INTERNEURONES INVOLVED IN STRIDULATORY PATTERN GENERATION IN THE GRASSHOPPER CHORTHIPPUS MOLLIS (CHARP.)

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1 The Journal of Experimenal Biology 199, (1996) Prined in Grea Briain The ompany of Biologiss Limied 1996 JEB ITEREUROES IVOLVE I STRIULATORY PATTER GEERATIO I THE GRASSHOPPER HORTHIPPUS MOLLIS (HARP.) W.-G. OKER A B. HEWIG* I. Zoologisches Insiu, Berliner Sraße 28, 3773 Göingen, Germany Acceped 3 Ocober 1995 In ehered grasshoppers, horhippus mollis, sridulaory leg movemens were elicied by d.c. brain simulaion. Sridulaory chirps comprise boh slow upand-down movemens and rapid oscillaions of he hindlegs. Inracellular recording, simulaion and saining of inerneurones wihin he meahoracic ganglion complex were performed simulaneously wih recordings of leg movemen. Five inerneurones were idenified in he meahoracic ganglion complex. The branching paerns of hese inerneurones were ypical of sridulaory inerneurones. Three of hese neurones had a srucure very similar o sridulaory inerneurones already characerized in he species Omocesus viridulus. uring sridulaion, he spike aciviy of all Summary inerneurones was phasically coupled o he chirp rhyhm; wo inerneurones addiionally exhibied coupling o he rapid leg oscillaions. Inracellular simulaion of inerneurones A1-A-2 and A1-AI-1 prolonged he duraion of he rapid leg oscillaions and influenced he generaion of he chirp rhyhm. s T3-LI-2 and T3-L-4 decreased he ampliude of he slow up-and-down movemen. The daa indicae ha a leas par of he meahoracic sridulaory nework of. mollis is organized in a srucurally and funcionally similar way o ha of O. viridulus. Key words: grasshopper, horhippus mollis, sridulaion, inerneurone, paern generaion. Inroducion Acousic communicaion sysems are presen in several groups of insecs (cicadas, bushcrickes, crickes, grasshoppers) (Elsner and Popov, 1978; Michelsen and Larsen, 1985). The differen species use species-specific sound paerns o recognize and localize heir maes. In gomphocerine grasshoppers, he sridulaory apparaus consiss of a cuicular vein on he forewings and a row of cuicular pegs along he proximal surface of he femora of he hindlegs. Sound is produced by rhyhmically rubbing he legs agains he vein of he wings (Elsner and Popov, 1978). Alhough he sridulaory apparaus in differen species is very similar, species-specific sound paerns are produced. This is due o species-specific moor paerns which move he hindlegs and acivae he sound-producing apparaus (Elsner, 1974a, 1975). The underlying sridulaory neuronal nework should hus show species-specific modificaions for he generaion of he paricular moor paern. Acridid grasshoppers, herefore, represen a axon in which he naural variey of sridulaory neworks may offer he chance o analyse species-specific adapaions a he neuronal level. In gomphocerine grasshoppers, he neuronal nework generaing he sridulaory moor paern is locaed in he meahoracic ganglion complex. The srucure, physiology and funcional significance of meahoracic sridulaory inerneurones have previously been analysed only in Omocesus viridulus (Gramoll and Elsner, 1987; Gramoll, 1988; Hedwig, 1992a,b). These auhors idenified sridulaory inerneurones ha drive he horacic nework, change he coordinaion of he hindlegs and modulae he shape of he sridulaory movemens. However, nohing is known abou he presence and funcion of hese inerneurones in oher species. We herefore carried ou he presen sudy for comparaive purposes. In he grasshopper O. viridulus, he sridulaory movemens during he calling and courship songs consis of raher simple and uniform up-and-down movemens. In horhippus mollis, however, sridulaory movemens are more complicaed. uring a chirp, a slow up-and-down movemen of he hindleg is followed by a period of rapid hindleg oscillaions (Elsner, 1974a). The differen sridulaory movemen paerns of O. viridulus and. mollis raised he quesions of wheher corresponding sridulaory inerneurones o hose in O. viridulus can be idenified in. mollis and/or wheher here are specific adapaions of he sridulaory neuronal nework. To *Auhor for correspondence.

2 654 W.-G. OKER A B. HEWIG answer his quesion, inracellular recording, saining and simulaion of meahoracic inerneurones were performed in sridulaing. mollis. Maerials and mehods Animals Male grasshoppers horhippus mollis (harp.) (Acrididae, Gomphocerinae) were colleced from bioopes in low mounain ranges in Franken, Germany, and were kep in he laboraory. A oal of 32 animals were used in experimens. Preparaion A preparaion was used which allowed inracellular recording, simulaion and saining of meahoracic inerneurones during sridulaion by he grasshoppers. Sridulaion was elicied by simulaing he brain wih direc currens slowly increasing o a maximum of 15 3 A. The curren was applied via a sucion elecrode aached o he proocerebrum. Simulaneously, he aciviy of meahoracic neurones and he sridulaory leg movemens were recorded. For inracellular simulaion of he neurones, depolarising curren pulses of 7 1 ms duraion were applied hrough he microelecrode and any simulaneous changes in he leg movemens were analysed. eails of he preparaion, experimenal procedures and daa analysis are given in Hedwig (1992a). eurones were named using he sysem described by Hedwig (1986a); for example, T3-LI-3 refers o he hird (3) idenified local inerneurone wih an ipsilaeral axon (LI) wihin he meahoracic ganglion (T3). The erms ipsilaeral and conralaeral are used wih respec o he soma posiion of he neurones. Resuls Sridulaory leg movemens of horhippus mollis uring a sridulaory sequence of he grasshopper. mollis, he chirps are generaed by wo differen movemen paerns of he hindlegs (paern I and paern II), which are performed simulaneously (Fig. 1). Paern I is characerized by a slow upand-down movemen followed by a phase of rapid leg oscillaions. A single slow up-and-down movemen lass for approximaely ms, whereas he rapid oscillaions are of 2 ms duraion and are performed for abou 3 ms. While one hindleg makes a clear slow up-and-down movemen (paern I) he conralaeral hindleg simulaneously produces only a slow upward movemen (paern II), which is hen followed by rapid oscillaions. There is a fixed assignmen of he movemen paerns o he hindlegs during a sridulaory sequence bu he assignmen may change occasionally beween sequences. eails of he sridulaory leg movemens of. mollis are given in Elsner (1974a). uring sridulaion elicied by brain simulaion, he differences beween he wo movemen paerns may no be clearly expressed, and boh hindlegs may perform movemens similar o paern I (see Figs 3 6). HL HL Paern I Paern II hirp cycle Rapid oscillaion cycle 2 ms Fig. 1. Sridulaory hindleg movemens of horhippus mollis: secion of a courship sequence. The sridulaory movemens during one chirp consis of a slow up-and-down movemen followed by a sequence of rapid leg oscillaions. Each hindleg (HL) performs a differen movemen paern (see ex). For calculaion of phase diagrams, he upper reversal poin of he slow up-and-down movemen was used as a reference, since i provided he bes riggering possibiliies. Srucure and aciviy of sridulaory inerneurone T3-LI-3 Inracellular recordings were obained from inerneurones in he meahoracic ganglion complex from locaions corresponding o he arborizaion paern of he sridulaory inerneurones in O. viridulus. Sridulaion could be elicied in abou 8 % of. mollis prepared for inracellular recordings. T3-LI-3 (LI: local ipsilaeral axon) has an anerior venral soma (Fig. 2A). A hin branch wih a varicose axonal appearance projecs very laerally in a poserior direcion and gives off small and shor collaerals. Profuse arborizaions wih a dendriic appearance occupy an area close o he dorsal surface of he ganglion and run parallel o he midline as far poserior as he second abdominal neuromere. aa on his inerneurone are based on seven recordings. uring sridulaion, he membrane poenial of inerneurone T3-LI-3 oscillaed in ime wih he chirp rhyhm (Fig. 2B). The inerneurone was depolarized above spike hreshold a he end of he rapid leg oscillaions and was maximally excied during he slow upward movemen. The corresponding discharge raes were abou 1 acion poenials per second (APs s 1 ) and 18 APs s 1, respecively. uring he downward movemen of he ipsilaeral leg, he depolarizaion rapidly decreased as he inerneurone received a barrage of IPSPs (arrows Fig. 2B). Phase diagrams of he chirp cycle and he rapid oscillaion cycle showed ha he discharge rae of he inerneurone was clearly coupled o he end of he chirp cycle and was in phase wih he slow upward movemen (Fig. 2, lef). There was no spike aciviy phasically coupled o he rapid oscillaion cycle (Fig. 2, righ). Simulaing he inerneurone wih injeced curren had no effec on sridulaory leg movemens. In resing. mollis, however, inracellular depolarizaion wih 3 na for 1 ms led o a discharge of 5 APs s 1 in he inerneurone and elicied a slow upward movemen of he ipsilaeral hindleg (Fig. 2). This demonsraes ha he inerneurone has access o he moor nework involved in he conrol of ipsilaeral hindleg movemens. Srucure and aciviy of sridulaory inerneurone T3-L-4 T3-L-4 (L: local conralaeral axon) was

3 Grasshopper sridulaory inerneurones 655 A T3-LI-3 Fig. 2. (A) Srucure of inerneurone T3-LI-3 of horhippus mollis in he meahoracic ganglion complex. oe he dense arborizaion paern of he neurone orienaed parallel o he midline of he ganglion. (B) Aciviy paern of he inerneurone during sridulaion (upper race). The lower races show he sridulaory paerns of he hindlegs. The arrows indicae a period of decreased polarizaion due o IPSPs. () iagrams giving he average insananeous spike frequency in he chirp cycle (lef) and during he rapid leg oscillaion cycle (righ). () epolarizaion of inerneurone T3-LI-3 in a resing grasshopper caused slow upward movemens of he ipsilaeral hindleg., neuron;, ipsilaeral hindleg movemen;, conralaeral hindleg movemen; Sim, simulus;, chirp cycle;, ime beween beginning of chirp cycle and he occurrence of an acion poenial,, phase; AP, acion poenial. B Frequency (APs s 1 ) =/ =/ 1 1 3nA Sim recorded and sained 12 imes; i has almos symmerical bilaeral arborizaions wihin he meahoracic ganglion complex (Fig. 3A). From he soma, which has a venral medial posiion, he primary neurie projecs dorsally and hen crosses he midline. The ipsilaeral arborizaions of he neurone have a smooh appearance characerisic of dendries. Mos ipsilaeral branches projec ino he laeral dorsal neuropile, bu some fine processes run parallel o he midline as far poserior 1 mv 1 mv as he abdominal neuromeres. In he conralaeral hemiganglion, he arborizaions exhibi a corresponding branching paern, bu have a varicose appearance which may indicae an axonal funcion (Hedwig, 1992a). The membrane poenial of he inerneurone oscillaed in ime wih he chirp rhyhm of he sridulaory movemens (Fig. 3B). uring he slow upward movemen of he leg, T3- L-4 was depolarized and hereafer discharged spikes a a rae of abou 8 9 APs s 1 during boh he downward movemen and he following rapid leg oscillaions. Spike aciviy sopped before he end of he rapid leg oscillaions and he membrane poenial of he inerneurone was a a minimum during he ransiion from he rapid leg oscillaions o he slow upward movemen. The phase diagrams for he chirp cycle and he rapid oscillaion cycle show ha he spike aciviy of he inerneurone was in phase wih he firs half of he chirp cycle (Fig. 3, lef) and ha here was also a coupling beween is spike aciviy and he rapid leg oscillaions (Fig. 3, righ). The discharge rae of T3-L-4 reached 55 APs s 1 in phase wih he upper reversal poin of he rapid oscillaions and only 11 APs s 1 a he lower reversal poin of he movemen. Thus, he spike aciviy was in phase wih boh he chirp cycle and he rapid leg oscillaions. In he grasshopper O. viridulus, inerneurone T3-L-4 is involved in he coordinaion of he sridulaory leg movemens of boh hindlegs (Hedwig, 1992a; Ocker, 1994). In. mollis, he effec of depolarizing he inerneurone was esed wih curren pulses of 1 ms duraion and 3 na ampliude. The simuli were applied during sridulaion while he ipsilaeral hindleg was producing paern II and he conralaeral leg was producing paern I. Simulaion of he inerneurone caused an increase in he ampliude of he ipsilaeral slow up-and-down movemens and simulaneously decreased he ampliude of he conralaeral slow up-and-down movemens (Fig. 3). These changes in movemen ampliude correspond o a funcional change in he sridulaory movemen paern, alhough i was no complee. Movemens of he ipsilaeral leg represen a ransiion from he low-ampliude paern II o he highampliude paern I, while he conralaeral changes represen a ransiion from paern I o paern II. Since his paricular animal sridulaed wih very small ampliude rapid leg oscillaions, we could no observe any influence on his secion of he chirp.

4 656 W.-G. OKER A B. HEWIG A T3-L-4 Fig. 3. (A) Srucure of inerneurone T3-L-4 in he meahoracic ganglion complex. Branches ipsilaeral o he soma had a smooh dendriic appearance, whereas conralaeral branches were more varicose. (B) Aciviy of he inerneurone (upper race) and movemens of he hindlegs (lower race) during sridulaion. () Phase diagrams giving he average frequency of spikes in he phase of chirps (lef) and during he cycles of he rapid leg oscillaions (righ). () epolarizaion of inerneurone T3-L-4 during sridulaion increased he ampliude of ipsilaeral slow up-and-down movemens and decreased he ampliude of conralaeral movemens. oed lines indicae he ampliude of he movemens before simulaion. The bridge circui was ou of balance during depolarizaion. Abbreviaions as in Fig. 2. B Frequency (APs s 1 ) =/ 1 1 3nA Sim Srucure and aciviy of sridulaory inerneurone T3-LI-2 Local inerneurone T3-LI-2 is resriced o one side of he meahoracic neuromere (Fig. 4A). The neurone has a very laeral soma posiion and he neurie projecs in a sligh curve o he poserior. The branch wih a dendriic appearance and he branch wih an axonal appearance hen separae. The main dendriic-looking branch projecs direcly poseriorly and gives off numerous side branches. The axonal branch, however, runs =/ 1 mv 1 mv owards he midline before urning back o ener he laeral neuropile of he ganglion. Here, he varicose arborizaions occupy a more dorsal posiion han he smooh dendriic branches. A oal of 17 recordings were obained from inerneurone T3-LI-2. The srucure of his inerneurone in. mollis closely resembles a corresponding inerneurone in O. viridulus (neurone ype 2; Gramoll and Elsner, 1987). uring sridulaion, spike aciviy of T3-LI-2 was coupled o he chirp cycle and o he rhyhm of he rapid leg oscillaions (Fig. 4B). epolarizaion of he inerneurone sared jus before he upper reversal poin of he slow up-and-down movemen and elicied a burs of spikes. epolarisaion and spike aciviy coninued unil he end of he rapid leg oscillaions. uring each rapid oscillaion of he hindleg, he inerneurone discharged wo acion poenials. Afer he rapid leg oscillaions, he depolarizaion decreased. The inerneurone did no spike during he slow upward movemen. This aciviy was refleced in he phase diagrams. The peak of aciviy, wih a discharge rae of abou 22 APs s 1, occurred a phase 1/, i.e. a he upper reversal poin of he slow movemen. uring he rapid oscillaions, he mean discharge rae fell from abou 2 APs s 1 o abou 1 APs s 1 (Fig. 4, lef). There was no aciviy during he slow upward movemen. The spike aciviy was also modulaed in he rhyhm of he rapid leg oscillaions. The discharge rae reached abou 12 APs s 1 direcly afer he upper reversal poin of each rapid oscillaion bu was only abou 75 APs s 1 a all oher phases (Fig. 4, righ). In O. viridulus, simulaion of inerneurone T3-LI-2 during sridulaion damps he movemen ampliude of he ipsilaeral hindleg (Hedwig, 1992b; Ocker, 1994). To es he funcional significance of his inerneurone in. mollis, he inerneurone was depolarized wih curren pulses of 9 ms duraion and 3 na ampliude (Fig. 4). This caused a disinc increase in spike aciviy. The inerneurone now discharged abou 6 9 APs (insead of wo) during every rapid leg oscillaion and some spikes now occurred during he slow upward movemen. As a consequence of he increased aciviy, he slow up-and-down movemen of he ipsilaeral hindleg was ransienly damped o abou 35 % of he normal ampliude. Afer he end of he simulus, he movemen ampliude reached is full magniude again. Alhough he inerneurone aciviy was coupled o he rapid leg oscillaions, we did no observe any change in he ampliude of hese movemens. There was no change in he

5 Grasshopper sridulaory inerneurones 657 A T3-LI-2 Fig. 4. (A) Srucure of inerneurone T3-LI-2 in he meahoracic neuromere. oe he hairpin-like course of he axon, which arborizes dorsal o dendriic branches. (B) Aciviy of he inerneurone (upper race) and movemens of he hindlegs (lower race) during sridulaion. Some spikes are clipped as a resul of he recording procedure. () Phase diagrams giving he average frequency of spikes in he phase of chirps (lef) and during he cycles of he rapid leg oscillaions (righ). () epolarizaion of inerneurone T3-LI-2 during sridulaion decreased he ipsilaeral slow up-and-down movemens bu had no effec on he conralaeral movemens. The ampliude of he movemen before simulaion is indicaed by he doed line. Abbreviaions as in Fig. 2. B Frequency (APs s 1 ) =/ =/ 1 1 Sim 3nA 1 Sim 3nA conralaeral movemens during depolarisaion of he inerneurone. Srucure and aciviy of sridulaory inerneurone A1-A-2 A1-A-2 (A: ascending conralaeral axon) has branches on boh sides of he meahoracic ganglion 1 mv 1 mv complex ha have srucural feaures ypical of sridulaory inerneurones (Fig. 5A). The soma occupies a laeral poserior posiion in he firs abdominal neuromere. Arborizaions ipsilaeral o he soma exhibi predominanly smooh dendriic srucures, whereas conralaeral branches are varicose. The dendriic and axonal arborizaions mainly occupy a medial region, parallel o he midline, in an area in which oher sridulaory inerneurones also have heir projecions. In he abdominal neuromeres, he laeral side branches form a ladder-like arborizaion paern. The axon ascends o anerior ganglia conralaerally wih collaerals projecing o he conralaeral medial neuropile. aa are from one recording. The membrane poenial of A1-A-2 exhibied oscillaions in ime wih he rhyhm of he sridulaory cycle (Fig. 5B). epolarizaion was sronges a he upper reversal poin of he slow up-and-down movemen, when he discharge rae of he neurone reached abou 12 APs s 1. The level of depolarisaion and spike aciviy hen decreased and he spikes sopped during he middle of he rapid leg oscillaions. epolarizaion reached a minimum a he ransiion from he rapid leg oscillaions o he slow upward movemen. There was no coupling of he spikes o he cycle of he rapid leg oscillaions. orrespondingly, he phase diagrams show maximal spike aciviy a phase 1/ and during he firs par of he chirp cycle (Fig. 5, lef). There was no coupling of he neurone aciviy in he rhyhm of he rapid leg oscillaions since he phase diagram shows no maximum (Fig. 5, righ). The effec of he inerneurone on he sridulaory movemens was esed during ongoing sridulaion (Fig. 5). The inerneurone was consanly hyperpolarized by 1.5 na and hen depolarized for 6 ms wih 2 na. uring hyperpolarizaion, here was lile spike aciviy during sridulaion. epolarizaion elicied a burs of spikes wih a discharge rae of abou 13 APs s 1. This significanly prolonged he ongoing rapid leg oscillaions of he sridulaory movemen paern. The chirp lengh was abou 424 ms before he simulus and increased o 1136 ms during depolarizaion. Such changes never occur during normal sridulaion. The ipsilaeral hindleg coninued performing rapid leg oscillaions afer he end of he depolarisaion, whereas he conralaeral leg immediaely began a slow up-and-down movemen. As a consequence, boh hindlegs moved ou of phase for one cycle. The subsequen ipsilaeral chirp cycle was disincly shorer

6 658 W.-G. OKER A B. HEWIG A A1-A-2 Fig. 5. (A) Srucure of inerneurone A1-A-2 in he meahoracic ganglion complex. oe he ladder-like dendriic arborizaions in he abdominal neuromeres and he dendriic and axonal arborizaions parallel o he midline of he ganglion. (B) Aciviy of he inerneurone (upper race) and movemens of he hindlegs (lower race) during sridulaion. () Phase diagrams giving he average frequency of spikes in he phase of chirps (lef) and during he cycles of he rapid leg oscillaions (righ). () A pulse of depolarisaion (2 na) applied o inerneurone A1-A-2 during sridulaion prolonged he duraion of rapid leg oscillaions and increased he chirp duraion. The neurone was consanly hyperpolarised by 1.5 na before and afer he pulse. Arrows mark he beginning of he chirp cycle prediced from he iniial rhyhm. Abbreviaions as in Fig. 2. B Frequency (APs s 1 ) Sim =/ nA 2nA na 2nA han he conralaeral cycle, bringing he legs ino phase. Thereafer, boh legs moved in phase again and normal chirp cycles were produced. These chirps were no in phase wih he chirps before he period of depolarisaion (arrows in Fig. 5 indicae he iniial chirp rhyhm). epolarisaion of his inerneurone herefore rese he chirp rhyhm. =/ 1 mv 1 mv Srucure and aciviy of he sridulaory inerneurone A1-AI-1 A1-AI-1 (AI: ascending ipsilaeral axon) has a median venral soma posiion and a complex srucure wihin he meahoracic ganglion (Fig. 6A). Ipsilaerally, he neurone exhibis a ladder-like arborizaion paern in he abdominal neuromeres. In he meahoracic neuromere, he main arborizaions projec laerally on he ipsilaeral side, alhough some projec more medially on he conralaeral side. The main neurie runs aneriorly and gives off numerous small smooh branches which form a profuse arborizaion parallel o he midline. Before he axon eners he ipsilaeral connecive, an axonal collaeral crosses he midline and urns back owards he abdominal neuromeres, giving off laeral varicose branches along is lengh. The daa are based on wo recordings. Recordings of he inerneurone were obained from he anerior axonal branches. A his recording sie, membrane poenial oscillaions caused by synapic aciviy are no apparen. However, inerneurone A1-AI-1 exhibied burss of spikes in phase wih he slow upward leg movemen. The aciviy of he inerneurone reached abou 22 APs s 1 and ceased before he upper reversal poin of he movemen. There was lile or no spike aciviy during he rapid leg oscillaions. The phase diagrams herefore show a peak in he discharge rae close o he end of he chirp cycle (Fig. 6, lef). There was only weak aciviy during he rapid oscillaion which was no phasically coupled o he rapid oscillaion cycle (Fig. 6, righ). uring ongoing sridulaion, he inerneurone was held hyperpolarized by injecion of.5 na and hen depolarized for 9 ms by 2 na (Fig. 6). This elicied a discharge rae of abou 125 APs s 1 in he inerneurone. The rapid leg oscillaions were now performed for much longer and, as a consequence, he ongoing chirp was prolonged from 49 o 78 ms. Spike aciviy of he inerneurone was inerruped before he end of he depolarisaion a he same ime as a slow upward movemen erminaed he rapid oscillaions. The firs chirp cycle following he depolarising pulse was shorened o 34 ms, bu subsequen chirps had a normal duraion. These chirps were, however, no in phase wih he chirp rhyhm before simulaion (arrows in Fig. 6 indicae he iniial chirp rhyhm). Simulaion of inerneurone A1-AI-1 herefore rese he iming of he chirp rhyhm.

7 Grasshopper sridulaory inerneurones 659 A B A1-AI-1 Frequency (APs s 1 ) Sim =/ 1 mv 2 ms 1 1 5nA 2nA.5 na 2nA Alhough he aciviy of A1-AI-1 was coupled o he slow up-and-down movemens, depolarisaion of he inerneurone suppressed his par of he movemen and prolonged he sequence of rapid leg oscillaions. iscussion The aim of he presen experimens was o analyse sridulaory inerneurones in he grasshopper. mollis and compare hem wih hose idenified in he grasshopper O. viridulus (Gramoll and Elsner, 1987; Gramoll, 1988; Hedwig, 1992a,b). Since. =/ 1 mv Fig. 6. (A) Srucure of inerneurone A1-AI-1 in he meahoracic ganglion complex. oe he ladder-like dendriic arborizaions in he abdominal neuromeres. (B) Aciviy of he inerneurone (upper race) and movemens of he hindlegs (lower race) during sridulaion. () Phase diagrams giving he frequency of spikes in he phase of chirps (lef) and during he cycles of he rapid leg oscillaions (righ). () epolarizaion of inerneurone A1-AI-1 during sridulaion prolonged he occurrence of rapid leg oscillaions, increased he chirp duraion and ransienly changed he chirp rhyhm. The inerneurone was consanly hyperpolarised by.5 na before and afer he depolarisaion. Arrows mark he beginning of he chirp cycle prediced from he iniial rhyhm. Abbreviaions as in Fig. 2. mollis performs compleely differen movemen paerns (Elsner, 1974a), i was our aim o gain insigh ino he organizaion of he sridulaory neuronal nework in. mollis. Wihin his conex, sridulaory inerneurones are defined as inerneurones which are phasically acive during he sridulaory moor paern. However, his does no exclude acivaion of he neurones during he generaion of oher moor paerns. Srucure of sridulaory inerneurones in horhippus mollis Four of he inerneurones idenified in. mollis (Figs 2, 3, 5, 6) arborize in a leas hree neuromeres of he meahoracic ganglion complex and reach from he meahoracic o he abdominal neuromere. All of hese inerneurones show, a leas o some degree, medial arborizaions ha run parallel o he midline of he ganglion. This is mos clearly expressed in inerneurone T3-LI-3. Alhough hese branches have eiher a smooh dendriic or varicose axonal appearance, hey may no, however, be inerpreed as sric inpu or oupu regions of he neurones (Wason and Burrows, 1985). Furhermore, he neurones exhibi ladder-like arborizaion paerns in he abdominal neuromeres. These general feaures of he arborizaion paern closely correspond o he organizaion of sridulaory inerneurones in O. viridulus, where he inerneurones also arborize in he meahoracic and abdominal neuromeres; hey exhibi ladder-like arborizaion paerns in he abdominal neuromeres and have a dense arborizaion paern parallel o he ganglionic midline (Fig. 7; Gramoll and Elsner, 1987; Hedwig, 1992a,b). Apar from he similar spaial arrangemen of he sridulaory nework wihin he meahoracic ganglion complex in boh species, here are also cerain similariies in a leas hree inerneurones. The arborizaion paerns of inerneurones T3-LI-3, T3-L-4 and T3-LI-2 very closely resemble hose of he corresponding inerneurones in O. viridulus (Fig. 7, see also Figs 5, 4, 7, respecively, in Gramoll and Elsner, 1987; Figs 2, 9 in Hedwig, 1992a; Fig. 4 in Hedwig, 1992b). Therefore, a leas hree inerneurones in he sridulaory neworks seem o be homologous in boh species, alhough direc evidence for homology can only be esablished by a developmenal analysis of cell lines. orresponding similariies have been repored for descending inerneurones of he subeosophageal ganglion and brain ha modulae or acivae he sridulaory paern (Lins and Lakes-Harlan, 1994; Hedwig, 1994; B. Hedwig, unpublished daa). The general

8 66 W.-G. OKER A B. HEWIG horhippus mollis Omocesus viridulus T3-LI-3 T3-L-4 T3-LI-2 Fig. 7. Srucure of he sridulaory inerneurones T3- LI-3, T3-L-4 and T3-LI-2 which exhibi similar branching paerns in he wo grasshopper species. mollis (lef) and O. viridulus (righ). srucural feaures of sridulaory inerneurones herefore seem o be preserved in he species-specific sridulaory neworks. learly, we have neiher idenified all he meahoracic sridulaory inerneurones nor undersand he funcional significance of minor changes in he arborizaion paerns or possible differences in membrane properies. We do no know a wha levels of he neuronal nework he species-specific characerisics ha lead o he observed species-specific moor paerns are expressed. Such adapaions may bes be idenified by analysing hose inerneurones ha are elemens of he cenral rhyhm-generaing sysem. However, in O. viridulus, we have no ye idenified elemens corresponding o inerneurones A1- AI-1 and A1-A-2, which in. mollis can modulae he rapid leg oscillaions and he generaion of he chirp rhyhm. Phase relaionships and funcional significance of inerneurone aciviy The sridulaory movemens of. mollis comprise a slow up-and-down movemen which is followed by a sequence of rapid leg oscillaions. Sridulaion always sars wih slow upand-down movemens, whereas he rapid oscillaions are gradually insered ino he inervals beween he slow up-and-

9 Grasshopper sridulaory inerneurones 661 down movemens. I herefore seems ha wo differen moorpaern-generaing mechanisms are involved in producing he sridulaory movemens. The phase relaionships beween he spike aciviy and leg movemens should make i possible o deermine o which nework each neurone probably belongs. As a consequence, his informaion may sugges which pars of he sridulaory movemen paern in differen species are homologous. The spike paerns of inerneurones T3-LI-3, A1-A-2 and A1-AI-1 (Figs 2, 5, 6) were only coupled o he slow up-anddown movemen. These inerneurones had differen phases of maximum aciviy wihin he chirp cycle, bu none of hem exhibied any coupling of heir spike aciviy wih he cycle of he rapid leg oscillaions. These neurones, herefore, are no likely o be involved in he generaion of he rapid leg oscillaions. The funcional significance of he inerneurones for he generaion of he sridulaory movemens was esed by eliciing an enhanced discharge rae during ongoing sridulaion. Ineresingly, hose neurones whose aciviy was coupled o he slow up-and-down movemen (A1-A-2 and A1-AI-1, Figs 5A, 6A) prolonged he sequence of rapid leg oscillaions when heir firing rae was arificially increased, bu did no influence he sridulaory movemen paern o which hey were phasically coupled. This may be explained, a leas parly, if neurones A1-A-2 and A1-AI-1 have an inhibiory effec on he slow up-and-down movemen and hereby suppor he occurrence of he rapid leg oscillaion. The effecs of inerneurone simulaion, however, indicae ha he slow upand-down movemen and he rapid leg oscillaions can be modulaed independenly and may hus be conrolled by differen neworks. T3-LI-3 in. mollis is coupled only o he chirp rhyhm. In O. viridulus, he corresponding inerneurone is involved in he iniiaion of he horacic sridulaory moor paern and is aciviy is srongly coupled o he sridulaory leg movemen. In O. viridulus, he movemens are raher simple, consising only of up-and-down movemens wih a cycle lengh of approximaely 8 ms. A comparison of he aciviy of inerneurone T3-LI-3 in he sridulaory neworks of boh species suggess ha he chirps of. mollis and he upand-down movemens of O. viridulus are corresponding acions. Wihin his conex, i would be ineresing o deermine which inerneurones are involved in he generaion of he rapid leg oscillaions in. mollis. As he phase diagrams indicae, inerneurones T3-LI-2 (Fig. 4) and T3-L-4 (Fig. 3) were coupled boh o he chirp rhyhm and o he rhyhm of he rapid leg oscillaions. These inerneurones may herefore be involved in he generaion of boh movemen paerns. I mus be considered, however, ha any coupling o he rapid leg oscillaions will also be expressed in he phase diagram as a coupling o he chirp rhyhm. We may herefore overesimae he funcional relaionship of hese neurones o he generaion of he chirp rhyhm. Inracellular depolarizaion of inerneurone T3-LI-2 (Fig. 4) decreased he ampliude of he ipsilaeral slow up-and-down movemens, whereas depolarizaion of T3-L-4 (Fig. 3) alered he coordinaion of he hindlegs. orresponding effecs can be demonsraed in he grasshopper O. viridulus (Hedwig, 1992a,b; Ocker, 1994). epolarisaion of inerneurone T3-LI- 2 decreased he ampliude of he ipsilaeral leg movemen. epolarisaion of inerneurone T3-L-4 changed he coordinaion of he sridulaory movemen paern of he hindlegs. The srucure of his inerneurone may be regarded as a prerequisie for his funcion, since he connecion of ipsilaeral dendriic arborizaions wih he conralaeral axonal branches suggess a direced flow of informaion beween boh sides of he ganglion. As in. mollis, neiher of hese inerneurones influenced he chirp rhyhm. Therefore, besides he similar srucural feaures in boh species, hese wo inerneurones also have a funcional similariy in heir effec on he sridulaory paern. We assume ha a leas pars of he sridulaory neworks in boh species are organized similarly in boh srucural and funcional respecs. Fuure prospecs The evoluion of species-specific sound paerns wihin he gomphocerine grasshoppers could heoreically come abou hrough changes in he sound-producing apparaus and/or he moor paern used for sound producion. Indeed a few species, such as Senobohrus rubicundus, have developed mixed wing sridulaion and leg sridulaion (Elsner, 1974b; Elsner and Wasser, 1995a,b). All species ha use leg sridulaion use a similar peripheral sound-producing mechanism ha shows only minor species-specific adapaion. everheless, even in closely relaed species, he moor paerns underlying sound producion may be compleely differen (Elsner, 1975), and hese differences in he iming of moor aciviy give rise o he species-specific sound paerns. Moor paerns underlying sound producion are herefore an imporan elemen in he evoluion of species-specific acousic signals. Since we now know some of he cenral sridulaory inerneurones, i will be ineresing o deermine he morphology and role of hese neurones in oher sridulaing and nonsridulaing grasshoppers o obain an insigh ino he evoluion of hese neuronal neworks (umon and Roberson, 1986). We are mos graeful o. Elsner, T. Maheson and P. ewland for criical commens on he manuscrip. W.-G.O. was an associae member of he Göinger Graduieren Kolleg Organisaion und ynamik euronaler ezwerke. Suppored by eusche Forschungsgemeinschaf He 218/1. References UMOT, J. P.. A ROBERTSO, R. M. (1986). euronal circuis: an evoluionary perspecive. Science 233, ELSER,. (1974a). euroehology of sound producion in gomphocerine grasshoppers (Orhopera: Acrididae). I. Song paerns and sridulaory movemens. J. comp. Physiol. 88, ELSER,. (1974b). eural economy: Bifuncional muscles and

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