Comp. Biochem. Physiol. Vol. 73A, No. 3, pp. 503 to 512, 1982 Printed in Great Britain.

0300-9629/82/l 10503-10$03.00;‘0 0 1982 Pergamon Press Ltd

A PERIPHERAL SENSORY-TO-ROTOR NEURON REFLEX ARC IN AN ARTHROPOD WALKING LEG

STANLEY G. RANE* and GORDON A. WYSE

Department of Zoology, University of Massachusetts, Amherst, MA 01003, USA

(Received 12 February 1982)

Abstract-L Claws of ~i~~~~s walking legs, when removed from the animal, close spontaneously or reflexively in response to tactile stimulation.

2. Electrical stimulation of claw sensory nerves evoked claw closing, as did stimulation of motor neurons in the large leg nerve.

3. Curare (lOme M) injected into the claw blocked the former but not the latter. 4. Acetylcholine (low4 M), injected into the claw, evoked claw closing and bursts of impulses. 5. These and other results suggest that peripheral chofinergic sensory-motor synapses in the claw

mediate reflexive and spontaneous closing. 6. Reflexive closing in the isolated claw was shown not to be an artifact of the dissection procedure, as

had been previously suggested.

INTRODUCTION

For both vertebrates and invertebrates, reflexes are mediated by peripheral sensory and motor neurons which communicate via synapses in the central ner- vous system (CNS). These peripheral neurons make and receive other synaptic contacts not directly involved in reflex circuits, but these other synapses are also restricted to the central nervous system. This report describes a reflex arc in the horseshoe crab, ~~~~~~s po~~p~~~~us, that is unconventional in that although the peripheral neurons involved extend to and from the CNS, their synaptic contacts mediating the reflex act are located in the periphery.

In arthropods there is little evidence for a per- ipheral reflex path (Bullock & Horridge, 1965), although there exist reports of reflex muscle contrac- tion in isolated limbs of some crustacea (Barnes, 1932), and of Lin~rl~s (Hoyle, 1958). Explanations of these apparent reflexes in the absence of the CNS have focused on the cut end of the mixed sensory and motor leg nerve. The cut was thought to have caused spontaneous discharge of motor neurons (Barnes, 1932; Parnas et 1*1., 1968) or to have produced an artificial synapse (ephapse) where sensory neurons, active either spontaneously or due to some stimulus,

could excite adjacent motor neurons (Barnes, 1932; Hoyle, 1958; Bullock & Horridge, 1965).

The claw of the walking leg of LimuEus exhibits reflexive closing even when the leg is removed from the animal. This reflex was reported to occur both spontaneously and in response to tactile stimulation of the claw. Hoyle (1958) termed this response a “pseudoreflex” since the severed leg was not in con- tact with synapses in the CNS. Hoyle (1958) and Parnas L’T al. (1968) proposed that the “pseudoreflex” was due to hyperexcitability and ephaptic contacts at

* To whom correspondence should be addressed.

the cut end of the leg nerve. Our study suggests, how- ever. that this “pseudore~ex” is due to a peripheral, sensory-motor neuron reflex arc mediated by cholin- ergic synapses.

MATERIALS AND METHODS

Adult horseshoe crabs (1416 cm across posterior edge of prosoma) were obtained from Marine Biological Labor- atory, Woods Hole, Massachusetts and from Gulf Speci- men Co., Panacea, Ftorida and were kept in a 200 gal recir- culating seawater (Instant Ocean) aquarium at 17-23’C. Animals were not fed and most were used within 3 months of arrival, Second, third and fourth walking legs from both sexes were used. Results did not vary with leg position or sex.

The following anatomical terms, suggested by Snodgrass (1952). are used in this report. The six leg segments, in proximal to distal order, are the coxa, trochanter, femur, patella, tibia and tarsus. The moveable tarsus and the stationary extension of the tibia, the index, are the digits of the claw or chela (Fig. 1). The tarsus and tibia correspond to the crustacean dactylopodite and propodite respectively. The tarsus closer (adductor) muscle is innervated by motor neurons in the large leg nerve (Fourtner & Sherman, 1972). The large leg nerve also contains many sensory axons which transmit information from receptors primarily in the index and tarsus (Wyse, 1971).

Legs were removed by a cut just distal to the COXa-

trochanter joint. A small strip of exoskeleton was removed from the posterior surface of the tibia to expose the opener (abductor) muscle and tendon. After the tendon was cut close to its attachment to the tarsus, it and the opener muscle were pulled free of the tibia and removed. The large leg nerve was exposed with a slit made along the posterior length of the leg, from the patella-tibia joint to the cut used to sever the leg. The nerve was separated from sur- rounding exoskeleton, connective tissue and muscle so that a 55570 mm length was exposed. In the index, branches of the large leg nerve were exposed for electrical stimulation by removal of a small piece of exoskeleton about 5 mm from the main body of the tibia.

503

504 STANLEY Ci. RANE and GORDON A. WYSE

CANNULAE I, I, EXPOSED INDEX

10 mm EX$OSED TAdSUS CLOSER MUSCLE

Fig. 1. Limulus claw closer muscle preparation. Two can- nulae were used for saline perfusion and drug injection. Hook electrodes could be placed on the large lea nerve or

I L

its branches in the index. The tarsus is adducted by closer muscle contraction,

Previous experimenters have kept their preparations in a seawater bath and have added drugs directly to this bath. For our work a cannula system was employed so that saline and drugs could be delivered to the interior of the tibia exclusively. Two holes (approx. 1.5 mm dia.) were bored in the anterior surface of the tibia, through which polyethylene tubes were inserted. Measured volumes of drugs were injected into the tibia through one tube by a syringe. The other tube ran from a 4-way valve through which passed a constant supply of either Chao’s (1933) Limulus saline (444 mM NaCl; 37 mM CaCl,: 9 mM KCI) or low-calcium, high-magnesium saline (444 mM NaCl: 9 mM KCI; 25 mM MgCl,; 12 mM MgSOJ. This per- fusion was stopped when drugs were injected. The claw with attached leg nerve was clamped in air. so that saline and drugs delivered via the cannulae could flow around the closer muscle and drip from the preparation through the hole cut for opener muscle removal (Fig. 1). The leg nerve was kept moist by resting it in a glass dish containing saline (changed periodically during an experiment), while a glass trough containing saline was used to moisten nerves exposed in the index.

For electrical stimulation and recording the leg nerve and leg nerve branches in the index were raised in air on silver bipolar hook electrodes. Stimuli of 0.05 msec dur- ation (longer durations noted) were applied in I set trains at 5 Hz and at various intensities from I to 15 V. Nerve activity was recorded with a suction electrode on the cut end of the leg nerve. Amplification and display were con- ventional. Adductor contraction produced claw closure (movement of tarsus) which was recorded with a force- transducer (Grass FT03) attached to the tip of the tarsus by thread.

Normal and low-calcium, high-magnesium salines con- tained Tris-acid maleate buffer (2.5 mM) and were always at pH 7.0. Curare and atropine were kept as stock solutions and diluted for use. while acetylcholine solutions were made fresh for each experiment. All solutions were stored refrigerated but were allowed to come to room tempera- ture before experiments.

RESULTS

The results of this study do not definitively prove the existence of a peripheral reflex circuit, but they argue against alternative explanations. For reasons of clarity. results are presented under headings that state their conclusions.

Rejexice cluw closing is not dependent on ephaptit mechanisms

When a walking leg is removed from a Limulus, the claw of the isolated leg often undergoes repeated, apparently spontaneous closing. Figure 2 shows

records of these spontaneous claw closing movements coincident with bursts of impulses recorded at the cut end of the leg nerve. KC1 (1.0 M) (a depolarizing blocker) eliminated the appearance of impulses at the cut nerve end but did not affect the associated claw closing. This finding indicates that spontaneous clos- ing of the isolated claw does not depend on conduc- tion to or from the cut nerve end, and further indi- cates that the impulse bursts associated with claw closing were initiated in the claw. Application of KC1 to the leg nerve, between a stimulating electrode and the claw, also blocked closer muscle contraction evoked by electrical stimulation of the leg nerve (not shown). Figure 2 also shows that saline rinse of the KC1 treated portion of nerve re-established nervous conduction and that prevention of claw closing with a wooden chock did not change the pattern of spon- taneous impulse bursts. The latter result suggests that the bursts were not generated by joint or muscle receptors activated by closing.

For the above preparaton and another exhibiting the spontaneous reflex, perfusion of the claw with low-calcium, high-magnesium saline simultaneously eliminated reflex claw closing and associated impulse bursts. Subsequent perfusion with normal saline re- stored both of these responses.

Acetylcholine evokes claw closing

Acetylcholine has been detected in Limulus leg nerve (Treherne, 1966) and is implicated as a sensory neuron transmitter in other arthropods (Florey & Biederman, 1960; Barker et al., 1972; Florey. 1973). It is not considered to be a candidate transmitter at arthropod peripheral neuromuscular junctions (Atwood, 1976). If sensory neurons synapse directly onto motor neurons in the claw. then they might use acetylcholine as an excitatory transmitter. Figure 3 shows that progressively larger concentrations of ace- tylcholine produced longer-duration bursts of im- pulses (recorded at the cut end of the leg nerve) associ- ated with more vigorous closer muscle contractions. For the four preparations tested these two effects always occurred together. The minimum effective con- centration of acetylcholine was 5 x lo-’ M. KC1 was used to block conduction of the acetylcholine-induced impulses to the cut end of the leg nerve. This conduc- tion block did not, however. prevent claw closing evoked by acetylcholine injection. Figure 4 shows that low-calcium, high-magnesium saline perfusion of the claw prevented acetylcholine-induced closer muscle contraction but did not prevent bursts of impulses. Since impulses were not due to muscular contraction. acetylcholine must have excited neurons directly.

Sen.sor_r axon stimulation ecvkes claw closing

The leg nerve sends two large branches, each com- prised of several axon bundles, into the index (Wyse. 1971). Since the index is the distal extreme of the leg and is devoid of muscle, it is unlikely that it contains any motor neuron axons. Electrical stimulation of nerves in the index. however, produced claw closing in all six preparations tested. Furthermore, injections of curare (10-‘~-10~5 M) into the claw reversibly blocked claw closing evoked in this manner. Figure 5 shows compound action potentials (CAPS) and force- transducer records for stimulation of nerves in the

Peripheral sensory-motor synapses in Limulu.s 505

NORMAL CLAW CHOCKED I

KCI BLOCK AT X KCI REMOVED

Fig. 2. Spontaneous impulse bursts and claw closing recorded with a suction electrode (R) and a force-tranducer (FT, up indicates closing) respectively. Note that blockade of nervous conduction with KCI did not disrupt claw closing. Saline rinse reestablished nervous conduction. Prevention of claw closing with a wooden chock did not alter the pattern of impulse bursts. Although not shown, low-

calcium. high-magnesium saline perfusion of the claw reversibly eliminated all spontaneous activity.

index. Recordings at two sites along the leg nerve showed that the direction of conduction for these CAPS was from the claw to the cut end of the leg nerve. Each record was made by superimposing CAP traces from five successive stimuli. Specific portions of the CAP (arrows in Fig. 5) appear blurred as a result of non-superimposition of impulses from successive stimuli. The variable latencies of these blurred im- pulses suggest that they were dependent on a tempor- ally varying synaptic process. Non-blurred portions of the CAP represent impulses with constant-velocity conduction. i.e. conducted without synaptic media- tion. The variable-latency impulses had velocities similar to the faster non-variable impulses, yet it is clear that they appeared at the recording electrodes much later than these non-variable impulses (Fig. 5). Based on the distances from stimulating to recording electrodes and on conduction velocities, the variable- latency impulses appeared at the cut end recording electrode (Rl) 3-5 msec later than expected. This con- duction delay could be attributed to a synaptic delay in the conduction path of the variable-latency im- pulses. Curare (6 of 6 experiments) reversibly blocked both claw closing and the variable-latency impulses (blurred areas of CAPS), but not the remainder of the impulses in the CAPS. For one experiment in which 10m6 M curare was effective, atropine (10m4M) also blocked both claw closing and variable-latency im- pulses. These results indicate that stimulation of leg nerve branches in the index produced claw closing and elicited some impulses, both via a cholinergic site of action. Low-calcium saline perfusion also blocked the variable-latency impulses and claw closing. The low-calcium block could result from effects on neuro-

muscular transmission, excitation-contraction coup-

ling, sensory-motor synapses, or more than one of these.

Leg nerve stimulation causes motor impulse initiation in the claw

Electrical stimulation of the leg nerve produced claw closer muscle contraction. Figure 6 shows records of muscle tension, nerve CAPS and individual long-latency impulses resulting from leg nerve stimu- lation. CAPS were conducted away from the point of stimulation in both directions and were recorded at two points (Rl. R2) with approximately equal latency. The groups of individual long-latency impulses (arrows) were recorded about 10 msec earlier at R2 than at Rl. Although not shown, KC1 applied to the leg nerve between the claw and electrode R2 did not affect CAPS but did reversibly block the long latency impulses (and claw closing) evoked by leg nerve stimulation. Long-latency impulses (LLI) then are action potentials initiated in the claw as a result of leg nerve stimulation proximal to the claw. They travel in the distal-proximal direction and characteristically appear at the cut end of the leg nerve 25-30 msec after the stimulus artifact. Curare (6 of 6experiments) or atropine (1 of 1) injected into the claw reversibly elim- inated LLI but not closer muscle contraction. Low- calcium perfusion blocked both the LLI and claw closing.

The above results suggest that LLI and the drug- sensitive, variable-latency impulses produced by stimulation of sensory nerves in the index were both initiated at the same cholinergic site in the tibia. If sensory neurons synapse en passant (in the tibia) with

R

FT

Fig.

3.

Sp

onta

neou

s an

d ac

etyl

chol

ine-

indu

ced

impu

lse

burs

ts

and

claw

cl

osin

g re

cord

ed

with

a

suct

ion

elec

trod

e (R

) an

d a

forc

e-tr

ansd

ucer

(F

T.

up

indi

cate

s cl

osin

g)

resp

ectiv

ely.

B

ars

show

du

ratio

n of

inj

ectio

n in

to

the

claw

of

0.5

ml

of e

ither

sa

line

or

acet

ylch

olin

e in

sa

line.

Pr

esum

ably

, ac

etyl

chol

ine

was

ac

ting

as

the

natu

ral

exci

tato

ry

tran

smitt

er

at

the

peri

pher

al,

sens

oryy

mot

or

syna

pses

. an

d th

e im

puls

es

wer

e an

tidro

mic

dhy

cond

uctin

g m

otor

sp

ikes

.

Peripheral sensory-motor synapses in Limulus 507

J

70 uv

5X10-4M ch NORMAL SALINE 1 SEC

5 X 1O-4 M Ach LOW Ca+*,HlGH Mg+* SALII\E

Fig. 4. Acetylcholine-induced impulse bursts and claw closing recorded with a suction electrode (R) and a force-transducer (FT, up indicates closing) respectively. Bars show duration of injection into the claw of 0.5 ml acetylcholine in either saline or low-calcium. high-magnesium saline. The lower record was made after 20 min of low-calcium perfusion. Since lack of calcium prevented claw closing and presum- ably synaptic transmission, acetylcholine is inferred to have initiated impulses by direct action on

neurons.

motor neutrons (see Fig. 5 and accompanying text), then leg nerve stimulation proximal to the claw could antidromically fire sensory neurons and via these synapses antidromically fire motor neurons. The ob- servations that follow support the hypothesis that LLI were antidromically travelling motor impulses.

CAPS evoked by leg nerve stimulation were com- posed of impulses exhibiting a range of conduction velocities (1.0 to 6.5 m/set) in proportion to their rela- tive sizes. Larger impulses had lower threshold volt- ages and probably were from large diameter motor axons, while small, high-threshold impulses were probably from small diameter sensory axons (Wyse, 1971). If leg nerve stimulation evoked LLI by per- ipheral synaptic excitation from relatively high- threshold sensory neurons, the thresholds for LLI should be above those required to evoke the first CAP components. For 9 of loexperiments LLI were evoked at thresholds 1.5-2.5 times higher than those required for generation of the first CAP components. This result is consistent with the hypothesis that LLI initiation is dependent on sensory axon stimulation.

Like the variable-latency, drug-sensitive impulses evoked by stimulation of nerves in the index, LLI exhibited variable stimulating-to-recording-electrode latencies for each stimulus in a train, while the CAP spikes evoked by leg nerve stimulation had consistent latencies. Increases in stimulus amplitude above the threshold for LLI generation progressively decreased the variations in LLI latency. presumably by recruit-

ing more sensory axons and producing sensory- motor synaptic excitation with less variable latency.

LLI are not generated by peripheral impulse rebound in motor neurons

Stephens & Atwood (1981) showed for the stretcher muscle of the crab, Pachygrapsus crassipes, that single shocks to the excitor axon caused multiple impulse generation at the periphery by a temperature-depen- dent mechanism. These peripherally-generated motor impulses travelled antidromically, as did the LLI evoked by leg nerve stimulation in this study. It is unlikely that the results reported here could be due to a similar mechanism, since curare blocked the in- itiation of LLI while it did not affect motor impulse conduction to the neuromuscular junction (Fig. 6). As a further test, conduction velocities were calculated for LLI (4.5-6.6 m/set) from records similar to Fig. 6. Also determined was the distance that impulses would travel if conducted from the stimulating electrode to the distal extreme of the claw and back to a recording electrode. This distance would be the distance trav- elled if these impulses were generated by a mechanism like that reported for Pachygrapsus. Expected travel times were calculated by dividing the round-trip dis- tance by the observed conduction velocities. These expected times were compared to the travel times actually recorded. The observed times were consist- ently 5-8 msec larger than the calculated times. Motor impulses generated by a mechanism like that

508 STANLEY G. RANE and GORDON A.WYSF

RI R2 S

-J

2OOuVl

'%Ei 77uv2

SALINE

CURARE

WASH

Fig. 5. Compound action potentials recorded at two sites along the leg nerve (RI, R2) and force- transducer records (FT) of claw closing. Nerves in the index (S) were stimulated with 1 set trains at 5 Hz, Electrode records were made by superimposing five traces from a train of five stimuli. Transducer records, each from a single trace at one-twentieth the sweep speed of the electrode traces, show tension response to the entire train. Stimulus intensities increase across a row of records. but are approximately equal in a column. Injection of 4 ml of lo-’ M curare into the claw reversibly blocked claw closing as well as some impulses (arrows). These impulses. which had variable post-stimulus latencies for successive stimuli, caused blurring of the CAPS. These may be motor impulses initiated at peripheral sensory motor synapses. while the remainder of the CAP represents sensory impulses initiated at the stimulating

electrodes.

described for Pachygrapsus would have had nearly identical observed and calculated travel times. The above results show that the LLI in Limulus were not motor impulses travelhng to and from the periphery along the same motor axon.

LLI are not due to proprioceptiue discharge

LLI and the variable-latency, drug-sensitive im- pulses observed during stimulation of nerves in the index might have been produced by proprioceptors sensitive to some aspect of claw closing. The tibia- tarsus joint does contain proprioceptors responsive to claw closing (O’Tanyi & Barber, 1966) but their axons are in the small leg nerve which also provides inner- vation of the claw opener muscle. The opener muscle and the proprioceptive organ at its tendon (Hayes & Barber, 1967) were removed with the small leg nerve in all experiments reported here. No claw propriocep- tive responses have been seen in large leg nerve (Wyse, 1971 and personal observation). Nevertheless,

if the large leg nerve contained axons from proprio-

ceptors sensitive to claw closing, then the inhibitory

action of curare on LLI might be explained in terms

of a block of proprioceptor response. Direct stimulation of closer muscle contraction

should have evoked discharge of proprioceptors (if present). Direct muscle stimulation was applied via two metal electrodes inserted into the closer muscle. Figure 7 shows impulses evoked by direct muscle stimulation, but they were not proprioceptive in nature. These impulses were probably from motor neurons since they only appeared coincident with closer muscle contraction, and they persisted even after contraction was prevented with low-calcium saline (not shown). Curare did not block initiation of these impulses. LLI were blocked by curare although they were probably also from motor neurons. This difference in pharmacological sensitivity was due to the different manners in which the two sets of im- pulses were evoked. LLI were presumably initiated at

Peripheral sensory-motor synapses in Limuius

RI S R2

SALINE

CURARE

Fig. 6. Compound action potentials and individual impulses recorded at two sites along the leg nerve (R 1, R2) and force-tr~sdu~r records (FT) of claw closing. The large leg nerve was stimulated (S) with 1 set trains at 5 Hz. Electrode records were made by su~rimposi~g five traces from a train of five stimuli. Transducer retards, each made from a single trace at one-tenth the sweep speed of the electrode traces. show tension response to train. Stimulus intensities increase across a row of records, but are approximately equal in a column. CAPS initiated at ‘3” propagated in both directions along the leg nerve. but long latency impulses (Ltl, arrows) were initiated in the claw and travelled to the cut end of the ieg nerve. fnjection of 4 ml 10W6 M curare into the claw reversibly biocked LLI but did not affect claw closing, LLI may be antidromically conducting motor impulses initiated at sensory-motor synapses

in the claw. Stimulation at 9’” causes antidromic sensory conduction to these synapses.

cholinergic sensory-motor synapses while the im- pulses in Fig. 7 were initiated by direct stimulation of motor neurons. Thus curare (and low-calcium saline) disrupts LLI initiation but not direct motor conduc- tion. Since direct stimulation of closer mu&e con- traction did not produce praprioceptive discharge, it appears that the large leg nerve does not transmit proprioceptive information about the claw joint. LLI then cannot be attributed to ~ropriocc~tive discharge.

Figure 8 shaws additional evidence that LLI were not generated by propriocepfors. Each record shows early CAPS (partially obscured by artifact) and two groups of LLI evoked by electrical stimulation of the leg nerve (same stimulus intensity for each record). The left record shows also the force-transducer record of claw closing. For the middle record the articular membrane of the tibia-tarsus joint was cut and the tarsus was detached from the closer tendon and re- moved. For the right record most of the closer muscle fibers were scraped free of their attachments to the

tibia1 exoskeleton so that leg nerve stimulation pro- duced little noticeable closer muscle movement. These manipulations should have altered any proprioceptive output; however, they clearly did not affect the LLI.

DlSCUSSION

The previous hypothesis that claws of isolated ~~?~~lffs walking legs retlexively close by ephaptic mechanisms (Hoyle, 19%; Farnas et ol., 1968) is inconsistent with the results of this study. Instead, this report presents two strong pieces of evidence for a peripheral, cholinergic sensory-motor reflex arc in the claw of the L&&s walking leg. First. stimulation of sensory axons in the distal, muscle-free index evokes claw closing even when the sensory and motor neurons have been cut off from the central nervous system. Second_ curare, which does not block claw closing evoked by motor neuron stimulation, blocks closing evoked by stimulation of presumed sensory

510 STANLEY G. RANE and GORDON A. WYSE

SALINE FT

RI R2

181350 MSEC 180 Uv 1 J lOOuV2

CURARE

WASH

Fig. 7. Impulses recorded at two sites along the leg nerve (RI. R2) and force-transducer records (FT) of claw closing. Metal electrodes were inserted into the claw closer muscle (S) for the purpose of direct stimulation. Stimuli were given in 1 set trains at 5 Hz. Individual stimulus durations here I msec. Electrode records were made by superimposing five traces from five stimuli in a train. Transducer records, each a single trace at one-twentieth the sweep speed of the electrode traces, show tension responses to train. Impulses (arrows) were probably from motor neurons backfired by the stimulating electrodes (see text). Curare did not affect these impulses, yet it did block LLI which were probably motor impulses also. This difference is explained by different modes of impulse initiation (see text). The absence of other impulses in the above records suggests that the large leg nerve does not transmit

impulses from claw proprioceptors.

RI S R2

NORMAL CLOSER TEN- CLOSER MUSCLE DON CUT, TAR- DETACHED FROM

SUS REMOVED EXOSKELETON

Fig. 8. Compound action potentials (obscured by artifact) and LLI (arrows) recorded at two sites along the leg nerve (Rl, R2). The leg nerve was stimulated (S) with 1 set trains at 5 Hz, at equal intensities for each of the three records. Electrode records were made by superimposing five traces from a train of five stimuli. The transducer record. a single trace at one-twentieth the sweep speed of the electrode traces, shows tension response to the train. LLI were not affected by destruction of the claw joint and removal of the tarsus (middle record), or by detachment of closer muscle fibers from their origins on the tibia1 exoskeleton (right record). The latter procedure eliminated almost all evoked closer muscle contraction.

If LLI were from claw proprioceptors. then either procedure should have affected them.

Peripheral sensory-motor synapses in ~~rnu/z~s 511

neurons. Other evidence, reported here, is consistent duced sensory-motor synaptic delays which were less with and supports this peripheral reflex hypothesis. variable.

The concentration of curare that blocked claw clos- ing and initiation of some impulses in this study can be compared to concentrations employed elsewhere. The concentrations used here were four orders of magnitude Iower than those reported to block neur- onal conduction in cockroaches (Friedman & Carl- son, 1970); two orders of magnitude less than those used to block iontophoretic acetylcholine excitation of lobster central ganglion somata (Barker et al., 1972); and equivalent to or less than those used to block cereal nerve-giant fiber synapse in desheathed cockroach sixth abdominal ganglion (Shankland et a[., 1971). The effects of curare reported here are thus assumed to be due solely to blockade of cholinergic synapses in the tibia.

Curare, atropine or low-calcium saline blocked both claw closing and some impulses evoked by stimulation of large leg nerve branches (sensory axons) in the index (Fig. 5). Since these impulses and claw closing were always blocked together, the im- pulses blocked were presumed to be from motor neurons. Stimulation of the leg nerve proximal to the claw (sensory and motor axons) evoked both claw closing and CAPS which conducted away from the stimulus site, but it also caused impulses (LLI) to be initiated in the claw segment. Curare, atropine or low- calcium saline blocked initiation of these impulses (Fig. 6); thus the LLI were probably similar to the drug-sensitive spikes evoked by stimulation of sensory nerves in the index. These results indicate that sensory axon stimulation. both orthodromic (applied at the index) and antidromic (applied proximal to the claw), evoked motor neuron impulses via peripheral. cholin- ergic synapses. The synapses must be en passant for motor neurons to be driven by both orthodromic and antidromic stimulation of sensory neurons. Curare did not prevent closer muscle contraction evoked by stimulation of the leg nerve (proximal to the claw) because stimulation here fired motor neurons directly. as well as via the peripheral sensory--motor synapses. Low-calcium saline prevented claw closing regardless of the point of stimulation, presumably by disruption of either neuromuscular transmission or excitation- contraction coupling. Low-calcium saline. however, blocked only those impulses which were also suscep- tible to cholinergic blockade. This result provides ad- ditional evidence that these impulses were initiated at a synaptic site in the tibia.

Although synapses are suggested here as sites for peripheral impulse initiation, there are other possible explanations for peripheral impulse initiation as a result of motor neuron stimulation proximal to the claw. Receptors sensitive to both muscle length and tension have been found in other Limu[us leg muscle (Eagles, 1978; Eagles & Gregg, 1979). It is therefore possible that impulses initiated in the tibia (LLI) could have been from proprioceptors in the closer muscle or the tibia-tarsus joint. Blockade of LLI by atropine or micromo~r concentrations of curare, however, seems inconsistent with this proposal. Ad- ditionally, LLI (coincident with closer muscle con- traction) persisted despite removal of the tarsus and prevention of tension development by freeing muscle fibers from their attachments to the tibia1 exoskeleton (Fig. 8). Thus it seems unlikely that LLI resulted from proprioceptive discharge. Direct stimulation of the closer muscle elicited only spikes that were inter- preted to be antidromic motor neuron impulses, as judged by their persistence after muscle contraction was blocked by a low-calcium saline. Since direct stimulation of closer muscle contraction elicited no other later impulses, the large leg nerve appears not to transmit proprioceptive information about claw closing.

A mechanism for peripheral generation of impulses in a motor neuron, subsequent to excitation of the same motor neuron at a more proximal site, has been reported for Puch_r~rap.su~s (Stephens & Atwood, 1981). The recorded latencies of peripherally gener- ated impulses (LLI) in our study appear to be 5-8 msec too long to have resulted from this mechan- ism (see “Results” section on peripheral impulse rebound). Moreover, peripheral initiation of impulses in Lirnuhrs could be pharmacologically blocked with- out impairment of motor impulse conduction (Fig. 7). Finally. peripheral impulse generation in fucl’t~- grcipsus vvas dependent on a 1.5 C increase between acclimation and experimental temperatures. For ex- periments reported here, temperature variations were minimal (1-3 C).

Both LLI and drug-sensitive impulses evoked by stimulation of nerves in the index exhibited variable post-stimulus latencies (Figs. 5 and 6). A synaptic ori- gin for these impulses could explain the variations in their post-stimulus latencies to successive stimuli. Travel times for impulses mediated by a synapse should be consistent (for successive stimuli) over the pre- and postsynaptic axons, but variations in synap- tic delay could change successive post-stimulus laten- ties over the entire path. Most CAPS observed in these experiments had consistent post-stimulus laten- ties. which suggest that they were conducted without synaptic mediation (Fig. 5). For leg nerve stimulation proximal to the claw, latency variations of LLI de- creased with increased stimulus voltages. This finding suggests that excitation of more sensory axons pro-

Although acetylcholine may act as a neuromuscular transmitter at some arthropod muscles (Futamachi, 1972: Marder & Paupardin-Tritsch, 1980), glutamate rather than acetylcholine is considered to be the major candidate arthropod neuromuscular trans- mitter. For Lirndus. acetylcholine elicited impulses that travelled in the distal-proximal direction, and also elicited claw closer muscle contraction. Curare blocked contraction evoked by sensory axon stimu- lation (Fig. 5). but not contraction evoked by stimu- lation of motor axons proximal to the claw (Fig. 6). Therefore acetylcholine probably does not act as the neuromuscular transmitter. Instead, it appears to mimic the excitatory transmitter at the peripheral sen- sory -motor synapses, causing motor impulses that travel to the neuromuscular junction as well as anti- dromically along the leg nerve. AcetylchoIine is present in Lit&us leg nerve and James & Walker (1980) found that it excited all central neuron somata on which it was tested; thus it may be a sensory neuron transmitter. Low-calcium saline blocked con- traction but not impulses elicited by acetylcholine, a

512 STANLEY G. RANE and GORDON A. WYSE

result which indicates that these impulses were due to a postsynaptic action of acetylcholine.

Since contraction was blocked presumably by dis- ruption of either neuromuscular transmitter release or excitation-contraction coupling, a direct excitatory effect of acetylcholine on muscle was not ruled out. Work in progress, however, suggests that an amino acid is the probable neuromuscular transmitter. Ace- tylcholine was reported to evoke muscle contraction in limbs of some crustacea (Katz, 1936; Florey & Florey. 1954), but its site of action was not deter- mined.

EAGLES D. A. (1978) Tension receptors associated with muscles in the walking legs of the horseshoe crab. Limulus polyphrmus. Mar. Behac. Physiol. 5, 215-230.

Eagles D. A. & Gregg R. A. (1979) Receptors sensitive to muscle length in the horseshoe crab. Mar. Brhar. Phy- dol. 6, 21 I-223.

In Limulus, then, there is a reasonable explanation for reflexive claw closing when motor and sensory neurons in the claw are isolated from synaptic con- tacts in the central nervous system, The explanation is peripheral synaptic communication between sensory and motor neurons. The previous explanations. which involved ephaptic sensory-motor transmission at the cut end of the leg nerve, appear untenable, both because blockade of neuronal conduction between the claw and the cut end does not block reflexive claw closing (Fig. 2), and because there is no electrophysio- logical evidence of impulse initiation at the cut end of the nerve. The function of a peripheral reflex path in the intact animal is not clear. In addition, it remains a moot point whether or not reflexes in excised limbs of some crustacea can be attributed to a peripheral reflex arc similar to that in Limulus.

FLOREY E. (1973) Acetylcholine as sensory transmitter in crustacea. J. camp. Physiol. 83,. 1~-16.

FLOREY E. & FLOREY E. (1954) Uber die mSgliche Bedeu- tung von Enteramin (5-Oxy-Tryptamin) als nerviiser Aktionssubstanz bei Cephalopoden und dekapoden Crustaceen. Z. Naturf: 9b, 58-68.

FLOREY E. & BIEDERMAN M. A. (1960) Studies on the dlstrl- bution of Factor I and acetylcholine in crustacean per- ipheral nerve. .l. yen. Physiol. 43, 509-522.

FOURTNER C. R. & SHERMAN R. G. (1972) A light and electron microscopic examination of muscles in the walking legs of the horseshoe crab. Lirnulrrs pd~~phrnws. C‘an. J. Zoo/. SO, 1447-1455.

FRIEDMAN K. J. & CARLSO~. A. D. (1970) The efiects of curare in the cockroach II. Blockade of nerve impulses by d-TC. J. exp. Biol. 52, 593-601.

FL~TAMATHI K. (1972) Acetylcholine: Possible neuromuscu- lar transmitter in crustacea. Scicnc,cj. .\‘.I: 175. 1373 1375.

HAYES W. F. & BARBER S. B. (1967) Proprioceptivc distri- bution and properties in Limul~rt walking legs. /. ‘.~p. Zoo/. 165, 195-210.

HOYLE G. (1958) Studies on neuromuscular transmission in Lirnulus. Biol. Bull. 115, 209-218.

KATZ B. (1936) Neuro-muscular transmission in crabs. J. Physiol.. Lond. 87, 199-221.

Acknowledgements-We are grateful to Bruce Winn for introducing us to the “pseudoreflex” in Limulus. This study was partially supported by a Faculty Research Grant from the University of Massachusetts.

MARDEK E. & PAUPARDIS-TRITSCH D. (1980) The pharma- cological profile of the acetylcholine response of a crus- tacean muscle. J. exp. Biol. 88, 147-159.

O’TANYI T. J. JR. & BARBER S. B. (1966) Stretch receptors in Limulus limbs. Am. Zoo/. 6, 519-520.

PARNAS I.. ABBOTT B. C., SHAPIRO B. & LANC; F. (1968) Neuromuscular system of Lirnulus leg closer muscle. C’omp. Biochem. Physiol. 26, 467-478.

SHANKLAXI) D. L.. ROSE .I. A. & DONNIGER C. (1971) The

REFERENCES

A~wo~I, H. L. (1976) Organization and synaptic physi- ology of crustacean neuromuscular systems. Proq. Nrurohiol. 7, 291-391.

BARKER D. L., HERBERT E., HILIX~RAN~ .I. G.. & KRAVIT~ E. A. (1972) Acetylcholine and lobster sensory neurons. J. Pk~siol. 226, 205-229.

BARNES T. C. (1932) Responses in the isolated limbs of crustacea and associated nervous discharge. Am. J. Phy- siol. 99, 321-331.

BULLOCK T. H. & HORRID~;~ G. A. (1965) .Srrucrurr ud ~unc~tion in rhe Nrrwu.s S~~sten~ of Inrrrtchrafe.\. W. H. Freeman. San Francisco.

cholinergic nature of the cereal nerve-giant fiber synapse in the sixth abdominal ganglion of the amerlcan cock- roach. Poiplunrtrr trn~cric~atur. J. Nrut~~hiol. 2(3). 247 262.

SNOIXRASS R. E. (1952) 4 Testhook of ilrthropod ,4rltrror?l\,. Cornell University Press. Ithaca.

S~I:~HENS P. J. 6t Arwooo H. L. (1981) Peripheral gener- ation and modulation of crustacean motor :txon activit> at high temperatures. J. Camp. PI7y.\io/. 142, 309 314.

TK~HCRNE J. E. (1966) The ,Ycfrrochc,,t~i,\~~~ of .l,fhropor/.\. Cambridge University Press. Cambridge.

WALTZER R. J. & JAMES V. A. (1980) Minirevien : The Cen- tral Nervous System of Lirfnd~rz pc~i~phrmu,~: Physiologl- cal and pharmacological studies. Co/rip. Bioc~hcm. Ph I viol. 66C. l21- 124.

CHAO I. (1933) Action of electrolytes on the dorsal median Wvst: G. A. (1971) Receptor organizallon and function in

nerve cord of the Limulus heart. Eiol. Bull. 64. 358-381. Lirm~lus chelae. Z. rerql Physiol. 73, 249- 273.