effects of pharmacological treatment and photoinactivation on the directional responses of an insect...

Effects of Pharmacological Treatment andPhotoinactivation on the Directional Responsesof an Insect Neuron

JORGE MOLINAy AND ANDREAS STUMPNER�

Johann-Friedrich-Blumenbach-Institut fur Zoologie und Anthropologie Abt.Neurobiologie, D-37073 Gottingen, Germany

ABSTRACT Soma-ipsilateral branches of the large segmental omega neuron of the phaner-opterid bush cricket Ancistrura nigrovittata have smooth endings, which extend through most ofthe auditory neuropile. Correspondingly, it shows a broad frequency tuning. Large excitatorypostsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) are observed whenrecording from soma-ipsilateral branches. Stimulation from the soma-ipsilateral side leads to astrong excitation. Soma-contralateral branches have a strong, beaded appearance. IPSPs, whichseem to be of soma-contralateral origin, can be recorded from these branches. Stimulation from thesoma-contralateral side leads to a strong inhibition of the omega neuron. Soma-contralateralstimulation must be 30–40 dB more intense than soma-ipsilateral stimulation to evoke similar spikenumbers in the omega neuron. The side-to-side difference is reduced to 10–15 dB after cutting theinput from the soma-contralateral leg (tympanic nerve). The thresholds for eliciting IPSPs by soma-contralateral stimulation correspond roughly to excitatory thresholds of the mirror-image omegawith the same stimuli. Pharmacological treatment with picrotoxin (PTX) or photoinactivation of theLucifer Yellow filled mirror-image omega neuron reduces contralateral inhibition considerably andeliminates all visible IPSPs. Nevertheless, an additional contralateral inhibition survives bothprocedures and is only eliminated after cutting the soma-contralateral tympanic nerve. These resultsdemonstrate that the mirror-image partners of the omega neuron mutually inhibit each other inbush crickets—as in crickets. This mutual inhibition is PTX-sensitive. At least one additionalelement exerts contralateral PTX-insensitive inhibition on the omega neuron. J. Exp. Zool.303A:1085–1103, 2005. r 2005 Wiley-Liss, Inc.

Neurobiological experiments with insects—when compared to vertebrates—provide the ad-vantage of working with individually identifiedcells. This allows for evaluation of intraspecificvariation and extended interspecific comparison.Since the size of the nerve cells largely determinesthe probability of successful recordings, data fromlarge nerve cells are more frequently published.One such prominent nerve cell is the auditoryinterneuron ON1 (omega neuron) of ensiferans.Since its first description as the ‘‘large segmentalneuron 1’’ in crickets by Popov et al. (’78), theON1 has been in the focus of cricket and later alsoof bush cricket research (Romer, ’85). The major-ity of data on the omega neuron concentrated ondirectional responses or took its frequency thresh-old curve as representative for overall hearing.Due to the obvious morphological similarity and tothe clear directional dependence in both groups,the omega neuron of certain bush crickets and theON1 of crickets were considered to be homologous

(Zhantiev and Korsunovskaya, ’83). However, incrickets a second omega-shaped neuron is found(ON2; Wohlers and Huber, ’82; Stiedl et al., ’97).Such a neuron has not been described for any bushcricket except for a weak hint in the haglidCyphoderris monstrosa (Mason and Schildberger,’93), which might however be more closelyrelated to crickets than bush crickets. Differencesbetween crickets and bush crickets might be due

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jez.a.228.

Received 9 March 2005; Accepted 10 August 2005

Grant sponsor: Deutsche Forschungsgemeinschaft Stu 189/1.

Abbreviations used: 5-HT serotonine dB SPL decibel soundpressure level EPSP excitatory postsynaptic potential GABA g-aminobutyric acid IPSP inhibitory postsynaptic potential ON1, ON2omega neurons 1 and 2 PTX picrotoxin.yCurrent address: Faculty of Life Sciences, University of Vienna,

Althanstr.14, A-1090 Vienna, Austria.�Correspondence to: Prof. Dr. Andreas Stumpner, Johann-

Friedrich-Blumenbach- Institut fur Zoologie und Anthropologie Abt.Neurobiologie, Berliner Str. 28, D-37073 Gottingen, Germany.E-mail: [email protected]

r 2005 WILEY-LISS, INC.

JOURNAL OF EXPERIMENTAL ZOOLOGY 303A:1085–1103 (2005)

to independent evolution of audition in bothgroups as discussed by Gwynne (’95) and Desut-ter-Grandcolas (2003).

Most data on the properties of the ON1 neuronin crickets are derived from Gryllus bimaculatusand Teleogryllus oceanicus and have been largelyfocused on ON1’s directional response throughmutual inhibition (Selverston et al., ’85; Wiese andEilts, ’85; Horseman and Huber, ’94). The trans-mitter of ON1 is still under debate: serotonin-likeimmunoreactivity has been described (5-HT-LIR;Hardt and Agricola, ’91; Horner et al., ’95), whilepharmacological experiments showed effects ofhistamine and blockers (Skiebe et al., ’90) and ofoctopamine (Luhr, ’97) on directional processing.Histamine-like immunoreactivity, however, hasnot been found (Hirtz, ’96). In direct experimentalmanipulation during or prior to behavioral tests,ON1 has been demonstrated to influence direc-tional behavior (Schildberger and Horner, ’88),although phonotaxis in Acheta domesticus wasvirtually unchanged when both ON1s were func-tionally eliminated (Atkins et al., ’84).

For the homologous neuron in bush crickets, lessdata are available. Data for Tettigonia viridissimaindicate a close correspondence to the cricket ON1neuron (Marquart, ’85a,b; Romer, ’85; Romeret al., ’88; Schul, ’97; Romer and Krusch, 2000).Here, we present data from a different group ofEnsifera (Phaneropteridae). There is some evi-dence that Phaneropteridae and Tettigoniidaediffer in their set of auditory neurons (e.g., bythe existence of more ‘‘ascending neurons’’ inPhaneropteridae, Stumpner, ’99a,b, 2001). In thecurrent work, we focus on the directional re-sponses of the omega neuron during pharmacolo-gical and photoinactivation experiments.

First, we tested the effect of picrotoxin (PTX), awell-known blocker of invertebrate g-aminobuty-ric acid (GABA)-gated chloride channels typicallyreferred to as GABAA receptors (Robbins and vander Kloot, ’58; Maynard and Walton, ’75). PTX hasalso been demonstrated to effectively reducespecific inhibition in locusts (Romer and Seikows-ki, ’85), crickets (Harrison et al., ’88; Faulkes andPollack, 2001) and bush crickets (Stumpner, ’98,2002). In an ascending neuron of Ancistruranigrovittata, PTX eliminated frequency-specificinhibition, while at least some directional inhibi-tion remained (Stumpner, ’98). This result in-dicates that the omega neuron inhibits ascendingneurons and omega’s effect on these neurons isnot sensitive to PTX as suggested by data fromHarrison et al. (’88) and Faulkes and Pollack

(2001). Response properties of an ascendingneuron (AN2) in T. oceanicus, which receivesstrong low-frequency inhibition, changed follow-ing PTX application (Harrison et al., ’88; Faulkesand Pollack, 2001). Response properties of ON1,the main candidate for directional inhibition incrickets, were apparently unaffected (Faulkes andPollack, 2001, who, however, did not specificallytest contralateral stimuli). Accordingly, we ex-pected the contralateral inhibition of the omeganeuron in the bush cricket A. nigrovittata to beunaffected by PTX.

Second, this paper presents data gained inphotoinactivation experiments. At first, one omeganeuron was filled with the fluorescent dye LuciferYellow. Then the response changes of the mirror-image omega neuron were monitored, while theomega filled with Lucifer Yellow was inactivatedby laser illumination (Miller and Selverston, ’79).The goal of these experiments was to test whetherthere is a mutual inhibition of the two omegas inbush crickets as has been described for crickets(Selverston et al., ’85). This was followed bycutting the contralateral leg and thereby thetympanic nerve providing the excitatory input tothe omega neuron that has been photoinactivatedto see if any contralateral inhibition remained.Cutting the whole leg is permissible, since incontrast to crickets the left and right auditorytracheae of bush crickets are not coupled. In linewith this, for a species closely related toA. nigrovittata, namely Poecilimon laevissimus,similar in body size and size of the trachealsystem, it has been demonstrated that at higherfrequencies (above 10–15 kHz) the excitation ofthe tympanic membrane is governed by the soundcoming through the ipsilateral auditory trachea(Michelsen et al., ’94; see also Stumpner andHeller ’92). Both pharmacological and photoinac-tivation experiments reveal considerable differ-ences from the data published for crickets anddemonstrate that putatively homologous neuronsmay have quite divergent properties in somerespects in spite of close correspondence in severalothers.

MATERIALS AND METHODS

Animals

F1- and F2-reared males and females ofA. nigrovittata (Brunner von Wattenwyl, 1878)along with a few individuals caught in the field inNorthern Greece were used for the experiments.The animals were reared in small cages

J. MOLINA AND A. STUMPNER1086

(7 cm� 7 cm� 15 cm; 10–15 individuals) undernatural light conditions and from the 4th instaron in larger cages (40 cm� 20 cm� 20 cm; 20 to 30individuals) at room temperature under a 12 hrlight, 12 hr darkness regime with heat from 250 Wlight bulbs for 2–3 hr per day. They were fed withplants collected in the field (different Rosaceae)and cricket chow (ENDERLE, Pforzheim, Ger-many). Altogether, data from 42 individuals ofeither sex were used: 10 individuals for morphol-ogy, 33 individuals for physiology (one for bothphysiology and morphology).

Stimulation

The experiments were performed in a Faradaycage. The walls were covered with sound insulat-ing material. Echo amplitudes were at least 30 dBbelow stimulus amplitudes. The stimuli weredelivered through two broad-band speakers (DY-NAUDIO DF 21, Skanderborg, Denmark;2–50 kHz) positioned at a distance of 37 cm tothe left and right of the animal. The stimuli hada duration of 50 msec for measuring frequencytuning and 100 msec for left–right tests. The riseand fall was 1.5 or 2 msec. The stimuli weresynthesized using a custom-built DA board andamplifier (Lang et al., ’93). Each stimulus wasrepeated 5 times with pauses of 250 msec. Theintensity increased from 30 to 90 decibel soundpressure level (dB SPL) (re 2� 10�5 Pa) in 10 dBsteps. Natural songs of A. nigrovittata males showa complicated temporal pattern (see Heller andvon Helversen, ’86). Carrier frequencies peakaround 15 (male) and 28 kHz (female, see Dobleret al., ’94). Calibration was done on a continuoussound wave using an amplifier (2610, BRUEL &KJAER, Quickborn, Germany) and Bruel & Kjaermicrophones (1/200 or 1/400). Repeated measure-ments showed an accuracy of at least 72 dB.

Recording and staining

A CO2-anesthetized animal was waxed ventralside up on a holder. All legs and the head werefixed with a wax resin mixture. The prothoracicganglion was exposed by removing the overlayingcuticle and was stabilized by a Nickel–Chromiumspoon from below and a steel ring from above. Inmost specimens, the dry sheath of the ganglionwas treated with collagenase (SIGMA, Taufkirchen,Germany) for 90–120 sec to facilitate insertion ofthe glass capillary or to allow diffusion of PTX intothe ganglion. Afterwards, the ganglion was rinsedwith saline (pH 6.8; Fielden, ’60). Recordings were

made with borosilicate capillaries (1.0/0.58 or0.75 mm o.d./i.d.; 100–160 MO) filled with neuro-biotin (5% in 1 M K-acetate, Vector, Burlingame,CA, USA) or Lucifer Yellow (Molecular Probes,Eugene, OR, USA; 5% in 0.5 M LiCl) and anintracellular amplifier (custom-built or BA-1S,NPI, Tamm, Germany). Staining of a cell wasdone by application of 0.3–1.0 nA depolarizing(neurobiotin) or 0.3–2 nA hyperpolarizing (LuciferYellow) current for 1–10 min. After the experi-ment, the ganglia were excised and fixed in para-formaldehyde (4 % in buffer, pH 7.4) for 60 min.Neurobiotin was visualized with the DAB method(DAKO, Hamburg, Germany, StreptAB-complex/HRP; VECTOR Vectastain elite ABC kit and DABsubstrate kit) or by coupling streptavidin-Cy3(Rockland, Gilbertsville, USA) to the neurobiotinafter treating the dehydrated ganglion with xylenefor 5 min and a mixture of collagenase andhyaluronidase for 1 hr. At the end of the proce-dure, the ganglia were dehydrated (15 min in 70%,90%, 96% and 100% ethanol and cleared inmethylsalicylate). Drawings of the cells were madeusing a LEICA (Bensheim, Germany) microscopeDialux 20 and a drawing tube. Photographs weremade either on the same microscope or on aconfocal microscope (LEICA TCS SP2 AOBS).

Pharmacological treatments

After successful penetration of an omega neuronit was characterized physiologically. Responses tostimuli from the left and the right with increasingintensity were recorded at 16 and 28 kHz (onlydata for 28 kHz are shown here). Additionally, thethresholds for stimuli at frequencies between 4and 46 kHz (4 kHz increments, pseudo-randomorder) from the soma-ipsilateral side of omegawere determined. Thereafter, 5 drops of 10�3 MPTX (SIGMA) were added to the mixture ofhemolymph and saline in the prothoracic cavity(for details see Stumpner, ’98). Changes ofomega’s responses to soma-contralateral stimuliwere continuously monitored. After 3–4 min, theresponses did not change anymore and the samephysiological tests were performed as before.Subsequently, the neuron was stained with neu-robiotin. Finally, the soma-contralateral leg wascut at the distal portion of the femur to remove allremaining input from that ear and the tests wererepeated. Another period of staining concluded theexperiment. The neuron then was visualized withthe DAB method. In one animal, in which theganglionic sheath was not treated with enzyme,

OMEGA NEURON OF A BUSH CRICKET 1087

PTX did not have any obvious effect (see alsoStumpner, ’98). Therefore, this experiment wasnot used for further evaluation.

Photoinactivation experiments

After finding an omega neuron and characteriz-ing it briefly (left–right tests, selected frequencyscans), the cell was filled with Lucifer Yellow for atleast 2 min. A new glass capillary filled withneurobiotin was used to search for the second cellon the opposite side and the mirror-image omeganeuron was penetrated. This neuron was char-acterized like the first one and stained briefly withneurobiotin. Then the complete ganglion wasilluminated for 90 sec by a blue light laser(Omnichrome, Series 56, He–Cd–laser, 442 nm,ca. 35 mW, Melles-Griot, Bensheim, Germany) bymeans of a fibre coupling system (output diame-tero1 mm). After illumination, the omega neuronwas characterized again (same tests as before) andstained with neurobiotin for 2–4 min. The experi-ment was concluded by cutting the leg soma-contralateral to the second omega neuron close tothe femoro-tibial junction, followed by anothercharacterization and more staining. After proces-sing and clearing (see above ‘‘Recording andstaining’’), a dual staining in the epifluorescencemicroscope was visible: the first omega neuron filledwith Lucifer Yellow, the second marked with Cy3.

Data evaluation

Data were stored on a DAT recorder (SONY,Berlin, Germany, 10 kHz sampling rate), digitized(DT 2128 F; STEMMER, Puchheim, Germany,Turbolab 4.0 or 4.2) and analyzed with theNEUROLAB program (Hedwig and Knepper,’92) and standard software. As threshold, wedefined an average response of one spike abovespontaneous activity in three out of five stimuli.Spikes were counted in a time window starting5 msec after stimulus onset and ending 30 msecafter stimulus offset. Latency was measured forthe first spike within this time window. Conse-quently, in spontaneously active neurons in somecases a spontaneous spike might have beencounted as stimulus evoked. In the diagrams,‘‘response %’’ means that for each individual thestrongest response to any stimulus of the serieswas set to 100% before averaging over individuals.For calculating a relative directional difference(Figs. 5 and 7), we counted the difference in spikesbetween left and right stimulation at a givenintensity and normalized it (in %) to the maximum

spike number with soma-ipsilateral stimulation.Then we subtracted this directional differenceafter the treatment from the difference before.A 100% change in difference means that beforetreatment the difference was 100% and aftertreatment the difference was zero (left and rightstimuli elicited identical spike numbers). A nega-tive value means that the difference after treat-ment was larger than it was before. ANOVAs wereperformed using the program Sigmastat.

RESULTS

Morphology

The omega neuron of A. nigrovittata is a largesegmental neuron with an anterior-dorsal somaand strong ipsilateral and contralateral branches,which are connected by a thick axon (close to themidline 12.272.7mm, n 5 10) lying most ventral ina commissure posterior to ventral commissure I(VCI) and right below the auditory neuropile.In crickets, this commissure has been namedomega commissure (Wohlers and Huber, ’82)and described to be inconspicuous, but inA. nigrovittata it is as large as VCI. The exactshape and diameter of the major branches is quitevariable (Figs. 1A and B). Whereas ipsilateraldendrites have smooth and very fine terminations,contralateral terminations are less dense but oflarger diameter and have a beaded appearance(Figs. 1C and D). The projections fill the wholeauditory neuropile on both sides with the excep-tion—at least in four out of five animals—of asmall most anterior region on the ipsilateral side(Fig. 1D).

Excitation and inhibition

Recordings of the soma-ipsilateral branches ofomega reveal large excitatory postsynaptic poten-tials (EPSPs) and action potentials when stimu-lated from the ipsilateral side (Figs. 2A and B, 4Aand B, and 6A and B). With soma-contralateralstimuli, the EPSP is much smaller and no or onlyone initial spike is elicited (Figs. 2A and 4A).Typically, compound potentials or clear inhibitorypostsynaptic potentials (IPSPs) can be seen.A small deflection in the rising phase of the EPSPin some recordings (Fig. 2B arrows) gives evidencefor inhibition with ipsilateral stimuli. This IPSP islost after cutting the contralateral tympanic nerve(respectively the whole leg; Fig. 2B). After cuttingthe soma-ipsilateral tympanic nerve, a strong


IPSP is seen and no excitation can be detected(Fig. 2A).

Latencies of graded potentials as well as of thefirst spike vary greatly between individuals.Average latencies (five stimuli at 28 kHz 90 dBSPL) of EPSPs vary between 10.8 and 19.7 msec(12.770.5 msec, n 5 9 individuals) and are fol-lowed by an action potential after approximately2 msec at high intensities (2.270.2 msec, n 5 7).Average latencies of IPSPs with soma-contralat-eral stimulation (soma-ipsilateral tympanic nervesevered) vary between 13.1 and 27.1 msec (mean:17.071.5 msec with 28 kHz 90 dB SPL, n 5 4).When EPSPs and IPSPs were obvious in the sameresponse at low intensities, the EPSP led the IPSPby approximately 3 msec (in five individuals the

values ranged between 1.7 and 4.7 msec). At highintensities, the latency of the EPSP (measured inthe intact animal; n 5 5) was 2.2–6.0 msec shorterthan the latency of the IPSP after cutting thesoma-ipsilateral tympanic nerve. Therefore, itseems safe to conclude that the fastest inhibitorypathway involves more synapses than the fastestexcitation.

Recordings from soma-contralateral branchesreveal large action potentials without any obviousEPSP. This is a clear indication that gradedpotentials from one side do not reach the oppositebranches. One might argue that EPSPs areshunted by the increased membrane conductancesduring action potentials. However, before the firstaction potential, no sign of an EPSP could be

Fig. 1. Morphology of the omega neuron of Ancistrura nigrovittata. (A) Wholemount view. (B) Main branches of four omeganeurons to show variability. (C) Cy3 staining of a horizontal section (20mm thickness) through the central area of the ganglion.The neurite is seen to the upper left. The two smaller photos to the right show 2.5 times enlarged portions from an ipsilateraland a contralateral region close to the midline. (D) Sagittal sections of the auditory neuropile close to the midline indicating thebranching area of the soma-ipsilateral and soma-contralateral dendrites. The lines in the schematized neuropile to the rightindicate the area, which was not occupied by ipsilateral dendrites (in one individual no larger area free of projections was found).Scale bars: 50 mm (large photo in C) and 20mm (small photos in C), 100 mm (A,D).


detected. In many contralateral recordings IPSPswere seen (Fig. 2C). These may be accompanied byaction potentials in the middle of large hyperpo-larizations, demonstrating that at the spike gen-erating zone, the membrane potential at thatspecific time was much more positive. Thisindicates that the IPSPs recorded contralaterallyare of contralateral origin. After cutting the soma-ipsilateral tympanic nerve, the action potentialswere gone, but the IPSP was still present (Fig.2D). The earliest IPSPs measured in the contra-lateral branch of a single individual were similarin latency (13.670.1 msec, n 5 5 stimuli) to theearliest IPSPs measured ipsilaterally in another

individual (13.170.2 msec, n 5 5 stimuli). While insoma-ipsilateral recordings, large EPSPs andIPSPs were detected in the same location, incontralateral recordings, large IPSPs were notaccompanied by EPSPs. In at least two experi-ments with a soma-ipsilateral cut, at low frequen-cies and higher intensities (80 dB SPL or higher)a small but reliable depolarization was observedin the soma-contralateral branch leading theIPSP. In summary, soma-ipsilateral branchesare the target of strong excitation and inhibi-tion, while soma-contralateral branches receivesome inhibition and potentially also some weakexcitation.

Fig. 2. Intracellular recordings in soma-ipsilateral (A,B) and soma-contralateral (C,D) dendrites. (A) Sample traces withipsilateral (many spikes) and contralateral stimulation (compound potential) and contralateral stimulation after the ipsilateraltympanic nerve was cut (IPSP). (B) Averaged sample traces (five repetitions) of another individual (spikes have been clipped).A small IPSP (open arrow) in the rising phase of the EPSP (closed arrow) is visible with ipsilateral stimulation, while withcontralateral stimulation a strong IPSP is obvious. After cut of the contralateral tympanic nerve, all IPSPs are gone.(C) Recorded spikes (ipsi) are not associated with any obvious EPSP, not even before the very first spike. IPSPs are seen withcontralateral stimuli and action potentials may occur during the hyperpolarization. (D) Responses to soma-ipsilateralstimulation at 28 kHz before (upper) and after cut of the soma-ipsilateral leg (middle). The lowest traces show an average (n 5 5)of recordings after the cut. Scale bars: horizontal: (A–D) 50 msec; vertical: (A, C and D) top row: 25 mV, (B and D) middle andbottom row: 5 mV.


Frequency tuning and directionalresponses

Omega’s responses to different frequencies arenot uniform. At all frequencies it has a largedynamic range covering 50 dB or more, if spikecount is measured (Fig. 3A for 28 kHz). Maximumspike frequency lies at around 350 spikes/sec.A phasic–tonic response pattern (most likelycaused by adaptation) is seen in all individuals(Figs. 4 and 6). It is most sensitive to frequenciesbetween 20 and 30 kHz (Figs. 3B–D), but shows arather broad tuning. Frequency tuning does notdiffer between males and females (Fig. 3B).

The inhibition from the soma-contralateral eardescribed above has considerable influence on thedirectional responses of omega. Contralateralstimuli elicit a much weaker response thanipsilateral stimuli, corresponding to a differenceof 40 dB or more (Figs. 3A, 5A, and 7A). When

cutting the soma-contralateral tympanic nerve,this difference is immediately reduced to 10–15 dBon average (Fig. 3A). The overall frequency tuningof omega is not affected by cutting the contra-lateral nerve (Fig. 3C; two-way ANOVA: df 5 1,F 5 2.86, P 5 0.094), but a slight increase ofsensitivity at lower frequencies is indicated (pair-wise multiple comparison procedure, 8 kHzP 5 0.048, 12 kHz P 5 0.023). If thresholds aremeasured for evoking an IPSP in omega after thesoma-ipsilateral tympanic nerve was cut, thiscurve is similar to thresholds of the contralateralomega in the same preparation (Fig. 3D). How-ever, thresholds of IPSPs are higher by up to12 dB. Around 8 kHz the average threshold of theIPSPs is below the excitatory threshold of thecontralateral omega. These results indicate thatthe omega neurons mutually inhibit each other,but also that additional inhibition, at least atlower frequencies, might exist.

Fig. 3. Directional responses and frequency tuning in omega neuron. (A) Normalized spike number of four omega (twomales, two females) for 28 kHz ipsilateral (solid lines) and contralateral (broken lines) stimulation. (B) Frequency tuning ofmales and females (n per data point is 9–11 in males, 8–11 in females). (C) Frequency tuning of five omega neurons (two males,three females) before (solid line) and following cut of the contralateral tympanic nerve (broken line). (D) Thresholds for elicitingan IPSP in omega after the ipsilateral tympanic nerve has been cut (broken line) and thresholds of the mirror-image omega inthe same preparation, also with the nerve cut (solid line). Data of three females and one male. Error bars give error of the meanin (A), standard deviation in (B, C and D).


Effects of picrotoxin applicationand leg cuts

Changes in the response (increased activity, lossof IPSPs) started approximately 2 min afterapplication of PTX in an estimated intraganglionicconcentration of 10�6–10�5 M (Stumpner, ’98).Responses reached a stable level soon thereafter.Controls with the AN1 neuron (Stumpner, ’98;experiments with the omega neuron and AN1started in the same year) showed earliest reap-pearance of IPSPs 12 or more minutes afterwashing. Long recordings (up to 45 min) of severalauditory neurons in untreated animals with acapillary filled with neurobiotin showed stableneuronal responses over the whole recording time,even with several replacements of saline and afterstaining with neurobiotin. This demonstrates thatsudden loss of inhibition cannot be explained bythe application of fluid or by longer intracellularrecording times.

Application of PTX clearly reduced the contra-lateral inhibition. A contralateral stimulus, whichin an untreated specimen evoked just one initialspike or only very few spikes, produced a phasic–tonic response with 10 or more spikes after PTXapplication (Fig. 4). In a single specimen, whichshowed only weak contralateral inhibition beforetreatment, the increase in spike number followingPTX application was minor (e.g., Fig. 4C). How-ever, even in this case a typical change in responsewas observed: a strongly phasic–tonic responsechanged to a pattern with a reduced phasic and anincreased tonic portion. This was also true foripsilateral stimuli, where the tonic portion (in thespike frequency and in the shape of the EPSP)usually became more pronounced (e.g., Figs. 4Band C). Contralateral stimuli, which before wereclearly inhibitory in their overall action, after PTXwere excitatory like ipsilateral stimuli of lowerintensity (e.g., Figs. 4A and B). This was trueirrespective of the increase of spontaneous activityoften evoked by the PTX application from 20 Hzon average (range 0–60 Hz) before application to35 Hz (range 0–80 Hz; see also Figs. 4A and C)thereafter. No obvious IPSPs were detectable afterapplication of PTX. Interestingly, PTX abolishedboth the strong IPSP, which can be recorded inthe soma-ipsilateral branches, and the IPSP seenon the contralateral branch. The latter was mostobvious in an individual that had a relatively highthreshold for contralateral stimulation even afterPTX application (Fig. 4D). With PTX, there wereno more IPSPs, while spikes were elicited only

occasionally, showing that the IPSP was not justcamouflaged by simultaneously occurring excita-tion (Fig. 4D).

A cut of the soma-contralateral leg (and therebyof the contralateral tympanic nerve) had a clear,though less dramatic effect than application ofPTX (Figs. 4 and 5). The response to contralateralstimuli increased further and correspondingly thedifference between left and right stimulationdecreased (Figs. 5A and B). In the graph ofFig. 5A, the effect of the cut may not seemimmediately obvious below 70 dB SPL, since theaverage response for contralateral stimuli in-creased only at higher intensities (for the highestintensity by 16–18%). Actually, when the contra-lateral responses are compared in an ANOVA, thedata before and after PTX differ significantly(Po0.001), while the difference between PTXand cut is not significant (P 5 0.18). Note, how-ever, that the average response to ipsilateralstimuli (and therefore also the relevant differencebetween ipsilateral and contralateral responsestrength) decreased at lower intensities after thecut. This is the result of a decrease in spikenumbers at most intensities in five individuals,whereas in three individuals spike numbersincreased. Average spike latencies with ipsilateralstimuli, on the other hand, were more or less thesame before and after the cut (average difference:0.3 msec longer after the cut). The same is true foraverage spontaneous activity. For clarification, thegraph in Fig. 5B shows the difference between thenormalized responses to ipsilateral and contra-lateral stimuli. There, the drop in directionaldifference by both treatments, PTX and cut, isobvious and significant (two-way ANOVA, effect ofPTX: Po0.001; effect of cut vs. PTX: Po0.01).The variability of the effects in individuals wasrather large. Figs. 5C and D show the calculatedchanges in difference (see arrows in Fig. 5B) forboth treatments and all individuals. Some indivi-duals showed large response changes induced byone and smaller changes induced by the othertreatment. In a pairwise comparison at all in-tensities above threshold, the difference in changewas highly significant (Po0.001; sign-test, Zar,’99) for both treatments (and also with 16 kHz,which evokes less directional difference, P waso0.001 for PTX and 0.017 for cut, data notshown).

While the responses to soma-contralateral sti-muli changed drastically following application ofPTX, the responses to soma-ipsilateral stimulichanged much less. Fig. 5E shows the frequency


tuning of omega before and after PTX application.As can be seen, the thresholds remain the same orwere slightly higher with PTX being applied.

Controls for photoinactivationexperiments

Photoinactivation of a Lucifer Yellow-filledneuron involves illumination of the complete

surface of the prothoracic ganglion. Therefore,unspecific effects, e.g., through heating, on neu-rons not filled with Lucifer Yellow are conceivable,at least if the exposure exceeds a certain timelimit. Since photoinactivation experiments havenot been performed on A. nigrovittata before, twotypes of control experiments were carried out.

At first, we tested how long the laser illumina-tion of the ganglion may last until an auditory

Fig. 4. Sample traces of four individuals of A. nigrovittata omega neuron at 28 kHz before and after treatment with PTX(A–D) and a subsequent cut of the contralateral leg (A–C). (A) and (B) show recordings in soma-ipsilateral dendrites, (C) and (D)show recordings in contralateral branches. With contralateral stimulation, PTX increases EPSP sizes (A,B) as well as spikenumber (A–C) and changes temporal pattern of spiking (A,B), but note especially (C). A leg cut may increase (A) or decrease(B,C) absolute spike numbers. Note in (D) that the IPSP recorded with soma-contralateral stimulation is lost after PTXtreatment. Scale bars: horizontal: 100 msec; vertical: 25 mV (A,C,D), 15 mV (B).


neuron, which is not filled with Lucifer Yellow,starts to change its responses. For this purpose,auditory neurons were recorded with electrodesfilled with neurobiotin and exposed to the laser

beam. In all cases (n 5 6, data not shown), theneurons did not show any change in spikingactivity or in membrane potential during the first3 min of exposure. It was only later that cells

Fig. 5. (A) Normalized response of the omega neuron (mean and standard error of the mean, n 5 8) to stimuli from thesoma-ipsilateral and soma-contralateral side before and following PTX application and following a cut of the soma-contralateralleg (including the tympanic nerve) at 28 kHz. (B) Relative directional difference between soma-ipsilateral and soma-contralateral stimulation (100% would mean no response to soma-contralateral stimuli) at 28 kHz before treatment, after PTXapplication and after soma-contralateral leg cut (means and S.E.; n 5 8). In (C) and (D), the individual changes in directionaldifference caused by PTX application (C) and leg cut after PTX (D) are shown as indicated by the arrows in (B). For bettercomparability, the same scaling of the Y-axis was used and therefore two graphs in (C) were truncated (but the maximum valueis indicated). (E) Frequency tuning of four omega neurons before and following PTX treatment (means and standard deviation).


showed first a reduction in action potential sizeand then a reduction in EPSP size. A change inmembrane potential (depolarization) was observedin only two cases (after more than 4 min ofexposure).

Secondly, we tested the time needed to eliminatethe physiological functionality of an auditoryneuron filled with Lucifer Yellow. This time spanis influenced by the amount of Lucifer Yellowinjected and the amount of saline present aroundthe ganglion (Atkins et al., ’84). Omega cells filledwith Lucifer Yellow and recorded during laserexposure (Fig. 6A) showed a gradual change(depolarization) of the membrane potential accom-panied with gradually increasing spontaneousactivity approximately 5 sec after the start of theexposure. A complete loss of spiking activityoccurred approximately 15–30 sec after the startof illumination (n 5 4, Fig. 6A).

The results obtained in the control experimentsindicate that in a time window between 30 sec and3 min after start of laser exposure, a cell filled withLucifer Yellow will be fatally damaged whereascells without Lucifer Yellow remain unaffected.Therefore, an intermediate time exposure of 90 secwas chosen to ensure that a cell filled with LuciferYellow is inactivated (killed or at least no longerfunctionally present in the network), while allcells without Lucifer Yellow are not affected.

The harmless illumination time of 90 sec forcells not filled with Lucifer Yellow was alsoconcluded from unchanged responses of variousneurons before and after laser illumination. Theseneurons were filled with neurobiotin and either noother cell had been successfully filled with LuciferYellow before or another neuron was filled withLucifer (auditory or non-auditory), but was notconnected to the neuron evaluated.

Effects of photoinactivation of one omegaon its mirror-image partner

In at least 13 experiments, the two omegas weresuccessively recorded in one individual. All pene-trations took place in the soma-ipsilateralbranches. The first omega was filled with LuciferYellow and the second with neurobiotin, whichwas later detected with streptavidin-Cy3 (Fig. 6C).In all cases, both neurons were identified by theirunique morphology after completion of the experi-ment. Intracellular recordings of the secondomega prior to laser exposure showed the typicalsoma-contralateral inhibition which can usuallybe seen as IPSPs, while soma-ipsilateral stimula-

tion evoked a phasic–tonic response (Fig. 6B). Thisled to a strong left–right difference in responsemagnitude over the whole intensity range tested(Fig. 7A). During the 90 sec of laser exposure, theactivity of the cell was constantly monitored usingsoma-contralateral stimulation. Often an increasein spontaneous activity was observed (perhaps dueto rising temperature in the ganglion). Afterapproximately 30 sec of exposure, the IPSPs wereprogressively replaced by EPSPs and concomitantspiking activity. After termination of the laserexposure, the cell showed an excitatory responseto both contralateral and ipsilateral stimulationwith phasic–tonic firing, which, however, was stillweaker to contralateral than to soma-ipsilateralstimulation (Figs. 6B and 7). The normalizedresponse function for soma-ipsilateral stimulationwith 28 kHz showed only minor changes withthe treatments (Fig. 7A), while the responses forsoma-contralateral stimulation clearly increased(two-way ANOVA; Po0.001). The highly effectivesoma-contralateral inhibition observed between50 and 80 dB SPL was replaced by spiking activity(Figs. 6B and 7). IPSPs in response to soma-contralateral stimulation were never detectedafter laser illumination. No differences betweensexes were seen.

Effects of leg cut after photoinactivation

In the majority of experiments, after photoinac-tivation and the subsequent characterization aswell as a short staining with neurobiotin, thesoma-contralateral leg was cut and with it thecomplete auditory input from the soma-contral-ateral side. In all (n 5 8) experiments there was anincrease in the spiking response of omega withsoma-contralateral stimulation as compared to thetest before the leg cut (Fig. 7; all contralateralresponses before and after laser and before andafter cut differ significantly, two-way ANOVA,Po0.001 in all cases). Consequently, there wasalso a significant decrease in the differencebetween responses to ipsilateral and contralateralstimuli (Fig. 7B; two-way ANOVA, Po0.001 forbefore vs. laser, P 5 0.017 for laser vs. cut).In some cases, the spontaneous activity increased(obvious in Fig. 6B). This might be due to somemechanical vibrations during the cutting proce-dure. The variability between individuals of theeffects of both laser illumination and leg cut wasquite large (Figs. 7C and D). However, it was clearthat both treatments had a definite effect. In apairwise comparison, the changes in directional


difference induced by the respective treatment(photoinactivation and leg cut) at all intensitiesabove threshold were highly significant (Po0.001

for all treatments; sign-test, Zar, ’99; this was alsotrue for 16 kHz, data not shown). Changes in theauditory tuning curve of ON1 were not observed

Fig. 6. (A) Sample traces of an omega neuron filled with Lucifer Yellow during photoinactivation in a control experiment.Most obvious are the increase in spontaneous activity, loss of membrane potential (depolarization) and of the spiking responsewithin 15 sec. (B) Sample traces of an omega with 28 kHz 70 dB SPL stimuli from the soma-ipsilateral and soma-contralateralside (lower two traces). Left: cell before any treatment (see the IPSPs with soma-contralateral stimulation); center: after 90 secof laser exposure (see the EPSPs and spiking activity with soma-contralateral stimulation); right: after additionally cutting thecontralateral leg (note the small difference between ipsi- and contralateral stimulation. (C) Confocal image of the two omeganeurons stained in one experiment (green: Lucifer Yellow, red: Neurobiotin-Cy3). Scale bars: horizontal: (A,B) 500 msec,(C) 40 mm; vertical: (A) 10 mV, (B) 25 mV.


after illumination or after cutting the soma-contralateral leg (data not shown).

DISCUSSION

General properties of the omega neuron ofbush crickets

Homology of the omega neuron described indifferent groups of ensifera is highly likely due tothe unique morphology and some basic physiolo-gical properties like its prominent contralateralinhibition. In A. nigrovittata, the soma-ipsilateraldendrites terminate in most of the neuropile (asseen in a parasagittal section close to the midline),usually leaving free a most anterior portion(Fig. 1). Such a free anterior portion has also beenfound in the omega neuron of T. viridissima andhas been correlated to the reduced sensitivity of

omega in the low-frequency range, since thisportion of the neuropile is the target area ofreceptor cells tuned to low frequencies (Romer,’85). In principle, the same argument holds truefor A. nigrovittata. Receptor cells tuned to highersonic frequencies do not send branches into themost anterior portion of the neuropile (Stumpner,’96, ’97, ’99a and unpublished). This area is alsothe target of the most distal cells of the proximalintermediate organ or subgenual organ (Ebendtet al., ’94; Stumpner, ’96; Stolting and Stumpner,’98). Such neurons are tuned to low frequenciesand in addition are vibration sensitive. Indeed,there is no indication that the omega neuron inany bush cricket is vibration sensitive. On thecontrary, ultrasonic frequencies yield the stron-gest responses, making the omega a neuron thatfocuses on ultrasound.

Fig. 7. (A) Normalized response of the omega neuron recorded with a neurobiotin electrode (mean and standard error of themean) to stimuli from the soma-ipsilateral and soma-contralateral side before and following photoinactivation of the mirror-image omega and following a cut of the soma-contralateral leg (including the tympanic nerve) at 28 kHz. Only those individualsare included (n 5 6) for which a complete set of data exists. (B) Relative directional difference between soma-ipsilateral andsoma-contralateral stimulation (100% would mean no response to soma-contralateral stimuli) at 28 kHz before treatment, afterphotoinactivation of the mirror-image omega and after soma-contralateral leg cut (means and SE; n 5 8). In (C) and (D), theindividual changes in directional difference caused by laser illumination (C) and leg cut after photoinactivation (D) are shownas indicated by the arrows in (B).


Since the bush cricket ear as a whole usually ismost sensitive at ultrasonic frequencies, omegastill works as a ‘‘broad-band neuron’’ with muchless specificity in tuning than other neurons (e.g.,Romer, ’85; Stumpner, 2002). Furthermore, inA. nigrovittata the omega neuron shows identicaltuning in males and females (Fig. 3B), eventhough the sexes use different carrier frequenciesfor their songs (Dobler et al., ’94). For thesereasons, the omega neuron is often taken asrepresentative for overall hearing of bush crickets(e.g., Romer et al., ’89). In T. cantans, a closerelative of T. viridissima, Hardt (’88) found amuch larger area of the anterior ventral auditoryneuropile to be free of ipsilateral omega dendritesthan in T. viridissima. Correspondingly, he noteda reduced sensitivity in the sonic frequency range.The significance of this species difference is notclear. It shows, however, that caution is neededwhen drawing conclusions for one (or many)species from data collected in another species.

The omega neurons of bush cricketsmutually inhibit each other

The omega neuron is excited by soma-ipsilateralinputs and inhibited by soma-contralateral inputs(Figs. 2 and 3). Directional inhibition of omegawas demonstrated earlier, e.g., for T. viridissima(Schul, ’97; Romer and Krusch, 2000) and Meco-poda elongata (Romer et al., 2002). In A. nigro-vittata, the inhibition is visible as large IPSPs inipsilateral recordings. IPSPs can also be observedin contralateral recordings, as has been describedfor the omega neuron in T. viridissima (Schul, ’97)and T. cantans (Hardt, ’88). In contrast, Shen andGuan (’90) did not see IPSPs, even with dendriticrecordings and contralateral stimulation. In A.nigrovittata, IPSPs on both sides have similarlatencies, but interindividual variation is verylarge. On average, IPSPs start 3 msec later thanEPSP onset. At lower intensities, this often leadsto complete suppression of spiking with soma-contralateral stimuli. At higher intensities, one ortwo initial action potentials may be produced.Obvious differences in EPSP-waveforms or EPSPlatency in response to different carrier frequencies(e.g., male song frequency vs. ultrasound) as seenin Teleogryllus (Pollack, ’94) have not beenobserved. In ipsilateral branches, large EPSPsand IPSPs (often compound potentials) can berecorded, while in contralateral branches onlylarge IPSPs can be detected, which may addition-ally be interrupted by action potentials in the

middle of the hyperpolarization (Fig. 2). EPSPsare not even seen at the beginning of a responsebefore the very first action potential. This leadsus to conclude that graded potentials recordedin soma-contralateral branches also have theirorigin on that side and are not coming from thesoma-ipsilateral side.

In A. nigrovittata, cutting the soma-contralat-eral leg immediately eliminates the large IPSPseen in ipsilateral dendrites (Fig. 2) and reducesresponse differences to left–right stimulation frommore than 40 dB to 10 or 15 dB (Fig. 3A) as istypical for auditory receptor cells (e.g., Stumpner,’97). After cutting the soma-ipsilateral leg, onlyIPSPs remain. The close (though not perfect)correspondence of the tuning of inhibition of oneomega to the tuning of excitation of the mirror-image partner (Fig. 3D) makes it likely that omegais the major source of contralateral inhibition ofits mirror-image partner.

This was corroborated with photoinactivationexperiments. The same type of experiments hasbeen conducted in crickets to evaluate the mutualinhibition between the mirror-image partners ofON1 (Selverston et al., ’85) and to evaluate thebehavioral significance of directional inhibitionduring positive phonotaxis (Atkins et al., ’84).In A. nigrovittata, as in crickets, the two mirror-image partners definitely inhibit each other.Inactivation of one omega reduces contralateralinhibition: All IPSPs visible with ipsilateraldendritic recordings are lost and an obvious inhibi-tion is replaced by a clear excitation (Figs. 6 and 7).

A consistent finding was also that after cuttingthe soma-contralateral tympanic nerve (leg) fol-lowing photoinactivation of one omega, the direc-tional difference decreased further (Fig. 7).Nevertheless, some directional difference re-mained, which is also seen in auditory receptorsand which is caused by biophysical sound shadow-ing (e.g., Stumpner and Heller, ’92; Michelsenet al., ’94; Stumpner, ’97). The observable effect ofa leg cut leads to the conclusion that directionalinformation at the level of the prothoracic audi-tory network is not only transmitted by the twoomegas, but must involve at least one additionalelement. One might argue that this additionalreduction of directionality indicates an incompleteeffect of the preceding photoinactivation. How-ever, in all control experiments, a cell which hadbeen successfully filled with Lucifer Yellow, lost allspiking activity and most of its negative mem-brane potential within the first minute of laserillumination (Fig. 6A). Moreover, there was not a


single experiment in which the leg cut was withouteffect. In conjunction with very similar effects ofthe PTX-treatments (see below), it can be con-cluded that this observed effect reliably indicatesthe existence of one or more additional PTX-insensitive elements which inhibit omega. Wehave no information whether the same is truefor crickets, because Selverston et al. (’85) did notperform anything comparable to a soma-contra-lateral leg cut after photoinactivation of oneomega, and because the experiments were per-formed with a different regime (closed fieldstimulation, acoustic trachea cut in the middle).

The mutual inhibition of the omeganeurons is sensitive to picrotoxin

treatment

PTX has been successfully applied in the bushcricket auditory system to trace the effects ofcertain inhibitions (Stumpner, ’98, 2002). Fre-quency-specific inhibitions were clearly affected byPTX and thresholds of IPSPs before applicationwere literally identical to thresholds for excitationafter application. Therefore, frequency-specificinhibition was completely eliminated by PTX. Atthe same time, direction-dependent inhibition ofan ascending neuron (AN1) was not, or notcompletely, lost. This indicated that the inhibitoryinfluences for frequency processing and direc-tional processing were independent, and differentneural elements had to be postulated as sources ofthe inhibition. Therefore, the working hypothesisfor A. nigrovittata was that the inhibition of theomega neuron (and therefore also the mutualinhibition) should be insensitive to PTX. Thisseemed to fit to data from crickets, since Faulkesand Pollack (2001) found only subtle effects ofPTX on ON1’s physiological responses inT. oceanicus (but contralateral inhibition has notspecifically been tested). Also, Skiebe et al. (’90)did not find any physiological effect of bath-applied GABA on the directional inhibition ofON1 in G. bimaculatus. This agrees with theimmunohistochemical finding that omega is nega-tive for GABA antibodies in both groups (e.g.,Watson and Hardt, ’96, Stumpner, unpublishedin A. nigrovittata).

After application of PTX in A. nigrovittata,soma-contralateral stimuli, which were mainlyinhibitory before, evoked strong excitatory re-sponses (Fig. 4). These responses were similar tothose with ipsilateral stimuli of lower intensity(some 25–40 dB lower, Fig. 5A). Therefore, the

hypothesis that contralateral inhibition in theomega neuron is not PTX sensitive, clearly hasbeen discarded by our experiments. Furthermore,it must be concluded that PTX influences ipsilat-eral and contralateral inhibition on omega, sinceIPSPs recorded on both the dendritic and axonalbranches of the omega neuron, and which mostlikely have separate sources (see above), wereabolished by PTX (Fig. 4). These separate sourcesmight, however, be output synapses of the mirror-image partner in both hemiganglia (which havebeen described for cricket ON1, Watson andHardt, ’96).

The combined data of photoinactivation andPTX treatment show that the omega neuronsmutually inhibit each other and that this inhibi-tion must be PTX sensitive. Inactivation of thecontralateral omega and application of PTX botheliminated a portion of the contralateral inhibitionand all IPSPs visible at the recording site. Thiscan only mean that these IPSPs are from omegaand are PTX sensitive. The PTX sensitivity ofomega–omega interaction does not mean thatomega must use GABA as transmitter. PTX haseffects on various chloride channels in vertebratesand invertebrates which use other ligands thanGABA (e.g., glutamate: Ikeda et al., 2003; glycine:Lynch et al., ’95; histamine: Nassel, ’99; seroto-nine: Das and Dillon, 2003).

Contralateral inhibition is not completelyexplained by omega’s action

The data also demonstrated that there must bean additional element which inhibits the contra-lateral omega neuron. This inhibition is PTXinsensitive. One has to assume that its inhibitiondoes not evoke visible IPSPs at the typicalrecording site of our experiments close to thebranching point of the major dendrites. Afterinactivation of the mirror-image omega or afterPTX –application, no IPSPs were detected. Never-theless, some contralateral inhibition still existedas demonstrated by cutting the tympanic nerve(actually the leg; however, since the two ears ofA. nigrovittata are physically not coupled, this isequivalent). This result might be interpreted inthree ways. (i) There is a direct synaptic inhibitionevoking IPSPs, but the synapses are too far fromthe recording site and the IPSPs are not con-ducted far enough. In another auditory interneur-on, the TN1 neuron (Stumpner, ’99a), a clearcontralateral inhibition is not visible in an intactanimal, since the simultaneously occurring EPSP


is too strong. (ii) There is a direct synapticinhibition, but the transmitter receptors and openion channels do not hyperpolarize the omeganeuron below its resting potential. This has beendescribed, e.g., for the primary afferent depolar-ization of many invertebrate mechanoreceptors(e.g., Burrows and Laurent, ’93; Poulet andHedwig, 2003). If this inhibition appeared as adepolarization instead of a hyperpolarization of aresting omega neuron, its action would be camou-flaged by the simultaneous excitation of omega. Tomake it visible, one would have to eliminate boththe ipsilateral excitation and the contralateralinhibition by the mirror-image omega. (iii) Thethird possibility is a presynaptic inhibition of theneurons providing the excitatory input of omega—be it sensory neurons or interneurons. Presynap-tic inhibition is quite common in mechanosensoryafferents and has also been found in variousOrthoptera-like crickets (Hardt and Watson, ’99;Poulet and Hedwig, 2003) and bush crickets(Hardt and Watson, ’95).

Unfortunately, there is no prime candidate forthe unknown inhibitory neuron. A group ofneurons, which have been hypothesized to providefrequency-specific inhibition (DUM cells, Stump-ner, ’98, 2002) receive excitation from both earsand show frequency-specific responses (Stumpner,’95, 2001). Therefore, they are not a likelycandidate for contralateral inhibition. The onlyother well-characterized local neuron (Stumpner,’95) is hemiganglionic and therefore might wellbe responsible for the IPSPs recorded in thesoma-contralateral branches of omega, but wouldneed an additional intermediary to affect spikingon the soma-ipsilateral dendrites—which thenagain is unknown.

There was a large variability in the data of bothPTX treatment and photoinactivation. This maybe due to the neurophysiological procedures likepenetration artefacts, but may also be caused byinterindividual differences, e.g., in the animal’sage. Our experimental animals were not agecontrolled—they were at least 1 week adult, buttypically were 2–6 weeks old. For the cricketA. domesticus, the strength of inhibition of theomega neuron on an ascending neuron increasedwith age (Stout et al., ’97). A similar effect cannotbe excluded for the contralateral inhibition of theomega neurons in A. nigrovittata and wouldcontribute to interindividual differences.

An increase in spontaneous activity after PTXapplication such as shown in Figs. 4A and 5A hasalso been observed in other studies with PTX (e.g.,

Faulkes and Pollack, 2001) and with otherneurons in A. nigrovittata (Stumpner, unpub-lished). It may indicate the existence of eitheradditional direct (not necessarily auditory) orPTX-sensitive inhibitory influences on the omeganeuron (e.g., by corollary discharge neurons,Poulet and Hedwig, 2003). Alternatively, it mayindicate more complex changes in the prothoracicnetwork elicited by PTX. So far, there is noindication that PTX evokes any unspecific effectsor a damage of the neurons (see Stumpner, ’98 fordetailed discussion).

Concluding comparison with crickets

In crickets and bush crickets the omega neurons(ON1 in crickets) inhibit each other. This inhibi-tion does not seem to be PTX-sensitive in crickets(indirect evidence). Also, there is no indication foradditional contralateral inhibition in crickets.ON1 activity may account for all directionalinhibition observed in the ascending neuronsAN1 and AN2 (Faulkes and Pollack, 2000; Reeveand Webb, 2003; see however Harrison et al. (’88),who did not find any signs of connectivity betweenAN2 (ANA) and both ON1 neurons in T. oceani-cus). In the bush cricket A. nigrovittata, theevidence is strong that there is at least oneadditional element which evokes contralateralinhibition in omega. These results indicate thathomologous neurons in related groups of animalsmay correspond closely in morphology and someaspects of physiology, but may clearly differ inother respects. It is not clear what the functionalsignificance of these species differences might be.The function of mutual inhibition of ON1 incrickets is generally seen as strengthening theleft–right difference for orientation. Contralateralinhibition has been found to be of major impor-tance in directional hearing, not only for variousinsects (Rheinlaender and Morchen, ’79; Rhein-laender and Romer, ’80; Wiese and Eilts, ’85), butalso for vertebrates including mammals (reviewedby Grothe, 2003). Alternative functions are dis-cussed like increasing the range of horizontalangles ascending cells like AN1 will respond towithout saturating (Reeve and Webb, 2003). Thisidea resulted from model calculations forG. bimaculatus phonotaxis. It is supported byseveral data: young A. domesticus show a weakcoupling of ON1 to ascending neurons but showprecise phonotaxis (Atkins et al., ’84). A. domes-ticus with one or even both ON1s killed showedvery little change in phonotactic orientation to a


range of frontal angles, where contrast enhance-ment would be needed most (Atkins et al., ’84;Stout et al., ’97). The inputs into ON1 ofT. oceanicus differ between low and high frequencies(Pollack, ’94). This might even indicate that ON1of crickets serves different purposes in differentbehaviors like positive phonotaxis to calling songand negative phonotaxis to bat echolocationsounds. In bush crickets it has been proposed thatthe omega neuron contributes to female prefer-ence of leaders in a chorusing species (M. elongata,Romer et al., 2002) or to reduce masking of a songcoming from one side by a song coming from theopposite side (Romer and Krusch, 2000). ForA. nigrovittata, most functions discussed areconceivable as well: Both sexes can performpositive phonotaxis (own observations). Densityin populations of Northern Greece and thereforesong overlap may be quite high. Predator avoid-ance behavior of this brachypterous species in-cludes escape jumps to the ground. Such behaviorhas been observed to rustling sounds (ownobservations). Preliminary data indicate that theomega neuron of A. nigrovittata not only inhibitsits mirror-image partner, but also some ascendingneurons (Molina, 2004). It seems plausible to usthat the original function of the omega neuron inearly auditory networks of Ensifera was contrastenhancement for directional processing, but thatadditional and potentially species-specific taskshave developed thereafter. More comparativestudies on the behavioral and neuronal level areneeded to test such hypotheses.

ACKNOWLEDGMENTS

We thank Matthias Hennig (HU Berlin) forcomments on the manuscript, Frank Lewitzka(Laser laboratory Gottingen) for help with initiallaser problems, Bernd Ronacher (HU Berlin) foradvice with statistics. We especially thank tworeferees for their extensive and very helpfulcomments.

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effects of pharmacological treatment and photoinactivation on the directional responses of an insect...

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