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AMER. ZOOL., 30:609-627 (1990) Nerve Cells and Insect Behavior—Studies on Crickets1 FRANZ HUBER Max-Planck-Institute fur Verhaltensphysiologie, D 8130 Seewiesen, Federal Republic of Germany SYNOPSIS. Intraspecific acoustic communication during pair formation in crickets provides excellent material for neuroethological research. It permits analysis of a distinct behavior at its neuronal level. This top-down approach considers first the behavior in quantitative terms, then searches for its computational rules (algorithms), and finally for neuronal implementations. The research described involves high resolution behavioral measurements, extra- and intracellular recordings, and marking and photoinactivation of single nerve cells. The research focuses on sound production in male and phonotactic behavior in female crickets and its underlying neuronal basis. Segmental and plurisegmental organization within the nervous system are examined as well as the validity of the single identified neuron approach. Neuroethological concepts such as central pattern generation, feedback control, command neuron, and in particular, cellular correlates for sign stimuli used in conspecific song recognition and sound source localization are discussed. Crickets are ideal insects for analyzing behavioral plasticity and the contributing nerve cells. This research continues and extends the pioneering studies of the late Kenneth David Roeder on nerve cells and insect behavior by developing new techniques in behavioral and single cell analysis. INTRODUCTION This report on nerve cells and insect behavior is dedicated to the memory of the late Kenneth D. Roeder, a founding father of neuroethology, the field of interdisciplinary research which aims to bridge the gap between behavioral strategies and the underlying neural substrates and mechanisms. Zoologists have the opportunity to select among the manifold behaviors formed by evolution, and to choose those where they think an answer is within reach when applying current technological know-how. Their approach is a comparative one and should be evolution-oriented. Zoologists are interested in individual, population and species solutions, in common principles as well as in differences. One should never forget that a cricket differs from a frog, and a crayfish differs from a bird in its demands. Neuroethologists working comparatively have to consider two equally important sets of questions which were formulated by T. H. Bullock (1984) (Table 1). 1 From the Symposium on Science as a Way of Knowing—Neurobiology and Behavior organized by Edward S. Hodgson and presented at the Centennial Meeting of the American Society of Zoologists, 27-30 December 1989, at Boston, Massachusetts. THE KIND OF APPROACH IN NEUROETHOLOGY Neuroethology as the study of the neural basis of behavior favors what I call the topdown approach: a distinct behavioral strategy has to be observed and analyzed first in the field under environmental constraints, then quantitatively studied in the laboratory, and finally explored at its neuronal and, if possible, molecular levels. This top-down approach is chosen because we strongly believe that it is the behavior, shaped and adapted by nature's abiotic and biotic forces, which leads us to pose the right questions to the nervous system. Thus, neuroethologists should become familiar with the concepts, methods, and data the study of behavior has to offer, as well as with the whole scenario of modern neurosciences, including approaches at the system, cellular and molecular levels (Huber, 1988, 1989). ACOUSTIC COMMUNICATION IN CRICKETS: A FAVORABLE STRATEGY Orthopteran and homopteran insects were among the first within the animal kingdom to have evolved hearing and sound production for intraspecific and interspecific interactions. For pair formation and reproduction, the main topics here, the information encoded in the send- 609 610 FRANZ HUBER TABLE 1. Aims of comparative and evolution-orientedbe considered veuroethology. the subsequent as a releasing stimulus for one (for literature see Loher 1. What are the neural correlates and causal rela- and Dambach, 1989). tionships to known behaviors and behavioral dif2. How to improve effective calling ferences among animals? and sound radiation 2. What are the behavioral correlates and causal relationships to known neural differences among anMole crickets have developed tactics to imals? make calling songs more efficient. They (Statements made by T. H. Bullock, 1984.) produce them in a burrow which they modify to an exponential horn which amplifies sounds of the correct carrier frequency. er's acoustic signals must be decoded by Flying conspecifics hear such signals already the receiver. The nervous system of the at distances of several hundred meters sender (usually the male) generates sound which guide their orientation (for literasignals which are species-specific in their ture see Bennet-Clark, 1989). frequency spectra and their temporal orgaMale tree crickets (Oecanthus burmeisteri) nization (Fig. 1) (for literature see Bennet- improve sound intensity and radiation by Clark, 1989). The nervous system of the a baffle. They cut a hole into a leaf, into receiver (usually the female) has to fulfill which they place themselves and sing (Protwo equally important tasks: it must be able zesky-Schulze et al., 1975). to discriminate conspecific songs from abiotic and biotic noises in order to rec- 3. Satellite behaviors ognize them (song-recognition), and it must In Gryllus integer only some males call localize the sender's position in space (song- and attract female crickets (also females of localization) (for literature see Schildber- the parasitoid flies, Euphasioptery ohracea ger et al., 1989). [Cade, 1975]), whereas other males are In insects, these distinct sender-receiver silent, surround the caller and are named interactions have evolved during the course satellites. If on her way to the singing male of phylogeny; they are formed during the female meets a satellite male, he is able ontogeny and based mainly on genetically to court and to mate with her (Cade, 1980). fixed patterns of behavior. In my report I The physiological conditions responsible will concentrate on crickets and consider for calling or noncalling are still unknown. Advantages and disadvantages for callers the topics listed in Table 2. and noncallers have been discussed, but will SOME BEHAVIORAL STRATEGIES IN not be mentioned here. CRICKETS Although crickets are best known and famous for their songs and acoustically mediated behavior, they have evolved other strategies which should interest us because they point to multisensory and multimodal conditions demonstrating that the cricket's world is not solely acoustic (Huber, 1988, 1989). Here are a few examples. 1. Behaviors involved in pair formation, reproduction and aggression During the reproductive season adult male and female crickets display sequences of distinct behavioral patterns which serve pair formation and mating as well as individual spacing, aggression and territorial defence. Each single behavioral event can 4. Prey-predator strategies Many cricket species are nocturnally active. Sound traps broadcasting the conspecific song attract flying males and females from far away (Ulagaraj and Walker, 1973; Walker, 1982). During their nocturnal flights these animals can be preyed upon by echolocating and hunting bats. Teleogryllus has evolved avoidance strategies (Moiseff et al., 1978). The animals hear ultrasonic sounds and process them in distinct neurons (Moiseffand Hoy, 1983) which control their turning away from the sound source (for literature see Pollack and Hoy, 1989). Acheta domestica in southern France is known as prey for a parasitic digger wasp 611 NERVE CELLS AND INSECT BEHAVIOR Calling songs Frequency spectra IHHtlHH* Gryllus campestns 2 4 6 8 1 0 1 2 4 6 8 10 12 14 k H z 1 6 6 10 12 14 kHz 1 4 1 6 kHz 2 0 Teleogryllvs oceanicus Melanogryftus desertus Oecanthus pellucens 2 2 4 FIG. 1. Calling song patterns and frequency spectra in different species of crickets (modified from Huber, 1990). of the genus Liris. The flying wasp patrols the cricket area then lands and approaches a cricket on foot. In the case of no escape, the wasp stings and paralyzes the cricket. The prey is then carried as food to the wasp's nest. But crickets have developed a warning system, consisting of an arrangement of filiform hair sensilla on their cerci. A flying and fast walking wasp creates air currents strong enough to stimulate the filiform hair sensilla and to elicit activity which is then transmitted to different ascending interneurons within the ventral nerve cord. Their activity controls quick defensive and escape responses, such as head stand, kicking with the hindlegs, and running away (Gnatzy and Heusslein, 1986; for literature see Gnatzy and Hustert, 1989). 5. Combined visual and acoustical cues for orientation Many crickets have well developed compound eyes and ocelli. Some Nemobius species use dark contours as landmarks to find their home territories. If they are prevented from seeing these landmarks, celestical cues suffice for orientation (for literature see Honegger and Campan, 1989). By using a closed loop walking compensator (Fig. 2), which allows the unrestrained animal to chose direction and speed of walking on the surface of a tread- mill while being kept in place by the counterrotating treadmill, we recently found that female Acheta domestica track optical targets such as black squares (Atkins et al., 1987). When given a choice between a black square and a conspecific calling song (Fig. 3), the female previously tracking a visual target switches to track the calling song, but only if its temporal pattern lies within the attractive range (Stout et al, 1987). During orientation to the sound source she performs a zig-zag walking course which is characteristic for phonotaxis and expressed as a pattern of several steps interrupted by short pauses. During orientation to the visual target she lacks that kind of walking mode (Weber et al, 1987). Thus, there seems to be a different interfacing between the visual and the acoustical recognition system and the walking generator. The shift in walking modes indicates the change in the cricket's attention and in the modality being processed. TABLE 2. TOPICS. A. B. C. D. E. Some behavioral strategies in crickets. The cricket's nervous system. Behavioral analysis of song recognition. Cellular correlates for song recognition. Behavioral and neuronal aspects of song localization. F. Song orientation in one-eared crickets. 612 FRANZ HUBER IR-Camera B o c n°-.Walking direction (°) —- L1 180°- n°30 60 90 120 Time (s) — L2 e-vector detection, receptors sensitive to blue light are required arranged in the dorsal rim area of the compound eyes. Orientation to e-vector apparently works already at illuminations as low as moon light (Labhart, 1988; Labhart et ai, 1984; Weber et ai, unpublished results). 6. Hormones and phonotactic behavior Hormonal effects have long been neglected in cricket acoustic behavior. Quite recently it was reported that adult female Acheta domestica lose phonotaxis and mating after removal of the corpora allata, i.e., glands that produce the juvenile hormone (JH). Both behaviors are restored after reimplantation of the glands or after application of JH (Stout et al., 1976; Koudele et al., 1987). However, subsequent work with female Gryllus bimaculatus showed that allatectomy in the last larval instar did not abolish phonotaxis in the adult (Fig. 5), although no JH was present in the hemolymph (Loher et al., unpublished results). 7. Aggression and phonotaxis Male crickets perform phonotaxis (Fig. 6A) (Weber, 1989; Weber and Hissmann, unpublished results). This strategy allows FIG. 2. Design and analysis for studying cricket unsuccessful calling males in the field to phonotaxis on the treadmill under closed-loop conditions. A. Experimental arrangement with the tread- leave their burrows and search for females mill (center), the infrared sensing and detecting device in the neighbourhood of other calling (IR camera), the electronics to control treadmill males. Moreover, males of many cricket movements (Comp. Electr.), and the broadcast of species are famous for their fights (Alexmodel calling song (L, Comput.). B. Section of a tracking record of a female to loudspeaker 1 (LI) with a ander, 1961). To our surprise we found switch to loudspeaker 2 (L2). Note the zig-zag course that a male which had won a fight displayed during tracking. C. x, y plots of tracking to LI or L2 phonotaxis (Fig. 6B), whereas in the loser respectively. Each trace represents walking for 20 sec. phonotaxis disappeared for several days (A, B, adapted from Kleindienst, 1987; C, adapted (Fig. 6C). Thus, aggression and pair forfrom Huber, 1987.) mation may be linked by a common neuronal and/or hormonal mechanism which has to be investigated. This finding invites Recently orientation to polarized light neuropharmacological studies to explore (e-vector) has been studied in the genus the physiological basis of winners and losGryllus (Fig. 4) (Brunner and Labhart, 1987; ers. Weber et ai, unpublished results). In comTo sum up: The world of crickets is not petition with conspecific calling songs, entirely an acoustical world. This should phonotactic orientation dominates over guide studies concentrating on multisene-vector orientation, and again the walking sory and multimodal information processmode changes when switching from the ing, which is corroborated by the finding e-vector to the sound source occurs. For that many identified brain neurons encode 613 NERVE CELLS AND INSECT BEHAVIOR cm/s 1 O ] WlWWrfcrWllWl^^ 360° optical target 1 min Square FIG. 3. Visual and acoustical orientation of female Acheta domestica. A. Arrangement of the treadmill with a horizontal platform leaving an area of ca. 20 cm in diameter of the sphere open (dashed circle) for the cricket's movement. The pulsed-infrared scanning system is shown above this area. The vertical cylindrical curtain provides homogenous illumination by a ring lamp on top of the scanning system. The curtain is acoustically transparent. The direction of acoustical stimulation (L) and the visual target (square) are shown for 90° separation. B. Tracking an optical target (position indicated by arrow head on the left). C. Tracking a model calling in the attractive range (60 ms syllable period SP) with a zig-zag course. The upper traces in B and C show the compensatory sphere velocity, and the different walking modes, i.e., more steps and longer pauses during tracking the square, and a spiky walking with fewer steps and shorter pauses during tracking the calling song. The lower traces show the direction of the sphere motion caused by the female's walking. In C the horizontal line denotes the direction of the loudspeaker (modified from Weber et al., 1987). multimodal stimuli (Schildberger, 1981, 1984a). THE CRICKET'S NERVOUS SYSTEM Crickets became suitable model systems for neuroethological research not only because of their clear cut and measurable acoustic behavior but also because of the organization of their nervous system which favors analysis of sound production, pair formation as expressed by phonotaxis, and avoidance behavior down to the single neuron level (for literature see Kutsch and Huber, 1989; Schildberger et al., 1989; Pollack and Hoy, 1989). As shown in Figure 7 the nervous system is divided into discrete ganglia. Moreover, each ganglion (and even nerve cells) has a bilateral and a mirror image arrangement (Huber, 1989). This facilitates studies of segmental motoneuronal (Bentley, 1969; Hennig, 1989) and neuromuscular interactions responsible for driving and controlling the forewings during stridulation. It enabled us to analyse plurisegmental interactions, especially the influence of the brain upon the thoracic song generator, and the effects of wing sensory systems on adapting wing handedness and tooth impact (for literature see Kutsch and Huber, 1989). In this respect one should never forget that large parts of the body are employed in a single behavioral act. When a male cricket calls it not only moves the forewings periodically, but also suppresses fast walking, lifts the antennae to a position characteristic of calling and raises the body from the ground. In addition, abdominal ventilation is synchronized with the chirp rhythm (Huber, 1960; Koch, 1983). These two rhythmically produced motor patterns are probably under the control of two sets of alternatively active plurisegmental nerve cells which feed information from the subesophageal ganglion down to the respective motor generators (Otto and Campan, 1978; Otto and Weber, 1982; Otto and Amon, 1986). Furthermore, cricket song is another case of a growing number of rhythmic behaviors organized by a central pattern generator (for definition see Selverston, 1980), which is efficiently controlled by sensory feedback from the wings (see Kutsch and FRANZ HUBER 614 homogeneous light -i- sound intact (control) 11, SO d8 L2. 50 dB 4 90° B f=T 1 180° L 1 270° =fO L2 polarized light + sound /////////////////? M/////////////!!'/////////J ////// lU^' 50 dB allatectomized FIG. 5. Phonotaxis of intact female Gryllus bimaculatus (control) and of adult females after allatectomy in the penultimate larval instar (allatectomized). For explanation see Figure 4 (courtesy of Loher et al., unpublished). Huber, 1989) and the cerci (see Dambach, 1989). BEHAVIORAL ANALYSIS OF SONG RECOGNITION In the past, my laboratory has concentrated mainly on high-resolution behavioral experiments developed to elucidate the acoustical constraints of female phonotaxis, a behavior, which expresses both song recognition and localization of the caller (for literature see Weber and Thorson, /////////f/l///!_ lm 1989). That behavior was combined with ///////////////Fiirrp a search for single nerve cell correlates and causal relationships (for literature see ////////////////7////777T7 FIG. 4. Relative frequency of tracking angles of female Gryllus bimaculatus to model calling songs and Schildberger et al, 1989). homogenous light (A), to model calling songs and polarized light of different e-vector orientations (B), and to polarized light of different e-vector orientations alone (C). The position of the sound source is marked by LI and L2. The experiments were carried out with changing loudspeaker positions from LI to L2 (A, B), and by increasing sound intensit) (from 50 to 80 dB SPL). Note that neither homogenous light nor polarized light at different e-vector orientations abolished phonotaxis (A, B). In C orientation to polarized light is demonstrated by changing the e-vector in steps of 45°. 1 m gi\ es a calibration for the tracking angles (courtesy of Weber et al., unpublished). NERVE CELLS AND INSECT BEHAVIOR 615 /////////////////////////////////// //////////'(/)itiff/fffffffflfffffiff //////////////////////////////////// 1 m / / / / / / / / / / / / / loser, 20 mm after combat FIG. 6. Male Gryllus campestris phonotaxis (A) persists in the winner of the combat (B) but is lost in loser (C). For explanation of the details compare Figures 2 and 3 (A) and Figure 4 (A, B). (Courtesy of Weber, 1989; Weber et al, unpublished.) 1. Phonotaxis in the field and in a closed-loop arrangement Crickets perform phonotaxis by means of walking or flying in the field (Klopffleisch, 1973; Walker, 1982). In order to evaluate the important calling song parameters for phonotaxis, a closed-loop setup was developed, as already described (Fig. 2). Using this experimental tool we found a threshold for phonotaxis to model calling songs usually around 50 dB SPL. By changing loudspeaker positions the female changed her orientation often within seconds and she performed a zig-zag course while tracking the sound source by meandering ca. 40° around the loudspeaker direction (Wendler et al., 1980; Weber et al., 1981). This phenomenon will be treated later. 2. Carrier frequency and walking angle Calling songs of the correct temporal pattern but with a wrong and higher than natural carrier frequency did elicit anom- FIG. 7. Aspects of the cricket's nervous system. A. CNS of Gryllus campestris with the ganglia, connectives and lateral nerves. B, brain; SEG, subesophageal ganglion; T l - 3 , thoracic ganglia; A3-7, free abdominal ganglia. In crickets Al and A2 are fused with T3. B. Prothoracic ganglion with the bilateral arrangement of the auditory nerve bundles (dotted areas) within the leg nerve (LN) and the left and right auditory neuropiles (LAN, RAN). C. Structure of the mirror image Omega cells (ONI L—black) (ONI R— hatched). Arrows indicate the excitatory auditory input to the left and right cell respectively. D. Scheme of a transversal section through the mesothoracic ganglion to demonstrate the bilateral arrangement of main neuropile areas (hatched vertically and horizontally) and several of the mirror image fiber tracts (hatched densely and oblique). DIT, dorsal intermedia! tract; DLT, dorsal lateral tract; DMT, dorsal medial tract; LVT, lateral ventral tract; VIT, ventral intermedial tract; VT, ventral tract. (Modified from Huber, 1990.) alous phonotaxis. The female tracked the sound source with an erroneous angle and this angle was carrier frequency dependent (Thorson et al., 1982). She behaved as if the sound source had changed in space. The mechanism for anomalous phonotaxis is not completely understood. However, anomalous phonotaxis clearly demonstrates that songs with wrong carrier frequencies but correct patterns do not abolish recognition but influence sound localization. 616 FRANZ HUBER song recognition, as discussed in the next section. CELLULAR CORRELATES FOR SONG RECOGNITION 1. Functional properties of cricket ears and prothoracic auditory interneurons The auditory pathway in crickets begins with the ears (tympanal organs) located within the proximal parts of the foretibiae. FIG. 8. Behavioral tuning (bandpass-property) to a specific range of syllable repetition rates (SRR) during Each auditory organ consists of about 50phonotaxis of female Cryllus campestris presented in 60 auditory sense cells arranged in rows a "to and fro" sequence. The dotted areas indicate and attached to the upper wall of the inner the degree of variation in the response of all females trachea (Eibl, 1978). This arrangement of tested with a preference near 30 Hz, and the black auditory sense cells reflects tonotopicity, dots denote the extreme values. (Modified from i.e., auditory receptors according to their Thorson etai, 1982.) location are tuned to different sound frequencies: proximal receptors respond 3. Bandpass-property for song recognition preferably to lower, distal receptors to By changing one of several temporal higher frequencies (Oldfield et al, 1986). parameters of the calling song, we found Thus, the ear analyzes frequencies, an abilone parameter especially important for ity required, for instance, to encode calling song recognition: the syllable repetition rate and courtship songs having different car(SRR) (Fig. 8) (Thorson et al., 1982), rier frequencies as well as ultrasonic sounds whereas even considerable changes in other (for literature see Bennet-Clark, 1989; Polparameters were much less critical. This lack and Hoy, 1989). indicates that at least Gryllus campestris and Sound intensity is encoded in the spike Gryllus bimaculatus have developed a win- frequency of the auditory sense cell. Moredow for attractive SRRs in phonotaxis, a over, auditory receptors at their best frebandpass, ranging from about 19-43 Hz, quency copy the temporal structure of the and peaking around 25-35 Hz. The prob- song, but, with an important restriction: lem of a trade-off strategy in song pattern they are not specifically tuned to the timing recognition, i.e., the evaluation and of the conspecific pattern (Esch et al., 1980). weighting of several parameters, is still This indicates that the ears of crickets cover unsolved and will not be discussed here a much wider range of sound frequencies (Stouts al., 1983, Doherty, 19856, c). and copy a broader range of patterns than used for intraspecific communication. From 4. Temperature: Sound production and a biological point of view, this is not at all phonotaxis surprising because the ears have also Crickets are poikilothermic animals. evolved as sensory devices for predator They have to match acoustic communica- avoidance where different sound frequention patterns with changes in ambient tem- cies and temporal patterns are used (Huber, peratures. "Hot" males produce faster 1989). Auditory nerve fibers project to the proSRRs than "colder" males, and equally acclimatized females are tuned to them, thoracic ganglion and terminate within a i.e., they shift their bandpass, respectively part of the ring tract, an area called the acoustic neuropile. Each ear is represented (Doherty, 1985a). To sum up: Our finding that the SRR is by fiber terminals only in the correspondthe most important recognition parameter ing hemiganglion (for literature see Schildencouraged us to search for neuronal cor- berger et al., 1989). Within the prothoracic relates and to propose a mechanism for ganglion auditory information is transmitS P [ms] S R R [Hz] 23 43 28 36 35 29 43 23 53 19 67 15 81 12 100 10 NERVE CELLS AND INSECT BEHAVIOR 617 ted to a family of neurons which exist as mirror image pairs, and some of them have been identified by intracellular recording and staining in several genera of crickets. We can distinguish local prothoracic interneurons such as the Omega neurons (ON) with arborizations restricted to both halves of the ganglion, ascending (AN) and descending (DN) plurisegmental neurons, projecting to the brain or to lower parts of the ventral nerve cord, and neurons with T-shaped structures (TN). Only for Teleogryllus commodus has monosynaptic transmission between auditory afferents and AN1 and AN2 neurons been substantiated (Fig. 9) (Hennig, 1988). But there is no indication of specific tuning in any of these prothoracic interneurons to SRRs necessary for the female to exhibit phonotaxis. This led us to search for cellular correlates of song recognition in the next station of the auditory pathway, the brain (Schildberger, 19846). 2. Song recognition by local brain neurons Based on behavioral studies, conspecific song recognition requires neurons sensitive to phonotactically effective SRRs. Auditory information is conducted to the brain via ascending interneurons (AN1, AN2 and probably others) and processed there by at least two classes of local brain neurons (BNC1, BNC2). According to anatomical arrangements and latency measurements, Schildberger (19846) proposed a cascade of events in each brain hemisphere: Ascending neurons feed song information into members of BNC 1 cells, and they and BNC2 cells are targets for further information processing. Within the classes of BNC 1 and BNC2 cells three functional types were discovered (Fig. 10A), (i) Neurons acting as highpass filters (HP-F) by responding to faster SRRs and (ii) neurons acting as lowpass filters (LP-F) by responding to slower SRRs, both covering a range inside and outside the phonotactic attractiveness, (iii) Within the class of BNC2 cells, a subclass was identified with a bandpass-property (BP-F), i.e., cells that responded only to those SRRs to which the female on the treadmill exhibited phono- FIG. 9. Indications for monosynaptic connections in Teleogryllus oceankus between auditory afferent fibers (HNF) and two ascending auditory interneurons (AN 1, AN2) located within the prothoracic ganglion, studied with electrical stimulation of the auditory nerve. A. Prothoracic ganglion with the structure of AN1 (left) and AN2 (right). Arrows indicate the position of the intracellular electrodes (for the auditory afferent fibers only shown left). B. Superimposed traces of spike potentials at expanded scale to demonstrate the time relationships between afferent auditory fiber spikes and postsynaptic responses in AN1 and AN2. Top to bottom: HNF, auditory afferent fiber; AN1, received excitatory input mediated by a low frequency (4 kHz) receptor fiber; AN2, received excitatory input from a high frequency receptor fiber. In each trace the initial deflection indicates the stimulus artefact. AN1 and AN2 exhibit short and constant latencies to the stimulus of 3.5 ms, and latencies to the onset of the receptor spike of ca. 1 ms (AN1) and 0.6 ms (AN2). (Modified from Henning, 1988.) taxis. But these cells showed no pattern copying of the syllable rhythm and they lost encoding of sound intensity at moderate and higher SPLs. Schildberger (see Huber and Thorson, 1985) proposed a model (Fig. 10B) about a cellular and network mechanism for song recognition in the brain, based on the ANDgate property. However, the remaining gap 618 FRANZ HUBER 75- 75 _ 3? x as O O c o 0) CD Q. CO 2 5 25 26 50 Thorax B 74 98 Syllable interval [ms] Brain BP-F Chirp • ••• SI Auditory Pathway ANDGate -Phonotaxis LP-F FIG. 10. Neuronal correlates for song recognition in the brain of Gryllus bimaculatus. A. Correlation of the phonotactic response (hatched area indicating a band-pass property, peaking around 30 Hz SSR) with the activity of bandpass neurons BP-F (marked by open circles for different females and by closed circles connected by lines in one female). Other local brain neurons of the same classes exhibit either highpass properties (HPF, triangles) or lowpass properties (LP-F, squares) responding preferably to faster or slower syllable repetition rates, respectively. Note that HP-F and LP-F form the boundaries of BP-F. B. Model to explain how the bandpass property for attractive SRRs could arise from AND-gating highpass and lowpass neurons. (A, modified from Schildberger, 19846; B, modified from Huber and Thorson, 1985.) involves our ignorance of the detailed neuronal implementation, especially with respect to synaptic mechanisms and connectivities among the cells. 1981). It allows sound to travel to the tympanum from outside and via the acoustic trachea from inside. Thus, vibrations of the tympanum, necessary for hearing (Kleindienst etal, 1983), are based on presBEHAVIORAL AND NEURONAL ASPECTS sure and phase differences of the sound OF SONG LOCALIZATION waves impinging on the tympana. 1. Cricket ears as pressure gradient Pressure gradient receivers have cardoid receivers directional characteristics, i.e., the sense The cricket ear is a pressure gradient cells are excited with different strengths receiver (Fig. 11 A) (for literature see Lar- depending on the angle of sound incidence sen et at, 1989). The tracheal system to (Fig. 11B) (Boyd and Lewis, 1983). Both which the tympana are connected acts as ears exhibit nearly mirror image direcan internal sound conducting pathway tional characteristics with a frontal and (Kleindienst, 1980, 1987; KleindienstWaZ., caudal intersection point. During phono- 619 NERVE CELLS AND INSECT BEHAVIOR cavity 2 step attenuator - stimulus generator inhibitory stimulus (dB) 65 75 85 95 FIG. 11. Cricket ears as pressure gradient receivers. A. Prothoracic segment opened and seen from a frontal view with the two ear bearing forelegs. ATY, anterior tympanum; PTY, posterior tympanum; AT, acoustic trachea connecting both ears; PTG, prothoracic ganglion; SR, SL, right and left stigma (lateral opening for sound entrance). B. Cardoid and mirror image directional characteristics of Gryllus campestris ears, obtained by recordings from whole auditory nerves of left (L) and right (R) ear. Polar plots show evoked responses to single sound pulses of 20 ms duration and 5 ms rise/fall time, averaged over 128 presentations and delivered at constant sound intensities (70 dB SPL) and of 4.8 kHz from different angles. The two directional curves cross frontal and caudal, and exhibit greatest left-right differences to lateral stimulation (adapted and modified from Boyd and Lewis, 1983). C. Diagram to explain binaural directional hearing based on the algorithm "turn to the side more strongly stimulated." According to the directional responses of the left and the right ear their information is fed into left and right central prothoracic ascending neurons (NL and NR), respectively. Their activity is compared by a central comparator (possibly located within the brain) which evaluates left/right excitation differences (AIR/L) for correcting the course, indicated by zig-zagging of the female during phonotaxis (adapted from Huber, 1987). tactic tracking crickets follow the algorithm "turn to the side more strongly stimu- lated." Their zig-zag course allows them to pursue the frontal crossover point where the left and right intensity and excitation differences are minimized (Fig. 11C) (for literature see Huber, 1987). E 4 1 45 ' 55 ' r—constant stimulus 65 dB *\ ' 0 65 excitatory stimulus (dB) FIG. 12. Quantitative analysis of contralateral inhibition in the Omega cell type 1 in response to 5 kHz sound signals. A. Closed sound field arrangement (leg phones = miniature sound chambers) for external and internal isolation of excitatory and inhibitory inputs to the ONI (fi). Ml, M2 microphones 1 and 2 acting as miniature loudspeakers. B. Response characteristic ( • — • ) and latency (O O) of the Omega cell for various excitatory and inhibitory stimulus settings. Symbols represent means of 30 consecutive sound presentations with standard deviations. Sound pulse duration: 50 ms, rise and fall times: 2 ms (adapted from Kleindienst el al., 1981). 2. Processing of monaural and binaural auditory input by prothoracic interneurons, and network analysis With the invention of the legphones (Fig. 12A) {i.e., miniature sound chambers around the ear) (Kleindienst et al., 1981), each ear could be stimulated separately after severing the acoustic trachea connecting both ears while recording simultaneously from different types of prothoracic interneurons. Thus, binaural and monaural inputs to these neurons and some network properties could be studied (Wohlersand Huber, 1982). 620 FRANZ HUBER pattern copying in these cells (for literature seeSchildberger^a/., 1989; Huber, 1989). 3. A direct approach to song localization by hyperpolarizing single ascending interneurons Network studies of prothoracic neurons and effects of cell killing (see also Atkins et al., 1984) led to the assumption that some of these neurons are involved in song localization. To test this hypothesis, an openloop arrangement was used to study phonotactic behavior and single cell responses simultaneously in female Gryllus 60 0 100 200 300 0 100 200 300 Time (s) Tims (ms) Time (mi) bimaculatus (Fig. 13) (Schildberger and Horner, 1988). FIG. 13. Correlation and causal relationship between phonotactic course and the activity of an ascending To obtain a causal relationship between auditory interneuron in the cricket, Gryllus bimacu- the phonotactic course and neuronal activlatus. A. Open-loop experimental arrangement for intracellular recordings from walking animals. The ities one needs reversible manipulation of animal is fixed on a holder and can only walk straight single neurons during the behavioral perforward, but the legs can turn an airsupported sty- formance which cell killing does not offer. rofoam ball. A special camera (IR) senses two com- The female cricket was mounted as shown ponents of the ball's (animal's) movement—rotation and translation—and thus its turning tendency. Call- in Figure 13A; it could only walk straight ing song is delivered by one of two loudspeakers (L, forward but turned an airsuspended ball R) 50° on either side of the longitudinal axis of the with its legs, and ball rotation was mearestrained animal. The computer controls model call- sured. One loudspeaker positioned 50° to ing song broadcast, and later evaluates tape recorded the left or to the right in azimuth to the movements of the animal and neuronal activities. B. Graph with rotation of the animal by sound delivered body axis of the female broadcast the callfrom the left speaker (a), from the right speaker (b), ing song while either AN1, ONI, or AN2 and to the right (c) after the left AN1 neuron had neurons were recorded intracellularly and been hyperpolarized, despite sound stimulation from later on manipulated electrically. With a the left. C. Outlines of the prothoracic ganglion with the mirror image AN1 cells (LAN], dendritic field model calling song presented via the left and axon on the left and RANI, dendritic field and loudspeaker, the left AN1 neuron (with the axon on the right). In both neurons the axon courses dendritic field on the left side) (Fig. 13C), to the brain. D. Response patterns of AN1 cells to a was more strongly excited and copied the four syllabic calling chirp (bottom trace). Further pattern (a, in Fig. 13D). The animal folexplanations are given in the text (adapted from lowed the algorithm "turn to side more Huber, 1989). strongly excited" and turned to the left sound source (a, in Fig. 13B). By considering the directional characteristic of the Between the mirror image ONI type right ear, under this condition the right neurons reciprocal inhibitory connections AN1 neuron must have been less excited were discovered which serve to increase (a', in Fig. 13D). When the calling song was the binaural contrast (Fig. 12B). This was broadcast via the right loudspeaker, the further established by selective cell killing animal turned to the right (b, in Fig. 13B). with photoinactivation (Selverston et al., While still recording from the left AN1 1985), which removed inhibition from one neuron it was now less excited than before ON 1 neuron upon the other. When testing (b, in Fig. 13D). On the other hand, the the animal after killing of one ON 1 neuron mirror-image partner cell on the contraon the treadmill, it tracked the sound lateral side was now more strongly excited source with a slightly erroneous angle. (b\ in Fig. 13D). Morever, ONI-neurons inhibit contralatThe crucial experiment for establishing eral AN1 and AN2 neurons, which may assist to enhance directionality as well as the hypothesis of binaural comparison was NERVE CELLS AND INSECT BEHAVIOR to hyperpolarize the left AN 1, i.e., to reduce its activity even below the level of its right mirror image cell (compare c with c' in Fig. 13D) and to broadcast the sound from the left, its excitatory side. An animal with a nonhyperpolarized left AN 1 cell turned to the left (a, in Fig. 13B). As predicted by the rule, the animal changed its course to the right when the neuron was hyperpolarized (c, in Fig. 13B). This indicates that the system responsible for orientation must have received "wrong directional information" due to the reduced activity of this single hyperpolarized cell. The effects of hyperpolarizing single AN2 and ONI neurons caused less dramatic changes in walking courses (Schildberger and Horner, 1988). 3. Pattern dependence of sound localization 621 and the activation levels of these two neurons corresponded only if pattern copying of the neurons was considered. With calling song from above and continuous tone from ipsilateral to the neurons recorded, their pattern copying was masked, whereas in the contralateral neurons pattern copying was still maintained. These results demonstrate that the overall activity of the two mirror image ascending neurons does not directly guide orientation. It first has to pass a filter tuned to the temporal structure of syllables and chirps. Thus, recognition of the conspecific song and localization of the sound source are not independent events. SONG ORIENTATION IN ONE-EARED CRICKETS 1. Walking courses in one-eared crickets When Pollack et al. (1984) studied sound The algorithm based on binaural comfrequency effects during tethered flight and parison predicts that animals with only one compensated walking in Teleogryllus oceani- ear ought to circle to the side of the cus, they found a larger shift in angular remaining ear. However, Huber et al. error of tracking at the same high fre- (1984), Schmitz et al. (1988) and Schmitz quency (15 kHz) after the sound pattern (1989) found that more than 30% of monhad changed from a four-pulsed calling aural females of Gryllus campestris and G. song to a sound containing a single pulse bimaculatus exhibited phonotaxis even with one ear. These females recognized the conof the chirp rhythm (2/sec). Stabel (1988) and Stabel et al. (1989) ana- specific song by input from one ear, and, lyzed phonotaxis of female Gryllus bimacu- even more surprising, they succeeded in latus with an open-loop device. They tracking the sound source. recorded the turning tendency of a tethIn adult female crickets, one foreleg ered female Gryllus bimaculatus on paired bearing an ear in the tibia was amputated tread wheels in a complex acoustic stimulus between coxa and femur. When the ampuparadigm. When calling song at the correct tation occurred before the 6th instar (4-5 carrier frequency was broadcast horizon- instars before imaginal molt) the leg regentally the female turned to its side as erated often to its full length and size, but expected. However, as soon as the same without an auditory organ. Several extercalling pattern was broadcast from above nal sensory systems reappeared and leg {i.e., without directional cues), and a con- muscle innervation was completed, as inditinuous tone of a slightly different carrier cated by the coordinated walking of the frequency was presented horizontally, the regenerated leg (Huber, 1987; Schildberfemale changed its direction and turned ger and Huber, 1988). away from the horizontal sound source. When a calling song was broadcast from This sign reversal in turning was then stud- a horizontal direction, some females kept ied at the level of two ascending auditory course, although with an error angle usuinterneurons, AN1 and AN2, recorded ally below 90°, which in nature would guide extracellularly from the cervical connec- them through an arc to the singing male tives while the animal walked. Both the (Fig. 14A, C). Phonotactic tracking was characteristic curves in turning tendency most commonly elicited in a smaller sound (toward the sound source or away from it) intensity range, preferably at lower inten- 622 FRANZ HUBER with the central neurons of the deprived side. Or, crickets can switch from the mechanism of binaural comparison to one with consecutive measurements of monaural input, that is, to a scanning mechanism, since auditory excitation varies according to the directional characteristics of the remaining ear. Here I only discuss results that favor the first alternative; for the second, refer to Schildberger and Kleindienst (1989). -1.2 FIG. 14. Phonotactic tracking in one-eared Gryllus bimaculatus females to model calling song. A. x, y plots of sound source dependent walking courses of an intact female. C. Change in walking angle of the same female after amputation of the right foreleg. Note the deviation from the sound source by about 70° toward the left ear. B. Precise phonotactic tracking of an adult female which lost the right foreleg in an earlier larval instar and regenerated it to its normal length, but without a functional auditory organ. D. No change in walking courses in the same female after amputation of the previously regenerated right foreleg. L 1, 2, loudspeakers 1 and 2, respectively, positioned 135° apart. Sound intensity was 70 dB SPL. Each trace represents a 20 sec walking (adapted from Huber, 1987). sides, and it could be observed in some adults already within 24 hr after ear loss. Course accuracy in some adults improved when the foreleg had been amputated in earlier larval instars (Fig. 14B, D), or in adults, after a week or two had elapsed between amputation and test (Schmitz et ai, 1988; Schmitz, 1989). Some females tracked the sound source as accurately as binaural animals. This led to the question, what kind of localization mechanism is involved in monoaural crickets. There are, in principle, two mechanisms which could account for tracking the sound source with only one ear. Either the cricket reorganizes the auditory pathway so that the remaining ear drives a central bilateral system which would allow central comparison. Such a mechanism would imply that the existing ear makes new connections 2. Bilateral central comparison due to changes in structure and function of central prothoracic auditory neurons In adults which had lost one foreleg in a larval instar and regenerated it, or in adults with enough time elapsed between amputation, bilateral comparison could result from two different mechanisms: (i) primary auditory fibers, known to terminate preferably within the ipsilateral auditory neuropile, could grow processes across the ganglionic midline to meet neurons with dendritic fields in the contralateral hemiganglion. Many cobalt backfills of auditory nerves in monaural crickets revealed that only very few primary auditory fibers crossed the ganglion midline, and perhaps not more than already seen in binaural animals (Schmitz, 1989). It is still unknown whether these fibers carry auditory information from the remaining ear to the contralateral central neurons deprived from their previous auditory inputs. A second alternative is that central auditory neurons, the target cells of primary auditory fibers, change their morphology and function after monaural deprivation. This was found in several of the identified local and ascending interneurons in different cricket species (for literature see Schildberger et ah, 1989). The previously deafferented neurons grew dendrites from their former input area which crossed the ganglion midline to invade the auditory neuropile of the intact side (cf, Fig. 15 A, B). These cross-grown dendrites made functional connections, very probably with primary auditory fiber terminations, which were manifested by their responses to stimulation of the remaining intact ear. Thus, NERVE CELLS AND INSECT BEHAVIOR they were rewired with the "wrong ear." According to their threshold curves (Fig. 15C) and intensity-response functions (Fig. 15D), the rewiring created functional properties very similar to intact cells, including the cell specific pattern copying (inset in Fig. 15C). Thus, one can state that the cross-grown dendrites must have "found" terminals of auditory sense cells of the wrong ear comparable to those with which these neurons were previously connected on their intact side, and even the synaptic organization must have been restored. This plasticity was unexpected in crickets and calls for future research in developmental and postembryonic neurobiology. 3. Constraints for phonotaxis with one ear and experimental proofs Despite this time dependent and unexpected reorganization within the prothoracic auditory pathway, the mechanism underlying monaural tracking is not yet explained. If localization in monaural animals is the result of a central bilateral comparison, then one should propose different thresholds, based on synaptic efficacies and/or combinations of excitation and inhibition. Furthermore different slopes of the intensity-response functions should be expected, resulting in different directional characteristics between neurons connected to the remaining ear, and those rewired to the wrong ear. For tracking, monaural animals need an intersection point of the intensity-response functions of a neuronal pair (similar to the frontal intersection point of binaural animals), which would enable them to set an equilibrium between the excitation of both auditory pathways necessary to perform phonotaxis, by following the rule similar to that used by intact binaural crickets (Fig. 16A). Such intersection points have recently been discovered in the intensity-response functions of the pair of AN2 neurons by Schildberger and Kleindienst (1989) (Fig. 16B). Only those females which exhibited phonotaxis on the treadmill (closed-loop) or showed a reverse in turning tendency in the open-loop condition at a distinct sound intensity, had an intersection point 623 FIG. 15. Structural and functional changes in prothoracic auditory neurons (Omega cells of type 1) in one-eared Gryllus bimaculatus, after the loss of the left auditory input in an earlier larval instar. A. Lucifer yellow fill of a left Omega neuron in the intact animal (horizontal view). B. Left Omega neuron with dendrites grown across the ganglion midline from the former input area and arborizations within the contralateral neuropile (which is normally the output area of the cell). C. Comparison of auditory thresholds in normal and deafferented ON 1 cells shows only slight differences within the tested frequency range. Inset: Pattern copying of the deprived ONI cell now wired with the wrong, intact ear. D. Intensity-response curves of intact and deprived ONI cells listed in C. The slopes are very similar except for a decrease in response in the deprived ONI at higher stimulus intensities (adapted from Huber, 1989). in the corresponding neuronal pair correlated within this intensity range. Nonorienting females lacked such an intersection point (cf Fig. 16B, lower left with lower right). Thus, it seems that the structural reorganization of the central auditory pathway in monaural crickets is accompanied by changes in some functional properties of the participating neurons. Both together allow a bilateral central comparison used for tracking. However, one should not forget that only some females tracked the sound source within a restricted intensity range while others, with probably a similar structural reorganisation but a missing functional repair did not (Schmitz et ai, 1988). This and the fact that some females tracked the sound source already several hours to one day after loss of one ear, where the described morphological changes have 624 FRANZ HUBER not been observed (Schmitz, 1989), pose questions regarding normal and regenerative development, functional plasticity in single neurons, and they even point strongly to a second sound orientation mechanism which has recently been found: a monaural scanning device (Schildberger and Kleindienst, 1989). CONCLUDING REMARKS FIG. 16. Evidences for a central bilateral comparison in one-eared crickets with loss of the right ear in an earlier larval instar and after subsequent regeneration of the right foreleg without its ear. A. Left, diagram of the situation: The intact left ear did not change its directional characteristic. Its input reaches both left (NL) and right (NR) prothoracic ascending neurons. They conduct song specific directional information to a central comparator (Com), which evaluates left/ right activity differences (AIL/R) and minimizes them to establish the course. A. Middle, hypothetical and rather parallel intensity—response functions of a neuronal pair with the NR newly connected. Turning tendencies are shown, if the animal follows the algorithm "turn to the side more strongly stimulated." A. Right, hypothetical intensity-response functions of the same neuronal pair with different slopes now intersecting. The intersection point would be a stable point for tracking. Turning tendencies are indicated below and above the intersection point. B. Experimental proof for A. Upper left, examples are shown for one animal (Animal A) which tracked the sound source under closed loop conditions at 60 dB SPL, and for a second animal (Animal B) with the same treatment but without phonotaxis. Upper right, Animal B followed the algorithm "turn to the side more strongly stimulated" by consistently turning to the left under open-loop conditions, whereas Animal A showed a reversal in turning in the range between 60 to 70 dB SPL. At low sound intensities it turned to the side of the deprived ear; at higher sound intensities it changed to the side of the intact left ear. Lower left, intensity—response curves of the mirror image AN2 neurons recorded extracellularly in Animal B with no phonotaxis indicate that the intact and normally wired left A\2-cell is more sensitive to sound within the whole intensity range tested. Thus the left AX2 seems to determine turning toward its side. Lower right, in Animal A, howeser, which exhibits phonotaxis under closed-loop conditions and shows a change in turning under open-loop conditions, an intersection point is seen in the intensity-response The top-down approach applied to song recognition and song localization in crickets was successful because results of highresolution studies of phonotactic behavior guided the questions addressed to single nerve cells. We first learned that the world of crickets is not solely an acoustic world which invites studies of multisensory and multimodal inputs and their processing within the CNS in the context of behavior. We have identified a cellular correlate in the brain with a bandpass-property for SRRs similar to the one obligatory for phonotaxis in the behaving animal. Without, however, knowing the neuronal hardware in detail, we even present a mechanism which could be responsible. Students of animal behavior would perhaps call the bandpass-cells in the brain an element of the innate releasing mechanism (IRM). For song localization the binaural algorithm "turn to the side most strongly stimulated" could be established at the level of a single ascending neuronal pair. However, the comparison and transfer of directional information (possibly within the brain) is still a matter of speculation. Other studies have shown that localization of a calling song source needs patterned information from the ventral nerve cord, which clearly suggests that recogni- curves (IR-function) in the two AN2 cells, within the corresponding sound intensity range (compare upper right with lower right). At lower intensities the deprived right AN2 cell is more strongly excited and determines turning to its side: at higher intensities the intact AN2 cell is more strongly excited which results in turning to the left as expected by following the algorithm (modified and adapted from Schildberger and Kleindienst, 1989). NERVE CELLS AND INSECT BEHAVIOR tion and localization are not based on two completely independent neuronal circuits. Finally, one-eared crickets with their ability to track a sound source have elucidated an unexpected plasticity in the behavior related to structural and functional changes of central neurons. These findings will certainly facilitate further studies on the development and the regeneration power of the auditory pathway, including sensory deprivation or overstimulation in single cells. Thus, the nervous system of the cricket is not completely hardwired. 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