Metathoracic Neurons IntegratingIntersegmental Sensory Information

in the Locust

TOM MATHESON*

Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom

ABSTRACTThis paper describes the morphology and physiology of five types of local interneurons

and three types of ascending intersegmental interneurons in the locust metathoracic gan-glion that are points of convergence of sensory information from the wings. Four types ofspiking local interneurons are members of a population with somata at the ventral midline.They are depolarised by stimulation of a metathoracic wing nerve, suggesting that theyencode a sensory representation of this appendage. Some are also depolarised with shortlatencies following stimulation of a mesothoracic wing nerve, indicating that they collateintersegmental as well as local information. All the local interneurons have branches in theanterior ventral association centre or around the roots of the nerve that carries wing sensoryneurons. This distinguishes them from other interneurons in the population. A fifth type oflocal interneuron that has unusual bilateral branching and is not a member of this populationis described for the first time. The ascending interneurons are members of three populations.Neurons of each population have a characteristic pattern of responses to stimulation of themesothoracic or metathoracic wing nerves, and some respond to tactile stimulation ormovements of a hind leg. These latter interneurons thus collate information from both wingsand legs. All three types of intersegmental interneurons have branches in the anteriorventral association centre or around the roots of the wing nerve. The responses of theinterneurons described here shed new light on both local and intersegmental networkfunction in this model system. J. Comp. Neurol. 444:95–114, 2002. © 2002 Wiley-Liss, Inc.

Indexing terms: scratching; grasshopper; Schistocerca gregaria; midline spiking interneuron;

intersegmental interneuron; local interneuron

To understand how nervous systems generate targetedlimb movements requires knowledge of the computationscarried out within individual neurons, between neurons ofa population, and between populations. In the locustSchistocerca gregaria, behavioural analyses have shownthat some of the neuronal interactions underlying tar-geted scratching movements of a hind leg are local to theganglion of a particular body segment, whereas others arespread across two or more ganglia (Berkowitz and Lau-rent, 1996a,b; Matheson, 1997, 1998). Although there issubstantial understanding of neuronal pathways that un-derlie some insect behaviours (e.g., walking, jumping, fly-ing, reflex leg withdrawal; see Burrows, 1996, for an over-view), little is known about the neuronal control of anytargeted limb movement (see, e.g., Brunn and Dean,1994). Understanding the control of such targeted limbmovements will provide important insights into how thenervous system resolves computational problems, such asthe integration of information encoded in different frames

of reference (Rosenbaum et al., 1996; Soechting et al.,1996) or redundancy (in which choices must be madeamong many motor solutions that could achieve a giventask). The control strategies used by insect nervous sys-tems may provide powerful “shortcuts” that can be imple-mented in autonomous robots (Cruse et al., 1998).

Locusts make precisely directed scratching movementsof one or both hind legs in response to tactile stimulationof the wings (Matheson, 1997). Behavioural analyses in-dicate the following key features. First, there is precise

Grant sponsor: BBSRC; Grant number: ARF/98/53; Grant sponsor: RoyalSociety (London); Grant number: RSRG17944.

*Correspondence to: Tom Matheson, Department of Zoology, Universityof Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom.E-mail: tm114@hermes.cam.ac.uk

Received 17 July 2001; Revised 18 October 2001; Accepted 20 November2001

Published online the week of January 21, 2002

THE JOURNAL OF COMPARATIVE NEUROLOGY 444:95–114 (2002)

© 2002 WILEY-LISS, INC.DOI 10.1002/cn.10140

somatotopic mapping of leg movements in response tostimuli at different locations on a wing (Durr and Mathe-son, 2001). Second, there is convergence of sensory infor-mation from the mesothoracic and metathoracic wingsonto metathoracic motor networks. Third, there is con-tralateral transmission from the wings on one side of thebody to the contralateral hind leg motor network (Mathe-son, 1997). The aim of this paper is to provide a descrip-tion of candidate interneurons that are likely to be in-volved in these pathways based on their patterns ofsensory inputs and morphological features.

I describe five classes of local and three classes of inter-segmental interneurons that receive sensory inputs fromthe wings and, in some cases, legs, which implicate themin the control circuits for targeted scratching movementsof the hind legs. Four types of spiking local interneuronswith somata at the ventral midline are members of apopulation that has been defined on the basis of bothmorphology and synaptic inputs from hind leg mechano-receptors (Burrows and Siegler, 1982, 1984; Siegler andBurrows, 1983, 1984; for review see Burrows, 1996). Othermembers of this population encode the location of tactilestimuli on a metathoracic leg and are therefore thought tobe involved in the local (segmental) generation of legavoidance reflexes. The neurons that I describe are depo-larised by stimulation of a metathoracic wing, suggestingthat they encode a sensory representation of this append-age as well.

One type of ascending intersegmental interneuron be-longs to a population of approximately 35 cells describedby Laurent and Burrows (1988) on the basis of their mech-anosensory inputs from a hind leg. I show here that someneurons in this population are depolarised by stimuli ap-plied to the wing.

Another type of metathoracic ascending interneuron issimilar to a population of bilaterally branching interneu-rons in the mesothoracic ganglion (Watson and Burrows,1983; Pearson et al., 1985). Such neurons have not previ-ously been described in the metathoracic ganglion, andinputs from the wings have not been investigated. Themorphology of the cells that I describe is also similar insome respects to a set of mostly unilateral flight interneu-rons described by Robertson et al. (1982) in the metatho-racic and fused abdominal ganglia, but there are also cleardifferences, so they cannot be the same cells. My analysesof local and intersegmental interneurons in the context oftargeted scratching movements broaden our understand-ing of how sensory inputs from different appendages of abody segment (wing, leg) or from different body segments(mesothoracic, metathoracic) are brought together in theventral nerve cord.

MATERIALS AND METHODS

Locusts (Schistocerca gregaria Forskål) taken from ourcrowded colony were restrained ventral side uppermostwith both left wings held out laterally. The right-handwings remained in their normal folded, resting position.All six legs were restrained at the femur, but the tibiaewere free to move. A pair of bipolar myogram electrodes(100-�m-diameter silver wire) was inserted into the lefthind leg tibial extensor muscle to permit stimulation ofthe muscle and thus evoke tibial extension. To record andstimulate wing sensory neurons, pairs of bipolar neuro-gram electrodes were inserted into the forewing subcostaor cubitus vein and the hind wing subcosta vein (Fig. 1).The nerves that run along these veins innervate campani-form, trichoid, and basiconic receptors spread across largeareas of the wing (personal observation). Throughout this

Abbreviations

aLAC anterior lateral association centreaVAC anterior ventral association centreCT C tractDCI–VI dorsal commissures 1–6 (of the metathoracic neuromere)DCI–VI(A1) dorsal commissures 1–6 of the first abdominal neuromereDIT dorsal intermediate tractdDMT dorsal part of dorsal medial tractDMT dorsal medial tractFETi fast extensor tibiae motor neuroniLVT inner part of lateral ventral tractLDT lateral dorsal tractlVAC lateral ventral association centreMDT median dorsal tractmeta metathoracicmeso mesothoracicmVAC medial ventral association centreMVT median ventral tractN1-5r roots of nerves 1–5oLVT outer part of the lateral ventral tractpLAC posterior part of lateral association centrepLAC(T3) posterior part of lateral association centre of the thoracic

neuromerePT P tractSMC supramedian commissuretr tracheaTT T tractVCI–II ventral commissure 1 or 2vDMT ventral part of dorsal median tractVIT ventral intermediate tractVLT ventral lateral tractvmVAC ventral part of medial ventral association centreVMT ventral median tractvVAC ventral part of ventral association centre

Fig. 1. Ventral views of the left forewing (A) and hind wing (B) ofan adult locust, Schistocerca gregaria, to show the neurogram record-ing sites in the subcosta veins. An alternative recording site inthe forewing cubitus vein was used in some experiments. Scale bar� 1 cm.

96 T. MATHESON

paper the phrase wing nerve is used to indicate theserecording sites to distinguish them from recordings madein nerve 1 (see below).

To gain access to the central nervous system, the ven-tral thoracic sclerites were cut away and underlying tra-cheal air sacs removed to reveal the mesothoracic andmetathoracic ganglia. Both ganglia were supported on awax-coated silver platform and treated with 0.1% (w/v)protease (Sigma type XIV, Sigma, St. Louis, MO) for 20seconds to soften their tough outer sheath. Pairs of bipolarhook electrodes (100 �m silver wire) were placed undermesothoracic nerve 1, which gives rise to the mesothoracicwing nerve, and under metathoracic nerve 1, which givesrise to the metathoracic wing nerve. Each nerve 1 containsnot only the axons of wing exteroceptors that pass into thecorresponding wing nerve but also axons of receptors atthe wing base and on the lateral thorax. Each pair ofelectrodes was insulated from the surrounding haemo-lymph with petroleum jelly. Throughout this paper theserecording sites are referred to as nerve 1 to distinguishthem from the wing nerve recording sites in the wingitself.

The signals from wing neurogram electrodes and nerve1 hook electrodes were amplified using Isleworth extracel-lular amplifiers. A Master 8 stimulator (A.M.P.I.) con-nected to each of the four electrodes via stimulus isolatorswas used to stimulate each recorded nerve independentlyand to stimulate the tibial extensor muscle (pulse dura-tion 1 msec, 0.5 Hz).

Recording and cobalt staining

Intracellular recordings were made from the somata ofmetathoracic neurons near the ventral midline using glassmicroelectrodes filled with 5% cobalt hexammine (Sigma).All recordings were made with a single-electrode bridgeamplifier with the bridge balanced. To improve recordingstability, all neurons were held hyperpolarised through-out recording by injection of a constant current of approx-imately –1 nA. Online signal averaging (Sigavg software;Cambridge Electronic Design, Cambridge, United King-dom) triggered by the stimulation of sensory nerves wasused to search for and characterise neurons with short-latency synaptic inputs from wing or nerve 1 sensoryneurons. All data were also stored on magnetic tape (RacalStore 7DS FM recorder) for later analysis using Spike 2software (Cambridge Electronic Design). After physiolog-ical characterisation, neurons were injected iontophoreti-cally with cobalt (250 msec pulses at 2 Hz for 20–40minutes; Brogan and Pitman, 1981) and silver intensified(Bacon and Altman, 1977).

Neuroanatomy and abbreviations

Morphological descriptions are derived from dorsalviews of whole ganglia mounted in Canada balsam andfrom 9 �m sections of ganglia embedded in paraffin wax,drawn using a Zeiss compound microscope and cameralucida. The nomenclature used to describe the ganglionictracts and neuropil follows Tyrer and Gregory (1982) andPfl¨uger et al. (1988). In the descriptions of physiologygiven below, the number of neurons responding in a par-ticular way is presented as “number responding/numbertested in this way.” Experimental constraints sometimesmeant that not all neurons of a type could be tested in allpossible ways. Unless stated otherwise, all stimuli werepresented to the side ipsilateral to the main field of neu-ronal branching. In cases where neurons branched bilat-erally, ipsilateral refers to the side on which the soma waslocated.

Photographic images were recorded at 1,312 � 2,000pixels using a Nikon D1 camera attached to the micro-scope. Selected areas of interest were cropped to size, andthe contrast and brightness adjusted manually using Can-vas 7 (Deneba). The results presented here are based onrecordings from 28 neurons that were stained and ana-lysed in detail and from more than 160 additional record-ings of less well characterised neurons in 110 animals.

RESULTS

Common features of the interneurons

Recordings were made from five types of metathoraciclocal interneurons and three types of ascending interseg-mental interneurons that all received synaptic inputs fol-lowing stimulation of a mesothoracic or metathoracicnerve 1 (N1) or stimulation of a wing nerve (Table 1). Allinterneurons that responded to stimulation of one N1 alsoresponded to stimulation of the other, ipsilateral N1.Many of the analysed neurons also received synaptic in-puts following tactile stimulation of the legs or body, andsome received inputs following movements of leg joints.All of the responses to stimuli of a wing nerve or N1 weredepolarising, often leading to spikes. All of the interneu-rons had anterior branches in and around the roots of N1or in the anterior ventral association centre (aVAC).

Metathoracic local interneurons

Five types of local interneurons responded to N1 stim-ulation (Table 1). Four of these (named here Types A–D)are previously undescribed members of a population ofventral midline spiking interneurons (Burrows and

TABLE 1. Key Features of the Interneurons1

Neuron typeNumberanalyzed

Meso N1input

Meso winginput

Meta N1input

Meta winginput

Wing baseinput

Hind legproprio input

Other tactileinput

Anterior ventralbranches

Local A 8 8/8 0/3 8/8 1/3 3/3 2/6 — yLocal B 3 3/3 1/3 2/3 0/3 — 2/2 — y2

Local C 5 3/3 1/3 4/4 3/4 — 4/4 — yLocal D 2 1/1 1/1 2/2 2/2 — 2/2 — yLocal E 2 1/1 0/1 1/1 0/1 — 0/1 — yInterseg A 4 2/3 2/3 4/4 4/4 — 1/1 Head/legs yInterseg B 2 — 2/2 — 2/2 — 0/1 Hind leg yInterseg C 2 2/2 0/2 2/2 2/2 1/1 0/1 — y

1Numbers indicate “number responding/number tested (not all neurones were tested in all ways).” Dashes � not tested.2The branches are restricted to the more posterior part of aVAC.

97INTEGRATION OF SENSORY INFORMATION

Siegler, 1982, 1984; Siegler and Burrows, 1983, 1984). Thefifth type (Type E) does not appear to belong to any pre-viously described population. All of the Type A–D localinterneurons have a soma near the ventral midline and amain neurite that crosses the midline in VCII as describedfor all other members of this population by Burrows andSiegler (1984). Where the soma lies particularly close tothe midline, as in the examples shown in Figures 3 and 6,the neurite leading from it joins VCII at the midline. Notethat other neurons of these types with somata fartherfrom the midline had neurites that crossed the midline.For consistency, my descriptions always refer to neurites“crossing the midline” even when the point of entry to thetract is right on the midline.

Type A local interneurons. Interneurons of Type Across the midline relatively posteriorly in VCII. Aftercrossing the midline, the main neurite (asterisk in Fig.2Cii) gives off prominent branches that turn anterior andposterior to the main neurite. The anterior branch givesrise to a ventral field of branching in aVAC (Fig. 2Ci),spanning horizontally from near the midline as far later-ally as the edge of the connective (solid arrow in Fig. 2A).Near the anterior margin of the neuropil, these branchesextend farther dorsally, to the level of VMT (arrow in Fig.2Ci). The posterior branch gives rise to a prominent ven-tral process that runs posteriorly for 100–200 �m almostparallel to the midline in MVT (solid arrowhead in Fig.2A,Ciii). Both anterior and posterior branches give rise toa sparse region of ventral branching near the main neuritethat begins in lVAC and extends dorsally near the midlineas far as the top of VIT (double arrowheads in Fig. 2A,Cii).

The main neurite continues laterally, giving rise to aprominent branch that curves farther laterally and poste-riorly towards the roots of nerve 5 near the edge of pLAC(Fig. 2A,Ciii). The main neurite ascends dorsally in PT(open arrow in Fig. 2A) and bifurcates several times togive rise to a widespread field of dorsal branches in aLACand pLAC (Fig. 2B, open arrowheads in Cii,iii). Anteriorly,the dorsal branches lie above DIT and DMT (arrowhead inFig. 2Ci).

Local interneurons of Type A were depolarised andspiked once or twice following electrical stimulation ofeither mesothoracic or metathoracic nerve 1 (Fig. 2Di,ii).The latencies of these depolarisations were 4.6–5.5 msecand 2.1–3.4 msec, respectively, in eight recordings. Touch-ing the wings or stimulating wing nerves had no effect intwo of the three (2/3) neurons tested in this way. Stimu-lation of the metathoracic wing nerve elicited a smalldepolarisation in 1/3 neurons. In contrast, even smallmovements (1–2° of arc) of either wing base powerfullyexcited 3/3 neurons (Fig. 2E). Passive movements of thehind leg tibia depolarised only 2/6 neurons, but activemovements of the tibia were accompanied by spiking in4/4 neurons. Injection of depolarising current into 1/1 neu-ron so that it spiked repeatedly caused no visible move-ment of the hind leg.

Type B local interneurons. The main neurite of TypeB local interneurons crosses the midline in VCII (Fig.3A,Di), before turning posteriorly and becoming thicker.This thick, main neurite continues laterally, giving offusually four posterior ventral branches, one anterior ven-tral branch, and a lateral ventral branch, before turningdorsally in PT (Fig. 3Dii).

The first posterior ventral branch has a large diameterand, after giving rise to sparse branching medially, be-

comes thinner and turns posteriorly near the midline(solid arrows in Fig. 3A,Dii,E). All four posterior branchescontribute to a sparse field of branching in vVAC andlVAC and as far posteriorly as pLAC (arrowheads in Fig.3A,Dii,iii).

The anterior branch from the main neurite extends only100 �m anteriorly, giving rise to a few short processes inaVAC (open arrows in Fig. 3A,Di). These branches are not,therefore, as far anteriorly as those of all the other neurontypes described here, and they do not mingle with theroots of N1. The most lateral ventral branch curves later-ally and posteriorly to follow the edge of the metathoracicneuropil toward the roots of N5 (double arrowheads in Fig.3A,Dii,iii).

The main neurite ascending dorsally in PT branches togive rise to three distinct regions of dorsal branching. Oneregion lies laterally (a in Fig. 3B,Diii), the second consistsof fine fibres that run medially as far as the midline inDCIII (b in Fig. 3B,Di,ii), and the third consists of anothergroup of fine fibres that run towards the midline in or justbelow DCV (c in Fig. 3C,Di,ii).

Local interneurons of Type B were depolarised by elec-trical stimulation of mesothoracic nerve 1 (Fig. 4A) withlatencies of 7.1–11 msec. Metathoracic nerve 1 stimula-tion also depolarised 2/3 neurons with latencies of 4.6 and7.4 msec (Fig. 4B). Only 1/3 neurons was depolarised bystimulation of the mesothoracic wing nerve, and none wasinfluenced by stimulation of the metathoracic wing nerve.Both active (Fig. 4C) and passive movements of the met-athoracic tibia were accompanied by depolarisation andspikes in 2/2 neurons. In contrast, when one animal ex-tended the metathoracic tibia against a fixed obstruction,there was no depolarisation in the neuron.

Type C local interneurons. The main neurite of TypeC local interneurons crosses the midline in VCII (Fig. 5A,solid arrow in Cii), before turning posteriorly and becom-ing considerably thicker. It continues posteriorly, givingoff usually five medial ventral branches, one prominentanterior ventral branch, and a lateral ventral branch,before turning dorsally into PT to give rise to a dorsal fieldof branches. The ventral field of branching is more wide-spread than that of Type A and B local interneurons.

The anterior ventral branch gives off several sidebranches that project laterally (arrowheads in Fig. 5A,Ci),before terminating in a sparse field of branching close to

Fig. 2. Morphology and physiology of a metathoracic midline spik-ing interneuron Type A. A: Ventral branches drawn from a wholemount. Solid arrow, anterior ventral branches; arrowhead, posteriormedial branch; double arrowhead, medial branches; open arrow, dor-sal process in PT. B: Dorsal field of branches. The ventral soma isdrawn as a dashed outline for reference, and the midline of theganglion is shown as a vertical dashed line. Ci–iii: Transverse sec-tions made at the levels indicated in A. Arrow, anterior ventralbranches; open arrowheads, anterior dorsal branches; double arrow-heads, medial branches; asterisk, main neurite; solid arrowhead, pos-terior medial branch. Di,ii: Electrical stimulation of mesothoracic (i)or metathoracic (ii) nerve 1 elicited a short-latency depolarisation andspikes (asterisks). Averages of 50 sweeps triggered from the stimulusartifact. The dashed lines indicate the baseline membrane potential.Ei–iii: Depressing either the mesothoracic (i) or metathoracic (ii,iii)wing elicited a burst of activity in the corresponding nerve 1 (lowertwo traces), and a depolarisation or depolarisation and spikes in theinterneuron (top trace). Scale bars � 100 �m in A–C, 5 mV, 20 msecin D, 5 mV, 1 second in E.

98 T. MATHESON

Figure 2

Figure 3

the midline in vmVAC and aVAC (double arrowheads inFig. 5Ci). The remaining ventral branches give rise towidespread arborisations throughout vVAC, lVAC, andventral pLAC (Fig. 5A,Ci–iii). A prominent process followsthe lateral edge of the metathoracic neuropil to terminatenear the roots of nerve 5 (asterisk in Fig. 5A,Ciii). Asecond process runs posteriorly along the medial edge ofthe neuropil, and a third process ascends medially to CT(open arrow in Fig. 5Cii) to give rise to branches dorsome-dially to VIT.

The dorsal process in PT gives rise to thick branchesthat run anteriorly and posteriorly at the level of (but

lateral to) VIT (arrowheads in Fig. 5Cii,iii). They give offmany side branches throughout the length and breadth ofaLAC and pLAC.

Local interneurons of Type C were depolarised by elec-trical stimulation of mesothoracic nerve 1 (6.1–6.6 mseclatency; Fig. 5Di; 3/3 neurons) or metathoracic nerve 1(3.5–5.3 msec; Fig. 5Dii; 4/4 neurons) and sometimes byelectrical stimulation of mesothoracic wing nerve (14.8msec; 1/3 neurons) or metathoracic wing nerve (5.6-11msec; Fig. 5Diii; 3/4 neurons). Touching the metathoracicwing elicited a depolarisation and spikes in 1/2 neuronstested in this way. In addition to these inputs from nerve1 or the wings, Type C local interneurons were powerfullyexcited by touching hairs on the metathoracic femur ortibia (Fig. 5E; 3/3 neurons) or passively extending thefemorotibial joint (4/4 neurons). Active movements of themetathoracic tibia were accompanied by depolarisationand spikes in 2/2 Type C local interneurons.

Type D local interneurons. Local interneurons ofType D have a ventral soma (soma in Fig. 6Cii) and aneurite that crosses the midline in VCII (Fig. 6A,B). Afterthe main neurite crosses the midline, it turns dorsally intoPT (arrow in Fig. 6Ci) and ascends almost to the level ofVLT (solid arrow in Fig. 6Cii), before giving rise to aprominent anterior branch (arrowhead in Fig. 6A), threemedial branches, a posterior lateral branch (Fig. 6A), anda thick dorsal lateral branch (Fig. 6B). This pattern is thusquite different from that of Type A-C local interneurons,which all give rise to their ventral branches, before turn-ing into PT. The anterior branch projects forward at thelevel of VIT, before plunging ventrally (arrowhead in Fig.6Ci) to give rise to an anterior ventral field of branches invVAC. The first medial branch gives rise to branches inand around VLT and MVT (Fig. 6Cii). The other medialbranches and the posterior lateral branch curve graduallydownward to form a dense field throughout much of ven-tral pLAC (Fig. 6A,Ciii). There are relatively few branchesin lVAC (Fig. 6Cii).

The thick dorsal lateral branch of Type D local inter-neurons curves laterally, posteriorly, and then medially(Fig. 6B) at the level of VLT (open arrow in Fig. 6Cii). Itgives off fine branches that ascend around the lateral edgeof aLAC and pLAC (arrowhead in Fig. 6Cii). The resultingdorsal field is sparser than the fields of Type A, B, and Clocal interneurons but still extends throughout the dorsalpart of pLAC (Fig. 6Ciii).

Type D local interneurons were depolarised by stimula-tion of the metathoracic wing nerve (7.1 msec; Fig. 6Di) ornerve 1 (6.8–8.5 msec; 2/2) and by stimulation of meso-thoracic nerve 1 (10.4 msec; 1/1; not shown). Touching themetathoracic wing elicited a strong depolarisation andburst of spikes (1/1; Fig. 6Dii), as did extending the met-athoracic tibia (2/2; Fig. 6Diii). Touching the mesothoracicwing had little or no effect (1/1).

Type E local interneurons. Bilaterally branching lo-cal interneurons with a soma near the ventral midlinewere stained in two animals (Fig. 7). Their morphology isquite different from that of local interneurons Types A–C,and they are clearly not members of the same population.The main neurite crosses the midline in VCII to enter TT,in which it runs dorsally (solid arrow in Fig. 7Bii), beforemaking an abrupt turn into DCIII (Fig. 7Bii, inset) andsplitting bilaterally. On the side ipsilateral to the soma,the main neurite gives off anterior, lateral, and posteriorbranches. The anterior and lateral branches give rise to a

Fig. 3. Morphology of a metathoracic midline spiking interneuronType B. The branches lie in three distinct dorsal-ventral planes (A–C).Di–iii: Transverse sections taken at the levels shown in A–C. E: Pho-tograph of the region outlined by a dashed rectangle in Dii to showbranching near VMT. All features marked with symbols are describedin the text. See Figure 4 for this interneuron’s physiological re-sponses. Scale bars � 100 �m.

Fig. 4. Metathoracic midline spiking interneurons Type B re-sponded to electrical stimulation of mesothoracic (A) or metathoracic(B) nerve 1 with depolarising postsynaptic potentials (averages of 50sweeps). The neurons were strongly depolarised and spiked duringactive movements of the metathoracic tibiae (shaded area; C). Scalebars � 1 mV, 20 msec in A,B, 5 mV, 200 msec in C.

101INTEGRATION OF SENSORY INFORMATION

Figure 5

102 T. MATHESON

region of arborisation around DIT and LDT near the rootsof N1 (solid arrowheads in Fig. 7A,Bi), and the posteriorbranch contributes to an ipsilateral posterior region ofbranching in posterior neuropil dorsal to DIT (solid doublearrowheads in Fig. 7A,Biii). A slender branch runs poste-riorly in LDT as far as the posterior edge of the metatho-racic neuromere. There are no branches in ventral neuro-pil on either side of the ganglion.

On the contralateral side, the main neurite in DCIIIcontinues laterally as far as LDT, first giving off a prom-inent posterior branch and then three thinner anteriorbranches, before turning dorsally and back on itself in aloop around LDT (open arrows in Fig. 7A,Bii). It thenthickens and runs posteriorly at the ventral edge of MDT(open double arrowhead in Fig. 7Biii).

The anterior branches on the side contralateral to thesoma all contribute to a dense arborisation around DIT,extending anteriorly as far as the edge of the neuropilnear the roots of nerve 1 (open arrowheads in Fig. 7A,Bi).The posterior branch splits into two processes, one ofwhich recrosses the midline in DCIV to contribute to theposterior ipsilateral field of branches, whereas the othergives rise to a contralateral posterior arborisation in andaround DMT (asterisk in Fig. 7Bii).

The physiology of only one Type E local interneuron wasanalysed. It was depolarised by electrical stimulation ofmesothoracic (7.4 msec; Fig. 7Ci) or metathoracic (3.95msec; Fig. 7Cii) nerve 1 contralateral to the soma. Thedepolarisation elicited by stimulation of metathoracic NIwas up to sixfold greater in amplitude than that elicitedby mesothoracic N1 stimulation. It did not respond totouching any of the legs.

Metathoracic ascending intersegmentalinterneurons

Three types of ascending intersegmental interneuronswith somata in the metathoracic ganglion were analysedin detail. The three types (A–C) have quite different mor-phologies and are clearly from different populations.

Type A intersegmental interneurons. Ascending in-terneurons of Type A have a ventral soma near the mid-line and a main neurite that runs posteriorly and dorsallyto give rise to widespread branching in dorsal and ventral

neuropils of the ipsilateral hemiganglion (Fig. 8A,B). Anaxon ascends as least as far as the mesothoracic ganglionin the ipsilateral connective, but the pattern of branchinghere is not known.

After leaving the soma (soma in Fig. 8Cii), the mainneurite ascends dorsally and posteriorly at the lateraledge of TT, giving off several fine processes into aVAC andposterior vmVAC below the level of VMT (solid arrow inFig. 8Cii). One of the fine processes turns forwards (solidarrow in Fig. 8B) to branch very anteriorly in aVAC (ar-row in Fig. 8Ci). At the level of VIT, the main neuriteturns laterally and splits to give rise to two thick mainprocesses, posterior and anterior, and a more slender ven-tral process (p, a, and v in Fig. 8B,Cii,iii).

The posterior process runs posterolaterally to end in aregion of branching at the level of VLT near the posterioredge of the metathoracic neuropil (arrowhead in Fig. 8A),giving off several side branches on the way. The first andmost prominent of these runs laterally above VIT (arrow-head in Fig. 8Ciii) to give rise to branches at the level ofDIT, before turning dorsally to give rise to furtherbranches near LDT. A second side branch runs posteri-odorsally medial to DIT (double arrowhead in Fig. 8Ciii) togive rise to a region of branching just lateral to MDT. Amedial branch runs posteriorly (double arrowhead in Fig.8A) near VIT, giving off fine branches into the surround-ing neuropil. The ventral process from the main neurite (vin Fig. 8B,Ciii) runs ventrolaterally between VMT andMVT to ramify across a large area of lVAC.

The anterior process from the main neurite (a in Fig.8B) curves forward and upward, medial to VIT (Fig. 8Cii).Just dorsal to VIT, it gives rise to the ascending axon,before turning laterally between DIT and VIT (a in Fig.8Cii) and splitting into two branches that reach to thelateral and ventral edges of the neuropil (open arrows inFig. 8A,Cii). These contribute to extensive dorsal and ven-tral branching near the margins of the neuropil (arrows inFig. 8Ciii). These branches intermingle with the roots ofnerves 2–5. Another fine branch runs anteriorly (asteriskin Fig. 8B) to contribute to an anterior field of branchingbetween MVT and VMT (asterisk in Fig. 8Ci).

The axon runs dorsally and medially to curve aroundDMT before turning laterally and entering LDT/MDT(axon in Fig. 8Cii). The path followed by the axon, as seenin whole mount, thus forms a characteristic medial, dor-sal, and then lateral loop, before bifurcating, with onebranch turning abruptly anterior in LDT/MDT to leavethe ganglion (via MDT) in the ipsilateral connective (axonin Fig. 8A), the other branch turning posteriorly (arrow-head in Fig. 8B) also in MDT to give rise to very dorsalbranching in and above DIT. The ascending axon givesrise to several short, fine branches in MDT before it entersthe connective.

Type A intersegmental interneurons were depolarisedby electrical stimulation of either the mesothoracic (2/3) orthe metathoracic (4/4) nerve 1 with latencies of 5.8–9.1msec or 4.5–7.6 msec, respectively. Similarly, electricalstimulation of the ipsilateral meso- or metathoracic wingnerve elicited depolarising postsynaptic potentials (PSPs)in the interneurons with latencies of 14.9–19.5 msec (2/3)or 11.7–16 msec (4/4), respectively (Fig. 8Di,ii). Touchingthe head or any of the wings or legs caused a barrage ofdepolarising synaptic inputs and sometimes spikes (1/1).Extending the tibia of the ipsilateral hind leg caused a

Fig. 5. Morphology and physiology of a metathoracic midline spik-ing interneuron Type C. A: Ventral branches drawn from a wholemount. Arrowhead, anterior lateral branches; double arrowhead, an-terior ventral branches; asterisks, posterior processes near the edge ofthe neuropil. B: Dorsal field of branches. The ventral soma is drawn asa dashed outline for reference. Ci–iii: Transverse sections made at thelevels indicated in A. Solid arrowhead, anterior lateral branches;double arrowheads, anterior ventral branches; solid arrow, main neu-rite; asterisk, posterior lateral process near roots of N5; open arrow,medial branch climbing to the level of VIT; open arrowheads, maindorsal branches running anteriorly and posteriorly. Di–iii: Electricalstimulation of mesothoracic (i) or metathoracic (ii) nerve 1 elicited ashort-latency depolarisation. Stimulating the metathoracic subcostanerve in the wing (see Fig. 1) elicited a burst of sensory activityrecorded in N1 and a depolarisation in the interneuron (iii). Averagesof 50 sweeps triggered from the stimulus artifact. E: Touching themetathoracic wing elicited bursts of sensory activity recorded in N1(lower trace) and large-amplitude depolarisations and spikes (uppertrace). Touching the metathoracic femur also generated bursts ofspikes. Scale bars � 100 �m in A–C, 1 mV, 20 msec in D, 2 mV, 1second in E.

103INTEGRATION OF SENSORY INFORMATION

Figure 6

depolarisation and spikes in the one neuron that wastested in this way (not shown).

Type B intersegmental interneurons. Ascending in-tersegmental interneurons of type B have a lateral soma,bilateral fields of branching, and contralaterally ascend-ing axons that branch in the mesothoracic ganglion beforecontinuing further anterior. Recordings were made fromtheir processes near the base of the ipsilateral connective.These interneurons probably belong to a population ofapproximately 35 cells, some of which were described byLaurent and Burrows (1988), although features of theiranatomy differ, and inputs from the wings have not pre-viously been detected.

The soma lies near the root of N1 (Fig. 9A), and from ita thick main neurite (arrow in Fig. 9Di) runs medially tocross the midline in DCIII, before splitting to give rise toa contralaterally ascending axon in DIT (solid arrowheadsin Fig. 9A,Di) and two prominent dorsal branches that runposteriorly; one in DIT (solid double arrowhead in Fig.9Dii) as far as abdominal neuromere 2 (double arrowheadin Fig. 9A), the other in DMT (asterisks in Fig. 9A,Dii).Both posterior neurites give off short branches, most ofwhich project dorsally to DIT and DMT, respectively. Theascending axon also gives off several small branchesaround and particularly dorsally to DIT before it entersthe connective (Fig. 9Di). The similar neurons describedby Laurent and Burrows (1988) have a contralateral axonin MDT, not DIT.

Ipsilaterally the main neurite gives rise to two posteriorventral branches (pv1, pv2 in Fig. 9B), a prominent ante-rior ventral branch (av in Fig. 9B), and three dorsalbranches (d1, d2, d3 in Fig. 9A). The anterior ventralbranch gives rise to a dense ventral region of branching inaVAC (open arrowheads in Fig. 9B,Di, inset). Some ofthese fine ventral branches cross the midline by up to 100�m. The similar neurons described by Laurent and Bur-rows (1988) do not have branches in aVAC. The firstposterior ventral branch (pv1 in Fig. 9B) contributes to themost posterior branches in aVAC and is the main source ofbranches in lVAC (open double arrowheads in Fig. 9B,Dii).The second posterior ventral branch runs ventrally in CT(pv2 in Fig. 9Dii) to contribute to the most posterior me-dial region of branching in lVAC.

The lateral dorsal branch from the main neurite (d1)gives rise to a very anterior dorsal region of branches thatintermingles with the roots of nerve 1 above and belowDIT (Fig. 9A). The medial dorsal branch, d2, gives rise tobranches in and around DMT anterior to the main neurite

(Fig. 9A,Di). The lateral posterior branch, d3, gives rise toa small region of branches in DIT (Fig. 9A), and the medialposterior branch, d4, gives rise to branches in DMT pos-terior to the main neurite (Fig. 9A,Dii).

The axon enters the mesothoracic ganglion in DIT andgives rise to several short branches that are mostly medialand dorsal to DIT and a prominent lateral branch (arrowin Fig. 9C) that gives rise to sparse branching in dorsalaLAC. The axon passes through the mesothoracic gan-glion in DIT and exits in the anterior connective. It is notknow whether the similar neurons described by Laurentand Burrows (1988) have this morphology.

Type B interneurons were depolarised by stimulation ofthe ipsilateral mesothoracic wing nerve with a latency of6.1–6.9 msec (2/2; Fig. 10A). Stimulation of the ipsilateralmetathoracic wing nerve also caused a depolarisation (2/2), with a latency of 3.4–4.3 msec (Fig. 10B). In one of theinterneurons, this input sometimes elicited a spike (notshown). Touching either ipsilateral wing elicited large-amplitude depolarisations and bursts of spikes (Fig.10C,D). Touching the tarsus or distal tibia of the ipsilat-eral metathoracic leg also elicited depolarisations andspikes (Fig. 10E). Touching elsewhere on the metathoracicleg, extending and flexing the tibia, or touching the otherlegs elicited little or no response. Possible inputs from thecontralateral wings were not tested.

Type C ascending interneurons. Type C ascendinginterneurons may be serial homologues of a type of neurondescribed from the mesothoracic ganglion by Watson andBurrows (1983) and Pearson et al. (1985), and they sharesome similarity with a population of flight interneuronsdescribed for the metathoracic and abdominal neuromeresby Robertson et al. (1982). The characteristic feature of allthese neurons is a prominent loop in the main neurite.Type C neurons have a ventral midline soma and a mainneurite that rises dorsally in TT, before bifurcating at thelevel of DCIII to send a process to the contralateral side ofthe ganglion (Fig. 11A,Cii,D). The main neurite on theipsilateral side (the side of main inputs from the wings)curves outwards in TT around DMT, giving off a promi-nent anterior branch (a in Fig. 11B,D) and two posteriorbranches into DMT (p1, p2 in Fig. 11B,D), before continu-ing without further branching laterally in TT as far asLDT (Fig. 11Cii). It then turns dorsally around LDT, giv-ing off a dorsal branch (solid arrows in Fig. 11A,B,Cii),before turning medially to enter DMT again. It thus formsan unusual and distinctive lateral loop that is prominentin both whole-mount and sectioned preparations. In DMTthe main neurite splits into two branches, an anterioraxon that exits the ganglion in the ipsilateral connective(axon in Fig. 11Cii) and a posterior neurite (p3 in Fig.11B,D) that runs in DMT as far as the third abdominalneuromere. This gives off fine branches that cross themidline by up to 110 �m in the metathoracic neuromereand 40 �m in abdominal neuromeres 1 and 2 (solid arrow-heads in Fig. 11A). The pattern of branching in the meso-thoracic ganglion is unknown.

The anterior branch from the main neurite (a in Fig.11B,D) runs anteriorly in DMT and turns laterally intothe roots of N1 to contribute to the dense anterior ventraland lateral regions of branching in aVAC and N1 roots(open arrows in Fig. 11A,Ci). The first two posteriorbranches of the main neurite (p1, p2 in Fig. 11B) runposteriorly in DMT, giving rise to dense branching in andnear this tract (Fig. 11Cii). At the posterior edge of the

Fig. 6. Morphology and physiology of a metathoracic midline spik-ing interneuron Type D. A: Ventral branches drawn from a wholemount. Arrowhead, anterior branch. B: Dorsal field of branches. Theventral soma is drawn as a dashed outline for reference. Ci–iii:Transverse sections made at the levels indicated in A. Solid arrow-head, anterior branch plunging ventrally; solid arrow, main neurite;open arrow, main dorsal neurite; open arrowhead, dorsal lateralbranches. Di: Electrical stimulation of the metathoracic subcostanerve elicited a barrage of sensory activity recorded in N1 (lowertrace) and a long-lasting depolarisation (upper trace). Average of 50sweeps triggered from the stimulus artifact. Dii,iii: Touching themetathoracic wing (ii) or extending and flexing the metathoracic tibia(iii) elicited large-amplitude depolarisations and bursts of spikes.Scale bars � 100 �m in A–C, 5 mV, 20 msec in Di, 5, mV, 100 msec inDii,iii.

105INTEGRATION OF SENSORY INFORMATION

Figure 7

metathoracic neuromere, a branch turns downward, me-dial to VIT, and curves laterally to enter MVT (doublearrowhead in Fig. 11A), where it gives rise to a fine neu-rite that projects up and down MVT, giving off only a fewshort side branches within the tract (open arrowheads inFig. 11A,Cii).

At the dorsal-lateral apex of the loop in the main neurite(solid arrows in Fig. 11A,B), a fine branch gives rise tomost of a sparse, almost uniform, network of branchesthat spans much of the dorsal lateral neuropil (Fig. 11B).Some of the more medial branches arise from the firstposterior branch of the main neurite (p1). All of the dorsalbranches are restricted to the most dorsal parts of thelateral association centres (aLAC, pLAC) or to more me-dial regions above DIT (double arrowhead in Fig. 11Cii).

On the contralateral side, the main neurite bifurcatesinto anterior and posterior branches in DMT that give riseto regions of branching that largely mirror those describedfor the ipsilateral side. There are many fine branches inthe roots of N1 (but not aVAC; Fig. 11A,Cii) and in DMT.The overall branching is less dense, the regular pattern ofdorsal branches is absent, and the fine ventral branch inMVT is also missing.

Two other intersegmental interneurons with inputsfrom the wings and a characteristic looping main neuritewere each recorded once. Despite the remarkable similar-ity of the paths followed by the main neurites in all thesecells (e.g., Fig. 11D), the remaining branching was sub-stantially different (not shown). One neuron branchedunilaterally in the metathoracic neuromere, whereas theother had more extensive ventral branches and fewer dor-sal branches than illustrated in Figure 11A, and the pos-terior contralateral projection in DMT was absent. To-gether with the Type C interneurons, these seem to bemembers of a small but diverse group characterised by theconvoluted path followed by their main neurite.

Type C ascending intersegmental interneurons weredepolarised with a latency of 16 msec following stimula-tion of the ipsilateral metathoracic wing nerve (2/2; Fig.12A). Stimulation of the ipsilateral mesothoracic wingnerve had little or no effect (2/2; not shown), but stimula-tion of the ipsilateral mesothoracic or metathoracic N1elicited clear depolarisations (2/2; Fig. 12B,C). Touchingthe lateral thorax near the metathoracic wing base elic-ited large-amplitude depolarisations and spikes (1/1; Fig.12D). Touching the contralateral wings also depolarised

the interneurons, but touching the legs had no effect (1/1;not shown).

DISCUSSION

I have described five types of local interneurons andthree types of intersegmental interneurons that receivesensory inputs from receptors in the wings. Some of theseinterneurons are members of known populations, but myanalyses reveal that their patterns of sensory inputs aremore widespread than has been recorded in the past.These neurons may be involved in pathways that generatescratching movements of a hind leg directed toward tactilestimuli on a wing.

To examine the patterns of synaptic inputs to the inter-neurons, electrical stimuli were applied to a wing nerve orto nerve 1, which innervates receptors of several types inthe wing and at the wing base. In freely moving locusts,electrical stimulation of a wing nerve elicits targetedscratching movements and, at high stimulus amplitudesand frequencies, other behaviours, such as struggling orwalking (Matheson, 1998). Stimulation of nerve 1 alsoelicits scratching, walking, or struggling (personal obser-vation) The electrical stimuli used here did not permit ananalysis of the size of the receptive fields on the wing, butin many cases direct mechanical stimulation confirmed aninterneuron’s response to tactile stimuli that normallyelicit scratching.

Common features of all the neurons

The observation that all of the neurons responded tostimuli of both mesothoracic and metathoracic N1 sug-gests that they are important points of convergence ofsensory information from different body segments andappendages. The universal presence of anterior branchesin and around the roots of N1 or in the aVAC suggests thatthis region is important for the processing of wing tactileinformation.

Metathoracic local interneurons

Local interneurons, named here Types A–C, are midlinespiking interneurons as defined by Burrows and Siegler(1982, 1984) and Siegler and Burrows (1983, 1984). Theseinterneurons have somata near the ventral midline of themetathoracic ganglion and branches that are separatedinto distinct dorsal and ventral fields by a process in theperpendicular tract (PT). The input and output propertiesof many of these cells have been described previously inthe context of local reflex movements of a hind leg elicitedby mechanical stimuli applied to the same leg (for reviewsee Burrows, 1989). These local interneurons receivemonosynaptic inputs from sensory neurons innervatingarrays of tactile hairs on a leg or innervating leg proprio-ceptors (not both). The patterns and strengths of theseconnections differ between interneurons so that the pop-ulation as a whole can encode a somatotopic representa-tion of the surface of the leg. The assumption is that thespecific patterns of outputs of the interneurons mean thattheir activity, driven by sensory inputs, elicits appropriatemovements of the leg to withdraw it from the stimulus (forreview see Newland and Burrows, 1997).

The dorsal and ventral fields of branches of Type D localinterneurons both arise from processes given off after themain neurite has climbed the P tract, so these neurons donot fit exactly the descriptions given by Burrows and

Fig. 7. Morphology and physiology of a metathoracic local inter-neuron Type E. A: Entire arborisation drawn from a whole mount.Solid arrowhead, anteriormost branch near roots of N1; double arrow-heads, posterior dorsal branches; arrow, main neurite turning dor-sally to loop around LDT; open arrowhead, branches in and aroundroots of N1; asterisk, posterior branches near DMT. Bi–iii: Trans-verse sections made at the levels indicated in A. Solid arrowheads andopen arrowhead, ipsilateral and contralateral branches near roots ofboth N1, respectively; solid arrow, main neurite in TT (also see inset;photomicrograph of the region indicated by a dashed rectangle in Bii);open arrow, main neurite looping around LDT; asterisk, branches inand around DMT; double arrowhead, dorsal branches above DIT.Ci,ii: Electrical stimulation of mesothoracic N1 (i) elicited a weak anderratic but long-lasting depolarisation, whereas electrical stimulationof metathoracic N1 (ii) elicited a consistently larger amplitude depo-larisation. Averages of 50 sweeps triggered from the stimulus artifact.Scale bars � 100 �m in A,B, 0.5 mV, 20 msec in Ci, 2 mV, 20 msecin Cii.

107INTEGRATION OF SENSORY INFORMATION

Figure 8

Siegler. Nevertheless, the overall patterns of branchingresemble closely those of other midline spiking interneu-rons, so I conclude that they too are members of thispopulation.

The observation that all local interneurons responded tostimulation of both mesothoracic and metathoracic N1provides the first reported evidence for convergence ofsensory information from two body appendages (wingsand legs) or two body segments (mesothoracic and met-athoracic) onto individual members of this population.Local interneurons of a different metathoracic populationhave receptive fields on both a hind leg and the ipsilateralmiddle leg (Nagayama, 1990). The inputs from the middleleg exteroceptors must be indirect, because none projectsinto the metathoracic ganglion.

The regions of neuropil containing the ventral inputbranches of local spiking interneurons are strongly corre-lated with the patterns of inputs that the neurons receivefrom tactile hairs on a leg (for review see Newland andBurrows, 1997). I show that local interneurons that aredepolarised by stimulation of a wing nerve or N1 haveanterior branches in aVAC or around the roots of N1.Some of the interneurons that respond to wing stimula-tion also respond to touching or moving a leg (see Table 1),and this is accompanied by branches in the ventral andlateral association centres to which leg hair afferentsproject, as well as branches in aVAC or around the roots ofN1. This pattern has not previously been illustrated. Thelocal interneurons, named here Types A–D, therefore ap-pear to represent previously undescribed subtypes of themidline spiking group. In the context of scratching behav-iour, they have the important property of collating extero-ceptive inputs from the wings (that could signal the loca-tion of a touch on the wing and hence a target site) andproprioceptive inputs from leg joints that could signal legposture (see Table 1). The convergence of these two keytypes of information is essential in the computation of alimb trajectory from a start posture to a particular targetsite, and it is this aspect that will be the subject of furtherdetailed analyses.

Local interneurons of Type E are not, to my knowledge,the same as any previously reported neuron. Their bilat-eral symmetry and posterior branches give them a super-ficial resemblance to some stridulatory interneurons ingrasshoppers (Ocker and Hedwig, 1996). Many interneu-rons in the flight system of a locust also have bilateralbranches, but most are intersegmental (see, e.g., Pearson

and Robertson, 1987) and none resembles the local neu-rons reported here. The main neurite follows a path sim-ilar to that of the Type C intersegmental interneurons, inthat it loops laterally around the lateral dorsal tract(LDT), but there are few other common morphologicalfeatures. The looped part of the neurite gives off severalsecondary branches, whereas that of the intersegmentalneurons is relatively bare; and the posterior route of themain neurite is in MDT, whereas that of the intersegmen-tal neurons is in DMT. Nevertheless, the similarity of theunusual looped main neurite is suggestive of commondevelopmental or functional constraints. Another similar-ity between Type E local and Type C intersegmental in-terneurons is that they both respond to N1 stimuli but notto touching or moving the metathoracic legs.

Intersegmental interneurons

Intersegmental interneurons characterised here includeone type with its metathoracic branches restricted to onehalf of the ganglion (Type A) and two types (B and C) withbilateral patterns of branching. The unilateral branchingof Type A neurons distinguishes them from most flightand stridulatory interneurons, which are almost all bilat-eral (see Burrows, 1996). Like the local interneurons,Type A intersegmental interneurons have prominent re-gions of branching in the aVAC, which is where they arelikely to receive their inputs from wing sensory neurons.Branches in the anterior lateral (aLAC) and lateral ven-tral (lVAC) association centres are presumably the sites ofinputs from leg receptors that give rise to the responsesrecorded in response to touching or moving the ipsilateralhind leg. The convergence of information from a hind legand both ipsilateral wings makes these interneuronspromising candidates for a role in scratching behaviour.

Intersegmental interneurons of Type B appear to belongto a population of approximately 35 cells described byLaurent and Burrows (1988) on the basis of their mech-anosensory inputs from a hind leg. The population con-tains ascending interneurons that receive direct synapticinputs either from sensory neurons innervating leg tactilehairs or from sensory neurons of a particular leg jointproprioceptor, the femorotibial chordotonal organ. Thosethat receive proprioceptive leg inputs have either an ipsi-lateral axon in the MDT or a contralateral axon in theVIT, whereas those that receive exteroceptive leg inputshave an ipsilateral axon in the VMT (Laurent and Bur-rows, 1988). The Type B interneurons described in thepresent paper are excited by touching the tibia or tarsus ofthe ipsilateral hind leg but not by movements of the fem-orotibial leg joint. The prediction would be that they havean ipsilateral axon in VMT, but this is not the case: Theyhave a contralateral axon in DIT and, overall, appear mostsimilar in whole mount to Laurent and Burrows’ proprio-ceptive neurons with a contralateral axon. The Type Binterneurons are depolarised following stimulation of ei-ther the mesothoracic or the metathoracic wing nerve (inthe wings) or by gently touching the wing with a softbrush, which indicates that they also receive inputs fromwing exteroceptors (the wings do not contain joint proprio-ceptors, and the stimuli used do not elicit movements ofthe legs that could lead to secondary feedback). Theseinputs are powerful enough to elicit spikes in the inter-neurons.

Type B interneurons have dense regions of branching inthe aVAC, which correlates well with what little is known

Fig. 8. Morphology and physiology of a metathoracic ascendingintersegmental interneuron Type A. A: Entire arborisation drawnfrom a whole mount. Arrowhead, posterior lateral branch; doublearrowhead, posterior medial branch; arrow, lateral branches. B: Il-lustration of the same neuron showing just the main branches. Arrow,anterior ventral branch; a, p, v, anterior, posterior and ventral mainbranches described in the text; arrowhead, posterior branch in MDT;asterisk, anterior branch near MVT. Ci–iii: Transverse sections madeat the levels indicated in A. Solid arrows; anterior ventral branches;asterisk, anterior branch near MVT; open arrows, lateral neuritegiving rise to branches in close association with roots of N2–N5;double arrowhead, branch climbing to level of MDT; arrowhead,branch climbing to level of LDT. Di,ii: Electrical stimulation of eithera mesothoracic wing nerve (i) or the metathoracic wing subcostalnerve (ii) elicited consistent depolarisations. Averages of 50 sweepstriggered from the stimulus artifact. Scale bars � 100 �m in A–C, 1mV, 20 msec in D.

109INTEGRATION OF SENSORY INFORMATION

Fig. 9. Morphology of a metathoracic ascending intersegmentalinterneuron Type B. A: Dorsal branches drawn from a whole mount.Arrowhead, ascending axon in DIT; double arrowhead, posteriorbranch in DIT; asterisk, medial branch in DMT; d1–d4, dorsalbranches referred to in the text. B: Ventral branches of the sameinterneuron. The soma and primary neurite are shown as dashedlines for reference. Arrowhead, dense branching in aVAC; doublearrowhead, posterior branches in lVAC, av, pv1, pv2, ventralbranches referred to in the text. C: Branches in the mesothoracic

ganglion drawn from a whole mount. Arrow, lateral branch to aLAC.Di,ii: Transverse sections made at the levels indicated in A (note thatthe plane of sectioning is slightly oblique, so the sections are notexactly symmetrical). Arrow; main neurite from soma; solid arrow-head, ascending axon in DIT; open arrowhead, dense branching inaVAC (also see inset; photomicrograph of the region indicated by adashed rectangle in Di); solid double arrowhead, posterior branch inDIT; open double arrowhead, posterior branches in lVAC; asterisk,posterior medial branch in DMT. Scale bars � 100 �m.

of the projections of wing sensory neurons (personal ob-servations). Further branching in the lVAC matches wellwith the projections of leg exteroceptors (Newland, 1991).Burrows and Newland (1993) have shown that the re-sponses of intersegmental interneurons from this popula-tion to stimulation of leg hairs can be explained by thesomatotopically organised overlap of the sensory and in-terneuronal branches. The present study indicates thatthis somatotopic mapping extends beyond the leg to in-clude the ipsilateral hind wing and possibly also the ipsi-lateral mesothoracic wing. I predict that these or similarinterneurons also receive inputs from exteroceptors on thethorax itself, so that together they encode a representa-tion of the entire surface of that body segment.

The ascending axon of Type B interneurons gives rise torelatively few branches in the mesothoracic ganglion. Sig-nificantly, there is no region of fine, dense branching inmesothoracic ventral neuropil that would be expected ifthese neurons receive direct exteroceptive inputs in thissegment. The conclusion must be, therefore, that the ex-citatory inputs following mesothoracic wing stimulationare mediated either by indirect pathways or by directconnections from mesothoracic wing sensory axons thatdescend to the metathoracic ganglion.

Laurent and Burrows (1988) hypothesise that the as-cending interneurons they describe could provide the an-terior legs with information about movements and thelocations of tactile stimuli on a hind leg, which could beused in the coordination of walking. The output effects areunknown, however, and depolarising the neurons so thatthey spiked did not lead to movements of the mesothoraciclegs. One interpretation given was that stronger parallelexcitation of several interneurons might be required. An-other explanation suggested by my data is that this pop-ulation (at least the Type B neurons) may be involved inother behaviours, such as scratching the wings. It wouldtherefore be valuable to search for output effects in themetathoracic segment. The new data for the first timereveal the pattern of branching in the mesothoracic gan-glion and show that the axons extend farther anteriorly,at least as far as the prothoracic ganglion.

Metathoracic ascending interneurons of Type C arereadily recognised by a prominent lateral loop in theirmain neurite, an ascending axon in the ipsilateral connec-tive, and their bilateral branching. The looped neurite issimilar to that of a set of mostly unilateral flight interneu-rons described in the metathoracic ganglion by Robertsonet al. (1982), but they are clearly not the same cells.Metathoracic Type C interneurons are similar to some of apopulation of bilaterally branching interneurons with alooping neurite that have been reported in the mesotho-racic ganglion (Watson and Burrows, 1983; Pearson et al.,1985). Pearson et al. show that there must be two to fivesimilar neurons (named by them Type 404) per mesotho-racic hemiganglion (four to ten per ganglion), and this issupported by my observation in the metathoracic ganglionthat there are at least three distinct neurons per side withthe lateral loop. The Type C interneurons described indetail here represent just one of these morphologies.

Several lines of physiological evidence suggest that me-sothoracic Type 404 interneurons are involved in the ini-tiation of flight, although they do not themselves makedirect output connections with flight motor neurons (Pear-son et al., 1985). Possible outputs onto metathoracic legmotor neurons have not been examined. The metathoracic

Fig. 10. Physiology of metathoracic ascending interneurons TypeB. Electrical stimulation of either a mesothoracic wing nerve (A) orthe metathoracic wing subcostal nerve (B) elicited barrages of sensoryactivity in the corresponding N1 (lower traces) and consistent depo-larisations (upper traces). Averages of 50 sweeps triggered from thestimulus artifact. Touching either wing (C,D) or the ipsilateral met-athoracic femur (E) elicited depolarisations and bursts of spikes.Repeated depolarisations in C correspond to repeated proximal-distalmovements of the brush across the surface of the wing. Scale bars �1 mV, 20 msec in A,B, 5 mV, 200 msec in C–E.

111INTEGRATION OF SENSORY INFORMATION

Fig. 11. Morphology of a metathoracic ascending intersegmentalinterneuron Type C. A: Ventral and midventral/dorsal branchesdrawn from a whole mount. Arrow, main neurite looping dorsallyaround LDT; arrowheads, fine posterior branches crossing the midlinein each neuromere; double arrowhead, posterior branch plungingdown into MVT. B: Dorsal branches of the same interneuron. Thesoma and primary neurite are shown as dashed lines for reference[the main anterior ventral (a) and three main posterior ventralbranches (p1–p3) are labeled here for clarity]. Arrow, main neurite

looping dorsally around LDT; Ci,ii: Transverse sections made at thelevels indicated in A. Open arrows; anteriormost branching in aVACand amongst the roots of N1; solid arrow, main neurite looping dor-sally around LDT; arrowhead, posterior branch in MVT; double ar-rowheads, very dorsal branches in aLAC and above LDT. D: Photomi-crograph of the region indicated by the dashed rectangle in Billustrating the distinctive lateral loop and branching of the mainneurite. The anterior ventral branch (a) and two of the posteriorventral branches (p1, p2) are labeled. Scale bars � 100 �m.

Type C interneurons are depolarised by stimuli of themetathoracic wing nerve (in the wing) and more power-fully by stimuli of metathoracic N1. These observations fitwell with the descriptions given for the mesothoracic Type404 neurons, which respond to touching the mesothoracicwing and to movements of the mesothoracic wing base(Watson and Burrows, 1983). The metathoracic neuronsare also depolarised by stimulation of mesothoracic N1,indicating that they collate information from both pairs ofwings. They also respond to tactile stimuli of the lateralthorax but not to stimuli of the legs.

Implications for understanding targetedlimb movements

Matheson (1998) used behavioural methods to analyseat a gross level the neuronal pathways underlying tar-geted scratching of the wings by the metathoracic legs.Matheson’s study indicated that sensory information froma mesothoracic wing must descend to the metathoracicmotor networks that drive hind leg movements and thatsensory information must cross the midline to reach themotor network of the contralateral hind leg. Furthermore,it suggested that there must be convergence of exterocep-tive wing sensory information with leg proprioceptive in-formation to permit computation of leg trajectories to-wards the target from different start positions.

The present paper takes Matheson’s (1998) analyses astep further, by identifying neurons that could fill some ofthese roles. First, there is convergence through short-latency pathways of sensory information from a wing anda leg of one body segment onto local spiking interneurons.This emphasises the need to examine the roles of theseneurons in behaviours other than local leg avoidance re-flexes, which have provided the key understanding of theirproperties so far. Second, sensory information from boththe mesothoracic and the metathoracic wings convergesthrough short-latency pathways onto the same metatho-racic local neurons. Such integration is an important fea-ture that provides a mechanism by which metathoracic legmotor networks could generate movements targeted to-ward equivalent sites on the two wings, which overlap intheir resting posture. Third, bilaterally branching localand intersegmental neurons are shown to receive short-latency inputs from the wings. They could carry this in-formation across the midline. Some of these neurons havepreviously been shown to influence a flight motor pattern,but previous studies have tended to assume that anyinterneuron with sensory inputs from a wing must beinvolved in flight (see, e.g., Pearson et al., 1985). Thisperspective might have prevented other possible roles,such as in scratching, from being fully explored. Fourth,some of the interneurons collate through short-latencypathways sensory information from exteroceptors on thewings and from joint proprioceptors of a metathoracic leg.These interneurons are likely to be key elements of apathway that generates targeted scratching movements ofa hind leg and will be the focus of further detailed anal-yses.

ACKNOWLEDGMENTS

The author thanks M. Burrows, M. Gebhardt, P.L. New-land, and S. Rogers for comments on a draft of the manu-script. The work was supported by a BBSRC Advanced

Fig. 12. Physiology of metathoracic ascending intersegmental in-terneurons Type C. A: Electrical stimulation of the subcostal nerve inthe ipsilateral metathoracic wing elicited a barrage of sensory activityin N1 (lower trace) and a small depolarisation that was followed by ahyperpolarisation in the interneuron (upper trace). Stimulation ofmesothoracic N1 (B) elicited a brief depolarisation in the interneuron,whereas stimulation of metathoracic N1 (C) elicited a large-amplitudeand long-lasting input with a superimposed spike (arrowhead). Thespike amplitude is small and is almost swamped by the synaptic inputas seen at the recording site in the soma. D: Touching the lateralthorax just ventral to the base of the wing elicited a burst of spikes inN1 and a depolarisation and spikes in the interneuron. Scale bars �0.2 mV, 20 msec in A,B, 2 mV, 20 msec in C, 5 mV, 50 msec in D.

113INTEGRATION OF SENSORY INFORMATION

Research Fellowship and a Royal Society (London) Re-search Grant to T.M. and a BBSRC Research Grant to M.Burrows.

LITERATURE CITED

Bacon JP, Altman JS. 1977. A silver intensification method for cobalt-filledneurones in wholemount preparations. Brain Res 138:359–363.

Berkowitz A, Laurent GJ. 1996a. Central generation of grooming motorpatterns and interlimb coordination in locusts. J Neurosci 16:8079–8091.

Berkowitz A, Laurent GJ. 1996b. Local control of leg movement and motorpatterns during grooming in locusts. J Neurosci 16:8067–8078.

Brogan RT, Pitman RM. 1981. Axonal regeneration in an identified insectmotoneurone. J Physiol (London) 319:34P–35P.

Brunn DE, Dean J. 1994. Intersegmental and local interneurones in themetathorax of the stick insect Carausius morosus that monitor middleleg position. J Neurophysiol 72:1208–1219.

Burrows M. 1989. Processing of mechanosensory signals in local reflexpathways of the locust. J Exp Biol 146:209–227.

Burrows M. 1996. The neurobiology of an insect brain. New York: OxfordUniversity Press.

Burrows M, Newland PL. 1993. Correlation between the receptive fields oflocust interneurons, their dendritic morphology, and the central pro-jections of mechanosensory neurons. J Comp Neurol 329:412–426.

Burrows M, Siegler MVS. 1982. Spiking local interneurones mediate localreflexes. Science 217:650–652.

Burrows M, Siegler MVS. 1984. The morphological diversity and receptivefields of spiking local interneurones in the locust with inputs from hairafferents. J Comp Neurol 224:483–508.

Cruse H, Dean J, Kindermann T, Schmitz J, Schumm M. 1998. Simulationof complex movements using artificial neural networks. Z Naturforsch53:628–638.

Durr V, Matheson T. 2001. Functional somatatopy of targeted limb move-ments in an insect. Proc 28th Gottingen Neurobiol Conf 2001. Elsner Nand Krentzberg GW, editors. Stuttgart. Thieme Verlag. p 319.

Laurent G, Burrows M. 1988. A population of ascending intersegmentalinterneurones in the locust with mechanosensory inputs from a hindleg. J Comp Neurol 275:1–12.

Matheson T. 1997. Hindleg targeting during scratching in the locust. J ExpBiol 200:93–100.

Matheson T. 1998. Contralateral coordination and retargeting of limbmovements during scratching in the locust. J Exp Biol 201:2021–2032.

Nagayama T. 1990. The organization of receptive fields of an anteromedialgroup of spiking local interneurones in the locust with exteroceptiveinputs from the legs. J Comp Physiol A166:471–476.

Newland PL. 1991. Morphology and somatotopic organisation of the cen-tral projections of afferents from tactile hairs on the hind leg of thelocust. J Comp Neurol 312:493–508.

Newland PL, Burrows M. 1997. Neuronal networks controlling leg move-ments of the locust. J Insect Physiol 43:107–123.

Ocker WG, Hedwig B. 1996. Interneurones involved in stridulatory patterngeneration in the grasshopper Chorthippus mollis (Charp). J Exp Biol199:653–662.

Pearson KG, Robertson RM. 1987. Structure predicts synaptic function oftwo classes of interneurons in the thoracic ganglia of Locusta migrato-ria. Cell Tissue Res 250:105–114.

Pearson KG, Reye DN, Parsons DW, Bicker G. 1985. Flight-initiatinginterneurones in the locust. J Neurophysiol 53:910–925.

Pfluger H-J, Braunig P, Hustert R. 1988. The organization of mechanosen-sory neuropiles in locust thoracic ganglia. Phil Trans R Soc London[Biol] 321:1–26.

Robertson RM, Pearson KG, Reichert H. 1982. Flight interneurons in thelocust and the origin of insect wings. Science 217:177–179.

Rosenbaum DA, Muelenbroek RGJ, Vaughan J. 1996. Three approaches tothe degrees of freedom problem in reaching. In: Wing AM, Haggard P,Flanagan JR, editors. Hand and brain. The neurophysiology and psy-chology of hand movements. San Diego: Academic Press. p 169–185.

Siegler MVS, Burrows M. 1983. Spiking local interneurons as primaryintegrators of mechanosensory information in the locust. J Neuro-physiol 50:1281–1295.

Siegler MVS, Burrows M. 1984. The morphology of two groups of spikinglocal interneurones in the metathoracic ganglion of the locust. J CompNeurol 224:463–482.

Soechting JF, Tong DC, Flanders M. 1996. Frames of reference in senso-rimotor integration: position sense of the arm and hand. In: Wing AM,Haggard P, Flanagan JR, editors. Hand and brain. The neurophysiol-ogy and psychology of hand movements. San Diego: Academic Press. p151–168.

Tyrer NM, Gregory GE. 1982. A guide to the neuroanatomy of locustsuboesophageal and thoracic ganglia. Phil Trans R Soc London [Biol]297:91–123.

Watson AHD, Burrows M. 1983. The morphology, ultrastructure and dis-tribution of synapses on an intersegmental interneurone of the locust.J Comp Neurol 214:154–169.

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