Brain Research, 109 (1976) 255-269 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

255

C E N T R A L G E N E R A T I O N OF L O C O M O T I O N I N T H E S P I N A L D O G F I S H

S. GRILLNER§, C. PERRET* ANDP. ZANGGER**

Kristineberg's Zoological Station, Fiskebiickskil and the Department of Physiology, University of G6teborg, GiJteborg (Sweden) (Accepted October 31st, 1975)

SUMMARY After a transection of the spinal cord a dogfish performs continuous swimming movements with a phase lag between adjacent segments. It is shown that the intersegmental coordination remains after an extensive dorsal root transection as well as after curarization. In the former case the m o t o r activity was recorded electromyographically in several segments along the body, in the latter case the intersegmental coordination was evaluated by recording the efferent activity in different ventral roots along the body. It was concluded that a spinal central network can account for the phase lag observed between successive segments during swimming. It was also shown that the efferent activity from parts of the spinal cord with no dorsal roots intact could be influenced by peripheral stimuli such as pressure on the pelvic fins; this result suggests that some afferent fibres reach the spinal cord via the ventral roots.

INTRODUCTION The question if locomotor movements are generated by a central network of neurones or are due exclusively to some sort of chain reflexes has received due attention for a long period of time. It is now established that there are central networks generating these movements in various types of invertebrates16,22, a~ and vertebrates 10 including mammals2,1~,la, z3, amphibiansla, a0 and also very likely bony fishes 15. Some elasmobranchs as the dogfish (Squalus acanthias, Scyliorhinus canicula) have been used extensively for studies of locomotion 7,19-21,z5 since they can perform con§ Present address: Dept. Physiology, GIH, LidingSv~igen, 11433 Stockholm, Sweden. * Present address: Universit6 Paris VI, U.E.R. de Physiologic Animale (61), Laboratoire de Neurophysiologie Compar6e, 9, Quai Saint-Bernard, Paris-5e, France. * * Present address: Institut dc Physiologie, Universit6 de Fribourg, P6rolles, 1700 Fribourg, Suisse.

256 tinuous swimming movements after transection of the spinal cord. These species have been claimed~1,~5 to be entirely dependent on peripheral phasic signals for the coordination of the segments along the body. Roberts 25 (see also refs. 27, 28) has recently advocated this view based on experiments in which he was unable to record coordinated rhythmic efferent activity along the body after abolishing all phasic inflow from the periphery by curarizing the animals. His conclusion implies that the dogfish should be an exception among vertebrates. However, his negative findings could be interpreted otherwise and be due to several other factors such as the abnormally low rate of burst activity obtained after curarization (0.05 Hz). In a study of the intersegmental coordination, during swimming, in the spinal dogfishs,9, it was found that consecutive segments were activated with a certain lag as the undulatory wave passed along the body. This time lag varied markedly with the speed of swimming but constituted always a certain part of each 'swimming cycle' (i.e., a constant phase coupling)9(see also ref. 17). This phase coupling can be due either to a central network (see ref. 9, 15) as in crustaceans ~9, or to some kind of detailed reflex organization as suggested by Roberts zS. The main aim of this investigation has been to reinvestigate this problem in the spinal dogfish, by comparing the electromyographical activity during swimming before and after dorsal root transection and by recording the efferent activity after curarization. METHODS

Spinalization. Thirty dogfish (Squalus acanthias, length 50-105 cm) were spinalized at segment no. 6-8 by a transverse transection through the laminae of the vertebrae and the spinal cord under visual inspection. The operation wounds were closed carefully with stitches (see ref. 9). Transection of the dorsal roots. The dogfish were further operated 1-4 days after the spinalization under anaesthesia (tricaine methane sulfonate, MS 222, Sandoz). The fish was on a table with only the head protruded into a box continuously perfused with fresh sea water allowing respiration by natural gill movements. From time to time anaesthetic was added to the water. The back of the fish was opened with an incision in the midline between the two dorsal fins, exposing the dorsal parts of the cartilagineous vertebrae. The dorsal and ventral roots enter through the vertebrae completely separated (see ref. 26). It was therefore possible to transect the dorsal roots just outside the vertebrae, which is proximal to the dorsal root ganglion. Bilateral transection of the roots was performed under a dissection microscope for 20-25 segments (usually segment no. 30-50) and sometimes extended rostrally over the first dorsal fin leaving up to 35 segments without intact dorsal roots. In 3 animals the deafferentation was performed after a laminectomy allowing intradural transection of the dorsal roots just at their entry into the spinal cord ~1. After closing the wound the animals were put back into the tank. To correlate the level of 'deafferentation' with the corresponding 'body sur-

257 face segments', we explored the EMG response of the red lateral muscle (above which the segmental borders are clearly delineated on the skin) to electrical stimulation of the most caudal or rostral ventral root in the part without intact dorsal roots (see ref. 1). We also explored the skin by fine stimulation with a needle, which gave a clear contraction only in the innervated segments. The border of the 'deafferentation' coincides well with these two tests. Additional spinal transections and spinal cord stimulation. In 14 experiments the spinal cord was also exposed in a later stage and cut at the level of the first and the last deafferented segment. The part remaining between the two transections was then deprived of intact dorsal roots and connexions with the rest of the central nervous system. After each experiment the completeness of the different transections was confirmed. In 6 such preparations (including one curarized) the effect of stimulating the cut rostral or caudal end of the spinal cord was tested. It was mounted on bipolar silver (Ag-AgC1) electrodes and stimulated with 10-100 Hz at 10-500/zA strength (pulse duration 0.5 msec). The efferent activity was recorded as electromyogram (EMG) or from the ventral roots. Recording of electromyographic activity. For recordings the head of the fish was fixed in a frame mounted in a tank (130 cm × 80 cm), continuously supplied with fresh sea water, while the body was free to exhibit swimming movements (see ref. 9). In this way the preparation could be kept alive in good condition for several days even after deafferentation. The EMG activity was recorded with small coaxial electrodes connected by thin cables via preamplifiers to an 8-channel inkwriter (Mingograph, linear frequency response up to 1200 Hz) and an oscilloscope. The electrodes were inserted just below the skin in the red musculature near the lateral line. Ventral root recordings under curarization. After exposing the vertebrae as described above, two or more ventral roots were dissected for approximately I cm from their exit through the vertebrae (segment no. 30-50). Each animal was then fixed by 4 clamps around the vertebral column with only the ventral part of the body in sea water. This procedure was used in the latter part of the experiments and was possibly of importance for normalizing the external pressure conditions around the body and thereby improving the circulation as compared to when the body was kept 'dry' on the table. For a control of the condition of the preparation the heart rate was monitored by ECG-recordings between two electrodes in the heart region. The normal rate was around 20 beats/min and its decrease clearly indicated the deterioration of the preparation. Injections were performed through a cathether in a subcutaneous vein located in the ventral midline or in the infraorbital sinus. After an i.v. injection of o-tubocurarine (5-10 mg/kg body weight) the mouth of the animal was perfused directly with sea water. The cessation of the gill movements was found somewhat unreliable as an indicator of a complete curarization, since it can occur at a relatively low dose of D-tubocurarine, which does not markedly influence the neuromuscular transmission in other parts of the body. We have therefore in addition tested the efficiency of the

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Fig. 1. EMG activity during swimming before and after dorsal root transection. The recordings arc from the same animal before (A) and after (B and C) cutting the dorsal roots of the segments no, 30-50. A and B show spontaneous activity, when the fish is swimming in the tank (see Methods), whereas C was recorded under tonic tail fin stimulation (clip at tip of the tail). The numbers of the segments recorded from a~e indicated to the left. In the recording in C there is in all likelihood some pick up from adjacent white muscle fibres.

curarization by (1) stimulating electrically the distal part of the ventral roots and checking for any EMG activity evoked in the red and white muscle fibres supplied by that particular root and (2) by observing if a stimulation of the spinal cord can elicit any detectable movement of any part of the body. Without curarization, very apparent movements over the entire body are elicited by such a stimulation. If these tests were negative the preparation was considered completely curarized. Recordings of the ventral roots were performed with bipolar Ag-AgCI electrodes or with suction electrodes. The efferent activity was recorded conventionally on an oscilloscope and moving film. RESULTS

(I) Is there an intersegmental coordination without phasic afferent input? In the swimming spinal dogfish with its dorsal roots intact there is in each segment an alternating activity of the two sides with a frequency that can be varied (see legend Fig. 1) within a large range (0.4-2.8 Hz). Along the body there is a lag between the activation of consecutive segments with the rostral segments leading (Fig. 1A). The duration of this lag varies with the 'speed' of swimming but it always occupies a given part of the cycle (or the burst discharge on one side), i.e. there is a phase coupling 8,9. The pattern of activity along one side of the body is shown in a schematical form in Fig. 2 (left graph). The bars indicate the period of E M G activity recorded at different locations simultaneously in 10 consecutive cycles and the dots the termination of each cycle. It is noticable that the bursts are somewhat shorter at more caudal segments. To elucidate how the intersegmental coupling is accomplished, we have performed 3 different types of experiments. (a) The effect of bilateral transection of the dorsal roots over 20-35 segments.

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Fig. 2. Comparison of the segmental muscle activity before and after dorsal root transection. In the two graphs to the left the period of EMG activity during 10 consecutive cycles of spontaneous swimming is plotted as bars with a dot to the right indicating the termination of the cycles. The two graphs compare the activity in 6 different locations (segment no. 17-56) along the same side of the body before (left) and after (middle) bilateral dorsal root transection of the segments no. 30-49. For the graph to the right, the individual values in the two graphs to the left are normalized (division by cycle length) and plotted as mean values (4- S.D.). The graph thus compares the average values before (upper) and after (lower) the dorsal root transection at the different locations.

Fig. 1B and C are recorded after bilateral transection of dorsal roots no. 30-50. The EMG activity in this region (segment 31-44) is still coordinated and a lag remains between the activation of e.g. segments 34 and 44, which resembles that recorded in the same fish before the dorsal root transection (Fig. 1A). At faster rates as in C the lag shortened as for the intact fish. The same type of data is compared for several cycles before and after deafferentation in Fig. 2 in the left and the middle graph (see above and legend). The pattern of activity in regards to the regularity of the burst activity, the intersegmental delay, and the period length appears virtually unchanged. This is also shown in the right graph in which the individual data from the same two sequences (in Fig. 2A and B) are pooled and normalized to a cycle duration of 1. A similar comparison between intact and 'deafferented' conditions in Fig. 3C shows the increasing ~0-1ag along the body under these two conditions. These results apply to 11 out of 12 operated fishes. To understand if this intersegmental delay varies with the speed of swimming also after dorsal root transection, the relation between the intersegmental delay is plotted versus the duration of burst discharges in Fig. 3A and B for two different fishes. The ordinate is normalized to delay/segment (i.e., the lag between two recording points is divided by the number of segments between them). The graphs show a clear dependence between the delay and the burst duration with a linear correlation similar to that observed in 'intact' spinal dogfish. It was often observed

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Fig. 3. Time lag between activation of segments with transected dorsal roots at different speeds. For two preparations (A and B) the time lag between two recording points (normalized to time lag per segment) during forward swimming is plotted versus the duration of burst discharges as the speed of swimming varies. The lines for linear regression are drawn together with the corresponding correlation coefficients. In C the phase lag (lag from onset of burst activity in segment 17 to onset of activity in segment 25-56 divided by period length) for the segmental muscle activity of one side is plotted versus the no. of the segments recorded from, before and after transection of the dorsal roots no. 3050. Each value is the mean (± S.D.) of 15 or more cycles recorded at various swimming velocities (frequency of burst discharge).

that the first 2-3 deafferented segments were less active than the other ones as e.g. in Fig. l, where the second electrode (no. 31) was just caudal to the transition between the afferented and 'deafferented' part. In about 50 % of the deafferented preparations the leading segment was shifted by the deafferentation to a position 5-10 segments caudal to the rostral 'deafferentation' border. The above results show that even without dorsal roots the segments along the body can still be well coordinated and their phase coupling remain (see Fig. 3A and B). In these preparations 'backwards swimming' (i.e., reversed coupling between segments) could also be induced as for spinal animals with intact afferents 9. (b) Bilateral transection of the dorsal roots combined with rostral and caudal spinalization. For the preparation described above it could be argued that the 'deafferented' spinal cord receives its phasic input via propriospinal fibres from the afferented neighbouring regions. We have therefore further operated 13 such 'deafferented' preparations by transecting the spinal cord at the level of the most rostral and most caudal cut dorsal root. The remaining piece of the spinal cord was consequently only neurally connected with the rest of the body via its ventral roots. This additional operation was performed 2-36 h after the first. Rhythmic alternating activity could be recorded in 12 out of 14 such preparations (Figs. 4A and 6A ventral root recording) within a few minutes after the spinal transection. This activity had a somewhat higher frequency at rest (range 0.3-1.8 Hz) than the ordinary spinal preparations (around 0.4-0.5 Hz). The burst duration was often very short as in Fig. 6A and B and could be below 10% of the cycle length. This value could vary during one experiment and probably reflects the general excitability level in the motoneurones. In other preparations the burst duration could be normal or even exceed 50 % of the cycle (Fig. 4A). Fig. 4A shows that in one such 'isolated' preparation the activity is well ¢o-

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Fig. 4. Coordinated muscle activity in a 35 segment preparation with complete dorsal root transection combined with rostral and caudal spinalization. In A each group of bars represents the muscle activity during 14 subsequent swimming cycles recorded in 5 different segments (number indicated to the left). The dots mark the cycle lengths. The leading segment (pacemaker) is probably slightly rostral to segment no. 34. The same delay between the onset of segmental muscular activity is plotted in B (expressed as phase lag) v e r s u s the number of the recorded segment. Only the part with 'forward' swimmingis taken (segments no. 34, 46, 49). Each value (O) is the mean 4- S.D. of 42 swimming cycles. The data obtained before the additional spinalizations at segments no. 17 and 53 are shown in the same way (X). • shows the phase lag for another "isolated" preparation (segments no. 30~9). C demonstrates the functional separation of the spontaneous rhythmic activity after an additional spinalization performed between the recording points (segments no. 23-36). The caudal part is an 'isolated' preparation (segments no. 30-49) from which 16 consequtive cycles are plotted (as in A) together with the ipsilateral activity at segment no. 23. ordinated between the different segments, and furthermore that there is a lag between the different segments with segment no. 34 leading the more rostral as well as the more caudal ones. In 4 out of the 12 preparations the caudal segments were leading the more rostral ones, whereas in the remainder the middle or the rostral segments were leading. It should be recalled that the spinal dogfish can exhibit forward swimming and under particular conditions also 'backwards' swimming and that it is common that one of the segments in the rostral third is the leading one 9. In the same preparation used in Fig. 4A the phase lag between segment no. 34 and more caudal segments is compared (Fig. 4B) before (open circles) and after (crosses) the spinal transection. This lag is longer in the isolated preparation, but this should be interpreted with some caution since the burst durations are unusually long in Fig. 4A. In other such preparations with short period length the lag was shorter. In general it can be stated that the muscle activity is more feeble in these 'isolated preparations' and it has often been difficult to decide the exact onset and termination of the period of E M G activity. The resulting movements were so weak that they were usually only detected if the preparation was watched carefully and thus the gross 'normal' swimming movements were lacking. The reason why Lissmann 2t reported that no rhythmical activity occurred after a complete deafferentation, was presumably that the mechanical recording techniques used were not sensitive enough. Fig. 4C shows that independent spontaneous activity can be exhibited by two adjacent parts separated by a spinal transection. The activity in one 'isolated' preparation (segment 36) is shown for 16 consecutive periods which is compared with the activity in segment no. 23 which has its dorsal roots intactL The period length at

262

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Is Fig. 5. Efferent activity recorded in two ventral roots after curarization. A shows the activity in two ventral roots supplying segments no. 35 and 45 during resting conditions. The rhythm was very stable, 0.56 ± 0.01 Hz. It is apparent that the activity in the two segments are tightly linked. B shows the effect of very light continuous pressure on the tip of the tail fin which increased the rate of activity markedly. The preparation was rigorously tested to show that the curarization was complete (see Methods). segment no. 23 is somewhat shorter and therefore we can observe that the E M G activity (23) starts in each cycle earlier than in the previous cycle when related to the activity at segment no. 36. These results allow us to conclude that the spinal cord devoid of its dorsal root inflow can generate rhythmic coordinated activity. However, unexpectedly, we found that this activity could still be influenced from the periphery and we therefore must assume that there are afferents entering the spinal cord via the ventral roots. These results are presented below in section II. (c) Ventral root recordings in the curarized spinal dogfish. The results with dorsal root transections discussed above effectively rule out the possibility that the intersegmental coordination should depend exclusively on the dorsal root fibres discussed by Roberts 25,27,2s. On the other hand, these experiments raise the possibility that the intersegmental coordination could depend upon phasic information from either ventral root afferents or mechanoreceptors within the spinal cord 2. It was therefore necessary to test if rhythmic coordinated activity could remain when all mechanical activity was suppressed. This was performed by blocking the neuromuscular transmission with tubocurarine and thereby the movement, while recording the efferent activity in the cut ventral roots (see Methods). Fig. 5A shows recordings from two ventral roots 10 segments apart when the preparation was left undisturbed after curarization. The efferent activity showed a very clear rhythmicity which was very regular (0.56 Hz ± 0.01 S.D. for 40 consecutive cycles). Activity in the same range could be recorded for several hours after the curarization (6 h). Whenever the tail fin was touched or stimulated by very light continuous pressure (between index finger and thumb) an immediate increase in the rate of burst activity (1.5 Hz) occurred (Fig. 5B). The same type of stimulus has a corresponding effect on the 'intact' swimming spinal dogfish 7,9,2°. The burst activity in the individual segment appears thus unchanged. It is apparent that the efferent

263

Fig. 6. Peripheral effects on the spontaneous rhythmic discharges after dorsal root transection combined with rostral and caudal spinalization. Preparation as described in Results in part lb. A • EMG recording from the left (L) and right (R) side of the red musculature belonging to segment no. 36 after 'isolation' of the spinal cord between segments no. 30--40. Bending to the left leads to an immediate and longlasting inhibition of the ipsilateral activity, whereas the stretched right side exhibits a tonic activity for 10-15 sec followed by the old rhythm (not shown). B and C: same arrangement as in A but in a different preparation. Recording electrode in segment no. 43. The graph in C shows the period length v e r s u s time for the activity recorded in B. The period length is influenced somewhat before the actual stimulation (arrow 1). Between arrow 2 and 3 strong tonic pressure is exterted on pelvic fin (the fin is pressed between one finger and the table surface beneath). Releasing the preparation from the stimulus (arrow 3) leads to reset to the old rhythm (0.8 Hz). D: similar representation as in C but another preparation; 1st and 3rd arrow indicate the effect of a short lasting bending of the body to the side recorded from followed by an immediate movement of the body back to a 'neutral' position. Note that the prolonged period is directly followed by a 'normal' cycle. A corresponding short lasting stretch of the recorded side results in a shortening of the cycle. activity in the two ventral roots are strongly coupled at resting rate as well as d u r i n g faster activity (Fig. 5A a n d B) a n d that there is a lag between the activation o f the two segments. The activity recorded in the same segment on the ipsi- a n d the c o n t r a lateral side is alternating. In 5 p r e p a r a t i o n s we have recorded resting rhythmic c o o r d i n a t e d activity which could be enhanced as described above by tonic stimuli such as light pressure on the

264 tail fin (see legend, Fig. 5). However, in several preparations only continuous or irregular activity could be recorded in the ventral roots but this was usually correlated with bad experimental conditions as revealed by low heart rate etc. (see Methods). We therefore conclude that after curarization the intersegmental coordination remains as well as an alternating activity between the two sides in each segment.

(II) Are there afferent fibers in the ventral roots of the dogfish? After a transection of the dorsal roots combined with a rostral and a caudal spinal transection (as in section I(b)), it would be expected that the motoneurones could not be influenced from the periphery. On the contrary Fig. 6A shows that the rhythmic alternating activity can be entirely silenced by bending the body to one side, whereas on the side that was stretched the E M G activity increased and became continuous. After 10 sec the burst activity reappeared but the burst on the 'stretched' side was then much greater than on the other side although the rhythm was unchanged. This pattern was very consistent and thus it was concluded that the position of the body could influence the electromyographical activity. The effect of phasic stimuli was tested in Fig. 6D by bending the body from one side to the other rapidly. It can be seen that when bending (lst and 3rd arrow) the body to the side of the recording electrode the subsequent burst is occurring with a long interval, but when this side is stretched instead a shorter interval is recorded (see also legend), whereas the subsequent burst has a normal period length. To assure ourselves that this activity was of central origin, we have also recorded from the central part of the cut ventral roots (the vertebral column was fixed above and below the recording site but the rest of the body could be moved). The efferent activity was influenced by the body position with the corresponding results as in Fig. 6A. Only in the pelvic region including the pelvic fins could we demonstrate that pinpricking or scraping the skin had an effect on the efferent discharge i.e., after dorsal root transection. When tonic pressure was exerted over the pelvic fins this caused a marked increase in the frequency of burst discharges as between arrow 2 and 3 in Fig. 6B and C. Hence it can be concluded that tonic stimuli from the body surface could influence the spinal cord and modify its output. Consequently information from the body surface must reach the spinal cord and it is difficult to see any other route than via the ventral roots. This last finding excludes the possibility that the effects could be entirely due to intraspinal stretch sensitive elements as described in the neural cords of invertebrates 3. To make sure that the dorsal root transection performed just outside the vertebrate was not leaving any fibres undamaged (see Methods) intradural transection of the dorsal roots was also performed (3 experiments)just at their entry into the spinal cord. The same results were obtained with this procedure. Based on this indirect evidence we therefore have to postulate that the ventral roots in the dogfish carry afferent fibres. However, no recordings have been performed from such afferents. Roberts 26 attempted to record afferent activity in the ventral nerves (continuation of the ventral roots) of the dogfish when manipulating the

265 body surface mechanically. He investigated the ventral nerves supplying part of the abdominal wall with negative results. We have repeated his experiments (n = 8) and in addition induced muscle contractions in the neighbouring segments by stimulating the adjacent ventral nerves while recording in one ventral nerve. We obtained the same negative findings. Therefore we made a few preliminary experiments (n = 3) with the ventral nerves supplying the more caudal pelvic regionr, 24 (no. 31-47) by dissecting them directly after their exit from the vertebral column. Also in these experiments we did not obtain any positive evidence for afferent fibres. Since all recordings were performed with ordinary bipolar silver electrodes, the chance of detecting thin myelinated or unmyelinated fibres is small. We therefore suggest that there are thin afferent fibres in the ventral roots of the dogfish. Since we have distinct effects only from the pelvic region (segment 31-47) it is possible that these afferents are limited to these segments. It is interesting to note that the rich contribution of unmyelinated afferent fibres recently found in the ventral roots (lumbar 7) of the cat originates to a large extent from pelvic organs like the bladder4,L

(III) The effect of electrical stimulation of the spinal cord on the rhythmic efferent activity To investigate if tonic stimulation of descending fibres in the spinal cord could influence the rhythmic efferent activity, we performed 5 experiments in the spinal 'isolated' preparation (as in section I(b) and II) and in one curarized preparation (as in section I(c)). Continuous (50 Hz) bipolar stimulation (10/~A) of the cut rostral end of the spinal cord resulted in an increased frequency of burst discharges as shown in, e.g., Fig. 7A and in addition a much larger level of activity in each burst. In Fig. 7B is shown a continuous recording from two segments with increasing strength of stimulation. It is apparent that the frequency of burst discharges increases (period length decreases) with stimulation strength (stimulation frequency kept constant at 50 Hz) as shown in the graphs of Fig. 7C based on the data in B. However, with stronger stimulation the bursts take a relatively larger part of the cycle duration. The frequency of the stimulation applied was usually 50 Hz but could be varied between 10-100 Hz. It is evident that a stimulation of the entire spinal cord is a very crude stimulus and different fibres with different or even competing functions are activated. Consequently it is not surprising that the effect of stimulation varied between different experimental conditions. In most experiments (n ---- 5) we could obtain results as in Fig. 7 but sometimes other effects as an inhibition or deceleration of the burst activity occurred. A detailed study of the effect of localized stimulation in different spinal cord tracts (fibres stimulated) is required to clarify the localization of the responsible fibres. Nevertheless, it can be concluded that a stimulation of the spinal cord can increase the rate of rhythmic activity (see also ref. 20, 21). It is possible that the responsible fibres could correspond to fibres from the brain that take part in the control

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o f the rate of swimming, i.e. fibres similar to the c o m m a n d fibres in invertebrates ~1 a n d possibly m a m m a l s l L The results reported in Fig. 7 could be m o s t easily explained if the s t i m u l a t i o n gave two different effects, possibly mediated by different systems: (1) to increase the level o f activity in the central n e t w o r k generating the b u r s t activity; a n d (2) to increase the excitability level in the m o t o n e u r o n e s a n d thereby increase the d u r a t i o n of each b u r s t discharge.

267 DISCUSSION

Central generation of locomotor rhythm Roberts 25 performed a study on the efferent rhythmic activity recorded in ventral nerves in the curarized dogfish. He summarized his results by stating that 'The spinal neurones on each side of the cord are sufficiently organized to discharge alternately but the longitudinal coordination of the locomotory wave is disrupted in the absence of phasic sensory input'. Our results (Fig. 5) show that this conclusion is erroneous and that the rhythmic efferent activity is still coordinated along the body in the curarized dogfish. Hence no phasic information from the periphery is required for generating this longitudinal coordination. It is important to note that the range of frequencies (of bursts) obtained in the curarized preparation coincides with that found in the swimming spinal dogfish, for resting conditions as well as during faster swimming. Roberts 25 recorded, on the other hand, rhythmic activity at a rate of 0.05 Hz which is l0 times lower than the frequencies found during undisturbed swimming (0.4-0.5 Hz). The explanation for his inabilityz5 to record 'curarized swimming' at a 'normal' rate and with intersegmental coordination might be related to one or several of the following factors. (1) The efferent activity was recorded in the ventral ramus of the ventral nerve supplying the abdominal wall. This branch can be expected to contain few fibres to red muscles as compared to the median branch supplying the bulk of the red muscle fibres from which the resting efferent activity can easily be recorded. It is thus possible that the ventral branch contains very few efferent fibres that could be expected to show rhythmic activity at rest. (2) During recording, Roberts' preparation 25 was lying on its back with an opened abdomen and a rather abnormal posture, which might give rise to afferent signals that could in some way prevent the motor rhythm. (3) When injecting o-tubocurarine i.v., we often observed a sudden drop in heart rate from around 20 beats/min to 1-2 beats/min, which was invariably accompanied by a rapid deterioration of the preparation. These (1-3) or other factors might cause the very low level of activity observed. The lack of intersegmental coordination 25 could be a direct consequence of these conditions or be due to that the neuronal mechanisms responsible for the phase coupling cannot operate at a rate reduced to 0.05 Hz. It is well known that peripheral stimuli can markedly enhance the rate of swimmingT M . On the other hand peripheral signals are apparently not required for a proper coordination of the swimming movements (Figs. 1-3 and 5). Roberts 28 described one subcutaneous mechanoreceptor which would have been well suited to provide the necessary afferent information for the intersegmental coordination. Although the central connexions of these dorsal root afferents are unknown, our findings do not deny the possibility that the afferents normally are connected in a way that would stabilize or strengthen the intersegmental coordination and perhaps compensate for imposed external perturbations (see ref. 10). Roberts z7 has even shown that the rhythmic swimming activity can under some conditions be made to follow a rhythm imposed on the body by the experimentor. The finding that the feeble rhythmic activity in the 'isolated' spinal preparation could be influenced

268 from the periphery (Fig. 6) indicates that even the 'presumed ventral root afferents' (see above) could be of some importance. How is the central intersegmental phase coupling achieved? It is known that if the spinal cord is cut in pieces of 8 segments or perhaps even less 9, rhythmic activity with maintained intersegmental coordination (within these parts) still remains. The capacity to generate locomotion is thus distributed throughout the spinal cord; afferents are not required (see above). A similar situation exists in crustaceans in which the different ganglia controlling the 'tail' are centrally phase coupled. It has been shown that this is achieved by a set of'coordinating neurones '29, whereby the activity in one generator in one ganglion can influence the generator in the adjacent ganglia and so forth. Our results could be similarly explained by one generator for alternating activity in each segment that is phase coupled with the generators in the adjacent s,9 segments. The evidence for the presumed ventral root afferents are evaluated in Results. ACKNOWLEDGEMENTS This work was supported by the Swedish Medical Research Council (Project No. 14X-3026) and Wilhelm and Martina Lundgrens Vetenskapsfond. C.P. was supported by a grant from the Swedish Natural Science Research Council (R 3531001) and P.Z. by a grant from the Swiss National Foundation. The hospitality of the staff of Kristinebergs Zoological Station is gratefully acknowledged.

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