Antidromic discharges in dorsal roots of decerebrate cats

Brain Research 846 Ž1999. 87–105 www.elsevier.comrlocaterbres

Research report

Antidromic discharges in dorsal roots of decerebrate cats I. Studies at rest and during fictive locomotion Irina Beloozerova 1, Serge Rossignol

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Centre de Recherche en Sciences Neurologiques, Faculte´ de Medecine, PaÕillon Paul-G.-Desmarais, 2960 Chemin de la Tour, UniÕersite´ de Montreal, ´ ´ Montreal, Canada H3T 1J4 ´ Quebec, ´ Accepted 10 August 1999

Abstract Spontaneous rhythmic antidromic discharges have previously been recorded in proximal stumps of cut dorsal roots during locomotion Žreal and fictive.. The goals of the present study were to elucidate Ž1. whether both orthodromic and antidromic discharges occur in the same dorsal root filament and Ž2. whether orthodromic discharges have an influence upon antidromic discharges of units in the same filament. Unitary activity was recorded in 70 uncut dorsal root filaments ŽL6-S1. in 15 decerebrate cats using bipolar AgrAgCl electrodes. Spikes with similar wave shapes were considered to represent the activity of single units. Spike-triggered averaging ŽSTA., local anaesthesia and transection of filaments were used to determine the direction of propagation of spikes. Spikes with different initial electrical polarities were found in most of the filaments and shown to propagate in opposite directions at rest and during fictive locomotion. On average, there were 38% " S.D. 23% antidromically discharging units per filament and their mean conduction velocity was 55 mrs " S.D. 25 mrs. After blocking orthodromic activity of the whole filament by a transection or local anesthesia applied distally to the recording site, changes were seen in the antidromic discharges of some units suggesting that spontaneous orthodromic discharges normally seen in the filament may influence the antidromic discharges of some units. Moreover, out of 27 antidromic units recorded during fictive locomotion, 12 were rhythmically modulated with peak discharges occurring in various parts of the locomotor cycle. We conclude that, in uncut dorsal roots, there is a normal coexistence of spontaneous orthodromic and antidromic discharges revealed by STA and that there is an interaction between spontaneous orthodromic and antidromic discharges. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Antidromic discharge; Dorsal root afferent; Cat; Locomotion; Presynaptic inhibition

1. Introduction Studies on locomoting preparations have suggested that sensory inputs from the limbs are subjected to phasic controls so that their influence on locomotion may depend on the phase of the step cycle in which they occur Žfor review see Refs. w65,67x.. This phasic control is undoubtedly exerted centrally at various points along sensory pathways by cyclical changes in the excitability of interneurones w1,37,38,45,55,58,69,70x. However, there is mounting evidence Žreviewed in Refs. w56,66x. that there are also, during locomotion, presynaptic mechanisms acting at the level of the primary afferents themselves that )

Corresponding author. Fax: q 1-514-343-6113; e-mail: [email protected] 1 Present address: Department of Psychology, University of Connecticut, 406 Babbidge Road, Storrs, CT 06269-1020, USA.

could potentially modulate the efficacy of sensory transmission. Using extracellular dorsal root recordings Ždorsal root potentials., as well as intracellular recordings Žprimary afferent depolarizations., it was indeed shown that most primary afferents were cyclically depolarized twice per locomotor cycle during fictive locomotion in thalamic or decerebrate as well as in spinal cats w2–5,29–32,40–43x. It was also striking that single sensory afferent units, recorded in the proximal stumps of cut dorsal root filaments, discharged rhythmically in synchrony with the step cycle Žsee also Refs. w4,5,33x.. Such antidromic discharges have also been seen during rhythmic behavior in other animals such as the crayfish w36x and neonatal rats w51x. These observations raise different issues as to the potential functional consequence of such antidromic discharges of primary afferents. Recent work suggests that indeed antidromic discharges of a single afferent evoked by passing current through an intracellular electrode may change dras-

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tically its orthodromic discharge frequency w39x. Therefore, antidromic discharges that may occur during locomotion could exert dramatic effects on the activity of sensory units Žas postulated before in Refs. w56,66x.. As one of the steps to eventually test our general hypothesis that these antidromic discharges exist in the normal walking cat and that they have a role to play in the overall flow of sensory transmission, it was felt necessary to show quite conclusively that the normal traffic of spikes in an intact dorsal root is made not only of orthodromic spikes but also of a sizable contingent of antidromic spikes. For this, we had to investigate the roots with a technique that could be eventually transferable to intact animals with intact roots. We have thus used, in the present study, spike-triggered averaging ŽSTA. to determine the direction of propagation of the various units recorded in uncut dorsal root filaments. Another issue addressed in the present paper was to find out if spontaneous orthodromic discharges of units through the intact dorsal roots, including the recorded rootlet, could influence antidromic discharges of single units since there are numerous potential interactions between sensory afferents through presynaptic mechanisms w47,71x. In our previous experiments, that question could not be evaluated because the dorsal root filaments were cut at the onset of recordings. In this paper we show that, in an intact dorsal roots, there is normally a traffic of both orthodromic and antidromic discharges. We also suggest that antidromic discharges in a filament may increase when the influences of orthodromic discharges are removed by transecting the filament or applying a local anesthetic distally to the recording site. In a forthcoming paper, results obtained with similar recordings of uncut dorsal root filaments during treadmill locomotion in decerebrate cats will be presented. Some of the results have been reported briefly w14,15,56,65–67x.

2. Materials and methods 2.1. Preparation The experiments were performed on 15 adult cats of both sexes Ž2.5–4.5 kg.. The animal was first put in a specially designed wood box and anesthetized with a mixture of Oxygen and Halothane 3%. A tracheotomy was then performed and a tracheal tube inserted for delivering the anesthetic ŽHalothane 1–2.5% and Oxygen.. A catheter

was inserted in one jugular vein for administering drugs and fluids. The blood pressure was monitored by cannulating one common carotid artery. The temperature was measured with a rectal thermometer and was maintained at around 38–398 by a feedback-controlled heating element using DC current. The end-expiratory pCO 2 was maintained between 3.5 and 4.5% using a Datex Monitor during assisted respiration. The spine of the cat was fixed in a spinal frame with two or three pairs of lateral pins and a laminectomy was performed between L4 and S2. The spinal cord was covered with warm paraffin oil. The head of the cat was fixed in a stereotaxic apparatus, and an extensive craniotomy was performed. The animal was decerebrated by a complete transverse section at a precollicular and premammilar level. All brain tissue rostral to the section was removed and the cranial cavity filled with hemostatic cellulose ŽSurgicel. and gauze. Thereafter, the anesthesia was discontinued. Six cats were paralyzed with gallamine triethiodide ŽFlaxedil; 5 mgrkg i.v. repeated when needed. and artificially ventilated. In these animals, the ipsilateral Sartorius nerve was dissected and its activity was recorded with AgrAgCl electrodes or using a monopolar recording electrode in a polymer cuff w57x. Paralyzed cats showed spontaneous locomotor activity in the muscle nerve although, in a few cases, locomotion was initiated by stimulation of the midbrain locomotor region. Filaments of L6, L7 or S1 dorsal root were carefully dissected one at a time, over a length of 3–4 cm, and placed on three bipolar AgrAgCl electrodes. Each electrode assembly was positioned 1 cm apart ŽFig. 1A. and the distance between the cathode and anode of each electrode pair was around 3–4 mm. 2.2. Recording and analyzes The activity of the dorsal root filaments and the Sartorius nerve was amplified with AC coupled amplifiers Žbandwidth 300 Hz–10 kHz. and recorded on a Honeywell tape recorder ŽModel 101. with a frequency response of 0–5000 kHz at the recording speed of 19 cmrs. Most of the experiments were played back on an electrostatic polygraph ŽGould ES 2000. to select sections for further off-line analysis. During the experiment, STA was used on-line to identify the direction of propagation of the recorded spikes. Firstly, a window discriminator was used to isolate a single spike recorded from one of the electrodes, usually the most

Fig. 1. Experimental set-up for recording and analyzing orthodromic and antidromic spikes in dorsal roots. ŽA. Dorsal root filaments were carefully separated and kept in continuity. Three bipolar AgrAgCl electrodes Ža1, a2 and a3. were positioned about 1 cm apart. ŽB. Activity of the dorsal root filaments and the ipsilateral Sartorius nerve ŽSrt n. was recorded during fictive locomotion and stored on a 7-channel tape recorder. ŽC. Schematic representation of the STA method. A window discriminator was used to isolate spikes with similar waveforms from the activity recorded in electrode a1. Such spikes were identified as ‘‘units.’’ These spikes served as triggers to accumulate 50–2000 traces recorded with other electrodes. Units were labeled as antidromically discharging when the average signal occurred later in the more distal electrodes then the spikes in electrode a1. Units were labeled as orthodromically discharging when the average signal occurred earlier in the more distal electrodes than the spikes in electrode a1.

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proximal electrode a1 Žsee Fig. 1A.. This spike triggered a 4-channel digitizing oscilloscope ŽTektronix TDS 420.. The signals from electrode a1 Žthe triggering spikes themselves. and from the other more distal electrodes Žsee Fig. 1A and B. were averaged for 50–2000 sweeps at a sampling frequency of 10–50 kHz depending on the time base used Žgenerally 1 msrcm time base and a digitizing frequency of 25 kHz per channel.. The scheme of Fig. 1C illustrates how, with this recording arrangement, it is possible to determine the direction of propagation of the spikes. For a given spike, potentials occurring in the distal electrodes a2 or a3 later than the triggering spike itself in electrode a1, indicate that the spike is propagating antidromically whereas potentials preceding the spikes on electrode a1 mean that the spike is propagating orthodromically Žsee Fig. 3B for real STAs.. It was also possible from such recordings to measure the conduction velocity of the spikes by dividing the latency by the distance between electrode pairs Žaround 1 cm.. All waveforms were stored in memory of the digitizing oscilloscope and printed out directly on the laser printer. Tape recorded data were not used for STA but were processed on a computer using custom-made software to establish the phase relationship of the discharges with the locomotor cycle. The locomotor cycle was defined as the interval between two successive bursts of activity in the ipsilateral Sartorius nerve. The cycle duration was divided in 256 equal bins and the frequency of the discharge in each bin was estimated by dividing the number of spikes by the duration of the bin. Post-event time histograms relative to the onset of the burst in Sartorius nerve were obtained over 10–200 locomotor cycles. The duration of the cycles was normalized to 100%.

3. Results 3.1. Spikes recorded in dorsal root filaments Altogether, the activity of 70 uncut dorsal root filaments Žof L6, L7 and S1 roots. was recorded. Fig. 2 illustrates typical examples of recordings taken from six different filaments from different experiments and displayed at a fast time base. All spikes recorded in the first filament ŽFig. 2A. had an initial negative Žup. wave which, in most spikes, was followed by a smaller positive deflection Ždown.. In the second filament ŽFig. 2B. the largest spike, which has an initial negative polarity was followed in one case by the single discharge of a spike with a predominant initial positive polarity. In filaments 3 to 6 ŽFig. 2C–F. there were different mixtures of spikes with positive–negative and negative–positive waveforms. Spikes of initial positive or negative polarities were found in all cats, although not necessarily in all recorded filaments. They could be seen in cats which had no locomotor

activity throughout the experiment or in cats in which there was spontaneous or evoked locomotor activity, either during the locomotor activity Žsee later. or in between bouts of rhythmic activity. Spikes with similar waveforms are assumed to originate from a single afferent fiber and are sometimes referred here as ‘‘unit’’. With the bipolar recording arrangement used here, the presence of spikes with different initial polarities in intact dorsal filaments already suggests the coexistence in the same filaments of units discharging in both antidromic and orthodromic directions. With the present arrangement it was not possible to determine if the same unit discharged in both directions during the same recording period. 3.2. STA STA was used on-line to determine the direction of spike propagation as detailed in Section 2. Fig. 3 illustrates a typical identification sequence with STA of spikes recorded in one L6 filament by three bipolar electrodes. The raw recordings of the three electrodes of Fig. 3A clearly contained spikes of different initial polarities. The spikes with the negative Žup. initial polarity recorded by the most proximal electrode a1 were used to trigger the averager ŽFig. 3B, left side.. The averaged spikes in the more distal electrodes a2 and a3 occurred before the triggering spike and therefore these spikes were classified as orthodromically propagating from the periphery towards the spinal cord. The second spike in the trace of electrode a1 had a positive–negative waveform and was also discriminated separately for averaging Žright side of Fig. 3B.. Since the averaged spikes in the distal electrodes a2 and a3 occurred after the triggering spike, they were classified as antidromically propagating spikes. For three units already shown to discharge antidromically by STA, the tissue surrounding the corresponding dorsal root ganglion was dissected away and electrode a3 was placed distally to the ganglion. STA records confirmed that the spikes passed the ganglion and most probably reached the peripheral nerve although this particular point was not verified in these experiments. 3.3. Local anesthetics applied to dorsal root filaments To further document the direction of propagation of spikes for 34 units, we applied a very small piece of cotton moistened with a 2% solution of the local anesthetic Lidocaıne ¨ to the most distal electrode. In this situation, orthodromic discharges should be stopped whereas antidromic discharges should persist. Fig. 4A represents the recording obtained from electrode a1 from one filament Žsee arrangement in Fig. 4C.. In the top panel, the recording is first shown on a slow time base and then, five times faster, to display the coexis-

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Fig. 2. Six samples of recordings obtained from uncut dorsal root filaments in six different animals. Spikes with an initial negativity Župward deflection. were identified by an open circle while those with an initial positivity Ždownward deflection. were labeled by a filled circle. All spikes recorded in filament A had an initial negativity followed by a smaller positivity. In filament B, one of the spikes had a positive wave first followed by a smaller negative one. Filaments C–F had a mixture of spikes with different initial polarities.

tence of spikes with opposite polarities. Using STA, the presence of orthodromic activity Župper left panel of Fig.

4B. as well as spikes with antidromic propagation Župper right panel of Fig. 4B. could be determined. Ten minutes

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after application of the local anaesthetic, there was a clear reduction of the overall activity of the root although some activity persisted as seen in the bottom record of Fig. 4A.

In the fastest time base display of that trace, the gain was increased to illustrate that all these spikes had a positive initial polarity compatible with their antidromic propaga-

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tion. With STA, it was shown that these spikes indeed propagated antidromically Žmiddle right panel of Fig. 4B.. No orthodromically propagating spikes were found in the records after local anesthesia. Note that in this record, electrode a3 was shunted by the cotton soaked in the anesthetic solution. The records of the middle left panel of Fig. 4B simply show the absence of orthodromic spikes after application of Lidocaıne. ¨ Ninety minutes after removal of the anesthetic, the ability of the filament to conduct spikes in both directions was restored, as shown in the two bottom records of Fig. 4B. This procedure was repeated for 34 dorsal root filaments Žin 6 cats. and yielded similar results: spikes with initial negative polarity disappeared from the recording of the proximal electrodes after application of anesthetic distally and only spikes with positive initial polarity propagating in the antidromic direction, as determined with STA, remained Ž1–5 per filament.. Thus, the data gathered from the polarity of the spikes, STA and local anesthesia all converged to indicate that there were units spontaneously discharging in the ortho and antidromic directions in the intact Žuncut. filaments and that the present recording set-up allows to study, up to some extent, the interactions between orthodromic and antidromic activity. 3.4. Transection of dorsal root filaments In some experiments, filaments were also cut to confirm the conclusions reached with the previous approaches. Fig. 5A shows again records at slow and fast time bases illustrating the presence of spikes with different initial polarities. The upper pair of STAs of Fig. 5B show spikes propagating in the orthodromic and the antidromic directions. After sectioning the filament as illustrated in Fig. 5C, only spikes with a positive initial polarity were present Žbottom trace of Fig. 5A.. The antidromic propagation of these spikes was confirmed by STA Žright hand side of the bottom panel of Fig. 5B.. Immediately after sectioning a dorsal root filament there could be a short period of great activity probably due to massive injury discharges. Thereafter, it was frequently observed that there was a depression of all discharges, and in some filaments no antidromic spikes could be recorded for some time. Usually, antidromic spikes appeared after and, in some cases, several minutes after the cut. In general, however, a lesser number of antidromically dis-

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charging units was seen in cut filaments than in Lidocaıne-blocked filaments. ¨ 3.5. New antidromic spikes appear after anesthetic block or section of dorsal root filaments Activity of 34 filaments was recorded both before and after distal anaesthetic blockade whereas 12 units were recorded both before and after transection of filaments. In 10 anesthetic-blocked filaments and in one cut filament, it was very clear that some of the antidromic spikes recorded in the proximal electrode were not present before the block or the cut. Fig. 6A presents recordings obtained with three electrodes from one filament, before and after application of Lidocaıne. ¨ Before Lidocaıne ¨ there were several spikes of relatively small amplitude ŽFig. 6A, left side.. The STA of the left panel in Fig. 6B demonstrates that these spikes with the negative Žup. initial polarity were propagating orthodromically. Approximately 40 s after the application of Lidocaıne ¨ ŽFig. 6A, right panel., a spike with much larger amplitude appeared in electrodes a1 and a2 Želectrode a3 was shunted by the Lidocaıne ¨ moistened cotton.. Because the amplitude of the newly appeared spikes was much larger than any of the spikes seen before the application of the anesthetic, it was concluded that these spikes were not present in the record before Lidocaıne. ¨ STA showed that these newly appeared spikes with the positive Ždown. initial polarity was propagating antidromically ŽFig. 6B, right side.. No wash out recordings were obtained as shown in Fig. 4 since the delay to get a reliable wash out can be quite long considering that these recordings are performed in an acute decerebrate cat. Fig. 6C presents a recording from one filament before Žintact, left side record. and after cutting Žon the right.. In the intact filament, the activity was abundant and there were spikes with various initial polarities. After cutting the filament, only spikes with positive initial polarity were present in the record and among them there was one new spike with a very large amplitude. That spike was definitely not present in the record in the intact filament. We believe that the abundant activity in intact filaments carrying a great number of orthodromic spikes masked the weaker antidromic activity so that it could only be revealed after blocking or cutting the filament. However, we are presenting, here, only spikes whose amplitude were large enough to ensure that they were not present before

Fig. 3. The use of the STA method for determination of the direction of propagation of spikes in dorsal root filaments. ŽA. Raw recordings obtained by three electrodes placed on one uncut dorsal filament as shown in C. There are two clearly identifiable units with spikes of different initial polarities. ŽB. STA of discharges of the two units with spikes of different initial polarities. When spikes with the initial negative Žup. polarity Žmarked by an open circle. triggered the averager, the average signal occurred in the more distal electrodes a2 and a3 came before the trigger. The spikes of this unit were first recorded by the most distal electrode Ža3. and propagated towards the spinal cord in the orthodromic direction and were successively recorded by electrodes a2 and a1. When spikes with the initial positive Ždown. polarity Žmarked by a filled circle. triggered the averager, the average signal occurred after the trigger in the more distal electrodes a2 and a3. Thus, spikes of this unit were first recorded by the most proximal electrode a1 and then were recorded in the antidromic direction away from the spinal cord. N s number of traces averaged. ŽC. Positioning of the electrodes on the dorsal root filament and directions of spike propagation are shown in a schematic form.

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the block or the cut. Because the filaments of L7 and S1 are long Ž4–5 cm., it was possible to cut the filament without disturbing its position relative to the proximal electrodes which could have, in theory, changed the characteristics of the recorded units.

3.6. Number and conduction Õelocity of antidromic and orthodromic units in the dorsal root filaments We can only approximate the number of units discharging antidromically within the overall activity of the dorsal

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root filaments. The number of units which could be seen in a given record depended very much on the size of the filament and, consequently, on our ability to isolate and discriminate the spikes in order to use STA. We have estimated the percentage of antidromically discharging units in 54 uncut filaments in which ortho- and antidromically propagating spikes could be clearly discriminated. This percentage of antidromic discharges in the 54 recorded filaments is illustrated in a histogram form in Fig. 7. The mean percentage of antidromically discharging units per filament was 38% " S.D. 23%. Two filaments had only antidromic activity; a few filaments had a high proportion of antidromic units Ž60–80% of all units within the filament.; about half of the filaments Ž25r54. had orthodromic and antidromic units in equal proportion, and of course, there were many filaments Ž19r54. with low antidromic activity Žonly 10–30% of all units in these filaments were discharging antidromically.. The conduction velocity of the antidromic and orthodromic units was estimated by measuring the time delay between spikes of the same unit recorded by STA with two electrodes spaced by 10 mm or 20 mm ŽFig. 8.. Because the distance between pairs of electrodes was small, the measurements of conduction velocities presented here should be considered to be strongly indicative but not very precise. In this distribution histogram, the number of larger units with higher conduction velocities are most probably overestimated because they likely have had spikes of larger amplitude and so could be studied with more confidence with STA. The distribution of conduction velocities is presented for 76 antidromic units in Fig. 8A and for 50 orthodromic units in Fig. 8B. The conduction velocities of antidromic units ranged between 14 and 130 mrs with a mean of 54.6 mrs Ž"S.D. 24.5 mrs.. On the other hand, the conduction velocities of the orthodromic units were distributed in two peaks, one with a mean of 33.0 mrs Ž"S.D. 8.6 mrs. and the other with a mean of 87.3 mrs Ž"S.D. 17.0 mrs.. 3.7. ActiÕity of antidromic units during fictiÕe locomotion Activity of 27 units was recorded in uncut dorsal root filaments during fictive locomotion in two cats. Twenty four of them were recorded in the proximal part of the filament after distal application of Lidocaıne ¨ whereas three more units were recorded in filaments without local anesthetics.

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The frequency of discharge of 11 of the 24 units recorded after Lidocaıne ¨ blockade was modulated with the locomotor rhythm. The activity of one of the three units recorded without Lidocaıne ¨ was also modulated during locomotion. So, in total, the activity of 12r27 Ž44.4%. units was rhythmically modulated in relation to the fictive locomotor cycle. Fig. 9 presents an example of an antidromically active locomotor-related unit. Fig. 9A shows the recording obtained from two electrodes proximal to the Lidocaıne ¨ block together with the ipsilateral Sartorius nerve, which displays a distinct rhythmicity of the unit’s discharge. Using STA ŽFig. 9B, it was shown that spikes of that unit Žlargest amplitude spikes. recorded by electrode a1 were propagating in the antidromic direction. These spikes could barely be seen on the recording of electrode a2 but the averaged record was further amplified and it could clearly be seen that spikes occurred later than in electrode a1. To better quantify the relationship of the antidromic discharge with the locomotor cycle, the spikes of electrode a1 are displayed in a raster format in Fig. 9C. Here the discharge is presented in relation to the Sartorius nerve bursts Žarrow 1 s onset of burst; arrow 2 s offset of burst and arrow 3 s onset of next burst. which determine the cycle. It is clear that the activity of the unit generally started somewhat before the onset the activity in Sartorius nerve and lasted throughout the burst, i.e., during the fictive flexor phase of the locomotor cycle. The unit then attained a mean frequency of about 2 Hz. The unit is practically silent between the periods of Sartorius discharge, i.e., during the extensor period of the locomotor cycle. The peak activity of different antidromic units could occur in different phases of the fictive step cycle. Fig. 10 shows four cases of locomotor-related units displayed as rasters and histograms aligned on the onset of activity in the Sartorius nerve. The peak of activity in three of the units ŽFig. 10A,B,C,D,G and H. fell fully in the flexor phase of the step cycle. On the other hand, the activity of the unit in Fig. 10E and F fell fully in the extensor phase of the step cycle. The average peak activity reached by all units but one was below 20 Hz. The activity of 7r12 units Ž58%. had a purely bursting pattern, i.e., the activity peaked during a given period of the cycle and there was none in the opposite phase ŽFig. 10C–D.. Just 5r12 Ž42%. units reached a peak of activity in one phase but continued to

Fig. 4. Effect of the Lidocaıne ¨ Ž2%. blockade. ŽA. Raw recordings of electrode a1 before Župper trace. and 10 min after application of Lidocaıne ¨ Žlower trace. reproduced from the electrostatic printer at low Žleft. and five times higher speed Žright. to better identify the units Žsee separate time bases.. ŽB. Determination of the direction of spikes’ propagation by STA. In ‘‘Control’’ conditions before application of Lidocaıne ¨ units with orthodromically and antidromically propagating spikes could be recorded by all of the electrodes. Ten minutes after application of Lidocaıne ¨ to the filament at the site of the electrode a3 Žsee scheme in C., only antidromic activity remained. The recording of electrode a3 is shunted by the moisten cotton. Ninety minutes after the removal of the anesthetic block on electrode a3, both orthodromically and antidromically discharging units could be identified by STA. ŽC. Positioning of the electrodes on the dorsal root filament and the site of Lidocaıne ¨ application as well as the direction of spikes’ propagation are shown in a schematic form.

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discharge at noticeable rates throughout the rest of the cycle ŽFig. 10F,H..

Fig. 11 summarizes the discharge patterns of all 12 locomotor-related units recorded. Each trace represents the

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activity of one antidromically discharging unit in a histogram form aligned to the onset of activity in Sartorius nerve. The main ‘‘burst’’ of activity for each cell was arbitrarily highlighted by blackening the period when the activity of the cell was above the minimal discharge during the cycle. The units have been ranked so that those bursting at the beginning of activity of Sartorius Žthe flexion phase. were put at the top of the figure, whereas those bursting later were represented progressively lower down. The activity of nine units out of started with that of the Sartorius and peaked during its activity. The bursts of all the units, except for two, lasted throughout the flexor phase. Only two units were still at their peak activity at the offset of the Sartorius burst, i.e., at the transition phase between the extensor and flexor periods. The activity of the 3 out of 12 units peaked between the bursts of activity in Sartorius nerve, i.e., during the period of activity in extensor nerves. These bursts were somewhat broader then those of the units which peaked during the flexor phase and occupied the whole period between the offset and onset of activity in Sartorius nerve. None of the units recorded were recruited for only part of the extensor period or had a discharged overlapping significantly the extensor and flexor period.

4. Discussion These results show that in decerebrate cats, at rest or during fictive locomotion, there is, in intact uncut dorsal roots, a normal traffic of orthodromic and antidromic discharge of primary afferents. This was ascertained by using STA, which clearly demonstrated the direction of propagation of different spikes recorded simultaneously in the same filament. The antidromic propagation of some of these spikes was confirmed by a dorsal root section and an anesthetic block distally to the site of recording. The proportion of antidromically discharging units was not negligible. It did represent, on average, 38% of the identifiable units per dissected dorsal root filament. Whereas the orthodromic discharges reflect mainly the activation of peripheral receptors, it could also represent some spontaneous activity of the dorsal root ganglion neurons Žsee later.. The antidromic discharges on the other hand reflect central spinal mechanisms capable of generating spikes in the terminals of afferents. Antidromic discharges have previously been observed in several conditions in the cat, either spontaneously in the dorsal roots Ždorsal root discharges w53,54,78–80x. or, classically, after stimulation of nerves or other dorsal

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roots, as part of dorsal root reflexes in cats w11– 13,17,22,34x, or in peripheral nerves during inflammatory processes in cats and monkeys w62,63,73x, in in vitro preparations w6–10x, in crayfish at rest and during locomotion w23–25x, and finally in locusts w19,20x. We have, at times, been led to think that these antidromic discharges are more or less experimental artifacts Žlow temperature, spinal cord deterioration. and that they do not exist under normal physiological conditions w17x. This is best captured by the following citation ‘‘All these results support the conclusion that antidromic impulses in the dorsal roots are unlikely to occur in the normal animal, and are therefore of only secondary physiological importance’’ w46x. As far as temperature is concerned, there is ample evidence that these antidromic discharges occur at normal temperature w34,64x although they may be more preeminent at low temperatures w75x. We also believe that fictive locomotion or decerebrate walking on a treadmill is hardly compatible with the notion of deteriorated preparations because, in decerebrate cats, locomotion will disappear before other signs of deterioration are apparent. It is thus likely that these discharges occur to some extent in normal circumstances. Why then are these antidromic discharges important to investigate? Two reasons have prompted the present study. First, numerous rhythmically organized antidromic discharges have been found during locomotion in either fictive conditions or during treadmill walking in decerebrate or intact cats w29–33,40,42,56,66x and rats w59,60x. However, because previous recordings were done in cut dorsal roots, it was important to find antidromically propagating discharges in intact roots since nerve section in the periphery has been shown to lead to the development of ectopic firing sites w27x. Secondly, recent studies in the crayfish w16x and also in the cat w39x indicate that antidromic discharges may exert a powerful control on orthodromic discharges. For instance, stimulation of the peripheral stump of a cut dorsal root can stop or delay the occurrence of the next orthodromic spike in units recorded also from the same distal portion of the cut root w39x. It was thus important to know better the circumstances in which antidromic spikes may occur since they may represent a very potent efferent control on peripheral afferent discharge. Finally, it was important to investigate whether orthodromic discharges have an influence upon antidromic discharges. 4.1. Fiber types with antidromic discharges It has been reported that antidromic discharges occur in cats in 100% of cut roots after a period of injury dis-

Fig. 5. Effect of transection of the filament distally to the recording electrodes. ŽA. Raw recordings obtained from electrode a1 in uncut Župper trace. and cut filament Žlower trace. reproduced at low speed on the left and at a five times faster time base Žsee separate time bases.. ŽB. Determination of the direction of spikes’ propagation by STA. In ‘‘Control’’ conditions there were units with spikes of different initial polarities and different directions of propagation along uncut filament. After the filament was transected, only units with antidromically propagating spikes remained. ŽC. Schematic drawing of the position of the electrodes on the dorsal root filament, the site of transection of the filament and the directions of spikes’ propagation.

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Fig. 7. Percentage of antidromically active units in dorsal root filaments in 54 dorsal root filaments. The number of filaments is given on the ordinate while the abscissa gives the percentage of antidromically discharging units Ždetermined by STA. in each group of filaments.

charges Ž50% in rats and 25% in frogs. w46x. Several other workers have reported that dorsal root reflexes occur in A-alpha to A-delta range fibers w21,22,74x. The mean conduction velocity of antidromically discharging units in cutaneous fibers of high spinal cats varies between 45 and 60 mrs w21x and, in low spinal cats, spontaneous dorsal root discharges in cutaneous and muscle nerves range from 7.5–97.5 mrs w53x with a mean value of 52.3 mrs Ž"5.3 mrs.. Although the above studies emphasized that antidromic discharges could be found in the A range only, experiments with experimentally induced inflammation of the knee in cats and monkeys showed that dorsal root reflexes involve fibers in the C range as well as in the A range w73x. Our data indicate that the population of units that we could clearly identify as antidromically discharging was rather homogenous. The mean conduction velocity of these units was 54 mrs Ž"S.D. of 24 mrs., ranging between 14 and 130 mrs Žmedian of 50 mrs., which coincide very well to the values reported above for the A range. On the other hand, two populations of fibers discharging orthodromically could be identified, one in the range of 70–110 mrs and another between 20 and 40 mrs. The fact that the distribution appeared different suggest that fibers in the middle range of conduction velocity may be more prone to discharge antidromically, whereas fibers in the A-alpha range may be less prone. On the other hand, there is a clearer overlap of fibers discharging in both directions in the population of lower conduction velocities. The above considerations also tend to suggest that if, in a given experiment, the same fibers had been found to discharge in both directions, two identical populations would have been produced. Therefore, without venturing

Fig. 8. Conduction velocity, in mrs, of the orthodromically Ž ns 50. and antidromically Ž ns 76. discharging units in all the 70 recorded dorsal root filaments, as derived from the time delay between the peaks of the averaged ŽSTA. spikes recorded and the distance between two electrodes placed on the same filament.

to the conclusion that fibers in a particular conduction velocity range only discharge antidromically, we could suggest that: Ž1. there is a predominance of medium conduction velocity fibers discharging antidromically in the A range and Ž2. that within a given recording time period, it is probable that the same unit does not alternatively discharge in one or the other direction. It is more likely that the fibers will discharge for a while in one mode and then switch to the other one. 4.2. Multiple sites of spike generation in primary afferents There is good evidence now in various neuronal systems that there can be multiple sites of spike generation in mollusks w48x and higher vertebrates w61x. The cell body of the neuron would not only generate orthodromic spikes to carry a message to a post-synaptic cell but would also receive a feedback through antidromic discharges of its axon which reflect the amount of activity at the site of axonal termination. Primary afferent neurons can generate spikes at multiple sites, besides the peripheral receptor. Recordings from the proximal stump of cut dorsal roots have shown that spikes

Fig. 6. New antidromically active units appear after Lidocaıne ¨ blockade and transection of the filament. ŽA. A unit with small spikes was recorded on electrodes a1 and a2 before the Lidocaıne ¨ blockade. Spikes of this unit disappeared after Lidocaıne ¨ blockade but large spikes of a new unit appeared in approximately 40 s after the application of the anesthetic. ŽB. After Lidocaıne ¨ application, it could be shown that the spikes of the new unit were antidromically propagating. ŽC. New large amplitude spikes with positive Ždown. initial polarity appeared after transection of a filament.

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Fig. 9. Activity of an antidromically discharging unit during fictive locomotion recorded in a filament blocked distally by Lidocaıne. ¨ ŽA. Raw record obtained from a filament with two electrodes during fictive locomotion together with the activity of the Sartorius nerve ŽSrt n.. ŽB. STA showed that spikes of the unit were propagating in the antidromic direction. ŽC. The discharge of the unit is rank-ordered in a raster format triggered on the onset of activity in Sartorius nerve. Arrow 1 indicates the onset of burst in Srt, arrow 2, the end of the burst and arrow 3, the onset of the next burst. N s number of steps.

can be generated by the central terminal, either spontaneously Ždorsal root discharges. or as a result of stimulation of an adjacent dorsal filament or a peripheral sensory nerve Ždorsal root reflexes.. As detailed before, such discharges have also been seen in other situations such as during locomotion Žfictive and real. and fictive scratching. Spontaneous and evoked spikes can also occur in dorsal root ganglions as shown by several authors in different preparations w18,27,28,49,72,76x. In the dorsal root ganglions there seems to be rather complex interactions between cells that may lead to mutual excitation or mutual inhibition. When a nerve is cut, the neuroma, which develops at the site of the lesion is also capable of generating spontaneous spikes. However, the nerve section

itself exerts even more profound effects on the neuron since the peripheral section will also enhance the spontaneous discharges of dorsal root ganglion neurons w77x. Finally, although it is well known that suprathreshold activation of peripheral receptor will lead to spike generation at the first node of Ranvier, it has been shown in recent years that peripheral pain receptors can be sensitized so that pain receptor which are unresponsive say to joint displacement become very responsive to the same joint displacement after chemical treatments leading to joint inflammation w26,44x. It was shown w22x that there is a clear interaction between dorsal root reflexes evoked in a nerve and the orthodromic stimulation of that nerve. In Sural nerve,

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Fig. 10. Four examples of antidromic activity in primary afferent fibers during fictive locomotion. A, C, E and G: the discharges of units are rank-ordered in a raster format triggered on the onset of the Sartorius nerve activity Žsee Fig. 9 for the meaning of arrows.. B, D, F and H: the discharges of the units are presented in a histogram format. Arrows 1 and 3 correspond to arrows 1 and 3 in the rasters. For these histograms, the locomotor cycle was normalized to 1 and the counts in every bin were divided by the bin duration to obtain the mean firing frequency of discharge.

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situation where when the orthodromic discharge decreases there is an increase of antidromic discharges probably leading to secretion of substances which maintain the inflammatory process in the knee. Similarly, in our study we could show an increase in the number of antidromically conducting units after the transection of the filament or after its blockade with a local anesthetic. This is indicative for an ongoing interaction between orthodromic and antidromic flows of the same filament. There are two possible mechanisms for such interaction. The first would relate to an interaction of orthodromic and antidromic flows inside a single afferent fiber. It is possible that when a unit tonically discharges orthodromically, it is for some reason less prone to antidromic firing. The second possibility related to the interaction between orthodromic and antidromic activity flows in different afferent fibers. Before cutting or blocking the filament it is likely that any particular afferent was influenced by other afferents through presynaptic mechanisms w47x. Removing this influence from other afferents by section or anesthetic block may result in an increased excitability of the fiber and a higher probability of generating antidromic spikes at the central terminal. 4.3. Mechanisms of interaction between multiple sites

Fig. 11. Distribution of the activity of 12 antidromically discharging locomotor-related units within the locomotor cycle. Each trace represents the activity of one unit shown in a histogram format for the average locomotor cycle, which is displayed twice Žfrom 0 to 1.0 and from 1.0 to 2.0.. The ‘‘burst’’ of activity in each cell is arbitrarily highlighted by blackening that section of the histogram in the second representation of the cycle. All histograms are aligned on the onset of averaged burst of activity in the Sartorius nerve Žtop trace.. The frequency scale applies to all traces.

stimulation of the nerve evokes dorsal root reflexes, which will be depressed by a continuous natural stimulation of its receptive field. Although this interaction between the ortho and antidromic firing could occur at the ganglion, it could also occur at the interneuronal level. In the model of knee inflammation w73x, it was also shown that there is an interaction between the ortho- and the antidromic discharges. The spontaneous discharges of the peripheral receptors are needed to maintain the dorsal root reflexes. The interaction Žcollisions. would lead to a

Collision w17,34x work on spinal cats showed that dorsal root discharges were irregular Žmean interval 6.1 ms " 0.5 ms. and were postulated to block orthodromic discharges by collision w80x. Bayev and Kostuyk also proposed blocking of orthodromic discharges by collision with high frequency antidromic discharge bursts during scratching w4x. However, it is probable that an other mechanism may have a more prolonged effect on the orthodromic discharge of a unit. We have shown in the cat that antidromic stimulation of the distal stump of a cut dorsal root can retard or even stop some units activated orthodromically by activation of the receptors w39x. Short trains of stimulation just at threshold and repeated at about once per second as expected during locomotion were very effective in reducing the firing rate of the orthodromic discharge. Changes in excitability of the spike-initiating region Žfirst node of Ranvier. could be responsible for such changes in firing rate w52x. Thus, it could be that a few or a train of antidromic spikes evoked by central excitability changes during rhythmic activities such as locomotion, could result in a significant alteration in the discharge frequency of afferent units. Such a mechanism has recently been demonstrated also in the crayfish w16x. 4.4. Antidromic actiÕity of primary afferents during fictiÕe locomotion Forty four percent of the antidromic units Ž12r27. recorded in uncut dorsal root filaments blocked with Lidocaine were modulated rhythmically during fictive locomo-

I. BeloozeroÕa, S. Rossignolr Brain Research 846 (1999) 87–105

tion. That number is higher than the one estimated for the cut filaments which is only 19% Ž37r194. of the units discharged in a rhythmical burst pattern during fictive locomotion w32x. It is possible that this is due to a sampling bias of units that could be recorded long enough for STA before and after anesthesia. A greater variety of units could probably be recorded in former studies since, de facto, all recorded units in cut filaments were assumed to be antidromic. The number of antidromically active units estimated in the experiments with the intracellular recordings Žuncut dorsal roots. from the muscle afferents showed that the activity of 29% Ž9r31. of the units was modulated with the step cycle w42x. In this case, the smaller percentage may be explained by the fact that intracellular studies will necessarily introduce a bias towards larger fibers and, from our present findings, the majority of antidromic units are in the middle range of conduction velocities. Although the number of the locomotor-related units found in filaments blocked with Lidocaıne ¨ during fictive locomotion may differ somewhat from that recorded in the cut dorsal filaments w32x, the patterns of locomotor modulation was very similar. The activity of different antidromic units could peak in different phases of the step cycle; however, the majority were found to be modulated in the period where flexors are active Ž8r12. during the fictive step cycle. The predominance of flexor-related antidromic units confirms previous findings w32,42x. Taken together with the above considerations, it is possible that these antidromic discharges occurring during locomotion represents a form of interaction between central and peripheral processes, whereby central mechanisms could control the excitability of peripheral receptors and lead to a specific phasic control of afferent inputs during locomotion. This possibility also suggests that the afferents themselves could serve as a very active comparator of peripheral and central processes. Thus we may speculate that the final pattern of discharge reaching the interneurons and the motoneurons would reflect some integration of central and peripheral events. If this were the case, the hypothesis that primary afferents may also serve as local interneurons would be strengthened w50x. This hypothesis coupled with the idea that presynaptic mechanisms from descending pathways may affect different collaterals of the same afferent w35x suggests that altogether presynaptic mechanisms may indeed be a quite sophisticated mechanism of sensori-motor integration w68x during movements such as locomotion.

Acknowledgements This work was supported by a Group Grant of the Canadian Medical Research Council. I.B. was supported initially by a fellowship from the Groupe de Recherche sur le Systeme ` Nerveux Central ŽGRSNC, a FCAR Center. and later on by a visiting scientist award from the FRSQ.

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