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

Metathoracic Neurons Integrating Intersegmental Sensory Information in the Locust TOM MATHESON* Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom

ABSTRACT This paper describes the morphology and physiology of five types of local interneurons and three types of ascending intersegmental interneurons in the locust metathoracic ganglion that are points of convergence of sensory information from the wings. Four types of spiking 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 they encode a sensory representation of this appendage. Some are also depolarised with short latencies following stimulation of a mesothoracic wing nerve, indicating that they collate intersegmental as well as local information. All the local interneurons have branches in the anterior ventral association centre or around the roots of the nerve that carries wing sensory neurons. This distinguishes them from other interneurons in the population. A fifth type of local interneuron that has unusual bilateral branching and is not a member of this population is 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 the mesothoracic or metathoracic wing nerves, and some respond to tactile stimulation or movements of a hind leg. These latter interneurons thus collate information from both wings and legs. All three types of intersegmental interneurons have branches in the anterior ventral association centre or around the roots of the wing nerve. The responses of the interneurons described here shed new light on both local and intersegmental network function 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 targeted limb movements requires knowledge of the computations carried out within individual neurons, between neurons of a population, and between populations. In the locust Schistocerca gregaria, behavioural analyses have shown that some of the neuronal interactions underlying targeted scratching movements of a hind leg are local to the ganglion of a particular body segment, whereas others are spread across two or more ganglia (Berkowitz and Laurent, 1996a,b; Matheson, 1997, 1998). Although there is substantial understanding of neuronal pathways that underlie some insect behaviours (e.g., walking, jumping, flying, reflex leg withdrawal; see Burrows, 1996, for an overview), little is known about the neuronal control of any targeted limb movement (see, e.g., Brunn and Dean, 1994). Understanding the control of such targeted limb movements will provide important insights into how the nervous system resolves computational problems, such as the integration of information encoded in different frames © 2002 WILEY-LISS, INC. DOI 10.1002/cn.10140

of reference (Rosenbaum et al., 1996; Soechting et al., 1996) or redundancy (in which choices must be made among many motor solutions that could achieve a given task). The control strategies used by insect nervous systems may provide powerful “shortcuts” that can be implemented in autonomous robots (Cruse et al., 1998). Locusts make precisely directed scratching movements of one or both hind legs in response to tactile stimulation of the wings (Matheson, 1997). Behavioural analyses indicate the following key features. First, there is precise

Grant sponsor: BBSRC; Grant number: ARF/98/53; Grant sponsor: Royal Society (London); Grant number: RSRG17944. *Correspondence to: Tom Matheson, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom. E-mail: [email protected] Received 17 July 2001; Revised 18 October 2001; Accepted 20 November 2001 Published online the week of January 21, 2002

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somatotopic mapping of leg movements in response to stimuli at different locations on a wing (Du¨ rr and Matheson, 2001). Second, there is convergence of sensory information from the mesothoracic and metathoracic wings onto metathoracic motor networks. Third, there is contralateral transmission from the wings on one side of the body to the contralateral hind leg motor network (Matheson, 1997). The aim of this paper is to provide a description of candidate interneurons that are likely to be involved in these pathways based on their patterns of sensory inputs and morphological features. I describe five classes of local and three classes of intersegmental interneurons that receive sensory inputs from the wings and, in some cases, legs, which implicate them in the control circuits for targeted scratching movements of the hind legs. Four types of spiking local interneurons with somata at the ventral midline are members of a population that has been defined on the basis of both morphology and synaptic inputs from hind leg mechanoreceptors (Burrows and Siegler, 1982, 1984; Siegler and Burrows, 1983, 1984; for review see Burrows, 1996). Other members of this population encode the location of tactile stimuli on a metathoracic leg and are therefore thought to be involved in the local (segmental) generation of leg avoidance reflexes. The neurons that I describe are depolarised by stimulation of a metathoracic wing, suggesting that they encode a sensory representation of this appendage as well. One type of ascending intersegmental interneuron belongs to a population of approximately 35 cells described by Laurent and Burrows (1988) on the basis of their mechanosensory inputs from a hind leg. I show here that some neurons in this population are depolarised by stimuli applied to the wing.

Abbreviations aLAC aVAC CT DCI–VI DCI–VI(A1) DIT dDMT DMT FETi iLVT LDT lVAC MDT meta meso mVAC MVT N1-5r oLVT pLAC pLAC(T3) PT SMC tr TT VCI–II vDMT VIT VLT vmVAC VMT vVAC

anterior lateral association centre anterior ventral association centre C tract dorsal commissures 1– 6 (of the metathoracic neuromere) dorsal commissures 1– 6 of the first abdominal neuromere dorsal intermediate tract dorsal part of dorsal medial tract dorsal medial tract fast extensor tibiae motor neuron inner part of lateral ventral tract lateral dorsal tract lateral ventral association centre median dorsal tract metathoracic mesothoracic medial ventral association centre median ventral tract roots of nerves 1–5 outer part of the lateral ventral tract posterior part of lateral association centre posterior part of lateral association centre of the thoracic neuromere P tract supramedian commissure trachea T tract ventral commissure 1 or 2 ventral part of dorsal median tract ventral intermediate tract ventral lateral tract ventral part of medial ventral association centre ventral median tract ventral part of ventral association centre

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

Another type of metathoracic ascending interneuron is similar to a population of bilaterally branching interneurons in the mesothoracic ganglion (Watson and Burrows, 1983; Pearson et al., 1985). Such neurons have not previously been described in the metathoracic ganglion, and inputs from the wings have not been investigated. The morphology of the cells that I describe is also similar in some respects to a set of mostly unilateral flight interneurons described by Robertson et al. (1982) in the metathoracic and fused abdominal ganglia, but there are also clear differences, so they cannot be the same cells. My analyses of local and intersegmental interneurons in the context of targeted scratching movements broaden our understanding of how sensory inputs from different appendages of a body segment (wing, leg) or from different body segments (mesothoracic, metathoracic) are brought together in the ventral nerve cord.

MATERIALS AND METHODS Locusts (Schistocerca gregaria Forskål) taken from our crowded colony were restrained ventral side uppermost with both left wings held out laterally. The right-hand wings remained in their normal folded, resting position. All six legs were restrained at the femur, but the tibiae were free to move. A pair of bipolar myogram electrodes (100-␮m-diameter silver wire) was inserted into the left hind leg tibial extensor muscle to permit stimulation of the muscle and thus evoke tibial extension. To record and stimulate wing sensory neurons, pairs of bipolar neurogram electrodes were inserted into the forewing subcosta or cubitus vein and the hind wing subcosta vein (Fig. 1). The nerves that run along these veins innervate campaniform, trichoid, and basiconic receptors spread across large areas of the wing (personal observation). Throughout this

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TABLE 1. Key Features of the Interneurons1 Neuron type Local A Local B Local C Local D Local E Interseg A Interseg B Interseg C 1 2

Number analyzed

Meso N1 input

Meso wing input

Meta N1 input

Meta wing input

Wing base input

Hind leg proprio input

Other tactile input

Anterior ventral branches

8 3 5 2 2 4 2 2

8/8 3/3 3/3 1/1 1/1 2/3 — 2/2

0/3 1/3 1/3 1/1 0/1 2/3 2/2 0/2

8/8 2/3 4/4 2/2 1/1 4/4 — 2/2

1/3 0/3 3/4 2/2 0/1 4/4 2/2 2/2

3/3 — — — — — — 1/1

2/6 2/2 4/4 2/2 0/1 1/1 0/1 0/1

— — — — — Head/legs Hind leg —

y y2 y y y y y y

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

paper the phrase wing nerve is used to indicate these recording sites to distinguish them from recordings made in nerve 1 (see below). To gain access to the central nervous system, the ventral thoracic sclerites were cut away and underlying tracheal air sacs removed to reveal the mesothoracic and metathoracic ganglia. Both ganglia were supported on a wax-coated silver platform and treated with 0.1% (w/v) protease (Sigma type XIV, Sigma, St. Louis, MO) for 20 seconds to soften their tough outer sheath. Pairs of bipolar hook electrodes (100 ␮m silver wire) were placed under mesothoracic nerve 1, which gives rise to the mesothoracic wing nerve, and under metathoracic nerve 1, which gives rise to the metathoracic wing nerve. Each nerve 1 contains not only the axons of wing exteroceptors that pass into the corresponding wing nerve but also axons of receptors at the wing base and on the lateral thorax. Each pair of electrodes was insulated from the surrounding haemolymph with petroleum jelly. Throughout this paper these recording sites are referred to as nerve 1 to distinguish them from the wing nerve recording sites in the wing itself. The signals from wing neurogram electrodes and nerve 1 hook electrodes were amplified using Isleworth extracellular amplifiers. A Master 8 stimulator (A.M.P.I.) connected to each of the four electrodes via stimulus isolators was used to stimulate each recorded nerve independently and to stimulate the tibial extensor muscle (pulse duration 1 msec, 0.5 Hz).

Recording and cobalt staining Intracellular recordings were made from the somata of metathoracic neurons near the ventral midline using glass microelectrodes filled with 5% cobalt hexammine (Sigma). All recordings were made with a single-electrode bridge amplifier with the bridge balanced. To improve recording stability, all neurons were held hyperpolarised throughout recording by injection of a constant current of approximately –1 nA. Online signal averaging (Sigavg software; Cambridge Electronic Design, Cambridge, United Kingdom) triggered by the stimulation of sensory nerves was used to search for and characterise neurons with shortlatency synaptic inputs from wing or nerve 1 sensory neurons. All data were also stored on magnetic tape (Racal Store 7DS FM recorder) for later analysis using Spike 2 software (Cambridge Electronic Design). After physiological characterisation, neurons were injected iontophoretically with cobalt (250 msec pulses at 2 Hz for 20 – 40 minutes; Brogan and Pitman, 1981) and silver intensified (Bacon and Altman, 1977).

Neuroanatomy and abbreviations Morphological descriptions are derived from dorsal views of whole ganglia mounted in Canada balsam and from 9 ␮m sections of ganglia embedded in paraffin wax, drawn using a Zeiss compound microscope and camera lucida. The nomenclature used to describe the ganglionic tracts and neuropil follows Tyrer and Gregory (1982) and Pfl¨uger et al. (1988). In the descriptions of physiology given below, the number of neurons responding in a particular way is presented as “number responding/number tested in this way.” Experimental constraints sometimes meant that not all neurons of a type could be tested in all possible ways. Unless stated otherwise, all stimuli were presented to the side ipsilateral to the main field of neuronal branching. In cases where neurons branched bilaterally, ipsilateral refers to the side on which the soma was located. Photographic images were recorded at 1,312 ⫻ 2,000 pixels using a Nikon D1 camera attached to the microscope. Selected areas of interest were cropped to size, and the contrast and brightness adjusted manually using Canvas 7 (Deneba). The results presented here are based on recordings from 28 neurons that were stained and analysed in detail and from more than 160 additional recordings of less well characterised neurons in 110 animals.

RESULTS Common features of the interneurons Recordings were made from five types of metathoracic local interneurons and three types of ascending intersegmental interneurons that all received synaptic inputs following stimulation of a mesothoracic or metathoracic nerve 1 (N1) or stimulation of a wing nerve (Table 1). All interneurons that responded to stimulation of one N1 also responded to stimulation of the other, ipsilateral N1. Many of the analysed neurons also received synaptic inputs following tactile stimulation of the legs or body, and some received inputs following movements of leg joints. All of the responses to stimuli of a wing nerve or N1 were depolarising, often leading to spikes. All of the interneurons had anterior branches in and around the roots of N1 or in the anterior ventral association centre (aVAC).

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

98 Siegler, 1982, 1984; Siegler and Burrows, 1983, 1984). The fifth type (Type E) does not appear to belong to any previously described population. All of the Type A–D local interneurons have a soma near the ventral midline and a main neurite that crosses the midline in VCII as described for all other members of this population by Burrows and Siegler (1984). Where the soma lies particularly close to the midline, as in the examples shown in Figures 3 and 6, the neurite leading from it joins VCII at the midline. Note that other neurons of these types with somata farther from 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 the tract is right on the midline. Type A local interneurons. Interneurons of Type A cross the midline relatively posteriorly in VCII. After crossing the midline, the main neurite (asterisk in Fig. 2Cii) gives off prominent branches that turn anterior and posterior to the main neurite. The anterior branch gives rise to a ventral field of branching in aVAC (Fig. 2Ci), spanning horizontally from near the midline as far laterally as the edge of the connective (solid arrow in Fig. 2A). Near the anterior margin of the neuropil, these branches extend farther dorsally, to the level of VMT (arrow in Fig. 2Ci). The posterior branch gives rise to a prominent ventral process that runs posteriorly for 100 –200 ␮m almost parallel to the midline in MVT (solid arrowhead in Fig. 2A,Ciii). Both anterior and posterior branches give rise to a sparse region of ventral branching near the main neurite that begins in lVAC and extends dorsally near the midline as far as the top of VIT (double arrowheads in Fig. 2A,Cii). The main neurite continues laterally, giving rise to a prominent branch that curves farther laterally and posteriorly 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 to give rise to a widespread field of dorsal branches in aLAC and pLAC (Fig. 2B, open arrowheads in Cii,iii). Anteriorly, the dorsal branches lie above DIT and DMT (arrowhead in Fig. 2Ci). Local interneurons of Type A were depolarised and spiked once or twice following electrical stimulation of either mesothoracic or metathoracic nerve 1 (Fig. 2Di,ii). The latencies of these depolarisations were 4.6 –5.5 msec and 2.1–3.4 msec, respectively, in eight recordings. Touching the wings or stimulating wing nerves had no effect in two of the three (2/3) neurons tested in this way. Stimulation of the metathoracic wing nerve elicited a small depolarisation in 1/3 neurons. In contrast, even small movements (1–2° of arc) of either wing base powerfully excited 3/3 neurons (Fig. 2E). Passive movements of the hind leg tibia depolarised only 2/6 neurons, but active movements of the tibia were accompanied by spiking in 4/4 neurons. Injection of depolarising current into 1/1 neuron so that it spiked repeatedly caused no visible movement of the hind leg. Type B local interneurons. The main neurite of Type B local interneurons crosses the midline in VCII (Fig. 3A,Di), before turning posteriorly and becoming thicker. This thick, main neurite continues laterally, giving off usually four posterior ventral branches, one anterior ventral branch, and a lateral ventral branch, before turning dorsally in PT (Fig. 3Dii). The first posterior ventral branch has a large diameter and, after giving rise to sparse branching medially, be-

T. MATHESON comes thinner and turns posteriorly near the midline (solid arrows in Fig. 3A,Dii,E). All four posterior branches contribute to a sparse field of branching in vVAC and lVAC and as far posteriorly as pLAC (arrowheads in Fig. 3A,Dii,iii). The anterior branch from the main neurite extends only 100 ␮m anteriorly, giving rise to a few short processes in aVAC (open arrows in Fig. 3A,Di). These branches are not, therefore, as far anteriorly as those of all the other neuron types described here, and they do not mingle with the roots of N1. The most lateral ventral branch curves laterally and posteriorly to follow the edge of the metathoracic neuropil toward the roots of N5 (double arrowheads in Fig. 3A,Dii,iii). The main neurite ascending dorsally in PT branches to give rise to three distinct regions of dorsal branching. One region lies laterally (a in Fig. 3B,Diii), the second consists of fine fibres that run medially as far as the midline in DCIII (b in Fig. 3B,Di,ii), and the third consists of another group of fine fibres that run towards the midline in or just below DCV (c in Fig. 3C,Di,ii). Local interneurons of Type B were depolarised by electrical stimulation of mesothoracic nerve 1 (Fig. 4A) with latencies of 7.1–11 msec. Metathoracic nerve 1 stimulation also depolarised 2/3 neurons with latencies of 4.6 and 7.4 msec (Fig. 4B). Only 1/3 neurons was depolarised by stimulation of the mesothoracic wing nerve, and none was influenced by stimulation of the metathoracic wing nerve. Both active (Fig. 4C) and passive movements of the metathoracic tibia were accompanied by depolarisation and spikes in 2/2 neurons. In contrast, when one animal extended the metathoracic tibia against a fixed obstruction, there was no depolarisation in the neuron. Type C local interneurons. The main neurite of Type C local interneurons crosses the midline in VCII (Fig. 5A, solid arrow in Cii), before turning posteriorly and becoming considerably thicker. It continues posteriorly, giving off usually five medial ventral branches, one prominent anterior ventral branch, and a lateral ventral branch, before turning dorsally into PT to give rise to a dorsal field of branches. The ventral field of branching is more widespread than that of Type A and B local interneurons. The anterior ventral branch gives off several side branches 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 spiking interneuron Type A. A: Ventral branches drawn from a whole mount. Solid arrow, anterior ventral branches; arrowhead, posterior medial branch; double arrowhead, medial branches; open arrow, dorsal process in PT. B: Dorsal field of branches. The ventral soma is drawn as a dashed outline for reference, and the midline of the ganglion is shown as a vertical dashed line. Ci–iii: Transverse sections made at the levels indicated in A. Arrow, anterior ventral branches; open arrowheads, anterior dorsal branches; double arrowheads, medial branches; asterisk, main neurite; solid arrowhead, posterior medial branch. Di,ii: Electrical stimulation of mesothoracic (i) or metathoracic (ii) nerve 1 elicited a short-latency depolarisation and spikes (asterisks). Averages of 50 sweeps triggered from the stimulus artifact. 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 (lower two traces), and a depolarisation or depolarisation and spikes in the interneuron (top trace). Scale bars ⫽ 100 ␮m in A–C, 5 mV, 20 msec in D, 5 mV, 1 second in E.

Figure 2

Figure 3

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

the midline in vmVAC and aVAC (double arrowheads in Fig. 5Ci). The remaining ventral branches give rise to widespread arborisations throughout vVAC, lVAC, and ventral pLAC (Fig. 5A,Ci–iii). A prominent process follows the lateral edge of the metathoracic neuropil to terminate near the roots of nerve 5 (asterisk in Fig. 5A,Ciii). A second process runs posteriorly along the medial edge of the neuropil, and a third process ascends medially to CT (open arrow in Fig. 5Cii) to give rise to branches dorsomedially to VIT. The dorsal process in PT gives rise to thick branches that run anteriorly and posteriorly at the level of (but

Fig. 3. Morphology of a metathoracic midline spiking interneuron Type 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: Photograph of the region outlined by a dashed rectangle in Dii to show branching near VMT. All features marked with symbols are described in the text. See Figure 4 for this interneuron’s physiological responses. Scale bars ⫽ 100 ␮m.

101 lateral to) VIT (arrowheads in Fig. 5Cii,iii). They give off many side branches throughout the length and breadth of aLAC and pLAC. Local interneurons of Type C were depolarised by electrical stimulation of mesothoracic nerve 1 (6.1– 6.6 msec latency; Fig. 5Di; 3/3 neurons) or metathoracic nerve 1 (3.5–5.3 msec; Fig. 5Dii; 4/4 neurons) and sometimes by electrical stimulation of mesothoracic wing nerve (14.8 msec; 1/3 neurons) or metathoracic wing nerve (5.6-11 msec; Fig. 5Diii; 3/4 neurons). Touching the metathoracic wing elicited a depolarisation and spikes in 1/2 neurons tested in this way. In addition to these inputs from nerve 1 or the wings, Type C local interneurons were powerfully excited by touching hairs on the metathoracic femur or tibia (Fig. 5E; 3/3 neurons) or passively extending the femorotibial joint (4/4 neurons). Active movements of the metathoracic tibia were accompanied by depolarisation and spikes in 2/2 Type C local interneurons. Type D local interneurons. Local interneurons of Type D have a ventral soma (soma in Fig. 6Cii) and a neurite that crosses the midline in VCII (Fig. 6A,B). After the main neurite crosses the midline, it turns dorsally into PT (arrow in Fig. 6Ci) and ascends almost to the level of VLT (solid arrow in Fig. 6Cii), before giving rise to a prominent anterior branch (arrowhead in Fig. 6A), three medial branches, a posterior lateral branch (Fig. 6A), and a thick dorsal lateral branch (Fig. 6B). This pattern is thus quite different from that of Type A-C local interneurons, which all give rise to their ventral branches, before turning into PT. The anterior branch projects forward at the level of VIT, before plunging ventrally (arrowhead in Fig. 6Ci) to give rise to an anterior ventral field of branches in vVAC. The first medial branch gives rise to branches in and around VLT and MVT (Fig. 6Cii). The other medial branches and the posterior lateral branch curve gradually downward to form a dense field throughout much of ventral pLAC (Fig. 6A,Ciii). There are relatively few branches in lVAC (Fig. 6Cii). The thick dorsal lateral branch of Type D local interneurons curves laterally, posteriorly, and then medially (Fig. 6B) at the level of VLT (open arrow in Fig. 6Cii). It gives off fine branches that ascend around the lateral edge of aLAC and pLAC (arrowhead in Fig. 6Cii). The resulting dorsal field is sparser than the fields of Type A, B, and C local interneurons but still extends throughout the dorsal part of pLAC (Fig. 6Ciii). Type D local interneurons were depolarised by stimulation of the metathoracic wing nerve (7.1 msec; Fig. 6Di) or nerve 1 (6.8 – 8.5 msec; 2/2) and by stimulation of mesothoracic nerve 1 (10.4 msec; 1/1; not shown). Touching the metathoracic wing elicited a strong depolarisation and burst of spikes (1/1; Fig. 6Dii), as did extending the metathoracic tibia (2/2; Fig. 6Diii). Touching the mesothoracic wing had little or no effect (1/1). Type E local interneurons. Bilaterally branching local interneurons with a soma near the ventral midline were stained in two animals (Fig. 7). Their morphology is quite 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), before making an abrupt turn into DCIII (Fig. 7Bii, inset) and splitting bilaterally. On the side ipsilateral to the soma, the main neurite gives off anterior, lateral, and posterior branches. The anterior and lateral branches give rise to a

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Figure 5

INTEGRATION OF SENSORY INFORMATION region of arborisation around DIT and LDT near the roots of N1 (solid arrowheads in Fig. 7A,Bi), and the posterior branch contributes to an ipsilateral posterior region of branching in posterior neuropil dorsal to DIT (solid double arrowheads in Fig. 7A,Biii). A slender branch runs posteriorly in LDT as far as the posterior edge of the metathoracic neuromere. There are no branches in ventral neuropil on either side of the ganglion. On the contralateral side, the main neurite in DCIII continues laterally as far as LDT, first giving off a prominent posterior branch and then three thinner anterior branches, before turning dorsally and back on itself in a loop around LDT (open arrows in Fig. 7A,Bii). It then thickens and runs posteriorly at the ventral edge of MDT (open double arrowhead in Fig. 7Biii). The anterior branches on the side contralateral to the soma all contribute to a dense arborisation around DIT, extending anteriorly as far as the edge of the neuropil near the roots of nerve 1 (open arrowheads in Fig. 7A,Bi). The posterior branch splits into two processes, one of which recrosses the midline in DCIV to contribute to the posterior ipsilateral field of branches, whereas the other gives rise to a contralateral posterior arborisation in and around DMT (asterisk in Fig. 7Bii). The physiology of only one Type E local interneuron was analysed. It was depolarised by electrical stimulation of mesothoracic (7.4 msec; Fig. 7Ci) or metathoracic (3.95 msec; Fig. 7Cii) nerve 1 contralateral to the soma. The depolarisation elicited by stimulation of metathoracic NI was up to sixfold greater in amplitude than that elicited by mesothoracic N1 stimulation. It did not respond to touching any of the legs.

Metathoracic ascending intersegmental interneurons Three types of ascending intersegmental interneurons with somata in the metathoracic ganglion were analysed in detail. The three types (A–C) have quite different morphologies and are clearly from different populations. Type A intersegmental interneurons. Ascending interneurons of Type A have a ventral soma near the midline and a main neurite that runs posteriorly and dorsally to give rise to widespread branching in dorsal and ventral

Fig. 5. Morphology and physiology of a metathoracic midline spiking interneuron Type C. A: Ventral branches drawn from a whole mount. Arrowhead, anterior lateral branches; double arrowhead, anterior ventral branches; asterisks, posterior processes near the edge of the neuropil. B: Dorsal field of branches. The ventral soma is drawn as a dashed outline for reference. Ci–iii: Transverse sections made at the levels indicated in A. Solid arrowhead, anterior lateral branches; double arrowheads, anterior ventral branches; solid arrow, main neurite; asterisk, posterior lateral process near roots of N5; open arrow, medial branch climbing to the level of VIT; open arrowheads, main dorsal branches running anteriorly and posteriorly. Di–iii: Electrical stimulation of mesothoracic (i) or metathoracic (ii) nerve 1 elicited a short-latency depolarisation. Stimulating the metathoracic subcosta nerve in the wing (see Fig. 1) elicited a burst of sensory activity recorded in N1 and a depolarisation in the interneuron (iii). Averages of 50 sweeps triggered from the stimulus artifact. E: Touching the metathoracic wing elicited bursts of sensory activity recorded in N1 (lower trace) and large-amplitude depolarisations and spikes (upper trace). Touching the metathoracic femur also generated bursts of spikes. Scale bars ⫽ 100 ␮m in A–C, 1 mV, 20 msec in D, 2 mV, 1 second in E.

103 neuropils of the ipsilateral hemiganglion (Fig. 8A,B). An axon ascends as least as far as the mesothoracic ganglion in the ipsilateral connective, but the pattern of branching here is not known. After leaving the soma (soma in Fig. 8Cii), the main neurite ascends dorsally and posteriorly at the lateral edge of TT, giving off several fine processes into aVAC and posterior vmVAC below the level of VMT (solid arrow in Fig. 8Cii). One of the fine processes turns forwards (solid arrow in Fig. 8B) to branch very anteriorly in aVAC (arrow in Fig. 8Ci). At the level of VIT, the main neurite turns laterally and splits to give rise to two thick main processes, posterior and anterior, and a more slender ventral process (p, a, and v in Fig. 8B,Cii,iii). The posterior process runs posterolaterally to end in a region of branching at the level of VLT near the posterior edge of the metathoracic neuropil (arrowhead in Fig. 8A), giving off several side branches on the way. The first and most prominent of these runs laterally above VIT (arrowhead in Fig. 8Ciii) to give rise to branches at the level of DIT, before turning dorsally to give rise to further branches near LDT. A second side branch runs posteriodorsally medial to DIT (double arrowhead in Fig. 8Ciii) to give rise to a region of branching just lateral to MDT. A medial branch runs posteriorly (double arrowhead in Fig. 8A) near VIT, giving off fine branches into the surrounding neuropil. The ventral process from the main neurite (v in Fig. 8B,Ciii) runs ventrolaterally between VMT and MVT 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 the lateral and ventral edges of the neuropil (open arrows in Fig. 8A,Cii). These contribute to extensive dorsal and ventral branching near the margins of the neuropil (arrows in Fig. 8Ciii). These branches intermingle with the roots of nerves 2–5. Another fine branch runs anteriorly (asterisk in Fig. 8B) to contribute to an anterior field of branching between MVT and VMT (asterisk in Fig. 8Ci). The axon runs dorsally and medially to curve around DMT before turning laterally and entering LDT/MDT (axon in Fig. 8Cii). The path followed by the axon, as seen in whole mount, thus forms a characteristic medial, dorsal, and then lateral loop, before bifurcating, with one branch turning abruptly anterior in LDT/MDT to leave the ganglion (via MDT) in the ipsilateral connective (axon in Fig. 8A), the other branch turning posteriorly (arrowhead in Fig. 8B) also in MDT to give rise to very dorsal branching in and above DIT. The ascending axon gives rise to several short, fine branches in MDT before it enters the connective. Type A intersegmental interneurons were depolarised by electrical stimulation of either the mesothoracic (2/3) or the metathoracic (4/4) nerve 1 with latencies of 5.8 –9.1 msec or 4.5–7.6 msec, respectively. Similarly, electrical stimulation of the ipsilateral meso- or metathoracic wing nerve 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). Touching the head or any of the wings or legs caused a barrage of depolarising synaptic inputs and sometimes spikes (1/1). Extending the tibia of the ipsilateral hind leg caused a

Figure 6

INTEGRATION OF SENSORY INFORMATION depolarisation and spikes in the one neuron that was tested in this way (not shown). Type B intersegmental interneurons. Ascending intersegmental interneurons of type B have a lateral soma, bilateral fields of branching, and contralaterally ascending axons that branch in the mesothoracic ganglion before continuing further anterior. Recordings were made from their processes near the base of the ipsilateral connective. These interneurons probably belong to a population of approximately 35 cells, some of which were described by Laurent and Burrows (1988), although features of their anatomy differ, and inputs from the wings have not previously been detected. The soma lies near the root of N1 (Fig. 9A), and from it a thick main neurite (arrow in Fig. 9Di) runs medially to cross the midline in DCIII, before splitting to give rise to a contralaterally ascending axon in DIT (solid arrowheads in Fig. 9A,Di) and two prominent dorsal branches that run posteriorly; one in DIT (solid double arrowhead in Fig. 9Dii) as far as abdominal neuromere 2 (double arrowhead in Fig. 9A), the other in DMT (asterisks in Fig. 9A,Dii). Both posterior neurites give off short branches, most of which project dorsally to DIT and DMT, respectively. The ascending axon also gives off several small branches around and particularly dorsally to DIT before it enters the connective (Fig. 9Di). The similar neurons described by Laurent and Burrows (1988) have a contralateral axon in MDT, not DIT. Ipsilaterally the main neurite gives rise to two posterior ventral branches (pv1, pv2 in Fig. 9B), a prominent anterior ventral branch (av in Fig. 9B), and three dorsal branches (d1, d2, d3 in Fig. 9A). The anterior ventral branch gives rise to a dense ventral region of branching in aVAC (open arrowheads in Fig. 9B,Di, inset). Some of these fine ventral branches cross the midline by up to 100 ␮m. The similar neurons described by Laurent and Burrows (1988) do not have branches in aVAC. The first posterior ventral branch (pv1 in Fig. 9B) contributes to the most posterior branches in aVAC and is the main source of branches 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 medial region of branching in lVAC. The lateral dorsal branch from the main neurite (d1) gives rise to a very anterior dorsal region of branches that intermingles with the roots of nerve 1 above and below DIT (Fig. 9A). The medial dorsal branch, d2, gives rise to branches in and around DMT anterior to the main neurite

Fig. 6. Morphology and physiology of a metathoracic midline spiking interneuron Type D. A: Ventral branches drawn from a whole mount. Arrowhead, anterior branch. B: Dorsal field of branches. The ventral soma is drawn as a dashed outline for reference. Ci–iii: Transverse sections made at the levels indicated in A. Solid arrowhead, anterior branch plunging ventrally; solid arrow, main neurite; open arrow, main dorsal neurite; open arrowhead, dorsal lateral branches. Di: Electrical stimulation of the metathoracic subcosta nerve elicited a barrage of sensory activity recorded in N1 (lower trace) and a long-lasting depolarisation (upper trace). Average of 50 sweeps triggered from the stimulus artifact. Dii,iii: Touching the metathoracic 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 in Dii,iii.

105 (Fig. 9A,Di). The lateral posterior branch, d3, gives rise to a small region of branches in DIT (Fig. 9A), and the medial posterior branch, d4, gives rise to branches in DMT posterior to the main neurite (Fig. 9A,Dii). The axon enters the mesothoracic ganglion in DIT and gives rise to several short branches that are mostly medial and dorsal to DIT and a prominent lateral branch (arrow in Fig. 9C) that gives rise to sparse branching in dorsal aLAC. The axon passes through the mesothoracic ganglion in DIT and exits in the anterior connective. It is not know whether the similar neurons described by Laurent and Burrows (1988) have this morphology. Type B interneurons were depolarised by stimulation of the ipsilateral mesothoracic wing nerve with a latency of 6.1– 6.9 msec (2/2; Fig. 10A). Stimulation of the ipsilateral metathoracic wing nerve also caused a depolarisation (2/ 2), with a latency of 3.4 – 4.3 msec (Fig. 10B). In one of the interneurons, this input sometimes elicited a spike (not shown). Touching either ipsilateral wing elicited largeamplitude depolarisations and bursts of spikes (Fig. 10C,D). Touching the tarsus or distal tibia of the ipsilateral metathoracic leg also elicited depolarisations and spikes (Fig. 10E). Touching elsewhere on the metathoracic leg, extending and flexing the tibia, or touching the other legs elicited little or no response. Possible inputs from the contralateral wings were not tested. Type C ascending interneurons. Type C ascending interneurons may be serial homologues of a type of neuron described from the mesothoracic ganglion by Watson and Burrows (1983) and Pearson et al. (1985), and they share some similarity with a population of flight interneurons described for the metathoracic and abdominal neuromeres by Robertson et al. (1982). The characteristic feature of all these neurons is a prominent loop in the main neurite. Type C neurons have a ventral midline soma and a main neurite that rises dorsally in TT, before bifurcating at the level of DCIII to send a process to the contralateral side of the ganglion (Fig. 11A,Cii,D). The main neurite on the ipsilateral side (the side of main inputs from the wings) curves outwards in TT around DMT, giving off a prominent anterior branch (a in Fig. 11B,D) and two posterior branches into DMT (p1, p2 in Fig. 11B,D), before continuing without further branching laterally in TT as far as LDT (Fig. 11Cii). It then turns dorsally around LDT, giving off a dorsal branch (solid arrows in Fig. 11A,B,Cii), before turning medially to enter DMT again. It thus forms an unusual and distinctive lateral loop that is prominent in both whole-mount and sectioned preparations. In DMT the main neurite splits into two branches, an anterior axon 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 abdominal neuromere. This gives off fine branches that cross the midline by up to 110 ␮m in the metathoracic neuromere and 40 ␮m in abdominal neuromeres 1 and 2 (solid arrowheads in Fig. 11A). The pattern of branching in the mesothoracic ganglion is unknown. The anterior branch from the main neurite (a in Fig. 11B,D) runs anteriorly in DMT and turns laterally into the roots of N1 to contribute to the dense anterior ventral and lateral regions of branching in aVAC and N1 roots (open arrows in Fig. 11A,Ci). The first two posterior branches of the main neurite (p1, p2 in Fig. 11B) run posteriorly in DMT, giving rise to dense branching in and near this tract (Fig. 11Cii). At the posterior edge of the

Figure 7

INTEGRATION OF SENSORY INFORMATION metathoracic neuromere, a branch turns downward, medial to VIT, and curves laterally to enter MVT (double arrowhead in Fig. 11A), where it gives rise to a fine neurite that projects up and down MVT, giving off only a few short side branches within the tract (open arrowheads in Fig. 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 to most of a sparse, almost uniform, network of branches that spans much of the dorsal lateral neuropil (Fig. 11B). Some of the more medial branches arise from the first posterior branch of the main neurite (p1). All of the dorsal branches are restricted to the most dorsal parts of the lateral association centres (aLAC, pLAC) or to more medial regions above DIT (double arrowhead in Fig. 11Cii). On the contralateral side, the main neurite bifurcates into anterior and posterior branches in DMT that give rise to regions of branching that largely mirror those described for the ipsilateral side. There are many fine branches in the roots of N1 (but not aVAC; Fig. 11A,Cii) and in DMT. The overall branching is less dense, the regular pattern of dorsal branches is absent, and the fine ventral branch in MVT is also missing. Two other intersegmental interneurons with inputs from the wings and a characteristic looping main neurite were each recorded once. Despite the remarkable similarity of the paths followed by the main neurites in all these cells (e.g., Fig. 11D), the remaining branching was substantially different (not shown). One neuron branched unilaterally in the metathoracic neuromere, whereas the other had more extensive ventral branches and fewer dorsal branches than illustrated in Figure 11A, and the posterior contralateral projection in DMT was absent. Together with the Type C interneurons, these seem to be members of a small but diverse group characterised by the convoluted path followed by their main neurite. Type C ascending intersegmental interneurons were depolarised with a latency of 16 msec following stimulation of the ipsilateral metathoracic wing nerve (2/2; Fig. 12A). Stimulation of the ipsilateral mesothoracic wing nerve had little or no effect (2/2; not shown), but stimulation of the ipsilateral mesothoracic or metathoracic N1 elicited clear depolarisations (2/2; Fig. 12B,C). Touching the lateral thorax near the metathoracic wing base elicited large-amplitude depolarisations and spikes (1/1; Fig. 12D). Touching the contralateral wings also depolarised

Fig. 7. Morphology and physiology of a metathoracic local interneuron Type E. A: Entire arborisation drawn from a whole mount. Solid arrowhead, anteriormost branch near roots of N1; double arrowheads, posterior dorsal branches; arrow, main neurite turning dorsally to loop around LDT; open arrowhead, branches in and around roots of N1; asterisk, posterior branches near DMT. Bi–iii: Transverse sections made at the levels indicated in A. Solid arrowheads and open arrowhead, ipsilateral and contralateral branches near roots of both 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 in and around DMT; double arrowhead, dorsal branches above DIT. Ci,ii: Electrical stimulation of mesothoracic N1 (i) elicited a weak and erratic but long-lasting depolarisation, whereas electrical stimulation of metathoracic N1 (ii) elicited a consistently larger amplitude depolarisation. 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 msec in Cii.

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

DISCUSSION I have described five types of local interneurons and three types of intersegmental interneurons that receive sensory inputs from receptors in the wings. Some of these interneurons are members of known populations, but my analyses reveal that their patterns of sensory inputs are more widespread than has been recorded in the past. These neurons may be involved in pathways that generate scratching movements of a hind leg directed toward tactile stimuli on a wing. To examine the patterns of synaptic inputs to the interneurons, electrical stimuli were applied to a wing nerve or to nerve 1, which innervates receptors of several types in the wing and at the wing base. In freely moving locusts, electrical stimulation of a wing nerve elicits targeted scratching movements and, at high stimulus amplitudes and frequencies, other behaviours, such as struggling or walking (Matheson, 1998). Stimulation of nerve 1 also elicits scratching, walking, or struggling (personal observation) The electrical stimuli used here did not permit an analysis of the size of the receptive fields on the wing, but in many cases direct mechanical stimulation confirmed an interneuron’s response to tactile stimuli that normally elicit scratching.

Common features of all the neurons The observation that all of the neurons responded to stimuli of both mesothoracic and metathoracic N1 suggests that they are important points of convergence of sensory information from different body segments and appendages. The universal presence of anterior branches in and around the roots of N1 or in the aVAC suggests that this region is important for the processing of wing tactile information.

Metathoracic local interneurons Local interneurons, named here Types A–C, are midline spiking interneurons as defined by Burrows and Siegler (1982, 1984) and Siegler and Burrows (1983, 1984). These interneurons have somata near the ventral midline of the metathoracic ganglion and branches that are separated into distinct dorsal and ventral fields by a process in the perpendicular tract (PT). The input and output properties of many of these cells have been described previously in the context of local reflex movements of a hind leg elicited by mechanical stimuli applied to the same leg (for review see Burrows, 1989). These local interneurons receive monosynaptic inputs from sensory neurons innervating arrays of tactile hairs on a leg or innervating leg proprioceptors (not both). The patterns and strengths of these connections differ between interneurons so that the population as a whole can encode a somatotopic representation of the surface of the leg. The assumption is that the specific patterns of outputs of the interneurons mean that their activity, driven by sensory inputs, elicits appropriate movements of the leg to withdraw it from the stimulus (for review see Newland and Burrows, 1997). The dorsal and ventral fields of branches of Type D local interneurons both arise from processes given off after the main neurite has climbed the P tract, so these neurons do not fit exactly the descriptions given by Burrows and

Figure 8

INTEGRATION OF SENSORY INFORMATION Siegler. Nevertheless, the overall patterns of branching resemble closely those of other midline spiking interneurons, so I conclude that they too are members of this population. The observation that all local interneurons responded to stimulation of both mesothoracic and metathoracic N1 provides the first reported evidence for convergence of sensory information from two body appendages (wings and legs) or two body segments (mesothoracic and metathoracic) onto individual members of this population. Local interneurons of a different metathoracic population have receptive fields on both a hind leg and the ipsilateral middle leg (Nagayama, 1990). The inputs from the middle leg exteroceptors must be indirect, because none projects into the metathoracic ganglion. The regions of neuropil containing the ventral input branches of local spiking interneurons are strongly correlated with the patterns of inputs that the neurons receive from tactile hairs on a leg (for review see Newland and Burrows, 1997). I show that local interneurons that are depolarised by stimulation of a wing nerve or N1 have anterior branches in aVAC or around the roots of N1. Some of the interneurons that respond to wing stimulation also respond to touching or moving a leg (see Table 1), and this is accompanied by branches in the ventral and lateral association centres to which leg hair afferents project, as well as branches in aVAC or around the roots of N1. This pattern has not previously been illustrated. The local interneurons, named here Types A–D, therefore appear to represent previously undescribed subtypes of the midline spiking group. In the context of scratching behaviour, they have the important property of collating exteroceptive inputs from the wings (that could signal the location of a touch on the wing and hence a target site) and proprioceptive inputs from leg joints that could signal leg posture (see Table 1). The convergence of these two key types of information is essential in the computation of a limb trajectory from a start posture to a particular target site, and it is this aspect that will be the subject of further detailed analyses. Local interneurons of Type E are not, to my knowledge, the same as any previously reported neuron. Their bilateral symmetry and posterior branches give them a superficial resemblance to some stridulatory interneurons in grasshoppers (Ocker and Hedwig, 1996). Many interneurons in the flight system of a locust also have bilateral branches, but most are intersegmental (see, e.g., Pearson

Fig. 8. Morphology and physiology of a metathoracic ascending intersegmental interneuron Type A. A: Entire arborisation drawn from a whole mount. Arrowhead, posterior lateral branch; double arrowhead, posterior medial branch; arrow, lateral branches. B: Illustration of the same neuron showing just the main branches. Arrow, anterior ventral branch; a, p, v, anterior, posterior and ventral main branches described in the text; arrowhead, posterior branch in MDT; asterisk, anterior branch near MVT. Ci–iii: Transverse sections made at the levels indicated in A. Solid arrows; anterior ventral branches; asterisk, anterior branch near MVT; open arrows, lateral neurite giving 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 either a mesothoracic wing nerve (i) or the metathoracic wing subcostal nerve (ii) elicited consistent depolarisations. Averages of 50 sweeps triggered from the stimulus artifact. Scale bars ⫽ 100 ␮m in A–C, 1 mV, 20 msec in D.

109 and Robertson, 1987) and none resembles the local neurons reported here. The main neurite follows a path similar to that of the Type C intersegmental interneurons, in that it loops laterally around the lateral dorsal tract (LDT), but there are few other common morphological features. The looped part of the neurite gives off several secondary branches, whereas that of the intersegmental neurons is relatively bare; and the posterior route of the main neurite is in MDT, whereas that of the intersegmental neurons is in DMT. Nevertheless, the similarity of the unusual looped main neurite is suggestive of common developmental or functional constraints. Another similarity between Type E local and Type C intersegmental interneurons is that they both respond to N1 stimuli but not to touching or moving the metathoracic legs.

Intersegmental interneurons Intersegmental interneurons characterised here include one type with its metathoracic branches restricted to one half of the ganglion (Type A) and two types (B and C) with bilateral patterns of branching. The unilateral branching of Type A neurons distinguishes them from most flight and stridulatory interneurons, which are almost all bilateral (see Burrows, 1996). Like the local interneurons, Type A intersegmental interneurons have prominent regions of branching in the aVAC, which is where they are likely to receive their inputs from wing sensory neurons. Branches in the anterior lateral (aLAC) and lateral ventral (lVAC) association centres are presumably the sites of inputs from leg receptors that give rise to the responses recorded in response to touching or moving the ipsilateral hind leg. The convergence of information from a hind leg and both ipsilateral wings makes these interneurons promising candidates for a role in scratching behaviour. Intersegmental interneurons of Type B appear to belong to a population of approximately 35 cells described by Laurent and Burrows (1988) on the basis of their mechanosensory inputs from a hind leg. The population contains ascending interneurons that receive direct synaptic inputs either from sensory neurons innervating leg tactile hairs or from sensory neurons of a particular leg joint proprioceptor, the femorotibial chordotonal organ. Those that receive proprioceptive leg inputs have either an ipsilateral axon in the MDT or a contralateral axon in the VIT, whereas those that receive exteroceptive leg inputs have an ipsilateral axon in the VMT (Laurent and Burrows, 1988). The Type B interneurons described in the present paper are excited by touching the tibia or tarsus of the ipsilateral hind leg but not by movements of the femorotibial leg joint. The prediction would be that they have an ipsilateral axon in VMT, but this is not the case: They have a contralateral axon in DIT and, overall, appear most similar in whole mount to Laurent and Burrows’ proprioceptive neurons with a contralateral axon. The Type B interneurons are depolarised following stimulation of either the mesothoracic or the metathoracic wing nerve (in the wings) or by gently touching the wing with a soft brush, which indicates that they also receive inputs from wing exteroceptors (the wings do not contain joint proprioceptors, and the stimuli used do not elicit movements of the legs that could lead to secondary feedback). These inputs are powerful enough to elicit spikes in the interneurons. Type B interneurons have dense regions of branching in the aVAC, which correlates well with what little is known

Fig. 9. Morphology of a metathoracic ascending intersegmental interneuron Type B. A: Dorsal branches drawn from a whole mount. Arrowhead, ascending axon in DIT; double arrowhead, posterior branch in DIT; asterisk, medial branch in DMT; d1– d4, dorsal branches referred to in the text. B: Ventral branches of the same interneuron. The soma and primary neurite are shown as dashed lines for reference. Arrowhead, dense branching in aVAC; double arrowhead, posterior branches in lVAC, av, pv1, pv2, ventral branches 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 that the plane of sectioning is slightly oblique, so the sections are not exactly symmetrical). Arrow; main neurite from soma; solid arrowhead, ascending axon in DIT; open arrowhead, dense branching in aVAC (also see inset; photomicrograph of the region indicated by a dashed rectangle in Di); solid double arrowhead, posterior branch in DIT; open double arrowhead, posterior branches in lVAC; asterisk, posterior medial branch in DMT. Scale bars ⫽ 100 ␮m.

INTEGRATION OF SENSORY INFORMATION

Fig. 10. Physiology of metathoracic ascending interneurons Type B. Electrical stimulation of either a mesothoracic wing nerve (A) or the metathoracic wing subcostal nerve (B) elicited barrages of sensory activity in the corresponding N1 (lower traces) and consistent depolarisations (upper traces). Averages of 50 sweeps triggered from the stimulus artifact. Touching either wing (C,D) or the ipsilateral metathoracic femur (E) elicited depolarisations and bursts of spikes. Repeated depolarisations in C correspond to repeated proximal-distal movements 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.

111 of the projections of wing sensory neurons (personal observations). Further branching in the lVAC matches well with the projections of leg exteroceptors (Newland, 1991). Burrows and Newland (1993) have shown that the responses of intersegmental interneurons from this population to stimulation of leg hairs can be explained by the somatotopically organised overlap of the sensory and interneuronal branches. The present study indicates that this somatotopic mapping extends beyond the leg to include the ipsilateral hind wing and possibly also the ipsilateral mesothoracic wing. I predict that these or similar interneurons also receive inputs from exteroceptors on the thorax itself, so that together they encode a representation of the entire surface of that body segment. The ascending axon of Type B interneurons gives rise to relatively few branches in the mesothoracic ganglion. Significantly, there is no region of fine, dense branching in mesothoracic ventral neuropil that would be expected if these neurons receive direct exteroceptive inputs in this segment. The conclusion must be, therefore, that the excitatory inputs following mesothoracic wing stimulation are mediated either by indirect pathways or by direct connections from mesothoracic wing sensory axons that descend to the metathoracic ganglion. Laurent and Burrows (1988) hypothesise that the ascending interneurons they describe could provide the anterior legs with information about movements and the locations of tactile stimuli on a hind leg, which could be used in the coordination of walking. The output effects are unknown, however, and depolarising the neurons so that they spiked did not lead to movements of the mesothoracic legs. One interpretation given was that stronger parallel excitation of several interneurons might be required. Another explanation suggested by my data is that this population (at least the Type B neurons) may be involved in other behaviours, such as scratching the wings. It would therefore be valuable to search for output effects in the metathoracic segment. The new data for the first time reveal the pattern of branching in the mesothoracic ganglion and show that the axons extend farther anteriorly, at least as far as the prothoracic ganglion. Metathoracic ascending interneurons of Type C are readily recognised by a prominent lateral loop in their main neurite, an ascending axon in the ipsilateral connective, and their bilateral branching. The looped neurite is similar to that of a set of mostly unilateral flight interneurons described in the metathoracic ganglion by Robertson et al. (1982), but they are clearly not the same cells. Metathoracic Type C interneurons are similar to some of a population of bilaterally branching interneurons with a looping neurite that have been reported in the mesothoracic ganglion (Watson and Burrows, 1983; Pearson et al., 1985). Pearson et al. show that there must be two to five similar neurons (named by them Type 404) per mesothoracic hemiganglion (four to ten per ganglion), and this is supported by my observation in the metathoracic ganglion that there are at least three distinct neurons per side with the lateral loop. The Type C interneurons described in detail here represent just one of these morphologies. Several lines of physiological evidence suggest that mesothoracic Type 404 interneurons are involved in the initiation of flight, although they do not themselves make direct output connections with flight motor neurons (Pearson et al., 1985). Possible outputs onto metathoracic leg motor neurons have not been examined. The metathoracic

Fig. 11. Morphology of a metathoracic ascending intersegmental interneuron Type C. A: Ventral and midventral/dorsal branches drawn from a whole mount. Arrow, main neurite looping dorsally around LDT; arrowheads, fine posterior branches crossing the midline in each neuromere; double arrowhead, posterior branch plunging down into MVT. B: Dorsal branches of the same interneuron. The soma and primary neurite are shown as dashed lines for reference [the main anterior ventral (a) and three main posterior ventral branches (p1–p3) are labeled here for clarity]. Arrow, main neurite

looping dorsally around LDT; Ci,ii: Transverse sections made at the levels indicated in A. Open arrows; anteriormost branching in aVAC and amongst the roots of N1; solid arrow, main neurite looping dorsally around LDT; arrowhead, posterior branch in MVT; double arrowheads, very dorsal branches in aLAC and above LDT. D: Photomicrograph of the region indicated by the dashed rectangle in B illustrating the distinctive lateral loop and branching of the main neurite. The anterior ventral branch (a) and two of the posterior ventral branches (p1, p2) are labeled. Scale bars ⫽ 100 ␮m.

INTEGRATION OF SENSORY INFORMATION

113 Type C interneurons are depolarised by stimuli of the metathoracic wing nerve (in the wing) and more powerfully by stimuli of metathoracic N1. These observations fit well with the descriptions given for the mesothoracic Type 404 neurons, which respond to touching the mesothoracic wing and to movements of the mesothoracic wing base (Watson and Burrows, 1983). The metathoracic neurons are also depolarised by stimulation of mesothoracic N1, indicating that they collate information from both pairs of wings. They also respond to tactile stimuli of the lateral thorax but not to stimuli of the legs.

Implications for understanding targeted limb movements

Fig. 12. Physiology of metathoracic ascending intersegmental interneurons Type C. A: Electrical stimulation of the subcostal nerve in the ipsilateral metathoracic wing elicited a barrage of sensory activity in N1 (lower trace) and a small depolarisation that was followed by a hyperpolarisation in the interneuron (upper trace). Stimulation of mesothoracic N1 (B) elicited a brief depolarisation in the interneuron, whereas stimulation of metathoracic N1 (C) elicited a large-amplitude and long-lasting input with a superimposed spike (arrowhead). The spike amplitude is small and is almost swamped by the synaptic input as seen at the recording site in the soma. D: Touching the lateral thorax just ventral to the base of the wing elicited a burst of spikes in N1 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.

Matheson (1998) used behavioural methods to analyse at a gross level the neuronal pathways underlying targeted scratching of the wings by the metathoracic legs. Matheson’s study indicated that sensory information from a mesothoracic wing must descend to the metathoracic motor networks that drive hind leg movements and that sensory information must cross the midline to reach the motor network of the contralateral hind leg. Furthermore, it suggested that there must be convergence of exteroceptive wing sensory information with leg proprioceptive information to permit computation of leg trajectories towards the target from different start positions. The present paper takes Matheson’s (1998) analyses a step further, by identifying neurons that could fill some of these roles. First, there is convergence through shortlatency pathways of sensory information from a wing and a leg of one body segment onto local spiking interneurons. This emphasises the need to examine the roles of these neurons in behaviours other than local leg avoidance reflexes, which have provided the key understanding of their properties so far. Second, sensory information from both the mesothoracic and the metathoracic wings converges through short-latency pathways onto the same metathoracic local neurons. Such integration is an important feature that provides a mechanism by which metathoracic leg motor networks could generate movements targeted toward equivalent sites on the two wings, which overlap in their resting posture. Third, bilaterally branching local and intersegmental neurons are shown to receive shortlatency inputs from the wings. They could carry this information across the midline. Some of these neurons have previously been shown to influence a flight motor pattern, but previous studies have tended to assume that any interneuron with sensory inputs from a wing must be involved in flight (see, e.g., Pearson et al., 1985). This perspective might have prevented other possible roles, such as in scratching, from being fully explored. Fourth, some of the interneurons collate through short-latency pathways sensory information from exteroceptors on the wings and from joint proprioceptors of a metathoracic leg. These interneurons are likely to be key elements of a pathway that generates targeted scratching movements of a hind leg and will be the focus of further detailed analyses.

ACKNOWLEDGMENTS The author thanks M. Burrows, M. Gebhardt, P.L. Newland, and S. Rogers for comments on a draft of the manuscript. The work was supported by a BBSRC Advanced

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Research Fellowship and a Royal Society (London) Research 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-filled neurones in wholemount preparations. Brain Res 138:359 –363. Berkowitz A, Laurent GJ. 1996a. Central generation of grooming motor patterns and interlimb coordination in locusts. J Neurosci 16:8079 – 8091. Berkowitz A, Laurent GJ. 1996b. Local control of leg movement and motor patterns during grooming in locusts. J Neurosci 16:8067– 8078. Brogan RT, Pitman RM. 1981. Axonal regeneration in an identified insect motoneurone. J Physiol (London) 319:34P–35P. Brunn DE, Dean J. 1994. Intersegmental and local interneurones in the metathorax of the stick insect Carausius morosus that monitor middle leg position. J Neurophysiol 72:1208 –1219. Burrows M. 1989. Processing of mechanosensory signals in local reflex pathways of the locust. J Exp Biol 146:209 –227. Burrows M. 1996. The neurobiology of an insect brain. New York: Oxford University Press. Burrows M, Newland PL. 1993. Correlation between the receptive fields of locust interneurons, their dendritic morphology, and the central projections of mechanosensory neurons. J Comp Neurol 329:412– 426. Burrows M, Siegler MVS. 1982. Spiking local interneurones mediate local reflexes. Science 217:650 – 652. Burrows M, Siegler MVS. 1984. The morphological diversity and receptive fields of spiking local interneurones in the locust with inputs from hair afferents. J Comp Neurol 224:483–508. Cruse H, Dean J, Kindermann T, Schmitz J, Schumm M. 1998. Simulation of complex movements using artificial neural networks. Z Naturforsch 53:628 – 638. Du¨ rr V, Matheson T. 2001. Functional somatatopy of targeted limb movements in an insect. Proc 28th Go¨ ttingen Neurobiol Conf 2001. Elsner N and Krentzberg GW, editors. Stuttgart. Thieme Verlag. p 319. Laurent G, Burrows M. 1988. A population of ascending intersegmental interneurones in the locust with mechanosensory inputs from a hind leg. J Comp Neurol 275:1–12. Matheson T. 1997. Hindleg targeting during scratching in the locust. J Exp Biol 200:93–100.

Matheson T. 1998. Contralateral coordination and retargeting of limb movements during scratching in the locust. J Exp Biol 201:2021–2032. Nagayama T. 1990. The organization of receptive fields of an anteromedial group of spiking local interneurones in the locust with exteroceptive inputs from the legs. J Comp Physiol A166:471– 476. Newland PL. 1991. Morphology and somatotopic organisation of the central projections of afferents from tactile hairs on the hind leg of the locust. J Comp Neurol 312:493–508. Newland PL, Burrows M. 1997. Neuronal networks controlling leg movements of the locust. J Insect Physiol 43:107–123. Ocker WG, Hedwig B. 1996. Interneurones involved in stridulatory pattern generation in the grasshopper Chorthippus mollis (Charp). J Exp Biol 199:653– 662. Pearson KG, Robertson RM. 1987. Structure predicts synaptic function of two classes of interneurons in the thoracic ganglia of Locusta migratoria. Cell Tissue Res 250:105–114. Pearson KG, Reye DN, Parsons DW, Bicker G. 1985. Flight-initiating interneurones in the locust. J Neurophysiol 53:910 –925. Pflu¨ ger H-J, Bra¨ unig P, Hustert R. 1988. The organization of mechanosensory neuropiles in locust thoracic ganglia. Phil Trans R Soc London [Biol] 321:1–26. Robertson RM, Pearson KG, Reichert H. 1982. Flight interneurons in the locust and the origin of insect wings. Science 217:177–179. Rosenbaum DA, Muelenbroek RGJ, Vaughan J. 1996. Three approaches to the degrees of freedom problem in reaching. In: Wing AM, Haggard P, Flanagan JR, editors. Hand and brain. The neurophysiology and psychology of hand movements. San Diego: Academic Press. p 169 –185. Siegler MVS, Burrows M. 1983. Spiking local interneurons as primary integrators of mechanosensory information in the locust. J Neurophysiol 50:1281–1295. Siegler MVS, Burrows M. 1984. The morphology of two groups of spiking local interneurones in the metathoracic ganglion of the locust. J Comp Neurol 224:463– 482. Soechting JF, Tong DC, Flanders M. 1996. Frames of reference in sensorimotor integration: position sense of the arm and hand. In: Wing AM, Haggard P, Flanagan JR, editors. Hand and brain. The neurophysiology and psychology of hand movements. San Diego: Academic Press. p 151–168. Tyrer NM, Gregory GE. 1982. A guide to the neuroanatomy of locust suboesophageal and thoracic ganglia. Phil Trans R Soc London [Biol] 297:91–123. Watson AHD, Burrows M. 1983. The morphology, ultrastructure and distribution of synapses on an intersegmental interneurone of the locust. J Comp Neurol 214:154 –169.