ORIGINAL PAPER

N. Hama Æ M. Takahata

Effects of leg movements on the synaptic activity of descendingstatocyst interneurons in crayfish, Procambarus clarkii

Received: 20 May 2003 / Revised: 8 September 2003 / Accepted: 15 September 2003 / Published online: 31 October 2003� Springer-Verlag 2003

Abstract Crustacean postural control is modulated bybehavioral condition. In this study, we investigated howthe responses of descending statocyst interneurons wereaffected during leg movements. Intracellular recordingwas made from an animal whose statoliths had beenreplaced with ferrite grains so that statocyst receptorscould be activated by magnetic field stimulation. Weidentified 14 morphological types of statocyst-drivendescending interneurons. Statocyst-driven descendinginterneurons always showed an excitatory response tostatocyst stimulation on either ipsilateral or contralat-eral side to the axon. The response of each statocyst-driven descending interneuron to statocyst stimulationwas differently modulated by leg movements in differentconditions. During active leg movements, six statocyst-driven descending interneurons were activated regardlessof whether a substrate was provided or not. In other twostatocyst-driven descending interneurons, the excitatoryinput during leg movements was stronger when a sub-strate was provided than when it was not. One statocyst-driven descending interneuron received an excitatoryinput only during leg movements on a substrate, whereasanother statocyst-driven descending interneuron did notreceive any input during leg movements both on asubstrate and in the air. These results suggest thatthe descending statocyst pathways are organized inparallel, each cell affected differently by behavioralconditions.

Keywords Crayfish Æ Descending interneurons Æ Legmovements Æ Response modulation Æ Statocyst

Abbreviations EMG electromyogram Æ NGI nonspikinggiant interneuron Æ SDI statocyst-driven descendinginterneuron

Introduction

Control of body posture generally depends on neuralsignals from sense organs of different modalities,including equilibrium, visual and somatosensory recep-tors (Horak and Macpherson 1996; Deliagina et al.1999). In fish, vestibular information makes a crucialinteraction with visual input to calculate the final bodyposture (von Holst and Mittelstaedt 1950; Ullen et al.1995; Deliagina et al. 2000). Sensory interaction hasbeen also reported to be crucial in the posture control ofinvertebrates (Schone et al. 1983; Okada et al. 1994). It isalso known in both vertebrates and invertebrates thatbehavioral context and ongoing activity significantlyaffect the operation of neuronal circuits controlling bodyposture (Davis et al. 1974; Everett et al. 1982; Deliaginaet al. 2000). However, the synaptic mechanism of thisinteraction among sensory systems and between sensoryand motor systems largely remains unknown.

In crustaceans, body tilting evokes equilibrium re-sponses that involve head and posterior appendagesincluding eyestalks, walking legs and uropods (Kuhn1914; Schone 1954; Davis 1968; Yoshino et al. 1980).These responses are primarily controlled by a pair ofstatocysts. It is also known well that these equilibriumresponses are extensively affected by leg proprioceptorand visual inputs (Kuhn 1914; Alverdes 1926; Schoneet al. 1976) as well as behavioral conditions (Takahataet al. 1984). Although previous studies have providedextensive descriptions of structural and physiologicalcharacteristics of statocyst-driven local and descendinginterneurons in the brain (Wiersma 1958; Takahata andHisada 1982; Nakagawa and Hisada 1989, 1990), syn-aptic mechanisms subserving their multimodal responsecharacteristics have been rarely addressed except in thenonspiking giant interneurons (NGI) that are presyn-

J Comp Physiol A (2003) 189: 877–888DOI 10.1007/s00359-003-0464-5

N. Hama (&) Æ M. TakahataAnimal Behavior and Intelligence, Division of Biological Sciences,Graduate School of Science, Hokkaido University, 060-0810Sapporo, JapanE-mail: hnori@sci.hokudai.ac.jpTel.: +81-11-7062753Fax: +81-11-7064923

aptic to eyecup motoneurons (Okada and Yamaguchi1988; Okada et al. 1994; Furudate et al. 1996).

Effects of leg-related signals on posture control areparticularly complex. Body rolling elicits steering re-sponses of uropods in different directions depending onwhether a substrate is provided or not during body rolling(Takahata et al. 1984). Passive or forced movements oflegs elicit equilibrium reflexes of various appendagesincluding eyestalks, antennae and uropods (Schone et al.1976; Clarac et al. 1976; Newland 1989). Active legmovements driven endogenously in the air facilitate uro-pod steering to be initiated by statocyst input (Takahataet al. 1984). Recent studies by Sakuraba and Takahata(1999, 2000) have shown that the central compensation ofappendage posture following unilateral statolith removal(Schone 1954; Yoshino et al. 1980) was observedwhen theexperimental animal was kept on a substrate but notwhenit was kept off the substrate during the post-operativeperiod of 2 weeks, suggesting that the central statocystpathway would be affected differently depending on theleg condition. It is therefore essential for understandingthe physiological mechanisms of not only posture controlin natural condition but also central compensation pro-cess to obtain knowledge on how the activity of statocyst-driven interneurons in the brain is affected synaptically byleg movements on and off the substrate.

In this study, we made intracellular recordings fromthe statocyst-driven descending interneurons in the brainof unanesthetized crayfish that could freely move theirlegs during experiment. By comparing the synaptic re-sponses of these interneurons recorded when the animalwas quiescent with those recorded when moving legs onand off the substrate, we analyzed how the activity of thedescending statocyst pathway was affected depending onthe leg movement condition.

Materials and methods

Animals and preparation

In all experiments, adult crayfish Procambarus clarkii of both sexesmeasuring 8–11 cm in body length were used. They were commer-cially obtained and kept in laboratory tanks before use. Four weeksbefore experiment, statoliths were replaced with ferrite grains onboth sides. The rostrum was cut away and the nonsensory hairscovering the opening of the statocyst were removed with fine for-ceps. The statolith was then washed out with water jet using a fineglass pipette. Ferrite grains were introduced into the statocyst lumenusing a fine needle. After this operation, animals were kept in lab-oratory tanks before experiment for 4 weeks. Before dissection, thechelipeds were cut away and each crayfish was anesthetized in cooledphysiological saline (van Harreveld 1936). A small portion of dorsalcarapace was removed to reveal the brain. The animal was then fixeddorsal side-up to a metal rod framework so that it was suspended offthe substrate. All legs and the abdomen were able to move freely.

The spike activity of interneurons descending from the brain toposterior ganglia and the behavioral state of the animal werecontinuously monitored throughout the experimental period. Torecord the descending activity, a single en passant suction electrodewas placed on the left circumesophageal commissure. The com-missure on the opposite side was also kept intact so as not tointerrupt the transmission of ascending leg information. The

behavioral state of the animal was monitored by electromyogram(EMG) recorded from the mero-carpopodite flexor muscle of the2nd or 3rd walking leg (Bush et al. 1978) on the right side using apair of Teflon-coated silver wires of diameter 125 lm. Since theflexor muscle is close to an extensor muscle, the possibility ofrecording from the extensor could not be excluded. Recording fromeither muscle, however, was purposive for monitoring the generalbehavioral state of the animal. A strain gauge-equipped substratemade of an elastic plastic plate could be either held close to ormoved away from the walking legs smoothly using a manipulator.In order to secure the measurement sensitivity as high as possible, apair of strain gauges were pasted on both sides of the plate to formtwo arms of a Wheatstone bridge circuit, the other arms of whichwere fixed resistances. This transducer system was used in order todetermine if the legs were being actively moved or not against thesubstrate during intracellular recording. The electrical signal wasfed to a high-input impedance amplifier with a high-frequency cutfilter (<0.3 kHz), and then to an oscilloscope and a data recorder(see below). The substrate was put against legs to yield about ahalf-magnitude of the force that would be experienced by the legswhen an animal with the body weight of 15–20 g stood quietly.Experimental set up is diagrammatically shown in Fig. 1.

Stimulation

In order to maintain stable intracellular recording while statocystreceptors were being stimulated, we applied magnetic field stimula-tion to the crayfish whose statoliths were replaced with ferrite grainson both sides.Methodological details have been provided previouslytogether with specific characteristics of the electromagnet (Muray-ama and Takahata 1992). Briefly, the stimulation was carried out bypassing a current of trapezoidal profile (maximum amplitude 0.1 A)to the energizing coil of an electromagnet which pulled ferrite grainsin the statocyst. The electromagnet was always placed lateral to thestatocyst on the left side with 45� to the horizontal plane and with10 mmdistance from the statocyst in this study. This combination ofcurrent, distance and direction mimicked body tilting in the left side-down direction by 90� (Murayama and Takahata 1992 ).

Intracellular recording and staining

Intracellular recording was made from the anterior part of the de-utocerebral segment of SDI dendrites inmost cases (Fig. 2).We usedglass microelectrodes filled with 5% lucifer yellow (LY) in 1 mol l)1

LiCl having resistances of 30–50 MW. The microelectrode was con-nected to a high input-impedance amplifier equipped with electricalcircuits for constant current injection and bridge balance adjustment(Axon Instruments, Axoclamp-2B). Physiological data were storedin a digital audio tape recorder (TEAC, RD-135T; DC-10 kHz) anddisplayed on a digital oscilloscope (Iwatsu, DS-9121; sampling rate12 kHz). A home-made computer program was used to obtainwaveform data from the oscilloscope that was connected with apersonal computer (Apple PowerMacintosh 7300) through a GPIBinterface. After physiological examination, impaled neurons wereintracellularly stained with LY through the recording electrode byapplying negative current pulses (6–10 nA, 500 ms duration, 1 Hzfor 10–15 min). For diffusion of LY, the ganglion was stored at 4�Cfor 1 h. Then the ganglion was dissected out, fixed, dehydrated inalcohol series, cleared in methyl salicylate and mounted on a fluo-rescence microscope (Olympus, BX-50). The neuronal structure wasobtained directly by camera lucida tracing.

Fig. 2 Structure of all 14 morphological types of SDIs. Thedendritic structure of each neuron is shown with its location inthe brain viewed dorsally. Gray circles indicate parolfactory lobeswhere primary statocyst afferents make axonal termination. Allneurons except SDI10 had its cell soma in the ventral paired medialcluster. ACC accessory lobe; ANT antennal lobe; OLF olfactorylobe; OPT optic lobe; PAR parolfactory lobe

c

878

Fig. 1 Experimental set up forintracellular recording from thestatocyst-driven descendinginterneurons (SDIs) in thebrain. Statocyst receptors wereactivated by magnetic fieldstimulation. The animal couldfreely move legs duringrecording. The descending spikesignals were recorded enpassant from thecircumesophageal commissureusing an extracellular suctionelectrode (left inset)

879

Results

We impaled 51 descending interneurons that respondedto statocyst stimulation using 268 animals, and classifiedthese statocyst-driven descending interneurons (SDIs)into 14 morphological types in this study. For each of 10out of 14 morphological types of cells, we could suc-cessfully make intracellular recording and staining fromthe same type in two or more different animals. Thestructure of all 14 types of interneurons is summarized inFig. 2 and their dendritic projection in Table 1. Thenomenclature of neuropiles and cell clusters is based onTautz and Tautz (1983). The input statocyst of each SDIas revealed by unilateral statolith removal experiment isalso shown in Table 1.

General features of SDI morphology

All SDIs except SDI10 had their cell body in the anteriorcluster (Fig. 2). Five of them were found to send an axonto the circumesophageal commissure on the contralat-eral side to the cell body whereas others to the ipsilateralcommissure. SDI10, having its soma in the ventralpaired medial cluster, sent an axon to the ipsilateralcommissure. Most SDIs projected their dendrites to theoptic lobe in the protocerebrum and to the parolfactorylobe in the deutocerebrum on either the ipsilateral orcontralateral side to the axon, or on both sides (Ta-ble 1). Almost no SDIs had dendritic projection to the

olfactory lobe. Projection to the accessory and antennallobes was occasional, more frequent on the ipsilateralside than on the contralateral side. As described in thefollowing sections, we could not associate any mor-phological property with physiological response char-acteristics of SDIs in this study.

Synaptic responses to statocyst stimulation

Of 51 SDIs, 41 cells sent their axon to the circum-esophageal commissure ipsilateral to the electromagnetwhile 10 cells to the contralateral one. They all showedan excitatory response, either subthreshold or supra-threshold depending on the cell, to statocyst stimulationexcept SDI3 that was encountered once on the ipsilateraland contralateral side, respectively, to show a decreasein spontaneous spike discharge frequency. No SDIshowed hyperpolarizing responses. The synaptic re-sponse of SDIs to statocyst stimulation could be reducedby depolarization and enhanced by hyperpolarization.In the SDI1 shown in Fig. 3a, both the peak amplitudeof discrete synaptic potentials and the baseline depo-larization was suppressed when the cell was depolarizedby current injection and enhanced when hyperpolarized(Fig. 3b). The insets between Fig. 3a and b show theshape of synaptic potentials at different membrane po-tential levels. The voltage dependence of synaptic inputindicated that the signal transmission from statocystsensory neurons to SDIs was mediated chemically.

Table 1 Summary of structural and physiological characteristics of 14 statocyst-driven descending interneurons (SDIs) identified in thisstudy

SDI OPT OLF ACC PAR ANT Type ofresponse

Synapticinput

Spikefiring

Inputside

Efferentcopy

Legproprioceptor

i c i c i c i c i c

1 + ) ) ) ) ) ) ) ) ) Tonic Discrete + i ) +2 + + ) ) + ) + ) + ) Tonic (complex) Continuous + b + )3 + + ) ) + ) + ) + ) Tonic (inhibition) Discrete + i + )4 + ) ) ) + ) + ) + ) Phasic Continuous + c + +5 ) + ) ) + ) + ) ) ) Phasic Discrete ) i ) )6 + + ) ) ) + ) + ) + Tonic Intermediate + b + )7 + ) ) ) ) ) + ) ) ) Tonic Intermediate + n n n8 + + ) ) ) ) + + ) + Phasic Continuous + c + +9 + + ) ) ) ) + + + + Phasic Intermediate ) i + )10 + ) ) ) ) ) + ) + ) Tonic Discrete + n n n11 + ) ) ) ) ) + ) ) ) Tonic Continuous ) n n n12 ) ) ) ) + ) + + + ) Tonic Continuous + b n n13 + ) + ) + ) + ) + ) Tonic Continuous ) n + )14 ) + ) ) ) ) ) ) ) ) Tonic Continuous + c + )

First 5 columns show the site of dendritic projection for each SDI:the subcolumns designated i indicate projection on the ipsilateralside to the axon, and c the contralateral sideACC accessory lobe; ANT antennal lobe; OLF olfactory lobe; OPToptic lobe; PAR parolfactory lobe+ projection of dendrites; ) noprojectionSynaptic input describes the nature of synaptic response to statocyststimulation that was either a train of discrete synaptic potentials ora slow, continuous potential each with or without spikesSpike firing indicates if the cell was spontaneously dischargingspikes or not. Input side indicates the input statocyst for each cell:

b bilateral statocysts; c contralateral to the axon; i ipsilateral to theaxon.Efferent copy indicates the effect of free leg movement in the air: +indicates enhancement of the synaptic response to statocyst stim-ulation, ) no enhancementLeg proprioceptor indicates the effect of leg movement on substrate:+ indicates further enhancement of the synaptic response com-pared with that during free leg movement in the air; ) no furtherenhancementn indicates that the synaptic response was not examined in theSDI

880

Experimental alteration of the membrane potential bycurrent injection enabled us to isolate the synaptic inputunderlying the spike response of SDIs to statocyst stim-ulation. Seven SDIs were spontaneously active, dis-charging spikes at the rate of 1–20 spikes s)1, while otherswere silent when the animal was at rest (Table 1). TheSDI14 shown in Fig. 3c was spontaneously active at rest.Statocyst stimulation caused an increase in the spike dis-charge rate with a slight indication of excitatory synapticinput. When a depolarizing current was injected intra-cellularly, the spontaneous spike discharge rate increasedand a very small increase, if any, was observed in the spike

activity in response to statocyst stimulation (Fig. 3d).Synaptic activity was hardly observed: it might bemaskeddue to high frequency of spike firing. When a hyperpo-larizing current was injected, by contrast, a large synapticinput superimposed with a few spikes was observed inresponse to statocyst stimulation. The sustained increaseof spike activity during stimulation at rest thus appearedto be due to continuous excitatory input from statocystreceptors. The spontaneous spike dischargewas abolishedcompletely during hyperpolarization.

The synaptic inputs to SDI1 (Fig. 3a) and to SDI14(Fig. 3c) were qualitatively different from each other inthat the former cell received a train of discrete synapticpotentials, whereas the latter received a slow and contin-uous input. Seven SDIs were found to receive continuousinputs while the other four SDIs received discrete inputs.The remaining three SDIs received intermediate inputs(Table 1). No correlation was observed between the syn-aptic input discreteness and the response profile charac-teristics: a train of discrete synaptic potentials wereobserved either transiently (SDI5) or steadily (SDI10).

Modulation of SDI responses to statocyst stimulationby leg movements

When an animal became active to move walking legseither against a substrate or in the air, the SDI responseto statocyst stimulation was affected in various waysdepending on the cell. The SDI14 illustrated in Fig. 4a

Fig. 3 Effects of current injection on the synaptic responses ofSDI1 (a, b) and SDI14 (c, d) to statocyst stimulation. a Changes inthe amplitude of synaptic responses of SDI1 during intracellularcurrent injection. The bottom trace (MFS) shows the currentflowing through the electromagnet that mimicked left-side downbody tilting. b Comparison of the amplitude of discrete synapticpotentials (filled circle) and the baseline depolarization (open circle)during current injection. Both were reduced by positive current andenhanced by negative current. Typical single responses at differentmembrane potentials are shown in the inset between a and b. cSeparation by negative current injection of the synaptic responseand the spike discharge that was superimposed on it. Statocyststimulation increased the frequency of spike firing. Negativecurrent ()1 nA) injection depressed the spontaneous spike dis-charge to reveal the synaptic responses (bottom trace) underlyingthe spike responses to statocyst stimulation at the resting potentiallevel (middle trace), while positive current (+1 nA) injectionincreased the spontaneous spike firing to obscure both synapticand spike responses (top trace). d Spike activity histograms for thedata shown in c

881

showed a transient suppressive response followed by atonic excitatory response consisting of a train of spikeswhen the animal was at rest. This cell also showed aninhibitory off-response after sensory stimulation. Whenthe animal became active to move legs in the air, theinterneuron showed a remarkable increase in the spon-taneous spike activity. But the sensory response tostatocyst stimulation increased very slightly, if any(Fig. 4b). When the animal became active in the condi-tion that a substrate was provided for the legs, it stret-ched and bent its legs in such a complex way that thesubstrate was either pushed downward by the legs orlifted upward by self-elasticity as indicated by themonitor trace (second from the top in Fig. 4c). Thespontaneous spike activity increased as in the case of freeleg movements without substrate (Fig. 4b), but thesynaptic response to statocyst stimulation also increasedsignificantly in this case (Fig. 4c). It is interesting to notethat this inhibitory off-response was clearly seen whenthe legs were provided with a substrate (Fig. 4c) but notseen at all when they were free in the air (Fig. 4b). Thus,the leg movements differently affected the synaptic re-sponse of SDI14 to statocyst stimulation depending onwhether the legs were provided with a substrate or not.

Different effects of leg movements on the sensoryresponse of SDIs

How the synaptic response to statocyst stimulation wasaffected by leg movements varied among SDIs. Typical

responses are shown in Fig. 5. The SDI1 illustrated inFig. 5a showed a phasico-tonic excitatory responseconsisting of a train of discrete synaptic potentials (up-per panel) when the animal was at rest. When activelymoved its legs in the air, the interneuron showed nonoticeable change in the spontaneous activity and thesensory response (middle panel). With a substrate pro-vided for the legs, however, the interneuron received acontinuous bombardment of synaptic inputs even in theabsence of sensory stimulation (the lower panel ofFig. 5a). The leg movement first caused lifting of thesubstrate from the resting level as indicated by upwarddeflection of the monitor signal (second trace). The legswere then extended to cause downward displacement ofthe substrate and suprathreshold excitation of theinterneuron. When the statocyst was stimulated duringthis excitation, the sensory response was found to makesynaptic summation with the excitation caused by legmovements (an arrowhead in the lower panel ofFig. 5a). Preceding this summation, several spikes wereobserved that were singly caused by strong excitationdue to irregular leg movements. The statocyst input itselfwas neither enhanced nor suppressed during the activeleg movements on the substrate. We confirmed thatsimply providing a substrate against the walking legswhen the animal was at rest had no noticeable effect onthe SDI1 response to statocyst stimulation. It thus ap-peared that SDI1 would act as the site of synapticsummation for statocyst and leg proprioceptor inputs.

The synaptic activity of SDI5, by contrast, was foundto be completely impervious to the animal’s behavioralstate (Fig. 5b). The interneuron received a train of discretesynaptic inputs when the animal was at rest (upper panel).It showed a phasic response to statocyst stimulation.When the animal became active without a substrate, theinterneuron’s spontaneous synaptic activity was hardlyaffected as well as its response to statocyst stimulation(middle panel). Although it appears that the synapticactivity is reduced in the middle panel, this was due totemporal variability in the spontaneous synaptic activityof the cell. Even when the animal was active with a

Fig. 4a–c Modulation of the SDI14 response to statocyst stimula-tion by leg movements. When the animal was at rest, statocyststimulation increased the frequency of spike discharges (a). Whenthe animal moved legs in the air without substrate, the frequency ofspontaneous spike discharge increased and statocyst stimulationelicited slightly stronger response than at rest (b). When a substratewas provided for the legs, the spike response was further enhanced(c). At the top of each panel is shown a spike activity histogram.The records below each histogram are composed of intracellular(top trace), substrate transducer (ST; second trace), leg electro-myogram (Leg EMG; third trace) and magnetic field stimulation(MFS; bottom trace) signals

882

substrate provided, the spontaneous activity was alsounaffected (lower panel). The peak amplitude of the sen-sory response was greater in the lower panel than in theupper panel, but the duration of excitatory response wasshorter. Thus, the sensory responsewas also unaffected byleg movements on a substrate. The SDI5 would functionin simply relaying the statocyst information to posteriorganglia regardless of the animal’s behavioral condition.

Some interneurons received excitatory synaptic inputduring active legmovements without a substrate as SDI14shown in Fig. 4. The SDI9 illustrated in Fig. 5c showed

no spontaneous spike discharge when the animal was atrest although it received continuous bombardment ofsubthreshold synaptic inputs (upper panel). When theanimal became active to move legs in the air, the inter-neuron showed spontaneous spike discharges togetherwith enhanced synaptic inputs (middle panel). The syn-aptic response to statocyst stimulationwas found tomakesummation with the background activity, causing morenumbers of spikes to discharge. It appeared that thestatocyst input was even more remarkable when the ani-mal was active than at rest, suggesting the presence of akind of activity-dependent boosting mechanism for thestatocyst input to SDI9. After the animal became active tomove legs against the substrate, the synaptic response tostatocyst stimulation wasmore remarked than at rest, butslightly less remarkable than during free movements(lower panel). The period of depolarization and spikedischarge was shorter in the lower panel than in the upperpanel. This response characteristic could be contrastedwith that of SDI14 in which the synaptic response tostatocyst stimulation was more remarked during legmovements against substrate than during free movements(Fig. 4).

Changes in membrane conductance of SDIs associatedwith leg movements and sensory responses

When the animal was at rest, the SDI8 shown inFig. 6a1 had an input resistance of 4.4 MW measured by

Fig. 5 Different effects of leg movements on the synaptic responseof SDI1 (a), 5 (b) and 9 (c) to statocyst stimulation. Upper panelsshow the synaptic response to statocyst stimulation when theanimal was at rest. Middle and lower panels show the responseswhen the animal moved legs in the air and on substrate,respectively. Statocyst stimulation induced a train of discretesynaptic potentials in SDI1. When the animal moved legs in the air,no remarkable effect was induced in the spontaneous activity ofSDI1. The synaptic response to statocyst stimulation also remainedunaffected, but when a substrate was provided, the excitatoryresponse to statocyst stimulation made synaptic summation withthe excitatory input due to leg movements on substrate to dischargespikes (arrowhead in the lower panel of a). The SDI5 showed samesynaptic responses to statocyst stimulation regardless of theanimal’s behavioral condition (b). The SDI9 responded with asmall depolarizing synaptic potential (<1 mV) to statocyststimulation when the animal was at rest (upper panel in c). Thisexcitation was enhanced when the animal actively moved legswithout substrate. It was also enhanced when the animal movedlegs on substrate. The enhancement was less remarkable during onsubstrate than during free movement of legs

883

intracellularly injecting constant current pulses of)1 nA. In response to statocyst stimulation, the inter-neuron discharged several spikes superimposed on a

depolarizing synaptic input. During this response, theinput resistance of SDI8 decreased to about 50.7% ofthe initial value (Fig. 6a2). This decrease in input resis-tance suggested that the statocyst signal activated SDI8chemically by synaptic excitation. When the animal ac-tively moved its legs in the air, the input resistancecontinuously decreased to about 90.0% (Fig. 6a3),indicating that the SDI8 received chemical excitatoryinput during free leg movements. The synaptic responseof SDI8 to statocyst stimulation was less remarkablethan that observed at rest. No noticeable change wasobserved in input resistance during the synaptic response(Fig. 6a4). It was noted here that the excitatory inputfrom the statocyst made no effective summation with thesustained excitatory input that the SDI8 received duringfree leg movement.

Occlusion of two excitatory inputs was also observedin SDI13 as shown in Fig. 6b. The cell showed tonicexcitatory response to statocyst stimulation when ananimal was at rest (Fig. 6b1). When the animal becameactive to move legs freely, the input resistance showed acontinuous decrease (Fig. 6b2). The rate of reduction ininput resistance was 31.6% (Fig. 6b3) and the differencewas statistically significant (P<0.01; Fig. 6b4). Whenthe animal was at rest, the input resistance was 9.7 and9.5 MW before and during stimulation, respectively. Thedifference was, however, statistically insignificant

Fig. 6a, b Membrane conductance change during leg movementand synaptic response to statocyst stimulation in SDI8. When theanimal was at rest (a, panel 1), statocyst stimulation induced phasicexcitation with spike firing in SDI8 (top trace). The leg EMG(second trace) showed a low spontaneous activity. Injection ofhyperpolarizing current pulses into the same cell (third trace)revealed that the input resistance was reduced to 50.7% during thesynaptic response. The part indicated by a thick line in a (panel 1) isexpanded in a (panel 2). When the animal actively moved legs in theair (a, panel 3), the cell showed spontaneous spike firing wasobserved (inset below a, panel 1 and a, panel 2; the arrow indicatesthe time when the animal started leg movements). The inputresistance was continuously reduced to 90.0% during leg move-ments. The spike response to statocyst stimulation, however,showed a slight decrease, and no noticeable change was observed inthe input resistance during statocyst stimulation. The part indicatedby a thick line in a (panel 3) is expanded in a (panel 4). Membraneconductance change during free leg movement and synapticresponse to statocyst stimulation in SDI13. Statocyst stimulationinduced a tonic depolarization in SDI13 when the animal was atrest (b, panel 1). The cell also showed sustained depolarizationwhen the animal became active to move legs in the air (b, panel 2).The input resistance was found to be significantly reduced duringfree leg movements. Typical single responses to current injection atrest and during leg movements (indicated by brackets in b, panel 1and b, panel 2) are shown in b (panel 3) with an expanded timescale. Statistical comparison of the input resistance is summarizedin b (panel 4)

884

(P>0.05). The input resistance also showed no changeduring the synaptic response to statocyst stimulationwhen the animal was actively moving legs in the air(Fig. 6b4).

Discussion

We systematically examined in this study, for the firsttime, how the activity of statocyst-driven descendinginterneurons (SDIs) in response to mimicked bodyrolling was affected by nerve signals associated with legmovements1. Fourteen morphological types of SDIspresently identified do not represent the whole popula-tion of descending interneurons that convey statocystinformation from the brain to posterior ganglia, since wedid not encounter those interneurons previously identi-fied either morphologically (Nakagawa and Hisada1989) or physiologically (Takahata and Hisada 1982).However, the fact that most of the possible combina-tions in the mode of interaction between statocyst andleg movement-related inputs have been observed in them(Table 1) suggests that basic principles in the synapticintegration of those inputs can be sought out based onthe results obtained in this study. We discuss in thefollowing sections possible mechanisms of multimodalsensory integration and its functional significance inbehavioral control.

Synaptic modulation of SDI responses during legmovement in different conditions

Eight of the 14 SDIs examined in this study showedsignificant modulation of their synaptic responses tostatocyst stimulation when the animal moved legs freelyin the air. Crustacean thoracic legs have a variety ofproprioceptors and stress detectors that monitor theposition and movement of each joint (Mill 1976) andmechanical stress of legs (Marchand et al. 1995). Duringleg movements initiated endogenously, the joints arebent and extended in a complex way so that joint pro-prioceptors would be activated extensively. It is wellknown, however, that the activity of proprioceptor andmechanosensory pathways is significantly modulatedduring locomotor behavior in vertebrates (Gossard et al.1991; El Manira et al. 1996; Sillar and Roberts 1988)and invertebrates (Murphey and Palka 1974; Sillar andSkorupski 1986; Wolf and Burrows 1995; Poulet andHedwig 2003). In the walking legs of crayfish, proprio-ceptor activity during locomotor behavior is effectively

suppressed by nerve signals from the central patterngenerator through presynaptic inhibition (Cattaert et al.1990, 1992; El Manira et al. 1991). It would be thereforesafe to assume that during free leg movement the pro-prioceptor inputs from thorax to the brain would befiltered and differently organized so that forced andvolitional movements could be centrally discriminated.A nerve signal that is used to anticipate and cancel outself-generated afferent effects due to volitional move-ments is called a corollary (Sperry 1950) or efferencecopy (von Holst and Mittelstaedt 1950) signal. Based onthis assumption, we hypothesized in this study that theexcitatory input the SDIs received during free legmovements was caused by a corollary signal issued fromthe locomotor center presumably located in the brain.

By contrast, two of those which showed responseenhancement (SDI4 and 8) and another that showed noresponse modulation during free leg movements (SDI1)received further modulatory signals during leg move-ments on a substrate: the synaptic response to statocyststimulation was more enhanced than during free legmovements (Fig. 5). In this situation, volitional legmovement is opposed by a substrate so that the acti-vation of joint proprioceptors would be less extensivethan in free movements. Such a situation also activatesvarious local reflex circuits to increase motor activityand enforce leg movement (Elson et al. 1992). Fur-thermore, cuticular stress detectors would be extensivelyactivated during restricted leg movements (Duysenset al. 2000; Leibrock et al. 1996). Accordingly, theproprioceptor input and the corollary signal that is tocounteract the former would strike a different balanceso that the activity of ascending pathways to SDIs inthe brain would be altered significantly. In this study,we tentatively supposed that the excitatory input theSDIs received during leg movements on substrate wascaused by ascending signals from the thorax in additionto corollary signals from the locomotor center in thebrain.

Synaptic mechanisms of SDI response modulationby leg movements

The synaptic activity of SDIs was affected differently byleg movements in the air and against substrate depend-ing on the cell: the synaptic response of SDI1 to stato-cyst stimulation, for example, was significantly affectedduring leg movement against substrate but not duringfree movement (Fig. 5a), whereas the response of SDI9was affected in either condition (Fig. 5c). These findingssuggest that the synaptic input to SDIs during legmovements is transmitted through different pathwaysdepending on whether a substrate is present or not,although the source of each synaptic input is as yetunidentified. The synaptic response of SDI14 to stato-cyst stimulation was enhanced by synaptic input duringfree leg movements (Fig. 4b), and further enhancedduring leg movements against substrate (Fig. 4c). It is

1Leg movements were seemingly similar to stepping during walk-ing, but our preliminary study with EMG recordings from all eightlegs showed that no bilateral coordination was observed during legmovements without a substrate, suggesting that the leg movementsobserved during intracellular recording was not necessarily thesame as stepping or walking. The phrase ‘‘leg movements’’ has thusno behavioral connotation other than ‘‘complex movements ofmany legs’’ throughout this study.

885

suggested that the enhancement in the former condition(Fig. 4b) is based on synaptic summation of statocystand efference copy input while that in the latter condi-tion (Fig. 4c) on summation of statocyst, efference copyand ascending input.

It should be noted here that, unlike in SDI14, thesynaptic response to statocyst stimulation is enhancedduring free leg movements, but not further enhancedduring on-substrate leg movements in SDI9 (Fig. 5clower panel). One possible explanation of this differ-ence between SDIs 9 and 14 would be that thearrangement of synaptic sites for ascending and effer-ence copy input is varied among each SDI. Since thesynaptic activity is based on transmembrane ionic flowthat was caused by conductance change due to trans-mitter action, interaction of excitatory inputs can beeither summatory or occlusive depending on thearrangement of synaptic input sites on the dendrite(Koch 1999; Shepherd 2001).

Thus, the finding that the input resistance of SDI8decreased during free leg movements when the synapticresponse to statocyst stimulation was also suppressed(Fig. 6a, panels 1–4) suggested that the synaptic site forefference copy input was electrotonically in close prox-imity to the site of electrode impalement: the corollaryinput caused reduction in the electromotive force for thesynaptic current due to statocyst input so that summa-tion of these two inputs was apparently absent. Thesame phenomenon was observed more clearly in SDI13(Fig. 6b, panels 1–4). Although the interneuron receivedexcitatory inputs during statocyst stimulation and freeleg movements, only occlusion was observed when bothwere present. It appeared to be interesting to note thatboth SDI8 and SDI13 had relatively simple dendriticmorphology (Fig. 2): they commonly lacked major sidebranches in their dendritic structure. We could not findany definitive correlation, however, between the mode ofsummation among statocyst, leg proprioceptor and ef-ference copy inputs in each SDI and its morphology:some SDIs including SDI14 that showed summation(Fig. 4c) were found to have large dendritic side bran-ches (Fig. 4), but those SDIs that did not show anynoticeable summation (e.g., SDI9) also had extensivedendritic branches. Further study is needed to identifythe sites of synaptic input from leg proprioceptors andthe locomotor center as well as to make out the site ofspike initiation in these cells.

Functional organization of descending statocystpathways

We showed for the first time that the synaptic responseof descending interneurons to statocyst stimulation wasaffected differently by leg movement depending on itssubstrate condition. Existence of substrate has signifi-cant effects on posture control of crustaceans in additionto direct elicitation of various appendage reflexes(Schone et al. 1976): thus, uropod steering as one form

of equilibrium reflexes of crayfish in response to imposedpostural change (Yoshino et al. 1980) was reversed in itsdirection when a substrate was provided during bodyrolling (Takahata et al. 1984). Central compensation ofeyestalk posture after unilateral statocystectomy wasmore significant when a substrate was present than not(Sakuraba and Takahata 1999, 2000). On the otherhand, the uropod steering response to body rolling wassignificantly facilitated when crayfish was actively mov-ing legs either in the air or on substrate (Takahata et al.1984). These previous findings suggest that some mech-anism should exist in the central nervous system ofcrayfish that distinguish between disturbed and undis-turbed movements of legs that were initiated endoge-nously. Although cellular basis of this mechanism is yetto be investigated systematically, the present studyclearly showed that the activity of SDIs, i.e., the outputof the brain directed to thoracic and abdominal ganglia,is modulated differently by leg movements depending onthe cell (Table 1).

It has been proposed that the central control ofbehavior is based on populational activity of nerve cellsin both vertebrates (Grillner et al. 1991; Sparks et al.1997; Deliagina et al. 2000) and invertebrates (Kien1983; Rowell 1993; Schildberger et al. 1988; Staudacherand Schildberger 1998). Descending statocyst pathwaysalso appear to be composed of a number of interneuronsorganized in parallel (Takahata and Hisada 1982;Nakagawa and Hisada 1989). In a previous study, adescending interneuron that was functionally identifiedas being statocyst-responsive showed similar spike re-sponses to body rolling whenever crayfish was at rest oractively moving legs in the air (Takahata and Hisada1985). In the present study, we could identify an inter-neuron (SDI5; Fig. 5b) the activity of which was alsounaffected by leg movements. On the other hand, manyother cells were found to change synaptic responses tostatocyst stimulation depending on the leg activity andsubstrate conditions.

It is thus suggested that the descending statocystpathway is organized in parallel not only for controllinga variety of statocyst-driven postural reflexes but alsofor a single behavioral act in different behavioral con-ditions. The present finding that not all descending in-terneurons were affected in the same way (Table 1),together with the previous finding that not all behavioralacts were affected similarly (see above) by behavioralcondition, points to a possibility that operational con-stituents of the descending motor control pathway arenot always the same when behavioral conditions aredifferent. A ‘‘consensus’’ (Sparks et al. 1997; Staudacher2001) of a combination of descending interneuronswould indeed subserve the control of a behavioral act ina specific behavioral condition, but a consensus of dif-ferent combinations of descending interneurons mightbe necessary for the control of the same act in a differentcondition depending on their response variability asso-ciated with behavioral context. Further study is neededto examine this possibility by neurophysiological anal-

886

ysis of the synaptic connection between each SDI andposterior motor centers.

Acknowledgements We thank Drs. Y. Okada and H. Furudate forcritical comments and helpful suggestions. This work was partiallysupported by a grant (No. 14340260) from the Ministry of Edu-cation, Science, Culture and Sports, Japan.

References

Alverdes F (1926) Stato-, Photo- und Tangoreakionen bei zweiGarneelenarten. Z Vergl Physiol 4:699–765

Bush BMH, Vedel JP, Clarac F (1978) Inter segmental reflex ac-tions from a joint sensory organ (CB) to a muscle receptor(MCO) in decapod crustacean limbs. J Exp Biol 73:47–63

Cattaert D, El Manira A, Marchand A, Clarac F (1990) Centralcontrol of the sensory afferent terminals from a leg chordotonalorgan in crayfish in vitro preparation. Neurosci Lett 108:81–87

Cattaert D, El Manira A, Clarac F (1992) Direct evidence forpresynaptic inhibitory mechanisms in crayfish sensory afferents.J Neurophysiol 67:610–624

Clarac F, Neil DM, Vedel JP (1976) The control of antennalmovements by leg proprioceptors in the rock lobster Palinurusvulgris. J Comp Physiol 107:175–192

Davis WJ (1968) Lobster righting responses and their neuronalcontrol. Proc R Soc Lond Ser B 170:435–456

Davis WJ, Mpitsos GJ, Pinneo JM (1974) The behavioral hierarchyof the molluscan Pleurobranchaea. I. The dominant position ofthe feeding behavior. J Comp Physiol 90:207–224

Deliagina TG, Orlovsky GN, Selverston AI, Arshavsky YI (1999)Neuronal mechanisms for the control of body orientation inClione. I. Spatial zones of activity of different neuron groups.J Neurophysiol 82:687–699

Deliagina TG, Zelenin P, Fagerstedt P, Grillner S, Orlovsky GN(2000) Activity of reticulospinal neurons during locomotion inthe freely behaving lamprey. J Neurophysiol 83:853–863

Duysens J, Clarac F, Cruse H (2000) Load-regulation mechanismsin gait and posture: comparative aspects. Physiol Rev 80:83–123

El Manira A, DiCaprio RA, Cattaert D, Clarac F (1991) Mono-synaptic interjoint reflexes and their central modulation duringfictive locomotion in crayfish. Eur J Neurosci 3:1219–1231

El Manira A, Tengner J, Grillner S (1996) Locomotor-relatedpresynaptic modulation of primary afferents in the lamprey.Eur J Neurosci 9:696–705

Elson RC, Sillar KT, Bush BM (1992) Identified proprioceptiveafferents and motor rhythm entrainment in the crayfish walkingsystem. J Neurophysiol 67:530–546

Everett RA, Ostfeld RS, Davis WJ (1982) The behavioral hierarchyof the garden snail Helix aspersa. Z Tierpsychol 59:109-126

Furudate H, Okada Y, Yamaguchi T (1996) Responses of non-spiking giant interneurons to substrate tilt in the crayfish, withspecial reference to multisensory control in the compensatoryeyestalk movement system. J Comp Physiol A 179:635–643

Gossard JP, Cabelguen JM, Rossignol S (1991) An intracellularstudy of muscle primary afferents during fictive locomotion inthe cat. J Neurophysiol 65:914–926

Grillner S, Wallen P, Bordin L (1991) Neuronal network generat-ing locomotor behavior in the lamprey: circuitry transmittersmembrane properties and simulation. Annu Rev Neurosci14:169–199

Harreveld A van (1936) A physiological solution for freshwatercrustaceans. Proc Soc Exp Biol Med 34:428–432

Holst E von, Mittelstaedt H (1950) Das Reafferenzprinzip: Wech-selwirkungen zwischen Zentralnervensystem und Peripherie.Naturwissenschaften 37:464–476

Horak FB, Macpherson JM (1996) Postural orientation and equi-librium. In: Rowell LB (ed) Handbook of physiology, section12. Exercise: regulation and integration of multiple systems.Oxford University Press, New York, pp 255–292

Kien J (1983) The initiation and maintenance of walking in thelocust: an alternative to command concept. Proc R Soc LondSer B 219:137–174

Koch C (1999) Biophysics of computation, Oxford UniversityPress, New York

Kuhn A (1914) Die reflektorische Erhaltung des Gleichgewichtesbei Krebsen. Verh Dtsch Zool Ges 24:262–277

Leibrock CS, Marchand AR, Barnes WJP, Clarac F (1996) Syn-aptic connections of the cuticular stress detectors in crayfish:mono- and polysynaptic reflexes and the entrainment of fictivelocomotion in an in vitro preparation. J Comp Physiol A178:711–725

Marchand AR, Leibrock CS, Auriac MC, Barnes WJP, Clarac F(1995) Morphology, physiology and in vivo activity of cuticularstress detector afferents in crayfish. J Comp Physiol A 176:409–424

Mill PJ (1976) Chordotonal organs of crustacean appendages. In:Mill PJ (ed) Structure and function of proprioceptors in theinvertebrates. Chapman and Hall, London, pp 243–297

Murayama M, Takahata M (1992) Multiple gate control of thedescending statocyst-motor pathway in the crayfish Procamb-arus clarkii Girard. J Comp Physiol A 170:463–477

Murphey RK, Palka J (1974) Efferent control of cricket giantfibres. Nature 248:249–251

Nakagawa H, Hisada M (1989) Morphology of descending stato-cyst interneurons in the crayfish Procambarus clarkii Girard.Cell Tissue Res 255:539–551

Nakagawa H, Hisada M (1990) Local spiking interneurons con-trolling the equilibrium response in the crayfish Procambarusclarkii. J Comp Physiol A 170:291–302

Newland PL (1989) The uropod righting reaction of the crayfishProcanbarus clarkii (Girard): an equilibrium response driven bytow largely independent reflex pathways. J Comp Physiol A164:685–696

Okada Y, Yamaguchi T (1988) Nonspiking giant interneurons inthe crayfish brain: morphological and physiological character-istics of the neurons postsynaptic to visual interneurons.J Comp Physiol A 162:705–714

Okada Y, Furudate H, Yamaguchi T (1994) Multimodal responsesof the nonspiking giant interneurons in the brain of the crayfishProcambarus clarkii. J Comp Physiol A 174:411–419

Poulet JFA, Hedwig A (2003) A corollary discharge mechanismmodulates central auditory processing in singing crickets.J Neurophysiol 89:1528–1540

Rowell CHF (1993) Intersegmental coordination of flight steeringin locusts. The Neurosciences 5:59–66

Sakuraba T, Takahata M (1999) Effects of visual and leg propri-oceptor inputs on recovery of eyestalk posture following uni-lateral statolith removal in the crayfish. Naturwissenschaften86:346–349

Sakuraba T, Takahata M (2000) Motor pattern changes duringcentral compensation of eyestalk posture after unilateralstatolith removal in crayfish. Zool Sci 17:19–26

Schildberger K, Milde JJ, Horner M (1988) The function of audi-tory neurons in the cricket phonotaxis. II. Modulation ofauditory response during locomotion. J Comp Physiol A163:621–631

Schone H (1954) Statocystenfunktion und statische Lageorientie-rung bei dekapoden Krebsen. Z Vergl Physiol 36:241–260

Schone H, Neil DM, Stein A, Carlstead MK (1976) Reactions ofthe spiny lobster, Parinurus vulgaris, to substrate tilt (I). J CompPhysiol 107:113–128

Schone H, Neil DM, Scapini F and Dreismann G (1983) Interac-tion of substrate, gravity and visual cues in the control ofcompensatory eye responses in the spiny lobster, Palinurusvulgaris. J Comp Physiol A 150:23–30

Shepherd GM (2001) The synaptic organization of the brain,Oxford University Press, New York

Sillar KT, Roberts A (1988) A neuronal mechanism, for sensorygating during locomotion in a vertebrate. Nature 331:262–265

Sillar KT, Skorupsky P (1986) Central input to primary afferentneurons in crayfish, Pacifastacus leniuscukus, is correlated with

887

rhythmic motor output of thoracic ganglia. J Neurophysiol55:678–688

Sparks DL, Kristan WBJ, Shaw BK (1997) The role of populationcoding in the control of movement. In: Stein PG, Grillner S,Selverston AI, Stuart DG (eds) Neurons, networks, and motorbehavior. MIT Press, Cambridge, MA, pp 21–32

Sperry RW (1950) Neuronal basis of the spontaneous optokineticresponse produced by visual inversion. J Comp Physiol Psychol43:482–489

Staudacher EM (2001) Sensory responses of descending brainneurons in the walking cricket, Gryllus bimaculatus. J CompPhysiol 187:1–17

Staudacher EM, Schildberger K (1998) Gating of sensory responsesof descending brain neurons during walking in crickets. J ExpBiol 201:559–572

Takahata M, Hisada M (1982) Statocyst interneurons in thecrayfish Procambarus clarkii Girard. I. Identification and re-sponse characteristics. J Comp Physiol A 149:287–300

Takahata M, Hisada M (1985) Interaction between motor systemscontrolling uropod steering and abdominal posture in crayfish.J Comp Physiol A 157:547–554

Takahata M, Komatsu H, Hisada M (1984) Positional orientationdetermined by the behavioural context in Procambarus clarkiiGirard (Decapoda: Macrura). Behaviour 88:240–265

Tautz J, Tautz RM (1983) Antennal neuropile in the brain of thecrayfish: morphology of neurons. J Comp Neurol 218:415–425

Ullen F, Deliagina TG, Orlovsly GN, Grillner S (1995) Spatialorientation in lamprey. II. Visual influence on orientationduring locomotion and in the attached state. J Exp Biol198:675–681

Wiersma CAG (1958) On the functional connections of single unitsin the central nervous system of crayfish, Procambarus clarkii(Girard). J Comp Neurol 110:421–471

Wolf H, Burrows M (1995) Proprioceptive sensory neurons of lo-cust leg receive rhythmic presynaptic inhibition during walking.J Neurosci 15:5623–5636

Yoshino M, Takahata M, Hisada M (1980) Statocyst control ofuropod movement in response to body rolling in crayfish.J Comp Physiol A 139:243–250

888