The central nervous organization underlying control of antagonistic muscles in the crayfish. II. Coding of position by command fibers

The Central Nervous Organization Underlying Control of Antagonistic Muscles in the Crayfish ’ 11. CODING OF POSITION BY COMMAND FIBERS

DONALD KENNEDY, WILLIAM H. EVOY,2 BENJAMIN DANE3 AND JOANNA T. HANAWALT Department of Biological Sciences, Stanford University, Stanford, California

ABSTRACT Single central interneurons that produce flexion or extension in the crayfish abdomen act in a coordinated fashion upon several ganglia. Each of several elements evoking a similar type of movement has a unique distribution of output to ganglionic centers: thus one of them may produce primarily rostral extension or flexion, another a primarily caudal movement, st i l l another a more general one. Each motor command therefore appears to code for a spec& abdominal geometry. Some units produce complex, cyclic motor outflow to postural muscles of the abdomen, or to the appendages.

The posture of the crayhh abdomen is controlled by the activity of tonic extensor and flexor muscles that operate each of the joints between the first five segments. The flexors, and probably the extensors as well, show serial homology in the arrangement of the muscle sheets and in the number, size, distribution and discharge patterns of motoneurons (Kennedy, Evoy and Fields, ’66). Kennedy and Takeda (’65) showed that five excitatory and one inhibitory axon supply slow flexors in the third segment. Their analysis of the distribution, sponta- neous activity and neuromuscular actions of these efferent neurons can be applied with only minor variations to the other abdominal segments.

We showed in the previous paper (Evoy and Kennedy, ’67) that a limited number of interneurons influence the discharge of tonic efferents in a particular segment. These “command fibers” act on the appro- priate postsynaptic cells to produce coordinated outputs which may be categor- ized as flexion, extension or suppression. Flexion elements, for example, excite flexor motoneurons, inhibit extensor motoneurons, and drive the peripheral inhibitor to the extensors. Within each category, there is some further differentiation. Command fibers for a particular function may differ in efficacy, or in the degree to which they selectively activate certain motoneurons, or

J. EXP. ZOOL., 165: 239-248.

in the characteristic temporal patterns of motor discharge they produce.

In the present experiments we ask whether such motor commands are also differentiated as to segmental distribution. In abdominal movements, the sequence and extent of activation of the different segments is critical. Crayfish hold their abdomens in a variety of shapes, as if the flexor/extensor ratio could be varied in- dependently in different segments. One might thus expect each command fiber to produce a unique distribution of motor outflow. For example, of six extension command units, one might excite all segments equally, one might affect only segments 1, 2 and 3, another 3, 4 and 5, another predominantly 2, and so on. Such a control system would involve an investment of only 20 or 30 interneurons in one half of the nerve cord, some of which might also control other motor systems. It would permit considerable flexibility, since com- binations of activity in two or more parallel elements could program a wide variety of movements.

Our results show that the command elements serving a particular function differ

1 Sup orted by grants from the U. S. Air Force Office of Scientific Research (AF-OSR 33466) and the U. S. Public Health Service (NB-02944).

2 Present address: Laboratory for Quantitative Biology, Department of Biology, University of Miami, Coral Gables Florzda.

3 Present Address: Department of Biology, Tufts University, Medford, Massachusetts.

239

240 D. KENNEDY, W . H. EVOY, B. DANE AND J. T. HANAWALT

in their region of influence. This organization is the counterpart, in the motor system, of the parallel arrangement of sensory interneurons that collect input from abdominal tactile receptors (Wiersma, ’58; Wiers- ma and Hughes, ’61). In that system, each element integrates a unique combination of sensory fields, though a particular locality may be represented by many different interneurons. The motor apparatus of each segment is, similarly, under the infiuence of many command interneurons. Each of the latter affects that segment’s ganglionic activity in some unique proportion with that of other segments. Thus the individual controlling elements may each be specific for some particular postural geometry.

Animals were prepared for electrophysio- logical experiments as previously described (Evoy and Kennedy, ‘67) except that the ganglionic roots of up to four abdominal segments were exposed for recording. Ac- tivity in these roots was recorded extra- cellularly with patinum wire electrodes, amplified, and displayed on a multiple channel oscilloscope. Small bundles of axons were dissected from interganglionic connectives for stimulation.

Changes in the shape of the abdomen were correlated with the activity of a single rostra1 flexor nerve in intact preparations. Animals were placed ventral side up in a lucite chamber, pinned in such a way that only the first 2 abdominal segments and the cephalothorax were immobilized. Ac- tivity in the superficial branch of a third root of ganglion 1 or 2 was recorded with a flexible suction electrode drawn from polyethylene tubing and applied to the end of the cut nerve. The posterior abdominal segments were left free and could flex and extend fully without disturbing the recording conditions. Command fibers were dissected from the connective anterior to the segment recorded. A 16 mm Arriflex camera was used to take color motion pic- tures of abdominal movements at 24 frames/second. A photocell connected to one channel of the oscilloscope recorded the opening and closing of the camera shutter, permitting correlation of electrical and cinematographic records. The periods of command fiber stimulation and oscillo-

MATERIALS AND METHODS

scope recording were indicated on the motion picture film by neon-bulb monitors, and movements were analyzed by project- ing the motion picture with a time-motion study projector which permitted frame-by- frame advance of the a m . The positions at the beginning and at the end of stimulation were traced as outlines, and inter- mediate stages were plotted by following the change in position of marks painted on the abdomen. In this way, the direction and velocity of movement of each segment could be determined for each period of stimulation, and correlated with the recorded neural discharge. An account of many of the photographic and analytical techniques may be found in Dane and Van der Kloot (’64). Purely behavioral observa- tions of movements in response to natural stimuli or centrally originating “sponta- neous” changes were made in the same way, using restrained but otherwise intact animals. A film demonstrating these experiments has been prepared, and can be made available for loan on request.

RESULTS

1. Segmental distribution of command fiber influence

Stimulation of single command fibers in a caudal interganglionic connective yielded coordinated output in each of a number of segments. In any particular segment, the flexor and extensor motoneurons showed reciprocal output, and the discharge frequencies in homologous roots on the two sides were similar (Evoy and Kennedy, ’67). A given command element produced re- sponses of the same type (e.g., flexion) in all of the ganglia it affected. When command fibers selectively excited certain motoneurons, they did so for homologues in all affected segments.

In addition to having the selective actions upon motoneurons which were observed in recording from a single segment (Evoy and Kennedy, ’67), command fibers also differ from one another in the degree to which they af€ect individual members of a whole series of segments.

Figure 1 illustrates such differences among five command fibers isolated from the 5-6 connective in a single preparation. Each of the elements, when electrically

ACTION OF COMMAND FIBERS IN CRAYFISH 241

Fig. 1 Distribution of effectiveness of five extensor command fibers in a single preparation. Right, records of discharge from the superficial branches of the third roots of ganglia 1 through 4 during stimulation of each command fiber. In each record the top trace indicates stimulation (at 100/ sec) of the command fiber. Second through fifth traces are the discharges of flexor efferents from ganglia 1, 2, 3 and 4. In each case the smaller units active before stimulation are inhibited and a new, larger unit is recruited; this is the flexor inhibitor. Left, plots of the average frequency of flexor inhibitor discharge in each segment for each of the five command fibers. The symbols COR@ spond to actual records as indicated on the right.

stimulated at 100/sec., evoked repetitive imal output from a different abdominal discharge in the flexor inhibitors of seg- region, and therefore should have produced ments 1-4. The output frequency of the maximal extension at a different point. flexor inhibitor was taken as a measure Larger numbers of command fibers were of the synaptic efficacy of the command in isolated from other single preparations, but that ganglion. The frequency plots show it was often impossible to determine the that each command element caused max- distribution of segemental effects for each

242 D. KENNEDY, W. H. EVOY, B. DANE AND J. T. HANAWALT

one because of the presence of other, com- peting elements with similar thresholds in the same bundle. However, enough preparations yielded series of either flexion or extension command fibers so that this distribution could be examined, and the results were qualitatively similar to those shown in figure 1.

Although an accurate count of the command fibers belonging to each class cannot be made with present techniques, accum- ulated estimates from a large number of preparations indicate that any category comprises at least six units, each one with its motor output distributed uniquely over the six abdominal segments. There may be further duplication related to specialization for other purposes, e.g., specificity of motoneuron excitation.

Attempts have been made to measure delay times for the interganglionic spread of command fiber effects. The onset of excitation is detectable only as the first of a series of shortened intervals when the recorded unit is spontaneously active, and it is therefore difficult to measure such delays in preparations that have high levels of endogenous discharge or weak central effects. In several preparations in which these measurements could be made, the interganglionic delay time was 30 t 10 msec. Measurements of conduction velocity have been made in a few of the larger command fibers (Kennedy, Evoy and Hana- Walt, '67). Using these data, the calculated conduction time between two adjacent ganglia is 1.5-2.0 rnsec, a figure that amounts

n

to less than 10% of the observed delay. In a few experiments, we stimulated single command fibers while recording from a motor root that was isolated from their direct influence: for example, an interneu- ron in the 3 4 connective was cut caudally, left attached rostrally, and stimulated to determine its effect on motor discharge from ganglia 3 and 4. Weak effects were sometimes seen caudally in such experiments, and these were quite different in pattern from those evoked by direct influence of the command element upon anterior ganglia. They may therefore have been due to the activation of one command channel by another.

2. Movements evoked by command fiber stimulation

The movements of the abdomens of unrestrained animals were analyzed to test the hypothesis that the unique segmental distribution of command elements enables them to produce specific configurations. Fibers were isolated from the connective between the last thoracic and the first abdominal ganglion; the first abdominal segment was pinned to permit recording from the flexor motoneurons comprising one of its superficial third roots and the rest of the abdomen was left free to move.

Repeated trains of stimulation applied to the same command fiber evoked movements that were nearly identical in ampli- tude and speed. The final positions were also congruent, provided that background activity permitted identical starting posi-

Fig. 2 Initial (solid) and final (dotted) positions of the abdomen, traced from cinematographic records at the beginning and at the end of stimulation of an extension command fiber at 100/sec. The left and right pairs represent repeats of the experiment with the same command element. Note that the initial and final positions in the two trials are identical, except that the telson and uropods were more flexed both at start and finish in the second.


tions. The repeatability of these actions is demonstrated in figure 2 which shows sketches of initial and final positions taken from the film analysis of two successive movements evoked by stimulation of the same command fiber. The initial positions were not quite identical; the final positions differed by an equivalent amount. In other experiments, extension and flexion command elements were isolated, placed on separate pairs of electrodes, and stimulated alternately. In this way, more identical starting positions could be provided for each succeeding movement. The results of these experiments confirmed that single command fibers consistently produce movements of a particular spatial and temporal character.

Since most commands affect a series of segments, changes in neural output recorded in the immobilized first segment were usually well correlated with the movements. Flexion at any point in the abdomen was accompanied by accelerated discharge in the flexor motoneurons of segment 1, and extension with activity in the flexor inhibitor. The frequency of discharge was also correlated with the rare and the segmental distribution of abdominal movements. In general, rapid movements were associated with high discharge frequencies, slow ones with lower frequencies. For movements of approximately the same speed, those appearing most strongly in the rostral segments naturally produced stronger motor output in segment 1 than did those involving primarily the caudal segments.

Figure 3 illustrates the differences in final position resulting from the stimulation of two different extension command fibers in the same preparation. The first one (3, top) caused extension predominantly in the rostral segments, while the second ( 3 , bottom) had a more generally distributed effect. The first of these movements was completed in 3.8 seconds, the second in 9.3 seconds. Figures 4 and 5 show records from the slow flexor efferents of ganglion 1 that correspond to the position changes illustrated in figure 3. Dis- charge in the flexor inhibitor developed rapidly and reached a very high maximum fresuencv during the quicker, more rostral

Fig. 3 Initial (solid) and final (dotted) positions of the abdomen, traced from cinematographic records at the beginning and at the end of stimulation of two different extension command fibers at 100/sec. Upper, a relatively rapid movement (see text) involving primarily the 2-3 joint (the more caudal joint angles change only a little). Lower, a slower extension involving a l l the caudal segments, produced by a second command fiber in the same preparation.

more generally distributed one (3, bottom; 5), it required a much longer time to reach its maximum frequency, and the latter had a lower value.

Similar differences in segmental involve- ment were shown for flexion command elements. The initial and final positions for two such fibers isolated in the same preparation are given in figure 6. As the outlines show, the first command fiber ( 6 , top) caused flexion in two anterior segments; these segments were not moved at all by the second command (6, bottom).

3. Complex effects Some command fibers were found to

evoke cyclic waves of excitation in the tonic flexor motoneurons. Sequences of motor activity progressed in a caudal- rostral direction, with delays of one second or more between the recruitment of adia-

movement (3, top; 4): In the slower and cent ganglia. One of these command fibers


Fig. 4 Records from the superficial branch of the third root supplying the left slow flexor muscles of segment 1, recorded simultaneously with the movement shown in figure 3 top. Lower trace indicates stimulus onset ( l O O / s e c ) and carries a 10 msec time mark. Records are continuous except that two seconds have been removed between the first and second rows. The arrow marks the time at which the movement was completed (3.8 seconds from the onset of stimulation). The unit driven at high frequency during the stimulus (and continuing to discharge afterward) is the flexor inhibitor.

was continuously stimulated to produce the response shown in figure 7. The ascending excitation was cyclically repeated with a period of several seconds. Such excitation first appeared in the ganglion anterior to the point of stimulation. When output in the most caudal ganglion did not develop fully, the activity failed to spread rostrally. It therefore seems likely that such sequential phenomena are due to secondary excitation of interneuronal connections between ganglia, which in turn activate motor outflow in the next anterior segment. Pro- gressive caudal-rostral flexion and extension movements have also been observed in the abdomens of intact, unrestrained prepa-

rations. These progress from one segment to another with approximately the time- course observed in figure 7 and may de- pend upon the same mechanism.

4. Control of other motor systems Some command fibers affected output to

other muscles in addition to influencing the system that controls the position of the abdominal segments. Continuous stimulation of one extension unit, for example, caused regular spreading, flexing and ex- tending of the telson and uropods that was repeated cyclically with a period of about one second. This “wagging” response oc- curred only when the rest of the abdomen


had completed a full extension. Since postural extension and the subsequent re- sponses of the appendages appeared at identical stimulation thresholds, it may be assumed that they were initiated by the same central command element. The al- ternative possibility that the appendages were driven by peripheral inputs active at full extension is unlikely, since other, equally effective extension command elements failed to produce the effect, Some extension or flexion command fibers caused complex, asymmetrical movements of the uropod blades. Like the “wagging” response, these movements were initiated at stimulation thresholds identical with those for the postural changes, and could be duplicated by repeating the stimulation se-

quence. Both these results confirm for the tail appendages those reported in the previous paper (Evoy and Kennedy, ’67) for the swimmerets. They indicate that there is overlap in the control of different motor systems by central interneurons, and that the same command element may produce bilaterally symmetrical output for one system and asymmetrical output for another.

The crayfish abdomen assumes an overall configuration which is determined by five joint angles. Each of these joint angles is set by two bilaterally symmetrical pairs of antagonistic postural muscles. Recip rocity is preserved in the motor output to these two muscle groups by a ganglionic

DISCUSSION

Fig. 5 Records as in figure 4, recorded simultaneously with the movement shown in figure 3 bottom. Two seconds of record were removed between the first and second rows. Discharge in the flexor inhibitor develops more slowly. The movement was not completed until approximately two seconds after the end of the record.


Fig. 6 Initial (solid) and final (dotted) positions of the abdomen, traced from cinematographic records at the beginning and at the end of stimulation of two flexion command fibers at 100/sec. Top, a movement involving primarily the 2-3 and more posterior joints; bottom, one involving primarily the 3-4 joints.

coordinating mechanism that distributes central excitation and inhibition to the ap- propriate efferent elements (Evoy, Kennedy and Wilson, '67; Evoy and Kennedy, '67). We have now shown that multisegmental series of these semi-autonomous ganglionic centers are under the control of single central command interneurons. Thus activity in one central neuron produces coordinated output of the same type in several segments, changing overall posture by moving a number of joints. Since it is known that five excitatory and one inhibitory efferent axon innervate each set of muscles on one side, those command elements with the broadest influence must S e c t the activity of 120 motoneurons.

Individual command fibers, moreover, distribute their effects in spatially unique ways. These experiments demonstrate that some elements in a given category act on only one to two segments, while others distribute their effects more broadly; they also show that units differ in the point at

which they exert maximal influence. Pre- sumably, these differences in effectiveness relate to the density of output connections made by a command fiber in each of several ganglia. We have not yet found any cases of strongly bimodal output, i.e., command fibers which produce vigorous effects at two points separated by a region of in- effectiveness. It seems likely that complex movements of this sort would be brought about by activity in sets of interneurons, thus minimizing the cellular investment necessary.

Single connectives may contain at least six command fibers that have the same motor effect but distribute it in unique ratios between the segments. This count must be expanded by a factor of two or three, since there are subclasses based upon the selectivity with which individual members of the motoneuron population are affected (Evoy and Kennedy, '67; Fields, Evoy and Kennedy, '67). It would be rea- sonable, however, to conclude that the en- tire central program for abdominal posture involves an investment of no more than 100 central command elements.

There is a striking resemblance between this motor organization and that found in primary sensory interneurons in the crayfish (Wiersma, '58; Wiersma and Hughes, '61; Wiersma and Bush, '63). The latter are long, and receive input connections in a number of ganglia. Frequently, for example, interneurons that respond to tactile hairs in the abdomen will respond to stimulation of serially homologous patches of receptors on each of several segments; but each member of such a set will have a unique segmental grouping of receptive fields. Like the output connections of command fibers, their inputs are spatially con- tiguous. Primary sensory interneurons receive a highly convergent input in any one ganglion, some of which may have orig- inated from extrasegmental sources (Ken- nedy and Mellon, '64).

Clarification of the relationship between command interneurons and primary sensory interneurons awaits the mapping of receptive fields for command units, a task currently in progress. In general, we have found the larger, more accessible sensory interneurons to be without effect on motor outflow. Command elements, on the other


Fig. 7 Discharge recorded continuously from the slow flexor efferents of ganglia 1 (top- trace), 2 (second trace), and 3 (third trace) during continuous stimulation (100/sec) of a command fiber in the 5-6 connective that produced cylic, sequential effects upon motor output.

hand, are often difficult to drive with natural sensory stimuli. They thus may represent a relatively high stage of synaptic in- tegration on the input side. Their output connections, however, follow a plan similar to that which regulates the connection of sensory fibers with first-order interneurons

The overlapping of command pathways for abdominal posture, appendage movements, and perhaps other systems is another indication of the polyvalent nature of arthropod neurons. The musculature of the appendages is richly innervated, and their cyclical operation requires the coordinated participation of a large number of motoneurons. A command fiber that controls appendages as well as the position of several abdominal segments, might in-

fluence as many as 300 efferent elements. The combination of different types of output suggests a new order of complexity in “simple” nervous systems which cannot be adequately described in hierarchical terms, as a set of parallel channels from receptors to motoneurons. Although many movements may be mediated by such direct cir- cuits, cross-connections appear to exist at several levels. As a result, the opportunity for permutation of output is enormous.

ACKNOWLEDGMENTS

The authors are grateful to Mrs. Herbert Pabst for preparation of the drawings in figures 2 and 3, and to Miss Anne Sturm- thal and Mr. William Bentley for technical assistance.


LITERATURE CITED Dane, B., and W. G. Van der Kloot 1964 An

analysis of the display of the golden-eye duck (BucephaZa cEauguZa (L.)). Behaviour, 22:

Evoy, W. H., and D. Kennedy 1967 The central nervous organization underlying control of antagonistic muscles in the crayfish. I. Types of command fibers. J. Exp. Zool., 165: 223-238.

Evoy, W. H., D. Kennedy and D. M. Wilson 1967 Discharge patterns of neurones supplying tonic abdominal flexor muscles in the crayfish. J. Exp. Biol., in press.

Kennedy, D., and DeF. Mellon 1964 Synaptic activation and receptive fields in crayfish interneurons. Comp. Biochem. Physiol., 13: 275- 300.

Kennedy, D., and K. Takeda 1965 Reflex control of abdominal flexor muscles in the crayfish. 11. The tonic system. J. Exp. Biol., 43: 229-246.

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Kennedy, D., W. H. Evoy and H. L. Fields 1966 The unit basis of some crustacean reflexes. Symp. SOC. Expt’l. Biol., 20: 75-109.

Kennedy, D., W. H. Evoy and J. T. Hanawalt 1966 Release of coordinated behavior in crayfish by single central neurons. Science, 154:

Wiersma, C. A. G. 1958 On the junctional connections of single units in the central nervous system of the crayfish, Procambarus cbrkii Girard. J. Comp. Neur., 110: 421-471.

Wiersma, C. A. G., and B. M. H. Bush 1963 Functional neuronal connections between the thoracic and abdominal cords of the cravfish.

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Wiersma, C. A. G., and G. M. Hughes 1961 On the functional anatomy of neuronal units in the abdominal cord of the crayfish, Procambarus clarkii Girard, J. Comp. Neur., 116: 209-228.

Documents

The central nervous organization underlying control of antagonistic muscles in the crayfish. II. Coding of position by command fibers