J Comp Physiol A (2003) 189: 143–151 DOI 10.1007/s00359-003-0388-0

ORIGINAL PAPER

G.S. Pollack Sensory cues for sound localization in the cricket Teleogryllus oceanicus: interaural difference in response strength versus interaural latency difference

Received: 1 July 2002 / Revised: 6 December 2002 / Accepted: 18 December 2002 / Published online: 18 January 2003 Springer-Verlag 2003

Abstract Two potential sensory cues for sound location timing as cues. Amplitude differences are generated by are interaural difference in response strength (firing rate reflection and diffraction of sound by the portion of the and/or spike count) and in response latency of auditory body interposed between the ears, or by anatomical receptor neurons. Previous experiments showed that specializations that deliver sound to each ear along these two cues are affected differently by intense prior multiple paths, resulting in direction-dependent inter- stimulation; the difference in response strength declines ference between these multiple inputs (Michelsen 1992). and may even reverse in sign, but the difference in la- Time differences arise because the path length, and thus tency is unaffected. Here, I use an intense, constant tone travel time, from the sound source to each ear varies in a to disrupt localization cues generated by a subsequent direction-dependent manner. Insects are small, resulting train of sound pulses. Recordings from the auditory in correspondingly small interaural time differences. nerve confirm that tone stimulation reduces, and some- For the cricket Teleogryllus oceanicus, for example, the times reverses, the interaural difference in response maximum difference in path length to each ear is strength to subsequent sound pulses, but that it en- approximately 1 cm, corresponding to an interaural hances the interaural latency difference. If sound loca- time difference of approximately 33 ls. Some animals, tion is determined mainly from latency comparison, then e.g., barn owls and humans, can detect microsecond- behavioral responses to a pulse train following tone level interaural differences in stimulus timing (Carr stimulation should be normal, but if the main cue for 1993). However, experiments on insects using dichotic sound location is interaural difference in response stimulation techniques suggest that they can detect only strength, then post-tone behavioral responses should much larger interaural differences in stimulus timing sometimes be misdirected. Initial phonotactic responses (von Helversen and Rheinlaender 1988; Rheinlaender to the post-tone pulse train were frequently directed and Mo¨ rchen 1979). In insects, sound localization is away from, rather than towards, the sound source, in- driven by the interaural difference in stimulus amplitude. dicating that the dominant sensory cue for sound Stimulus amplitude affects the responses of auditory location is interaural difference in response strength. receptor neurons in two ways: response strength (i.e., spike count or firing rate, as well as number of receptors Keywords Adaptation Æ Binaural cues Æ responding) increases with increasing amplitude, and Communication Æ Hearing Æ Insect response latency decreases (Imaizumi and Pollack 2001). Interaural difference in either of these response features might underlie sound localization, and it is not clear whether one or the other is most important for this task. Introduction One way to determine this is to observe orientation responses to sound when these two candidate cues Animals estimate the azimuth of a sound source using are dissociated, for example, with the receptor neurons the interaural difference in stimulus amplitude and/or of one ear responding earlier, yet more weakly, than those of the other ear. The straightforward way to create G.S. Pollack such conditions, independent control of stimulus timing Department of Biology, McGill University, and amplitude at each ear through dichotic stimulation, 1205 Avenue Docteur Penfield, Montreal, is technically difficult in behaving insects (but see Montreal, Quebec, H3A 1B1, Canada E-mail: [email protected] von Helversen and Rheinlaender 1988). Here, I exploit Tel.: +1-514-3986418 a feature of the physiology of receptor neurons, Fax: +1-514-3985069 intensity-dependent response depression, to uncouple 144 interaural differences in response strength and response intensity-dependence of the response decrement, the latency. decrease will be greater in the ipsilateral ear, where Auditory receptor neurons exhibit sensory habitua- stimulus intensity is higher. The binaural difference in tion upon rapidly repeated stimulation, that is they response strength will thus be reduced or, as in the case produce fewer and less frequent action potentials in re- shown in Fig. 1, even reversed in sign. A constant tone sponse to succeeding stimuli (Sippel and Breckow 1984; lacks the temporal pattern of attractive stimuli (Doolan Coro et al. 1998; Givois and Pollack 2000). The response and Pollack 1985; Pollack and El-Feghaly 1993; Hennig decrement is greater the higher the stimulus amplitude, and Weber 1997), and thus no systematic phonotaxis and thus is more pronounced for receptor neurons in the response is expected. The stimulus is then switched to an ear ipsilateral to the sound source. This bilateral asym- attractive temporal pattern, i.e., a series of sound pulses metry in the amount of sensory habituation reduces the presented at an appropriate repetition rate. If the effects interaural difference in response strength. Under ex- of the conditioning tone persist throughout at least the treme conditions, the difference may even reverse in sign; initial portion of the pulse train, then this treatment the more pronounced decrement of the ipsilateral re- should dissociate the two potential binaural cues for sponse may cause it to become weaker than the con- sound direction at the time when a behavioral decision is tralateral response. Repeated stimulation also affects made. Specifically, the interaural difference in response response latency, which increases for successive re- latency should be relatively unaffected by the condi- sponses. However, unlike the change in response tioning tone (because the increase in latency produced strength, the change in latency is independent of stim- by prior stimulation is intensity-independent), whereas ulus amplitude, and thus the interaural latency differ- the interaural difference in response strength should be ence remains constant (Sippel and Breckow 1984; Givois reduced and sometimes reversed in sign. If crickets use and Pollack 2000). latency difference as the main cue for sound direction, In the present paper, intensity-dependent response then post-tone phonotaxis responses to the attractive depression is used as a tool to uncouple interaural dif- pulse train should be directed towards the sound source, ferences in response strength and latency, and the con- as usual. However, if the main cue for sound direction is sequences of this for sound localization are determined the difference in response strength, then post-tone re- by measuring phonotactic responses of crickets. The sponses should occasionally be directed away from, experimental strategy is illustrated in Fig. 1. An intense rather than towards, the sound source. (100 dB SPL) constant tone, with the carrier frequency of cricket song, is played from one side. If a constant tone causes response depression similar to that induced Materials and methods by rapidly repeated sound pulses then, because of the Animals

Crickets, Teleogryllus oceanicus, were reared in the laboratory. Virgin females were used, generally within 2 weeks of the final molt. Crickets were maintained on a 12 h:12 h photoperiod, and behavior experiments were performed within the first 2–3 h of scotophase. Physiology experiments were performed within the first 8 h of scotophase. Except for a few cases, behavior and physiology experiments were performed on different animals.

Stimuli

Digitized sound stimuli were prepared in Microsoft wave file for- mat (sampling rate 44.1 kHz, 16-bit resolution) using custom- Fig. 1 Strategy for uncoupling interaural differences in response written programs. Carrier frequency was 4.5 kHz. Three temporal strength and latency. Top lines are schematic representations of patterns were used: (1) 40-ms pulses (including 5-ms linear on- responses of ipsilateral (black) and contralateral (gray) auditory set and offset ramps) presented once per second; (2) a 10-s train of nerves; bottom line represents stimulus protocol. A constant tone 40-ms pulses, repeated at a rate of 16 pulses s)1; (3) a 60-s tone (60 s, 100 dB SPL) is broadcast from one side, and is then replaced followed, after a 5-ms pause, by the same 10-s pulse train. The pulse by a behaviorally attractive train of sound pulses. The decrement in duration used, 40 ms, is approximately the upper limit of behav- response strength during the tone is greater the higher the stimulus iorally acceptable pulse durations for T. oceanicus (M. Hennig, intensity, resulting in a decrease, and perhaps reversal, of the personal communication). These longer-than-optimal pulses were interaural difference in response strength, both during the tone and used because pilot experiments showed that this helped to maintain during the initial portion of the pulse train. Both the magnitude the effect of the conditioning tone on responses to subsequent and duration of the difference in response strength shown here are pulses. Sounds were played through a Riptide sound card (Rock- exaggerated for clarity. The shift in response latency due to prior well) driven by Windows Sound Recorder (in a few experiments a stimulation is independent of intensity, and consequently the Cirrus sound card was used, driven by Linux 2.4.19) . Following interaural latency difference for responses to the pulse train is not power amplification (S1-1050G, Sanken-Allegro) and attenuation affected. Immediately following the switch from tone to pulse train, (HP 350B), stimuli were broadcast from either of two loudspeakers interaural latency difference is normal, but interaural difference in (Tandy 40-1310) on the left and right sides of the cricket, perpen- response strength is reduced or, as in this example, reversed in sign dicular to its long axis, at a distance of 45 cm from the midline. 145

Sound level was measured using Bruel and Kjaer instruments (4135 Determination of response direction microphone, 2610 measuring amplifier) and is reported as the RMS level of the stimulus at peak amplitude (i.e., during the constant Recordings of abdominal position were contaminated by noise tone, or during the constant-amplitude portion of the trapezoidal related to the beating of the wings (frequency ca. 30 Hz) and to sound pulses). respiration (0.5–2 Hz), and by slow drift of the baseline position of the abdomen. Wingbeat-related noise was reduced by con- volving the recordings with a 100-ms-wide rectangular window, Behavior experiments but it was sometimes difficult to distinguish phonotactic responses from the other sources of noise. To ensure objective determina- Behavioral responses were monitored by observing abdominal tion of response direction and latency, responses were identified steering movements during flight phonotaxis (Moiseff et al. 1978). by a computer program that scanned the recordings, searching for Crickets were attached at the pronotum to a wooden applicator the first point following onset of the pulse train at which the stick using a mixture of bees’ wax and violin-bow rosin and were voltage representing abdominal position deviated from the mean placed, ventral side uppermost, in front of a fan that produced a pre-pulse-train voltage (calculated for segments ranging from 1.1 gentle wind stream (ca. 1–3 m s)1) to induce tethered flight (either to 10 s) either by three standard deviations, or by 75% of the pre- full flapping flight or flight posture, in which the legs and antennae pulse-train range, whichever was greater. These limits were se- are held in the positions characteristic of flight, and flight muscles lected because the response latencies and directions that they are rhythmically active, but the wings are not opened; see Pollack yielded for control stimuli (pulse-train alone) matched closely and Plourde 1982 for further description and comparison of pho- with judgments based on visual inspection of the recordings. A notactic responses during full flight and flight posture). A fiber- second criterion for response identification was that latency be optic light source cast a shadow of the cricket’s abdomen on a pair between 0.01 and 1 s. The lower limit was set to exclude three of photocells situated dorsal and posterior of the tip of the abdo- ‘‘responses’’ with latencies shorter than those of first-order audi- men. The photocells were joined end to end and masked so that a tory interneurons. The upper latency limit was set because ex- butterfly-shaped region surrounding the junction was left exposed. amination of the distribution of all response latencies showed that Because of the shape of the mask, the amount of the exposed region the majority (75%) were strongly clustered at values well below that is covered by the shadow varies with the left-right position of 1 s, suggesting that the occasional ‘‘responses’’ with longer la- the abdomen. The two photocells were wired together so that tencies might be spurious measurements due either to respiratory movement of the shadow towards the cricket’s right produced an movements or to slow drift of the baseline position beyond the increase in the output voltage, and movement to the left produced a limits described above (as visual inspection of the recordings decrease. Experiments were performed in a chamber suggested in many instances). As expected for spurious move- ments, these were distributed nearly equally between movements (122 cm·61 cm·61 cm) lined with echo-attenuating foam wedges n (Ilbruck). towards and away from the sound source ( =6 and 8, respec- Crickets were selected to show clear responses to the pulse- tively). train stimulus at 80 dB SPL as judged by visual inspection. After tests with the pulse trains, crickets were tested with the tone fi pulse-train stimulus, at 100 dB SPL, presented one to Measuring interaural differences in auditory nerve responses four times (usually once) from each side in alternation, with a 60-s rest between trials. Although stimulus level was nominally The tone fi pulse-train stimulus used in behavior experiments was 100 dB SPL, recordings from the auditory nerve (see below) presented at 100 dB SPL, five times from each side in alternation, showed that the ipsilateral response amplitude to the first few with rest periods of at least 60 s between trials. Interaural differ- sound pulses following the conditioning tone was equivalent to ences were estimated by comparing the responses of a single nerve non-decremented responses to 80–86 dB SPL (mean equivalent to ipsilateral and contralateral stimuli. Digitized responses were levels of 1st and 16th post-tone sound pulses: 79.9±8.5 dB and high-pass filtered (5th order digital Butterworth filter, cutoff fre- 86.2±9.7 dB SPL, respectively, n=15, determined by linear quency=100 Hz) to remove any d.c. offset, full-wave rectified, and interpolation of level-response curves for single sound pulses smoothed by convolution with a Gaussian kernel with standard ) presented at 1 pulse s 1). Following the tests with the tone fi trill deviation of 1 ms. The five responses to stimuli from each side were stimulus, some crickets were tested again with the 80-dB SPL then averaged. Further analyses were performed on these processed pulse-train stimulus. recordings. Interaural differences in response strength and latency were computed for each sound pulse following the tone. Difference in Neurophysiology experiments response strength was measured as the difference of the integrals of the responses to ipsi- and contralateral stimuli over a 50-ms time Neurophysiological recordings were performed in the same cham- window beginning at the onset of each sound pulse. Latency dif- ber as the behavior experiments. Whole-nerve recordings of audi- ference was measured by cross-correlating responses to ipsilateral tory receptors were made with Teflon-insulated silver wires (o.d. and contralateral stimulation over a 20-ms portion of the recording 114 lm) implanted in the femur. The cricket was mounted in a beginning at the onset of each sound pulse. Latency difference was position similar to that adopted during flight, except that the taken as the lag value for which the cross-correlation coefficient forelegs were held away from the pronotum to provide access to the was maximal. anterior surface of the femur for electrode implantation. Further Data analysis was performed using Scilab (http://www.rocq- details are given in Pollack and Faulkes (1998) and Givois and inria.fr/scilab). Statistical tests were performed using the program Pollack (2000). R (http://www.r-project.org).

Data analysis Results

Voltage signals (representing abdominal position, auditory-nerve Nerve recordings responses, and stimulus monitor) were digitized using Digidata 1320A hardware and Axoscope 8.0 software, both from Axon Instruments. Sampling rate for auditory-nerve responses was Figure 2A shows rectified and smoothed responses 10 kHz; abdominal-position traces were sampled at either 1, 5, (see Materials and methods) of the auditory nerve to or 10 kHz. ipsi- and contralateral stimulation with the tone fi 146 b Fig. 2A,B Receptor responses to the tone fi pulse-train stimulus. A Responses to ipsilateral (black) and contralateral (gray) stimulation. The recordings have been rectified, smoothed, and averaged as described in Materials and methods section. Bottom trace shows stimulus pattern. B Three examples of responses to the first two post-tone sound pulses, shown on a magnified time scale

and after approximately 30 s of stimulation the two re- sponses converge. When the stimulus is switched to the pulse train, responses increase in size as the system re- covers partially from the severe depression induced by the tone. In Fig. 2B, three examples of the initial portion of the response to the pulse train are shown on an expanded time scale. Response latency for ipsilateral stimulation is shorter than that for contralateral stimulation, but the overall response to contralateral stimulation is larger than that to the ipsilateral stimulus. Results of 15 experiments are summarized in Fig. 3, where interaural differences in response strength and la- tency are plotted for each of the 160 post-tone sound pulses (10 s at 16 pulses s)1). The absolute magnitudes of the recordings varied widely between experiments, according to the proximity of the recording electrode to the nerve. To facilitate pooling of these results, response–strength differences were normalized, within each experiment, to the mean difference (n=10–20) gen- erated by single, 100-dB sound pulses presented at 1 pulse s)1. Immediately following the tone, the mean difference in response strength is slightly negative. The sign of the first response was reversed (contra>ipsi) in 8 of 15 recordings. The response difference gradually increases until, after approximately 1–1.5 s (16–24 sound pulses) it stabilizes. The interaural difference in response latency, shown in Fig. 3B, is largest shortly following the tone. It decreases gradually as stimulation continues, stabilizing at a value of approximately 1.4 ms.

Behavioral responses to post-tone pulse train

The results shown in Figs. 2 and 3 demonstrate that presentation of a constant tone prior to the pulse train does differentially affect interaural differences in re- sponse strength and in latency. The difference in re- sponse strength is reduced, and sometimes reversed in sign, whereas the latency difference retains its normal sign and is largest shortly after the tone. The behavioral consequences of these alterations in localization cues are shown in Fig. 4. Figure 4A shows phonotactic steering responses of three individuals. In each case, the steering response to the pulse train alone was directed towards the sound source. When the pulse train was preceded by the conditioning tone, the initial response was directed away from the sound source. Results from 25 crickets are summarized in Fig. 4B, pulse-train stimulus. The initial response to tone onset is which plots the proportion of responses (ignoring trials considerably larger for the ipsilateral stimulus. However, on which no response occurred) that were initially mis- the response decrement is also greater for this stimulus, directed for both stimuli. For the control stimulus, 7 of 147

57 responses were classed as misdirected according to initially misdirected. The difference between these is the criteria described in Materials and methods. For the highly significant (P<0.0001, Fisher’s exact test). tone fi pulse-train stimulus, 28 of 40 responses were In the examples shown in Fig. 4A, initially misdi- rected responses were later replaced by ‘‘corrected’’ responses towards the sound source. Similar corrections occurred in 19 of the 28 cases where initial responses

Fig. 3A,B Summary of post-tone binaural response differences. A Mean (±SE) of the interaural difference in response strength, for responses to each of the 160 post-tone sound pulses (10 s at 16 pulses s)1). To facilitate pooling of recordings that differed in absolute amplitude (according to proximity of the recording electrodes to the nerve), data were normalized, for each experiment, with respect to the mean interaural difference generated by 100-dB pulses presented at 2 pulses s)1. B Mean (±SE) latency difference for post-tone responses. n=15 for both graphs

c Fig. 4A,B Phonotaxis responses to stimuli preceded by silence and by conditioning tones. A Recordings of abdominal movements in response to the pulse train alone (black) and to the pulse train preceded by the conditioning tone (gray). Upward movements of the traces represent flexion of the abdomen towards the sound source, indicting an attempt to steer towards the source; downward movements represent flexions away from the sound source. Examples from three crickets are shown. In each case, the initial movement of the abdomen following onset of the pulse train alone was towards the sound source, and the initial movement following onset of the pulse train that was preceded by a conditioning tone was away from the sound source. B Summary of effects of the conditioning tone on steering responses to the pulse train. Graph shows proportions of responses that were directed away from the sound source, for control stimuli, in which the pulse train was preceded by silence, and for trials when it was preceded by the conditioning tone. Trials with no response are omitted 148 were misdirected. The mean (±SD) latency of the cor- source at the onset of the post-tone pulse train. For the rected responses (from onset of the pulse train) was third set, flexion was towards the sound source at tone 2.13±1.94 s. onset, and away at pulse-train onset. Figure 5B compares responses to tone onset and to onset of the post-tone pulse train for the 40 trials in which both re- Misdirected positive phonotaxis or properly sponses could be measured (in the remaining trials the directed negative phonotaxis? response to the onset of the tone was not recorded). Although crickets often did react to the onset of the Flying crickets steer away from ultrasound stimuli tone, these movements were directed randomly (18/40 (Moiseff et al. 1978) and also, occasionally, from intense towards the sound source, 12/40 away, P=0.248, Fish- low-frequency stimuli (Nolen and Hoy 1986). This raises er’s exact test). There was a trend towards increased the possibility that post-tone responses that were probability of non-response at tone onset compared to directed away from the sound source were properly the onset of the post-tone pulse train (10/40 versus 3/40, directed negative responses, rather than misdirected P=0.066). Most importantly, the frequency of responses positive responses. Negative phonotaxis is a short- directed away from the sound source at tone-onset was latency escape response. For T. oceanicus, latency is 25– significantly lower than that at onset of the post-tone 70 ms (Nolen and Hoy 1986). Latencies for phonotaxis pulse train (12/40 versus 25/40, P=0.007). responses in the experiments reported here were much longer, on the order of 350–400 ms (Table 1). Latencies of post-tone responses directed away from the sound Discussion source did not differ from latencies of responses to the control stimulus, either when compared for the data set Pre-conditioning the auditory system with a long-last- as a whole, or within individual crickets in a pair-wise ing, intense, constant tone diminished, and sometimes manner. Latencies were also similar for post-tone re- reversed, the interaural difference in response strength of sponses that were directed away from and towards the the auditory nerve to subsequent sound pulses. By sound source. contrast, the interaural latency difference was enhanced. If post-tone responses were attempts to steer away If latency difference were the main cue for sound local- from the sound source, then they should be even more ization, then steering responses to the post-tone pulse frequent at the onset of the tone, when the auditory train should have been directed towards the sound system is unadapted and the stimulus will be perceived source with similar frequency as for control stimuli, but as of greater intensity (see Materials and methods). if the main cue were the difference in response strength, Figure 5 compares responses at tone onset with those at then misdirected responses should have occurred more the onset of the post-tone pulse train. Figure 5A shows frequently than for the control. Misdirected responses abdominal-movement recordings for three individuals. were frequent following the conditioning tone, suggest- For the top two sets of traces, there was no response at ing that interaural difference in response strength, rather the onset of the tone, and flexion away from the sound than in latency, is the main directional cue.

Table 1 Latencies of phonotaxis responses.Values (in ms) are mean±standar ddeviation. For the pair-wise comparisons, trials for each response class were averaged within each cricket; the values shown are the means of these per-cricket averages. Probabilities were calculated using unpaired (all trials)and paired (pair-wise) t-tests

All trials

Control Post-tone, away Post-tone, towards from sound source sound source

362±188 (n=50) 370±176 (n=28) 382±122 (n=12)

‹ P=0.852 fi‹P=0.817 fi

Pair-wise comparisons

Control Post-tone, away Post-tone, towards from sound source sound source 371±119 (n=17) 379±72 (n=17)

‹ P=0.870 fi

401±373 (n=5) 373±69 (n=5)

‹ P=0.797 fi 149 b Fig. 5A,B Comparison of responses at onset of tone and at onset of post-tone pulse train. A Abdominal-movement traces from three crickets, at the onset of the conditioning tone (black) and at the onset of the post-tone pulse train (gray). Upward movements of the traces represent flexion of the abdomen towards the sound source; downward movements represent flexions away from the sound source. For the top two sets of traces, there was no response to the onset of the tone, and movement away from the sound source at the onset of the pulse train. For the third set, the abdomen flexed towards the sound source at tone onset, and away at onset of the pulse train. B Summary of responses to tone onset and pulse-train onset for 40 trials in which both responses could be measured (in some trials the response at the onset of the tone was not recorded). There were significantly fewer (P=0.007, Fisher’s exact test) movements away from the sound source at tone onset than at onset of the post-tone pulse train

the tone was consistently reduced, but was reversed in sign only in about half the cases. Yet behavioral responses were initially directed away from the sound source with higher frequency (70%). Some of these may have been due to spurious abdominal movements that were nevertheless detected as valid responses by the algorithm used to score behavior (as was presumably the case for the 12.3% of control responses that were tallied as misdirected). It is also possible that the neurophysi- ological measurements underestimated the interaural difference in responses during behavior. To allow access of the recording electrodes to the auditory nerve, the legs were fixed nearly at right angles to the cricket’s long axis, rather than parallel to, and close to, the lateral surface of the pronotum, where they are held during flight. Thus, the acoustical situations during the physi- ological and behavioral measurements were imperfectly matched, and interaural difference in stimulus magni- tude during the physiology experiments may have been less than that during behavior. Another shortcoming of the neurophysiological data is that the recordings in- clude spontaneous activity of non-auditory sensory neurons with axons in the ‘‘auditory’’ nerve. Moreover, the intense stimuli used here would stimulate auditory receptor neurons tuned to mid- and high sound frequencies in addition to the low-frequency-tuned receptors that presumably drive positive phonotaxis (Imaizumi and Pollack 1999). An additional possible explanation for the mismatch between neurophysiolog- ical and behavioral results is that the decrement in response strength measured here for receptor neurons might be amplified at the interneuron level. Recent experiments show that this is the case for responses to ultrasound of receptor neurons and of an identified ultrasound-sensitive interneuron, AN2 (Samson and Pollack 2002); the decrement of AN2 responses induced by repeated stimulation is substantially greater than the decline in receptor responses. The neurophysiological results, nevertheless, do Behavioral reversal is more frequent than expected show clearly that the conditioning tone affects interaural based on neurophysiology difference in response strength and interaural latency difference in opposite ways; interaural strength differ- The neurophysiological data show that the interaural ence becomes weaker, and latency difference becomes difference in response strength immediately following stronger. 150

Post-tone responses are misdirected tone pulse train should have been sufficient to allow positive phonotaxis them to determine correctly the side of stimulation. Latency differences on the order of 1–2 ms are known to It is unlikely that the post-tone responses that were di- be resolved by other insect nervous systems. Under con- rected away from the sound source were genuine nega- ditions approaching dichotic stimulation, the grasshopper tive responses, rather than misdirected positive Chorthippus biguttulus turns reliably (75% accuracy) to- responses. Negative phonotaxis is most readily elicited wards the side where stimulation is earlier with delays as by ultrasound stimuli, but can also be evoked by intense short as 400 ls (von Helversen and Rheinlander 1988). low-carrier stimuli (Nolen and Hoy 1986), presumably Shifts in interaural timing of 2 ms (the smallest value because these stimuli also excite ultrasound receptors tested) strongly affect the responses of binaural auditory (Imaizumi and Pollack 1999). In T. oceanicus, negative interneurons of Locusta migratoria (Rheinlaender and phonotaxis is elicited by 4.5-kHz stimuli only at sound Mo¨ rchen 1979). The fly Ormia ochracea can apparently levels >95 dB SPL, and even then only in about 20% of localize sound based on interaural latency difference of crickets (Nolen and Hoy 1986). Although the tone fi - <10 ls (Mason et al. 2001); however other possible cues, pulse-train stimulus in the present experiments was e.g., activation of different numbers of receptor neurons in presented at 100 dB SPL, the response of the auditory the two ears, have not been ruled out entirely in this case. nerve to the initial portion of the pulse train was equivalent to the unadapted response to a stimulus that was 15–20 dB lower in sound level (see Materials and Behavioral implications methods). The greatest portion of this decrease was al- most certainly in the intensely activated low-frequency- Givois and Pollack (2000) postulated that, because of its tuned receptors, however ultrasound receptors would relative immunity to the effects of sensory habituation, also have been somewhat adapted. The expected fre- interaural latency difference might be the preferred cue quency of genuine negative phonotaxis in the current for sound localization. The present experiments show experiments would thus be less than 20%, whereas the that, despite its sensitivity to sensory habituation, the observed frequency of misdirected responses was 70%. main cue is instead interaural difference in response A second argument that post-tone responses were strength. This might have implications for acoustic sig- misdirected attempts to steer towards the sound source naling. Sexual selection theory posits that females is that the latencies of these responses were indistin- should prefer songs that demonstrate, by their structure, guishable from those of positive phonotaxis responses to that they emanate from males of high genetic quality the control stimulus, as well as from post-tone responses (Brown 1999). Such songs would demand high expen- that were directed towards the sound source (Table 1). diture of energy and would be characterized by high Response latencies in the current experiments (Table 1) intensity, high sound-pulse rate, and long duration. were, on average, five to ten times as long as latencies of Several studies show that females prefer songs with these negative phonotaxis (25–70 ms, Nolen and Hoy 1986). characteristics (Hedrick 1986; Simmons 1988; Shaw and Even the shortest latency observed for a post-tone mis- Herlihy 2000). These same features, however, would directed response (118 ms) was beyond the range of la- most effectively induce sensory habituation of receptor tencies reported by Nolen and Hoy (1986) for genuine neurons, and would thus have the largest effect on sound negative phonotaxis. localization cues. If selection pressure favoring energy- Finally, responses directed away from the sound intensive songs went unchecked, a paradoxical situation source were half as likely at the onset of the tone than at might arise in which the most desirable males would also the onset of the post-tone pulse train (Fig. 5). This op- be the most difficult to locate. It seems possible that posite to what would be expected for authentic negative sensory habituation of receptor neurons, and the con- phonotaxis, which should be more frequent at tone sequent effects on the encoding of localization cues, may onset, when the auditory system is unadapted. exert an opposing selective force, limiting the extent to which song structure might reflect male quality. Alter- natively, females might adopt behavioral strategies that Comparison with other studies offset the deleterious effects of sensory habituation. Fe- males approach a sound source along a characteristically The size of the interaural latency difference following zig-zag path (Weber et al. 1981). Zig-zagging appears to tone stimulation, approximately 2 ms for the first post- be an intrinsic feature of the walking pattern during tone responses, eventually stabilizing at ca. 1.4 ms, is phonotaxis (Bo¨ hm et al. 1991). As pointed out by Givois similar to the latency difference for responses that are and Pollack (2000), zig-zagging would cause frequent not preceded by a conditioning tone, for pulse rates alternation in which ear is ipsilateral and which is con- ranging from 1 to 28 pulses s)1 and sound levels ranging tralateral, and the resulting ‘‘resetting’’ of binaural dif- from 80–100 dB SPL (range of latency differences: 1.3– ference in response strength might serve to allow 1.8 ms; Givois and Pollack 2000). Thus, if crickets accurate sound localization despite sensory habituation. normally rely on latency difference to localize sound, Finally, crickets might determine the direction of sound then the information available to them during the post- source while they are still sufficiently distant that sound 151 level is low, and thus sensory habituation is negligible Imaizumi K, Pollack GS (1999) Neural coding of sound frequency (Givois and Pollack 2000). As they approach the source by cricket auditory receptors. J Neurosci 19:1508–1516 Imaizumi K, Pollack GS (2001) Neural representation of sound and sensory habituation becomes more severe, they amplitude by functionally different auditory receptors in might then rely increasingly on visual cues that had been crickets. J Acoust Soc Am 109:1247–1260 associated with the acoustically localized source. The Mason AC, Oshinsky ML, Hoy RR (2001) Hyperacute direc- ability to use visual cues to track a source that was ini- tional hearing in a microscale auditory system. Nature 410: 686–690 tially located acoustically has been demonstrated for the Michelsen A (1992) Hearing and sound communication in small tettigoniid, Poecilimon affinis (von Helversen and Wen- animals: evolutionary adaptations to the laws of physics. In: dler 2000). It is not yet clear whether crickets possess the Webster DB, Fay RR, Popper AN (eds) The evolutionary bi- same ability. ology of hearing. Springer, Berlin Heidelberg New York, pp 61–77 Moiseff A, Pollack GS, Hoy RR (1978) Steering responses of flying Acknowledgements Supported by the Natural Sciences and Engi- crickets to sound and ultrasound: mate attraction and predator neering Research Council of Canada. I thank Ms. Cheryl Vienneau avoidance. 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