temporal and spatial spread of an intersegmental reflex in crayfish

J. exp. Bio!. 113, 109-122 (1984) Printed in Gnat Britain © The Company of Biologists Limited 1984

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TEMPORAL AND SPATIAL SPREAD OF AN INTERSEGMENTAL REFLEX IN CRAYFISH BY CHARLES H. PAGE, DOMINICK GADALETA AND ANTHONY IACOPINO Department of Biological Sciences and Bureau of Biological Research, Rutgers University, Piscataway, NJ 08854, U.SA. Accepted 23 March 1984

SUMMARY

An intersegmental reflex initiated by flexing a crayfish leg at the merocarpal joint was examined by recording reflex response latencies of cephalic, thoracic and abdominal muscles. The first response was an intrasegmental resistance reflex in the extensor muscle of the stimulated leg. Spread of the intersegmental reflex occurred in both cephalic and caudal directions. Activation of the cephalic appendages occurred first (antennal levators, with latencies of 14-18 ms) while the abdominal musculature was activated last (superficial extensors, with latencies of 51-62ms). Shift of the stimulus site from the second to the fifth leg resulted in shorter latencies of the fifth leg and abdominal muscle responses while the latency for second leg extensor muscle discharge increased. Low correlation coefficients between the response latencies of pairs of cephalic and abdominal muscles, and thoracic and abdominal muscles, indicate that reflex pathways which initiate the abdominal responses are different from those that evoke the cephalic and thoracic responses. High correlation coefficients indicate common reflex pathways for activating either ipsilateral or contralateral pairs of extensors or flexors in the second and fifth legs. High correlations suggesting common reflex pathways or cross-coupling were also obtained for bilateral pairs of abdominal extensors. Low correlation coefficients for the response latencies of paired muscles indicate separate reflex pathways for (1) bilaterally homologous leg muscles and (2) first-fifth segment abdominal extensors. Surgical isolation of the supraoesophageal ganglion from the ventral nerve cord eliminated the responses of the antennal levators while producing a substantial increase in the response latencies of both maxilliped extensors and the contralateral second leg extensor and flexor, as well as a small but significant decrease in the contralateral first abdominal extensor. The synaptic input zones of the interneurones that mediate the thoracic and abdominal responses are located in the ventral nerve cord and not in the supraoesophageal ganglion.

INTRODUCTION

Analysis of a behaviour pattern requires a quantitative description of the sequence in which the various muscles that produce the behaviour are activated. Such a description Key word*: Intersegmental reflex, crayfish, behaviour pattern.

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can be obtained most readily by recording electrical activity from the muscles involved^ in the behaviour. Muscle responses have been recorded for rhythmic behaviour patterns including those that produce locomotion, ventilation, respiration, singing, etc. in vertebrates and invertebrates (Bentley & Konishi, 1978). Detailed descriptions of muscle activities that generate non-rhythmic behaviour patterns such as escape, defence, equilibrium or reproductive responses are rare. While the lack of repetition in their motor activity patterns makes them more difficult to analyse, many nonrhythmic behaviour patterns have the experimental advantage of being initiated by a restricted set of sensory stimuli. Most reflex responses of individual appendages and body segments are mediated by reflex pathways whose central components are contained within the ganglion that innervates the muscle or muscles affected by the intrasegmental reflex (Evoy & Ayers, 1982; Page, 1982). In contrast, since intersegmental 'whole animal' reactions involve the activation of the cephalic, thoracic and abdominal muscles, the neural pathways that mediate these responses extend to all levels of the crustacean nerve cord — supraoesophageal, suboesophageal, thoracic and abdominal ganglia. Although detailed analyses of motor activities are available for subcomponents of. several intersegmental responses (equilibrium reactions of swimmerets, Davis, 1971; uropods, Yoshino, Takahata & Hisada, 1980; and abdomen, Page & Jones, 1982a,6; abdominal escape responses, Wine & Krasne, 1972, 1982) little is known about the cephalic and thoracic components of these responses. In crayfish, flexion of a second leg will initiate a full abdominal extension with a latency of about 50ms (Page & Jones, 1982a,b). Analysis of motoneurone responses suggests that the abdominal extension is produced by the activity of several descending 'command fibre' interneurones (Page & Jones, 1982a,6). This abdominal extension is part of an equilibrium reaction that includes movements of most of the cephalic, thoracic and abdominal appendages (Page, 1981). This paper reports on a series of experiments in which the initial responses of cephalic, thoracic and abdominal muscles to leg flexion were recorded. Correlations between appendage and abdominal muscle responses were determined by simultaneous recordings of the response latencies of selected muscle pairs. The effects of surgical isolation of the supraoesophageal ganglion from the ventral nerve cord were examined to determine whether the supraoesophageal ganglion contains reflex pathways that are essential for mediation of the reflex responses. In addition to providing a description of the temporal spread of reflex activity through the thoracic, cephalic and abdominal body segments, the results indicate that the reflex pathways which lead to activation of the cephalic and thoracic muscles are separate from those that transmit reflex activity to the abdominal nerve cord. METHODS

The experimental preparation Medium sized crayfish, Procambarus clarkii, were obtained from Monterey Hydroculture Farms, Soquel, California, maintained in aquaria, and fed with dried dog food. Two weeks before experimentation, the first (chelipeds), third and fourth pairs ofj

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Ijegs were autotomized to reduce contact between adjacent legs, which inhibits the reflex response (Page, 1981). The antennae were shortened to prevent them from contacting the walls of the experimental chamber. The animals recovered from the effects of leg removal within 1 week. Several days before an experiment a plastic mount was attached to the dorsal carapace of the thorax with Eastman 910 adhesive (Eastman Chemical Products). Only healthy, vigorous animals which exhibited a normal range of behaviour patterns (feeding, walking, defence and escape) were used in these experiments. The crayfish was suspended from a metal rod that was screwed into the plastic mount in the centre of a water-filled chamber large enough to allow unobstructed leg and abdominal movement. The eyes were occluded with a mixture of petroleum jelly and powdered charcoal to prevent visual stimulation from interfering with the response (Page, 1981). The water (20-24°C) in the chamber was aerated continuously.

AN — M on

AN

Moff L5

Fig. 1. The leg flexion stimulus. (A) Animal suspended in experimental chamber with second leg held in an extended position preparatory to stimulation. (B) Animal following release of leg by turning off electromagnet. Leg was drawn into flexed position by Sg weight. AN, antenna; MX, maxillipeds; L5 is fifth leg; Al and A5 are first and fifth abdominal segments; M indicates electromagnet used to hold leg in extended position; W is 5g weight suspended from middle of carpus.

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Leg stimulation The equilibrium reaction was evoked by flexion of a second or fifth walking leg (Page, 1981). The proximal segments of the stimulated leg were held in a horizontal position by a clamp attached to the middle of the merus (Fig. 1). Two minutes prior to stimulation the more distal segments of the leg (carpus, propus, dactylus) were drawn into a horizontal position by a thread tied around the middle of the carpus and attached to a lever system that was located outside the experimental chamber. The leg was flexed by turning off an electromagnet which released the lever. To ensure rapid and complete flexion of the merocarpal joint a 5 g weight was suspended from a tie attached to the middle of the carpus. Successive trials were separated by a 2-min recovery period. Recording muscle activity Muscle potentials were recorded by inserting pairs of 100/im diameter insulated copper wire electrodes into selected muscles. These comprised the antennal levator (Schmidt, 1915), the merocarpal extensor in the third maxilliped (Wales, Clarac, Dando & Laverack, 1970), the merocarpal extensors and flexors in the second and fifth legs, and the superficial tonic extensor muscles in the first and fifth abdominal segments. To record from the antennal levators, electrodes were inserted through the lateral aspect of the joint between the basal segment (protopodite) and the flagellum of the antenna. Electrodes were implanted into the other muscles through pinholes made in the cuticle overlying the muscle. The positions of the inserted electrodes were verified by correlating muscle potentials with appendage and abdominal movements and by post mortem dissection. Usually the responses of two to three muscles were recorded simultaneously. Sets of simultaneous recordings were obtained for pairs of: antennal levators and abdominal extensors; maxilliped extensors and abdominal extensors; second and fifth leg extensors; second and fifth leg flexors; first and fifth segment abdominal extensors; bilateral pairs of antennal levators, maxilliped extensors, second and fifth leg extensors and flexors and first and fifth segment abdominal extensors. The oscilloscope trace was triggered by the switch that turned off the electromagnet. Muscle potentials were differentially amplified with a Grass P15 preamplifier (10Hz— 1 kHz bandwidth), displayed on a storage oscilloscope and photographed. Interruption of circumoesophageal connectives After cooling the animal in ice a small piece of cuticle was removed just anterior to the mandibles to expose the circumoesophageal connectives (Gordon, Larimer & Page, 1977). The connectives were pulled to the surface with fine hooks and sectioned. Care was taken to avoid damaging adjacent 'vital' structures (green glands, stomach, ventral artery). The wound was sealed by inserting a piece of tissue paper saturated with petroleum jelly into the cuticular incision. Each crayfish was placed in a small aquarium to recover. For the first day the aquaria were filled with crayfish saline (van Harreveld, 1936) after which the saline was replaced with dechlorinated water that was change^

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wdaily. After a 1-week recuperation period 90 % (27 out of 30) of the animals produced vigorous thoracic and abdominal responses to the leg flexion stimulus. Data analysis Latencies were measured directly from the storage oscilloscope screen as the time interval from the beginning of the oscilloscope trace to the start of the initial muscle response evoked by leg stimulation. Because of the 'spontaneous' discharge of small potentials in many muscle recordings (Figs 2, 3) a set of criteria were used to standardize the identification of the initial muscle response. The initial muscle response was the first muscle potential following leg stimulation whose amplitude equalled or exceeded five times the background noise level of the oscilloscope trace. Significant differences in the mean latency values of each experiment were determined with Student's f-test. Correlation coefficients were calculated for pairs of muscles from which simultaneous recordings were obtained, and significance was determined by use of the r-test (Ott, 1977). RESULTS

Muscle responses Each of the appendage and abdominal muscles responded to the flexion of the second leg (Figs 2, 3). The mean latencies ( ± S . E . ) of these responses are summarized in Fig. 4. The sequences of muscle activation were consistent between successive trials. For example, comparison of the order in which the responses of ipsilateral and cE2

iE2

iES

-vr-

cE5

cF2

iF5

10 ms Pig. 2. Responses of pairs of leg muscles to the leg flexion stimulus. E2 and E5 are the extensors and F2 and F5 the flexors in the second andfifthlegs; i is ipsilateral to the stimulated leg, c is con trilateral. Simultaneous recordings from iE2-LE5, iF2-iF5, cE2-cE5, cF2-cF5. Arrows mark beginning of response. The potentials were retouched for photographic reproduction.

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iAN

LAI

cAN

iMX

iAS

cMX

cAS

20 ms Fig. 3. Responses of pairs of antennal, maxilliped and abdominal muscles to the leg flexion stimulus. AN, antennal levator; MX, maxilliped extensor; Al and A5, the superficial extensors in the first and fifth abdominal segments; i is ipsilateral to the stimulated leg, c is contralateral. Simultaneous recordings for iAN-cAN, LMX-cMX, iAl-cAl, iA5-cA5. Arrows mark beginning of response. The potentials were retouched for photographic reproduction.

contralateral members of homologous muscle pairs were initiated shows that the ipsilateral muscle was excited before its contralateral counterpart in 70% (maxillipeds) to 100% (second and fifth legs) of their responses (Table 1). The extensor muscle of the stimulated second leg was the first to be excited. It produced a burst of potentials with a mean onset latency of 11-9 ms (Figs 2, 4). The extensor muscles in the non-stimulated (second and fifth) legs responded with irregular discharges that had mean latencies of 21 -6-29-8 ms. The ipsilateral extensor of the fifth leg and the extensor in the contralateral second leg were always activated before the extensor in the contralateral fifth leg (Fig. 4, Table 1). In each leg an interval of more than 10 ms elapsed between the initiation of responses in the extensor and flexor muscles (Fig. 4). The activation sequence of the leg flexors was identical to that described above for the extensors. The first to respond was the flexor in the stimulated second leg; then the ipsilateral fifth and Table 1. Sequence of activation for responses in bilaterally homologous muscles pairs Muscle Antennal levators Maxilliped extensors 2nd leg extensors 2nd leg flexors 5th leg extensors 5th leg flexors 1st abdominal extensors 5th abdominal extensors

% Ipsilateral

% Contralateral

N

86 70 100 100 100 100 87 98

14 30 0 0 0 0 13 2

90 92 84 84 84 84 83 82

Percentage of times in which a response first appeared in the ipsilateral or in the contralateral member of a homologous muscle pair. N = total number of responses recorded from seven different animals.

Crayfish intersegmental reflex AN

MX

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*

e e

E2

F2

E5

- * •

-

*

-

F5

Al

- * -

AS

•9-

20

40 Time (ms)

60

Fig. 4. Mean latencies ± standard error of the mean for 84 responses recorded from seven animals. AN, antennal levator muscle; MX, extensor of maxilliped; E2 and F2, second leg extensors and flexors; E5 and FS, fifth leg extensors and flexors; Al and A5, first and fifth abdominal segment superficial extensors; x, ipsilateral; o, contralateral to the stimulated leg.

contralateral second legflexorsandfinallytheflexorin the contralateralfifthleg (Fig. 4). With the exception of the extensor in the stimulated leg, the antennal levators and maxilliped extensors had the shortest latencies of any of the muscles which we examined (mean latencies of 14-4-184ms) (Figs 3, 4). Both muscles responded to leg flexion with a brief initial burst that was followed by a lower frequency discharge of small and large muscle potentials (Fig. 3). In most instances the ipsilateral muscles began to discharge before their contralateral counterparts (Table 1). Of the muscles whose responses were monitored, the superficial abdominal extensors were always the last to be activated by the leg stimulus (mean latencies of 51-6-62-9ms). They responded with a vigorous burst of potentials that slowly declined "in frequency (Fig. 3). In 78% (128 out of 165) of the trials, abdominal extension responses were recorded from the fifth segment muscles before they were detected in the muscles of the first abdominal segment (Fig. 4). In most instances the ipsilateral extensors responded before their contralateral homologues (Table 1). The slowest responding muscle in this study was the contralateral extensor in the first Abdominal segment (mean latency of 62-9 ms).

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Correlations between latencies of muscle pairs Correlation coefficients were calculated for the latencies of the reflex responses of selected muscle pairs to determine whether the cephalic, thoracic and abdominal muscles were activated by common neuronal pathways. Statistically significant (high) correlations were obtained for bilaterally homologous (ipsilateralcontralateral) pairs of abdominal muscles, while ipsilateral-contralateral pairs of antennal, maxilliped and leg muscles had lower correlations (Table 2). High correlations were calculated for extensors and flexors in the same leg (examined for the contralateral second leg only) and for pairs of second and fifth leg extensors or flexors located on the same side of the animal. In contrast, there was no significant correlation between response latencies of the first and fifth abdominal extensors. Comparisons of the responses of the cephalic and thoracic appendages with those in the first abdominal segment always produced a low correlation. Correlations between antennal, maxilliped and leg muscles were not calculated because simultaneous recordings from the appropriate muscle pairs (antennal-maxilliped, antennal-leg, maxilliped-leg) were not obtained. Since a significant (high) correlation indicates that the response latencies of the two muscles being compared have relatively constant ratios, the high correlations described above can be interpreted as reflecting the presence of a high proportion of common synapses in the reflex pathways which activate the two muscles and/or cross-coupling between the motoneurones that innervate the muscles. An alternative interpretation is that the high correlation coefficients result from the excitation of the two muscles by independent reflex pathways each of which has invariant temporal characteristics. However, this interpretation seems unlikely because the responses of most of the muscle pairs exhibiting high correlations are characterized by relatively long latencies and large standard errors. For example, correlations for the pairs of bilaterally homologous muscles with the shortest response latencies (ipsicontralateral pairs of antennal levators and maxilliped extensors, Fig. 4) were Table 2. Correlation coefficients for response latencies in specified muscle pairs Pairs

Correlation

IPSI-CONTRA iAN-cAN 0-6 iMX-cMX 0-7 iE2-cE2 0-2 iF2-cF2 0-6 iES-cES 0-5 iF5-cF5 0-6 iAl-cAl 0-8* iAS-cAS 0-9*

Pairs

Correlation

INTRA-LEG cE2-cF2 0-9* INTRA-THORACIC iE2-iE5 0-8* iF2-iFS 0-8» cE2-cE5 0-7* cF2-cFS 0-8* INTRA-ABDOMINAL iAl-iA5 0-5 CEPHALIC-ABDOMINAL iAN-cAl -0-2 cAN-cAl -0-1

Pairs

Correlation

THORACIC-ABDOMINAL iMX-cAl -0-2 cMX-cAl -0-3 iE2-cAl 0-1 iF2-cAl 0-3 cE2-cAl 0-2 cF2-cAl 01 iES-cAl 01 iFS-cAl 01 cES-cAl 0-2 cFS-cAl 0-4

Correlation at 95 % significance level (•). 84 trials with seven animals. AN, antennal levator muscle; MX, extensor of maxilliped; E2 and F2, second leg extensor and flexor; ES and F5, fifth leg extensor and flexor; A1 and AS, first and fifth abdominal segment superficial extensors; i, ipsilateral; c, contralateral to the stimulated leg.

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Table 3. Latencies of muscle responses to fifth leg flexion Muscle

2nd leg extensor 2nd leg flexor Sth leg extensor 5th leg flexor 1st abdominal extensor

Ipeilateral

Contralateral

Latency (ms)

% Change

Latency (ms)

% Change

19-4 ± 1-3 295 ±1-0 10-9111 25-5 ±1-0

+ 63* +2 -SO* -25*

24-412-2 32-8 ±0-8 14-8 ±1-3 290 ±0-6 36-6 ±2-8

+8 -1 -SI* -30* -42*

Each latency is a mean value ( ± standard error) for 84 responses of seven different animals. Responses were not measured for ipsilateral 1st abdominal extensor. % Change indicates percentage difference from latencies evoked by second legflexion(Fig. 4). Significant change at 99% (•), Student's Mest.

significantly lower (Table 2) than those obtained for bilateral pairs of abdominal superficial extensors which had much longer response latencies (Fig. 4). Responses to fifth leg stimulation To test whether the response latencies were affected by changing the segmental level at which the stimulus was applied in the thoracic nerve cord, we examined the responses evoked by flexing a fifth leg. When compared with the latencies measured during second leg stimulation (Fig. 4), stimulation of the fifth leg produced a decrease in response latencies for the fifth leg muscles and the contralateral first abdominal extensor (Table 3). One of the four muscles in the second leg had longer response latencies; the other three were unchanged. Interruption of circumoesophageal connectives Responses evoked by legflexionwere recorded from maxilliped, leg and abdominal muscles in animals whose circumoesophageal connectives had been previously sectioned. Only the responses of the antennal levators were blocked by surgical isolation Table 4. Effects of cutting both circumoesophageal connectives upon muscle response latencies Muscle

Maxilliped extensor 2nd leg extensor 2nd leg flexor 5th leg extensor 5th leg flexor 1st abdominal extensors 5th abdominal extensors

Contralateral

Ipsilateral Latency (ms)

% Change

Latency (ms)

% Change

27-2±M 11-5 ± 0-3 301 ±0-3 22-1 ±0-4 34-6 ±0-4 52-3 ±1-6 55-7 ±2-4

+ 77* -3 +5 +2 +3 -4 +8

29-2 ±1-9 40-0 ±0-2 45-4 ±0-6 31-4±0-3 41-7 ±0-4 56-5 ±1-9

+ 68* + 77* + 37* +5 +1 -10*

Each latency is a mean value ( ± standard error) for 60 responses offiveanimals. Responses were not measured for contralateral Sth abdominal extensor. % Change indicates percentage difference from mean latency value for intact animals (Fig. 4). Significant change at 99 % level (•), Student's Mest.

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of the supraoesophageal ganglion from the ventral nerve cord. Response latencies ofj the ipsilateral leg and abdominal muscles were unchanged from those measured in th * intact animal (Table 4). In contrast, there was a significant increase in the response latencies for muscles in both ipsilateral and contralateral maxillipeds and in the contralateral second leg, while the extensors in the contralateral first abdominal segment responded with a shorter latency. DISCUSSION

The earliest observed response to the leg flexion stimulus is an excitation of the extensor muscle in the stimulated leg. This reaction resembles other resistance reflexes in crustacean legs. These responses are believed to be initiated by stimulation of chordotonal organs and to result from activation of a monosynaptic reflex arc (Bush, 1965; Clarac, 1970; Lindsey & Gerstein, 1979; Wiens & Gerstein, 1976). All other responses to the flexion stimulus are produced by activating specific interneuronal pathways which: (1) ascend to the suboesophageal and supraoesophageal ganglia; (2) connect to the other thoracic ganglia; and (3) descend to the abdominal ganglia. The order in which appendage and abdominal muscle responses were initiated in response to flexion of a second leg (Fig. 4) was nearly identical to that reported by Page (1981) for appendage and abdominal movement responses. The temporal relationships of the reflex pathways'that mediate appendage and abdominal muscle responses are summarized in Fig. 5. The latencies for the antennal and maxilliped responses were only 2-4 ms longer than the latency of the resistance reflex. The shortness of these latencies indicates that reflex activity is transmitted over fast conducting pathways with few if any synaptic interruptions to the antennal and maxilliped motor centres in the supraoesophageal and suboesophageal ganglia respectively. Since the initiation of the antennal and maxilliped responses is nearly simultaneous it is probable that the antennae and maxillipeds are activated by different reflex pathways. An alternative explanation for the short latencies of the antennal and maxilliped responses is that the antennal and maxilliped responses are initiated by excitation of vibration sensitive mechanoreceptors located in the anterior cephalothorax rather than within the stimulated leg. Three observations contradict the hypothesis that some reflex responses are initiated by excitation of receptors located outside the stimulated leg: (1) interruption of all neural connections between the antennal motor centres and the thoracic nerve cord eliminates the reflex responses of the antennal levators; (2) responses of ipsilateral muscles usually precede those of their contralateral homologues (Table 1); (3) shifting the stimulus from a second to a fifth leg increases the response latency of the ipsilateral second leg while the latencies for the fifth leg and abdominal muscles decrease (Table 3). Although the number of interneurones that compose the reflex pathways indicated in Fig. 5 is not known, correlation coefficients for response latencies recorded simultaneously from muscle pairs provide clues concerning the organization of these reflex pathways. Low correlations between the cephalic and abdominal and between the thoracic and abdominal muscles indicate that the abdominal muscles are activated by different reflex pathways from those that excite the cephalic and thoracic appendages.

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S

he variability in the order in which the first and fifth segment abdominal extensors e excited (in 78 % of the trials the fifth segment extensors were excited before those in the first segment) and their relatively low correlation coefficients imply that different interneurones conduct reflex activity to the anterior and posterior abdominal segments. At least six fibres have been identified in the abdominal nerve cord which when stimulated electrically initiate abdominal postural extension (Fields, Evoy & Kennedy, r

SP

1-9

B

Su

14-4

4-0

-ft.

10-7

STIM

T2

1

8-2