished). The L5 r o o t was cut bilaterally a n d m o u n t e d o n r e c o r d i n g electrodes to m e a s u r e the c o m p o u n d action potential ( C A P ) arriving f r o m the sciatic nerve (Fig. 2). In all r e c o r d i n g situations, p a i r e d recordings were m a d e on the lesion side a n d on the intact side o f the cord. I n each case the same r e c o r d i n g electrode pair was used for the two sides to ensure identical electrode separation. (3} The DRP on the L5 dorsal root (R3 in Fig. 1). The L5 D R was cut a n d carefully dissected free u p to the d o r s a l r o o t entry zone. One r e c o r d i n g electrode was placed u n d e r the r o o t as close as possible to the c o r d w i t h o u t t o u c h i n g it. T h e other
T8
~
R1
Fig. 1. Diagram of the stimulus, S, and recording sites, R. All pairs of electrodes were placed in symmetrical locations on the lesion side and on the intact side. S is the stimulus point on the sciatic nerve 15 mm proximal to the lesion on one side and at a similar point on the intact nerve. The recording points are: RI, electrodes on the transected spinal cord at T8; R2, on the dorsal roots of L2 and/or L3 with the proximal electrode close to the cord and the distal electrode on the cut end; R3, similar to R2 but placed on the cut L5 dorsal root; R4, on the distal part of the cut dorsal root to record the input volley; R5, on the cut posterior biceps nerve to monitor stimulus spread from the main part of the sciatic nerve.
99 was placed on the cut end of the root. A fixed interelectrode distance o f 5 mm was used (Fig. 6). (4) The DRP on the L2 and L3 dorsal roots (R2 in Fig. 1). These roots were cut peripherally near the dorsal root ganglion and dissected free up to the cord. Electrodes were mounted on the roots as for L5 and the DRPs evoked by sciatic stimulation were recorded. (5) The tract volleys in the thoracic spinal cord (R1 in Fig. 1). The spinal cord was transected at T8. The dorsal and ventral roots of T9 were cut bilaterally and the entire cord was gently elevated and placed on an insulating sheet of parafilm. A single pair of recording electrodes was placed on the cord. One electrode rested against the cut end and the other was placed either 5 mm or 10--15 mm caudally on the surface of the dorsal columns in the midline. Stimuli were delivered to the intact or severed sciatic nerve and in some experiments, to the central branch of dorsal roots. Examples of spinal tract recording are shown in Fig. 4. The CAP, DRP and tract volley recordings were led through a high impedance head stage to conventional amplifiers with filters set to pass 0.1 Hz to 10 kHz. In general, 16 or 32 responses were averaged and written out on a chart recorder. The heights and areas of responses to be reported were measured from these records; heights from peak to baseline and area from the initial inflexion until the trace crossed the baseline once again. In order to ensure that each recording was made with a minimal interruption of the input, measurements were made in the following order. First the spinal cord tract volley on R1 was measured with all roots intact. Then dorsal roots L2 and L3 which do not contain sciatic afferents were cut and their DRPs measured, R2. Finally dorsal root L5, which contains many sciatic afferents was cut and the L5 DRP and the input CAP were measured, R3 and R4. In some experiments a terminal check of the input CAP on dorsal root L4 was made. All statistical evaluations were based on two-tailed Student's t-tests. RESULTS
The first 10 days During the first 10 days after operation the input CAP and the tract volleys were relatively unaffected while striking changes occurred in the DRPs. We will discuss the effects of section and ligation of the sciatic nerve and then contrast this with the effect of crushing the sciatic nerve. (1) The input as measured on dorsal root L5 (Figs. 2 and 3) The lesioned and intact sciatic nerves were stimulated and recordings were made on the cut L5 dorsal root (R4 in Fig. 1). The pairs of stimulating and recording electrodes were placed in as symmetrical locations as possible on the two sides. Examples of the recordings obtained are shown in Fig. 2 from an animal 5 days after sciatic section. The maximal volley, the top line, shows the CAP, which includes all types of A fibres. The middle line is the CAP produced when the stimulus current was
100
m '
Fig. 2. The input compound action potentials, CAPs. The upper three records came from stimulation of the intact sciatic nerve recorded on the ipsilateral L5 dorsal root, R4 in Fig. 1. The largest volley is generated by a maximal stimulus for myelinated afferents. The middle sweep shows the shape of the CAP when the stimulus was reduced to reduce the CAP height by 50 % and the smallest CAP has 25 of the maximal height. The lower three records are the same as the upper three except that the stimuli were applied to the sciatic whichhad been cut and ligated 5 days previouslyand recorded on that nerve's ipsilateral L5 dorsal root. Time, 0.5 msec. Height of maximal volley upper records, 8 mV.
reduced to give a reduction of 50 ~ of the maximal CAP amplitude (about 30 ~ of maximal CAP area), and the lower line was produced by a stimulus sufficient to produce 25 ~ of the maximal CAP amplitude (about 15 ~ of maximal CAP area). As expected, the reduced stimuli produced little change of the initial latency but the later parts of the CAP disappeared since this is produced by the slower fibres with higher thresholds. The best measure of the total number of nerve impulses in the volley is the area under the curve 10,12. In the upper graph of Fig. 3 we show the ratio of the area of the CAP recorded on L5 after stimulating the lesioned nerve over the CAP area from the intact side. This ratio is somewhat variable around the 1.0 line for the first 10 days (mean ratio 0.95 4- 0.13) and then there is a decline. There are two reasons for this variability in the first 10 days. The first and most important is that the contribution of the sciatic nerve to the L5 dorsal root varies somewhat between animals and between sides in the same animal. In Sabra rats, the great bulk of the sciatic afferents enter the spinal cord over dorsal roots L4 and 5 with minor contributions in L6. However the relative fraction shared between L4 and 5 varies and this fraction need not be precisely the same on the two sides (Devor, in preparation). We had to leave the major input L4 intact since our main interest was to study the central effects of the arriving volley. A second smaller source of variation is that the exact position and the contact resistance of the recording electrodes may vary between the two sides 12.
101 The latency o f the beginning o f the C A P and the conduction distance were used to calculate conduction velocity o f the fastest fibres in the nerve. This measure is not subject to the sources o f variability o f the C A P area. Again, as in all subsequent graphs, we plot the ratio o f the measure from the lesioned nerve over that f r o m the intact nerve. The conduction velocity o f the fastest fibres in the lesioned nerve began to decline by 5 days after nerve section and ligation. Crushed rat nerves behaved similarlyL It is noteworthy that the decline in C A P area begins a b o u t 5 days after that o f C A P conduction velocity. We suspect that the retrograde reduction in axon diameter, which partly accounts for C A P deceleration, moves centrally at this pace. Only when axon diameter at the recording site falls does the c o m p o u n d extracellular field potential, the C A P area, do so.
1.0 0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
_z
,1, .60
\
~,
0 ILl
.40 0
t
1..00
@
'
i
I
I
I
I
I
I
" ~ ° . % - ° % . . . . . . -I. . . . . . . . . . . . . . .
\ ' .~ ~
.80
CAP
•
VELOCITY
L..
0
0
.60
~
.40
.20
0
' 2
' ,
6
POST
' 810
20
,
i O
OPE R A T I V E
6
I0
i. 80
,
DAYS
Fig. 3. The ratio of the input on the L5 dorsal root from the lesioned sciatic nerve over that from the intact nerve. Each data point represents results from a single rat, and the solid line joins the mean ratio for each survival interval checked. Upper graph, the area under the CAP from the first inflexion until the curve returned to baseline was measured on each side from 16 averaged responses. Lower graph, the velocity of the beginning of the CAP was compared between the lesion and intact side. In this and subsequent graphs a vertical line marks the 10th postoperative day and the scale is expanded for the first 10 days.
102
m
Fig. 4. The ascending volleys recorded on the transected spinal cord at T8 (RI, Fig. 1). The wave shows three components. Wave 1 disappeared when the dorsal columns were cut. Waves 2 and 3 were signs of volleys ascending in the axons of post-synaptic spinal cord cells excited by the sciatic afferents. The upper trace was evoked by maximal stimulation of the intact sciatic while the lower trace was produced by a maximal stimulus to the sciatic nerve which had been cut and ligated 5 days previously. Each is an original record and not a product of averaging. Time, 2 msec; wave 3 peak voltage, 200/~V.
(2) The spinal tract volleys at T8 (Figs. 4 and 5) When the sciatic nerve is maximally stimulated and the entire cut spinal cord at T8 is mounted on recording electrodes, a characteristic wave is observed (Fig. 4). It has three major components which we have labelled waves 1, 2 and 3. The first wave is produced by action potentials travelling in the dorsal columns, especially the ascending collaterals of the entering primary afferent fibers. The evidence for this is: (i) the height of wave 1 was unaffected when the stimulus frequency was increased to over 200 Hz; and (ii) the wave disappeared ifa complete lesion was made of dorsal columns by a jewellers forceps crush at L1 or L2 leaving other white matter intact. Waves 2 and 3 were not affected by dorsal column transection and decreased their amplitude if the frequency of stimulation was raised to 20 Hz or more. We propose that waves 2 and 3 represent the massed activity in various post synaptic ascending systems which originate in cord cells excited by the arriving sciatic nerve volley20. We use them as a general quantitative measure of the amount of post synaptic firing in certain systems without attempting to dissect their origin in detail. It is worth noting that wave 3 can first be detected (with averaging) with threshold input volleys and that it continues to increase in area until the input CAP is fully saturated for its A component. The height of wave 1 was measured in all animals in which it was discernible and the ratio of the two sides is shown day by day in the upper graph of Fig. 5. There was no significant change of the ratio in the first 10 days (days 0-4 versus 5-10, P > 0.2). The ratio of the height of wave 2 varied between rats (range -1- 20 ~o to ---14 ~ ) but here, too, there was no significant trend over the first 10 days (days 0--4 versus 5-10, P :> 0.2). Finally, the ratio of the area under the entire evoked potential, reflecting in particular the size of wave 3, was measured. In spite of its variation (range -t-20 ~ to --12 ~ ) there was no significant change during the first 10 days (days 0-4 versus 5-10, P ~- 0.2). In 8 rats, the central portion of DR's L2, L3 or L5 was stimulated. L5 contains sciatic fibers, L2 and L3 do not. Root stimulation resulted in spinal tract volleys of similar form to that shown in Fig. 4 for stimulation of the sciatic nerve but with a
103 shorter latency due to the shorter conduction distance. There was no significant difference (P > 0.2) between the waves evoked by stimulation o f the L2 and L3 dorsal roots on either side during the first 10 days after unilateral nerve section (mean ratio of ligated over intact was 1.02, range 0.92-1.26 in 4 rats), or even, during the following 58 days (mean ratio 1.07, range 0.94-1.18 in 4 rats). Waves evoked by stimulation o f the L5 dorsal root were also the same (P > 0.2) on the two sides for first 10 days (mean ratio 1.04, range 0.88-1.20 in 3 rats). These data add to the evidence already shown that the response o f dorsal column afferents and relay cells to a single stimulus is not affected during the early period. 1.20
1.00
-
WAVE
I
.80
I-- .60 U 1.20
i
i
i
i
i
i
i
i
!
i
!
z 1.00 •
ILl
>
0 .80~ a
~.60
0 ~
.40
~ 1.20 r
I
WAVE
3
.80
.60
.40
.20
•
•
•
i
~.
4
6
8
,
I0
POST
20
,
,
40
60
OPERATIVE
i
80
100
i
i
120 130
DAYS
Fig. 5. Ratio of the three ascending volleys from the lesioned nerve. Each data point represents results from a single rat. All three waves appear to be unaffected by the peripheral lesion until 10-20 days after the nerve has been cut. For waves 1 and 2 we measured the peak amplitude. For wave 3 we measured the area under the entire curve from the first inflexion until the curve returned to baseline from averaged records of 16 or 32 responses.
104
L_ Fig. 6. The ipsilateral dorsal root potential (DRP) recorded on the cut dorsal root L5, R3 in Fig. 1. The upper trace shows the DRP evoked by stimulation of the intact nerve. It has two obvious components: first a brief positive (downward) deflection,DRP IV19,next a prolonged negative (upward) wave, DRP V14. It was the area under this long negativeDRP V averaged from 16 or 32 stimuli, which was used in the subsequent graphs for comparison of the two sides. On the rising phase of the negative DRP from the intact side (upper trace) a ragged dischargeis visible whichis the dorsal root reflex, DRR. The lower trace is the ipsilateral DRP evoked from the sciatic nerve which had been cut and ligated 5 days previously. The DRR has disappeared and the area of the negative wave is smaller than that produced by the intact nerve. Time, 20 sec; voltage 200 #V.
(3) Dorsal root potentials (Figs. 6 and 7) The shape of passive D R P has been described by Lloyd 19. The first three components, D R P I, II and III, are not easily distinguishable when peripheral nerve is stimulated since the arriving volley is not sufficiently synchronized. The first obvious wave (Fig. 6) is Lloyd's D R P IV where the proximal electrode goes positive. This is rapidly followed by a prolonged negative wave, D R P V, which peaks at about 15 msec and then slowly declines to the original baseline. The late positive D R P VI of Lloyd is not present in animals anesthetized with barbiturate. On the rising phase of the negative D R P V in Fig. 6 top, a brief antidromic volley of action potentials, the dorsal root reflex (DRR) is recorded. (a) DRPs evoked on dorsal roots L2 or L3 - - top graph, Fig. 7. The two sciatic nerves were stimulated maximally and DRPs on the ipsilateral dorsal roots L2 or L3 were recorded and averaged. In some animals both roots were inspected one after the other. L2 and L3 roots were selected as the closest dorsal roots rostral to the entry point of sciatic afferents which contain no significant number of sciatic axons (ref. 16 and Devor unpublished). The area under the negative wave D R P V was measured and the ratio of the lesioned side over the intact side was calculated (Fig. 7, top). No significant difference between the two sides was observed over the first 10 days (mean ratio ----- 1.0 q- 0.1, days 0--4 versus 5-10, P > 0.2).
(b) DRPs on the L5 dorsal root evoked by a sciatic volley--mid and bottom graphs, Fig. 7. The L5 dorsal root contains about half of the total of sciatic afferents (ref. 16 and Devor in preparation). The afferent volley on these fibers was recorded on the peripheral part of the cut dorsal root and used to construct Figs. 2 and 3. The evoked D R P was recorded on the central part of the same root. There was no change in the D R P for the first 3 days after the sciatic nerve had been sectioned, but by day 4 the cut nerve evoked a 25 ~ smaller ipsilateral D R P than that evoked by the intact
105 nerve. This fall continued so that by day 8 the ipsilateral L5 D R P from the cut nerve
50 % of that from the normal side. A s the D R P fell, the D R R (Fig. 6). A D R R was seen o n the experimental side in only 1 o f the 15 rats
was only
disappeared studied 4 or
more days after nerve section.
1.20
1.00
'•"••IX DRP
.80
.60
on
L2
or L 3
~ r
a
.4O
z uJ >
I
I
I
|
a
,
1.00
,80 MAX
DRP
on
L5
0 t'~ uJ t-
0m
~)
.60
.40 .30
*
i
|
,
I.O0[ . . . . . . . . .
~ . . . . . . . . . . . . . . . . . . . . . . . . . .
0
1/4
~_ .80
DRP
on
L5
tv
.60
/
o i tj
.20
0
2
4
6
8
I0
POST
• I
.40
610
OPERATIVE
800
I
IO0
I
120
140
DAYS
Fig. 7. The ratio of DRPs produced by the lesioned nerve over that produced by the intact nerve. In the upper graph, the ratio is shown for the DRPs recorded on dorsal roots L2 or L3, R2 in Fig. 1. The comparison is made using maximal sciatic volleys and it is evident that a striking decline begins only after 22 days. In the middle graph, the results are shown for the DRPs on dorsal root L5 evoked by maximal sciatic volleys, R3 in Fig. 1. The DRP produced by cut nerves declines by 4 days after the lesion. DRPs produced after nerve crush are shown by the open circles. These do not change from the normal range. The lower graph shows the ratio of DRPs on L5 produced by a sciatic input volley reduced to 25 % of maximal height. The changes here are more marked than those produced by the maximal input.
106 u.l
O 1.20
CONTRALATERAL
DRPs
0 1.oo
~
.80
Z u~. .60
O O
~-..40
I 2
POST
i 4
i 6
OPERATIVE
i 8
i I0
112
, 14
DAYS
Fig. 8. DRPs were recorded from the L5 dorsal root contralateral to the stimulated nerve. The area of the contralateral DRP evoked by stimulation of the intact nerve was divided by the area of the contralateral DRP evoked by stimulation of the injured nerve. The results show that by 4 days the intact nerve produces a smaller contralateral DRP than that produced by the cut nerve. After recording the L5 D R P to a maximal input volley, stimulus strength was lowered until the height of the CAP recorded on the peripheral part of the cut L5 dorsal root was a quarter of its maximum (see Fig. 2). The decline of the D R P on the operated side was more dramatic, but had the same time course as when maximal input volleys were used. (c) Contralateral DRPs evoked on dorsal root L5 - - Fig. 8. It is evident that a sectioned nerve was unable to evoke the normal D R P on a root which contained many axons from the sectioned nerve, L5, and yet was still able to evoke normal D R P s on nearby roots which contained few if any sectioned axons, L2 and L3. It was of interest, therefore, to observe DRPs evoked by the two nerves on contralateral dorsal roots. The contralateral passive D R P in rats has a single negative component which begins some 5 msec later than the ipsilateral negative D R P V, and is smaller in area than the ipsilateral DRP. By 4 days after unilateral sciatic nerve transection the intact sciatic nerve evoked a smaller D R P on the opposite (experimental) L5 dorsal root than the lesioned nerve evoked on its opposite (intac0 L5 dorsal root. Put another way, the mechanism for depolarising the terminals of primary afferent fibres that have been cut in the periphery is defective whether the afferent volley is carried by peripherally cut afferents or by intact afferents. (d) DRPs evoked by stimulation o f a nerve which has been crushed. In 6 animals, we crushed the sciatic nerve rather than cutting and ligating it. Sensory testing in the first 10 days after operation showed that the sciatic area of the foot was completely anestehticS,L Signs of reinnervation of the foot from the sciatic nerve did not begin for 18-20 days. As after cut and ligation, the area of the afferent CAP recorded on the L5 dorsal roots following maximal sciatic stinmlation did not change within the first 10 days. The initial conduction velocity of the CAP, however, fell to an average of 83 ~ (range 81-86 ~o) of normal by the 10th day (see also ref. 5). The size of the three waves on the cut cord representing the tract volleys also did not differ from normal (P > 0.1) within the first 10 days: wave 1 averaged 1 0 2 ~ of normal (range
107 100-104~o); wave 2, 98~o (range 96-107~o) and wave 3, 97~o (range 91-103~o). In striking contrast to cut and ligated nerves, however, the crushed nerves were capable of producing normal DRPs throughout the first 10 postoperative days and beyond (Fig. 7 middle). The average size of the DRP generated by crushed nerves was 101 ~ of normal (range 98-102~) in the period 5-10 days after crush. In contrast, the DRP generated by cut nerves in the same period was 62 ~ of normal (range 46--86 ~o).
The period after 10 days After the 10th postoperative day, the size of the arriving volley from the cut nerve began to decline and reached 40 ~ of normal by the 80th day (Fig. 3, top). The conduction velocity of the CAP continued its decline which began on day 5 and reached 40 ~oof normal by day 80 (Fig. 3 bottom). These data are consistent with earlier reports of retrograde conduction change after axotomy especially in sensory axonslL It is therefore of no great surprise that the various central effects of the afferent volley also declined. The details of these later developments can be seen to the right of the vertical line in Figs. 5 and 7. DISCUSSION We conclude that following peripheral nerve section there is an early failure of the mechanism which produces dorsal root potentials (DRPs) and depolarization of the terminals of primary afferents (PAD) in the dorsal horn. This occurs at a time when the afferent volley carried centrally by the peripherally cut axons, and many postsynaptic responses to this volley are unchanged. This finding is interesting because it coincides with changes in the chemistry of afferent terminalsl,Z, 2a and with changes of synaptic connectivitys. The DRP has previously been shown to be a widespread and stable phenomenon19,25-27. We have made two direct measures of the size of the afferent volley from the cut nerve. The first is the area of the CAP recorded on the L5 dorsal root. The second, a direct measure of a special fraction of the afferent volley, is the first wave of the cord tract volley which is generated by action potentials in branches of peripheral axons which ascend in dorsal columns. We observed no consistent changes in either of these measures during the first 10 days after sciatic nerve cut and ligation. Yet during that time the DRP fell to 50 ~o of normal (Fig. 7). It is possible that there was a small drop in the afferent volley that we failed to discriminate, and that this drop accounts for the observed decrease of the DRP. We obviously need to know how sensitive the DRP is to the size of the afferent volley. We obtained the necessary data by recording from the L5 dorsal root on the intact side of operated animals and comparing the area of the DRP evoked by a maximal input volley, a half amplitude volley and a quarter amplitude volley (Fig. 2). The half maximal volley (about 30~ of maximal area) produced a DRP 69 ~o of maximal (n -- 14, range 38-98 ~o). The quarter maximal volley (about 15~ of maximal area) produced a DRP 62~ of maximal (n -- 14, range 28-95 ~). It is apparent that the size of the DRP is not extremely sensitive to the
108 size of the input volley. In order to produce the 50 ~ drop of DRP observed 10 days after nerve section, it would be necessary to reduce the input volley to below 25 ~ of its maximum amplitude and/or to below 1 5 ~ of its maximal area. In spite of the variability of the input measure, it is quite clear that we would have detected such a massive shift. The one change in the afferent volley which coincides with the change in the DRP is the early reduction in conduction velocity (Fig. 3 bottom, also see ref. 4). We must obviously ask if this could be the cause of the decreased DRP, perhaps by desynchronizing the afferent volley. We can dismiss this possibility for two reasons. If an animal is cooled from 38 °C to 30 °C, there is a larger decrease of afferent conduction velocity than observed here but there is a marked increase of both the D R R and the DRP 19. A more powerful argument comes from our own results on crushed nerve. At least for the first 10 days the conduction velocity of the crushed afferents falls in exactly the same manner as after cut and ligation 5. Yet, as we have shown, there was no change in the ability of the central part of a crushed nerve to produce a DRP (Fig. 7, middle). Other evidence that the failure of the DRP generating mechanism is not simply due to failure of input comes from the fact that the contralateral DRP generated by intact nerve also failed in the region of terminals of peripherally cut axons (Fig. 8). The failure of the intact nerve to produce a contralateral DRP begins at the same time, 4 days, as the cut nerve's failure to produce a normal ipsilateral DRP. At the time of the DRP failure in the region of the terminals of cut afferents, the cut nerve produced normal sized DRPs on the adjacent intact ipsilateral roots, L2 and L3 (top line, Fig. 7). Furthermore, the cut nerve produced a much larger crossed D R P than did the intact nerve. This suggests that the failure is related to elements other than the afferents themselves. Another indicator of successful synaptic transmission of primary afferent information into the spinal cord is provided by the size of waves 2 and 3 (Fig. 5). These waves, which remained intact during the first 10 days, represent the firing of structures in the lumbar cord which send projecting axons as far as the lower thoracic cord. The simplest interpretation of the preservation of the spinal tract volleys is that the cut nerve has retained its normal postsynaptic excitatory power on the cells from which these axons originate. Alternatively, it could be that the excitatory effect of the input volley decreased, but that this was exactly counteracted by a rise of postsynaptic excitability. This possibility was checked by measuring the size of waves 2 and 3 evoked by stimulation of dorsal roots L2 and L3. Since many of the axons in roots L2 and L3 synapse on the same cells that receive sciatic afferents6, postsynaptic denervation supersensitivity would be expected to increase the postsynaptic response to this convergent input. No such increase was observed at least for the first 68 days after nerve section (P > 0.1). Since there are changes in the proximal part of the cut fibers as judged by their reduced conduction velocity, a possible explanation for the collapse of the DRP is a decrease in the space constant of the central terminal arborization. If this occurred, the normal depolarization of the terminals would no longer be conducted as far as previously out onto the dorsal roots. Czeh et al. 3 made intracellular recordings from
109 primary sensory neuron somata after peripheral axotomy and failed to find a change in the passive properties of the membranes. We can also dismiss this possibility on the basis of experiments we have done 29 in which we stimulated the terminals of the cut nerves with local microelectrodes. This technique25 generates an antidromic volley recorded on the peripheral nerve. Changes in the size of this antidromic volley provide a measure of excitability change at the terminals. The results of this test show that by 8 days after nerve section, the afferent volley from the cut nerve produces no signs at all of excitability change (i.e. PAD) in its own axon terminals. The same volley, however, produces the full expected increase in the excitability (PAD) in nearby axon terminals whose parent axons have not been cut in the periphery. Similar results are reported by Horch and Lisney14 for cat. This result fits well in another way with the results reported here. We show by comparing the area of the CAP on the L4 and L5 dorsal roots as evoked by maximal stimulation of the dorsal roots themselves with the CAP evoked by stimulation of the sciatic nerve, that approximately 50 ~ of the fibres in these roots are of sciatic origin (Devor, in preparation). Therefore if a volley from a cut sciatic nerve produced no DRP in its own afferents in the L5 dorsal root and a normal DRP in intact L5 dorsal root afferents one would expect the observed 50 decrease of the overall DRP. We conclude that peripheral nerve section and ligation leads to a failure of the DRP generating mechanism around the central terminals of the cut fibers. Nerve crush does not do so. Let us consider other central correlates of these two peripheral operations. We will concentrate on the first 10 days after injury since the DRP changes in this time while the afferent volley remains normal. The following three changes appear identical in crushed and sectioned nerves: (i) decrease of conduction velocity5; (ii) disappearance of fluoride-resistant acid phosphatase from the primary afferent terminals2,4,23; (iii) appearance of reactive cells in the dorsal horn 11. Since these alterations occur with both operations we can tentatively eliminate them as being causally related to the DRP change which occurs only after nerve section. We have recently found that ligation and section produces a marked loss of substance P in the central terminals of sectioned axons whereas we have been unable to detect any loss during the first ten days after crush 1. Finally, the two operations have different effects in the periphery. For example, ligation and crush differ in the degrees to which they disrupt axoplasmic transport 17, the blood-nerve barrier 21 and, of course, the outgrowth of regenerating sprouts 24. What significance might a failure of the DRP or PAD have on other aspects of spinal cord physiology? PAD has been associated with presynaptic surround inhibition or blockg, 27. Its failure might therefore be associated with disinhibition and an amplification of otherwise weak afferent signals. In fact, with respect to peripheral nerve injury, the disappearance of PAD might represent part of a homeostatic process whose net result is to increase the effect of whatever afferent inputs might have survived the injury. We have recently reported on the synaptic reorganization of a second order sensory neurons whose major primary afferent input had been cut by peripheral nerve sections. Specifically within 21 days following cut and ligation of foot nerves in cats and by 4 days in rats, cells formerly responding exclusively to foot stimulation began
110 to r e s p o n d to stimulation o f n o r m a l l y innervated thigh skin. The change o f receptive fields was n o t observed if the nerves were crushed rather t h a n cut. These d a t a suggested to us either that new connections from thigh afferents s p r o u t within the spinal cord, or that previously existing b u t ineffective thigh afferents become effective. W e have so far thiled to find evidence o f central s p r o u t i n g 4. O u r present observations o f D R P changes lend s u p p o r t to the alternative hypothesis o f disinhibition. The possible linkage between the D R P change a n d the receptive field change is s u p p o r t e d by the fact t h a t they b o t h occur with the same time course after nerve section and b o t h changes fail to occur after crush injuries. It is quite clear that the physiological changes within the spinal cord that follow peripheral nerve injury are n o t limited to the terminals o f the axons which have been cut. ACKNOWLEDGE MENTS W e w o u l d like to t h a n k Dr. M a r i a F i t z g e r a l d for her help with the early experiments a n d D i n a h Schonfeld a n d R u t h G o v r i n - L i p p m a n n for technical assistance. The w o r k was s u p p o r t e d in L o n d o n by the M e d i c a l Research Council a n d the N a t i o n a l Institutes o f H e a l t h a n d in J e r u s a l e m b y the Thyssen F o u n d a t i o n a n d the l s r a e l - U . S . Binational Science F o u n d a t i o n .
REFERENCES 1 Barbut, D.,Polak, J. M. andWalI, P.D.,SubstancePinspinalcorddorsalhorndecreasesfollowing peripheral nerve injury, Brain Research, in press. 2 Csillik, B. and Knyihar, E., Biodynamic plasticity in the Rolando substance, Progr. NeurobioL, 10 (1978) 203-230. 3 Czeh, G., Kudo, N. and Kuno, M. Membrane properties and conduction velocity in sensory neurons following central or peripheral axotomy, J. Physiol. (Lond.), 270 (1977) 165-180. 4 Devor, M. and Claman, D., Mapping and plasticity of acid phosphatase afferents in the rat dorsal horn, Brain Research, 190 (1980) 17-28. 5 Devor, M. and Govrin-Lippmann, R., Selective regeneration of sensory fibers following nerve crush injury, Exp. Neurol., 64 (1979) 260-270. 6 Devor, M., Merrill, E. G. and Wall, P. D., Dorsal horn cells that respond to stimulation of distant dorsal roots, J. PhysioL (Lond.), 270 (1977) 519-531. 7 Devor, M., Schonfeld, D., Seltzer, Z. and Wall, P. D., Two modes of cutaneous reinnervation following peripheral nerve injury, J. comp. Neurol., 185 (1979) 211-220. 8 Devor, M. and Wall, P. D., Reorganisation of spinal cord sensory map after peripheral nerve injury, Nature (Lond.), 276 (1978) 75-76. 9 Eccles, J. C., Presynaptic inhibition in the spinal cord. In J. C. Eccles and J. P. Schad6 (Eds.), Physiology of Spinal Neurons, Progr. Brain Res., Vol. 12, Elsevier, Amsterdam, 1964. 10 Gasser, H. S. and Grundfest, H., Axon diameters in relation to the spike dimensions and conduction velocity in mammalian A fibers, Amer. J. Physiol., 127 (1939) 393414. 11 Gilmore, S. A. and Skinner, R. D., Intraspinal non-neuronal cellular responses to peripheral nerve injury, Anat. Rec., 194 (1979) 369-388. 12 Hoffer, J. A., Stein, R. B. and Gordon, T., Differential atrophy of sensory and motor fibers following section of cat peripheral nerves, Brain Research, 178 (1979) 347-361. 13 Horch, K. W., Ascending collaterals of cutaneous neurons in the fasciculus gracilis of the cat during peripheral nerve regeneration, Brain Research, 117 (1976) 19-32. 14 Horch, K. W. and Lisney, S. J. W., Changes in primary afferent depolarisation of myelinated cutaneous sensory neurones during nerve regeneration in the cat, J. PhysioL (Lond.), in press.
111 15 Jessell, T., Tsundo, A., Kanazawa, I. and Otsuka, M., Substance P depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurones, Brain Research, 168 (1979) 247-259. 16 Kaizawa, J. and Takahashi, I., Fiber analysis of the lumbar spinal roots and their sciatic branches in rats, Tohoku J. exp. Med., 100 (1970) 61-74. 17 Karlstron, L. and Dahistr6m, A., The effect of different types of axonal trauma on the synthesis and transport of amine storage granules in rat sciatic nerves, J. NeurobioL, 4 (1973) 191-200. 18 Lieberman, A. R., The axon reaction, Int. Rev. NeurobioL, 14 (1971) 49-124. 19 Lloyd, D. P. C., Electrotonus in dorsal roots, ColdSpr. Harb. Symp. quant. BioL, 17 (1952) 203-219. 20 Lundberg, A., Ascending spinal hindlimb pathways in the cat. In J. C. Eccles and J. P. Schad~ (Eds.), Physiology of Spinal Neurons, Progr. Brain Res., I/oL 12, Elsevier, Amsterdam, 1964. 21 Mellick, R. and Cavanagh, J. B., Longitudinal movement of radioiodinated albumin within extravascular spaces of peripheral nerves following three systems of experimental trauma, J. NeuroL Neurosurg. Psychiat., 30 (1967) 458. 22 Mendell, L. M., Manson, J. B. and Scott, J. G., Connectivity changes in la afferents on axotomized motoneurons, Brain Research, 73 (1974) 338-342. 23 Schoenen, J., Budo, C. and Poncelet, G., Effet de la section du sciatique sur l'activit6 de l'isoenzyme fluororesistant de la phosphatase acide dans la moelle epiniere du rat, C.R. Soc. BioL, 162 (1968) 2035-2037. 24 Sunderland, S., Nerves and Nerve Injuries, 2nd edn., Churchill Livingstone, Edinburgh, 1978. 25 Wall, P. D., Excitability changes in afferent fiber terminations and their relation to slow potentials, J. Physiol. (Lond.), 142 (1958) 1-21. 26 Wall, P. D., The origin of a spinal cord slow potential., J. PhysioL (Lond.), 164 (1962) 508-526. 27 Wall, P. D., Presynaptic control of impulses at the first central synapse. In J. C. Eccles and J. P. Schad6 (Eds.), Physiology of Spinal Neurones, Progr. Brain Res., VoL 12, Elsevier, Amsterdam, 1964. 28 Wall, P. D., The role of substantia gelatinosa as a gate control, Res. PubL Ass. nerv. rnent. Dis., 58 (1980) 205-231. 29 Wall, P. D., Changes in primary afferent depolarisation following peripheral nerve injury, submitted.