Neurobiology of acoustically mediated predator detection.

Ultrasound-driven avoidance responses have evolved repeatedly throughout the insecta as defenses against predation by echolocating bats. Although the auditory mechanics of ears and the properties of auditory receptor neurons have been studied in a number of groups, central neural processing of ultrasound stimuli has been examined in only a few cases. In this review, I summarize the neuronal basis for ultrasound detection and predator avoidance in crickets, tettigoniids, moths, and mantises, where central circuits have been studied most thoroughly. Several neuronal attributes, including steep intensity-response functions, high firing rates, and rapid spike conduction emerge as common themes of avoidance circuits. I discuss the functional consequences of these attributes, as well as the increasing complexity with which ultrasound stimuli are represented at successive levels of processing.

Selective phonotaxis to high sound-pulse rate in the cricket Gryllus assimilis.

Calling song of the cricket Gryllus assimilis is unusual among Gryllus spp. in the high sound-pulse rate, ca. 80 Hz, within its chirps. We asked whether, as in other cricket species, females were able to analyze such a high pulse rate. In phonotaxis experiments, females failed to respond to stimuli with pulse rates substantially higher or lower than the species-typical value, demonstrating that they are indeed selective for this parameter. We also examined how pulse rate was represented by modulation in firing rate of the neuron AN1, the main carrier of information about cricket-song-like stimuli to the brain. For attractive stimuli, i.e. with high pulse rates, modulation of AN1 firing rate through time was surprisingly modest. This suggests that the brain circuits that analyze AN1 spike trains might be more sensitive to slight variations in AN1 firing rate than their counterparts in more slowly singing species.

Bursting neurons and ultrasound avoidance in crickets.

Decision making in invertebrates often relies on simple neural circuits composed of only a few identified neurons. The relative simplicity of these circuits makes it possible to identify the key computation and neural properties underlying decisions. In this review, we summarize recent research on the neural basis of ultrasound avoidance in crickets, a response that allows escape from echolocating bats. The key neural property shaping behavioral output is high-frequency bursting of an identified interneuron, AN2, which carries information about ultrasound stimuli from receptor neurons to the brain. AN2's spike train consists of clusters of spikes - bursts - that may be interspersed with isolated, non-burst spikes. AN2 firing is necessary and sufficient to trigger avoidance steering but only high-rate firing, such as occurs in bursts, evokes this response. AN2 bursts are therefore at the core of the computation involved in deciding whether or not to steer away from ultrasound. Bursts in AN2 are triggered by synaptic input from nearly synchronous bursts in ultrasound receptors. Thus the population response at the very first stage of sensory processing - the auditory receptor - already differentiates the features of the stimulus that will trigger a behavioral response from those that will not. Adaptation, both intrinsic to AN2 and within ultrasound receptors, scales the burst-generating features according to the stimulus statistics, thus filtering out background noise and ensuring that bursts occur selectively in response to salient peaks in ultrasound intensity. Furthermore AN2's sensitivity to ultrasound varies adaptively with predation pressure, through both developmental and evolutionary mechanisms. We discuss how this key relationship between bursting and the triggering of avoidance behavior is also observed in other invertebrate systems such as the avoidance of looming visual stimuli in locusts or heat avoidance in beetles.

Recognition of variable courtship song in the field cricket Gryllus assimilis.

We analyzed the courtship song of the field cricket Gryllus assimilis. The song comprises two elements: groups of ca. 10 pulses (chirps) with low fundamental frequency (3.5-3.7 kHz) alternating with high-frequency (15-17 kHz) pulses (ticks) that usually occur as doublets. Some elements of courtship song are quite variable (high coefficient of variation) both within and between males, whereas others are more stereotypical. In experiments with playback of synthesized courtship songs, we studied the importance of several song parameters for mating success, which we evaluated as the probability with which females mounted muted, courting males. Altering some features that show little variability, such as chirp-pulse rate or carrier frequency of ticks, resulted in significant decreases in mounting frequency, consistent with the notion that trait values showing little variability are constrained by stabilizing selection exerted by females. However, alteration of one invariant trait, the occurrence of both song components, by omitting either component from test songs only slightly affected female responsiveness. Alteration of a variable song trait, the number of ticks per song phrase, had no effect on female response rate, thus failing to provide support for the idea that variable traits provide a substrate for sexual selection. An unusual characteristic feature of the song of G. assimilis is that chirp pulses often contain substantial high-frequency power, and indeed may entirely lack power at the fundamental frequency. Playback experiments showed that such songs are, nevertheless, behaviorally effective. To understand the neural basis for this, we recorded the responses of the two principal ascending auditory interneurons of crickets, AN1 and AN2. Our results suggest that the frequency selectivity of the neurons is sufficiently broad to tolerate the spectral variability of courtship chirps.

The structure and size of sensory bursts encode stimulus information but only size affects behavior.

Cricket ultrasound avoidance is a classic model system for neuroethology. Avoidance steering is triggered by high-firing-rate bursts of spikes in the auditory command neuron AN2. Although bursting is common among sensory neurons, and although the detailed structure of bursts may encode information about the stimulus, it is as yet unclear whether this information is decoded. We address this question in two ways: from an information coding point of view, by showing the relationship between stimulus and burst structure; and also from a functional point of view by showing the relationship between burst structure and behavior. We conclude that the burst structure carries detailed temporal information about the stimulus but that this has little impact on the behavioral response, which is affected mainly by burst size.

Temporal coding by populations of auditory receptor neurons.

Auditory receptor neurons of crickets are most sensitive to either low or high sound frequencies. Earlier work showed that the temporal coding properties of first-order auditory interneurons are matched to the temporal characteristics of natural low- and high-frequency stimuli (cricket songs and bat echolocation calls, respectively). We studied the temporal coding properties of receptor neurons and used modeling to investigate how activity within populations of low- and high-frequency receptors might contribute to the coding properties of interneurons. We confirm earlier findings that individual low-frequency-tuned receptors code stimulus temporal pattern poorly, but show that coding performance of a receptor population increases markedly with population size, due in part to low redundancy among the spike trains of different receptors. By contrast, individual high-frequency-tuned receptors code a stimulus temporal pattern fairly well and, because their spike trains are redundant, there is only a slight increase in coding performance with population size. The coding properties of low- and high-frequency receptor populations resemble those of interneurons in response to low- and high-frequency stimuli, suggesting that coding at the interneuron level is partly determined by the nature and organization of afferent input. Consistent with this, the sound-frequency-specific coding properties of an interneuron, previously demonstrated by analyzing its spike train, are also apparent in the subthreshold fluctuations in membrane potential that are generated by synaptic input from receptor neurons.

Release from bats: genetic distance and sensoribehavioural regression in the Pacific field cricket, Teleogryllus oceanicus.

The auditory thresholds of the AN2 interneuron and the behavioural thresholds of the anti-bat flight-steering responses that this cell evokes are less sensitive in female Pacific field crickets that live where bats have never existed (Moorea) compared with individuals subjected to intense levels of bat predation (Australia). In contrast, the sensitivity of the auditory interneuron, ON1 which participates in the processing of both social signals and bat calls, and the thresholds for flight orientation to a model of the calling song of male crickets show few differences between the two populations. Genetic analyses confirm that the two populations are significantly distinct, and we conclude that the absence of bats has caused partial regression in the nervous control of a defensive behaviour in this insect. This study represents the first examination of natural evolutionary regression in the neural basis of a behaviour along a selection gradient within a single species.

Behaviorally relevant burst coding in primary sensory neurons.

Bursts of action potentials in sensory interneurons are believed to signal the occurrence of particularly salient stimulus features. Previous work showed that bursts in an identified, ultrasound-tuned interneuron (AN2) of the cricket Teleogryllus oceanicus code for conspicuous increases in amplitude of an ultrasound stimulus, resulting in behavioral responses that are interpreted as avoidance of echolocating bats. We show that the primary sensory neurons that inform AN2 about high-frequency acoustic stimuli also produce bursts. As is the case for AN2, bursts in sensory neurons perform better as feature detectors than isolated, nonburst, spikes. Bursting is temporally correlated between sensory neurons, suggesting that on occurrence of a salient stimulus feature, AN2 will receive strong synaptic input in the form of coincident bursts, from several sensory neurons, and that this might result in bursting in AN2. Our results show that an important feature of the temporal structure of interneuron spike trains can be established at the earliest possible level of sensory processing, i.e., that of the primary sensory neuron.

Flight behaviour attenuates the trade-off between flight capability and reproduction in a wing polymorphic cricket.

Flight-dimorphic insects have been used extensively to study trade-offs between energetically costly traits. Individuals may develop and maintain structures required for flight, or alternatively they may invest in reproduction. Previous experiments have not examined whether flight itself might affect investment into reproduction. As in other Gryllus species, flight-capable individuals of the wing polymorphic cricket, Gryllus texensis, incur an apparent reproductive penalty for being able to fly, expressed as smaller ovaries in females and lower courtship propensity in males, than their flight-incapable counterparts. We find that a short bout of flight eliminates the trade-off. Two days after the flight, the ovaries of flight-capable females were comparable with those of short-winged females. Similarly, flight markedly increased the probability of courtship behaviour. Our results suggest that the impact of the flight-reproduction trade-off described in earlier studies may have been overestimated.

Developmental control of ultrasound sensitivity by a juvenile hormone analog in crickets (Teleogryllus oceanicus).

Cricket ears are sensitive to ultrasound as well as to lower, cricket-like sound frequencies. Ultrasound stimuli evoke negative phonotaxis in flying crickets, a behavior that has been interpreted as a defensive response against predation by echolocating bats. A recent study on a wing-dimorphic species, Gryllus texensis, showed that short-winged individuals, which are incapable of flight, are less sensitive to ultrasound, but not to lower sound frequencies, than their long-winged counterparts. The developmental decision to develop as a long- or short-winged individual is made during the last two larval instars, and there is some evidence suggesting that juvenile hormone (JH) has an instructive role, such that high levels of JH result in short-winged individuals. We show that treatment of last-instar larvae of a monomorphic long-winged species, Teleogryllus oceanicus, with a JH analog causes a decrease in sensitivity to ultrasound, but not to the lower sound frequency used for intraspecific communication.

Carrier-dependent temporal processing in an auditory interneuron.

Signal processing in the auditory interneuron Omega Neuron 1 (ON1) of the cricket Teleogryllus oceanicus was compared at high- and low-carrier frequencies in three different experimental paradigms. First, integration time, which corresponds to the time it takes for a neuron to reach threshold when stimulated at the minimum effective intensity, was found to be significantly shorter at high-carrier frequency than at low-carrier frequency. Second, phase locking to sinusoidally amplitude modulated signals was more efficient at high frequency, especially at high modulation rates and low modulation depths. Finally, we examined the efficiency with which ON1 detects gaps in a constant tone. As reflected by the decrease in firing rate in the vicinity of the gap, ON1 is better at detecting gaps at low-carrier frequency. Following a gap, firing rate increases beyond the pre-gap level. This "rebound" phenomenon is similar for low- and high-carrier frequencies.

Flight and hearing: ultrasound sensitivity differs between flight-capable and flight-incapable morphs of a wing-dimorphic cricket species.

We studied frequency sensitivity of flight-capable and flight-incapable forms of the wing-dimorphic cricket Gryllus texensis, using both behavioral and neurophysiological measurements. Behavioral thresholds for negative phonotaxis in response to ultrasound stimuli are lower for long-winged (i.e. flight-capable) crickets than for short-winged (flight-incapable) individuals, whereas thresholds for positive phonotaxis in response to a calling-song model do not differ. Similarly, thresholds of the identified interneurons ON1 and AN2 differ between flight morphs for high sound frequencies but not for the frequency of calling song. Our results show that sensitivity to ultrasound is closely linked to flight ability, and thus to the risk of predation from aerially hawking bats. We suggest that sensitivity to ultrasound is one of a suite of flight-associated characteristics, the development of which may be under common hormonal regulation.

A behavioral role for feature detection by sensory bursts.

Brief episodes of high-frequency firing of sensory neurons, or bursts, occur in many systems, including mammalian auditory and visual systems, and are believed to signal the occurrence of particularly important stimulus features, i.e., to function as feature detectors. However, the behavioral relevance of sensory bursts has not been established in any system. Here, we show that bursts in an identified auditory interneuron of crickets reliably signal salient stimulus features and reliably predict behavioral responses. Our results thus demonstrate the close link between sensory bursts and behavior.

Central projections of auditory receptor neurons of crickets.

We describe the central projections of physiologically characterized auditory receptor neurons of crickets as revealed by confocal microscopy. Receptors tuned to ultrasonic frequencies (similar to those produced by echolocating, insectivorous bats), to a mid-range of frequencies, and a subset of those tuned to low, cricket-like frequencies have similar projections, terminating medially within the auditory neuropile. Quantitative analysis shows that despite the general similarity of these projections they are tonotopic, with receptors tuned to lower frequencies terminating more medially. Another subset of cricket-song-tuned receptors projects more laterally and posteriorly than the other types. Double-fills of receptors and identified interneurons show that the three medially projecting receptor types are anatomically well positioned to provide monosynaptic input to interneurons that relay auditory information to the brain and to interneurons that modify this ascending information. The more laterally and posteriorly branching receptor type may not interact directly with this ascending pathway, but is well positioned to provide direct input to an interneuron that carries auditory information to more posterior ganglia. These results suggest that information about cricket song is segregated into functionally different pathways as early as the level of receptor neurons. Ultrasound-tuned and mid-frequency tuned receptors have approximately twice as many varicosities, which are sites of transmitter release, per receptor as either anatomical type of cricket-song-tuned receptor. This may compensate in part for the numerical under-representation of these receptor types.

Effect of the temporal pattern of contralateral inhibition on sound localization cues.

We studied the temporal coding properties of identified interneurons in the auditory system of crickets, using information theory as an analytical tool. The ascending neuron 1 (AN1), which is tuned to the dominant carrier frequency (CF) of cricket songs, selectively codes the limited range of amplitude modulation (AM) frequencies that occur in these signals. AN2, which is most sensitive to the ultrasonic frequencies that occur in echolocation calls of insectivorous bats, codes a broader range of AM frequencies, as occur in bat calls. A third neuron, omega neuron 1 (ON1), which is dually tuned to both ranges of carrier frequency, was shown previously to have CF-specific coding properties, allowing it to represent accurately the differing temporal structures of both cricket songs and bat calls. ON1 is a source of contralateral inhibition to AN1 and AN2, enhancing binaural contrast and facilitating sound localization. We used dichotic stimulation to examine the importance of the temporal structure of contralateral inhibition for enhancing binaural contrast. Contralateral inhibition degrades the coding of temporal pattern by AN1 and AN2, but only if the temporal pattern of inhibitory input matches that of excitation. Firing rate is also decreased most strongly by temporally matched contralateral inhibition. This is apparent for AN1 in its mean firing rate; for AN2, high-frequency firing is selectively suppressed. Our results show that the CF-specific coding properties of ON1 allow this single neuron to enhance effectively localization cues for both cricket-like and bat-like acoustic signals.

Temporal and directional processing by an identified interneuron, ON1, compared in cricket species that sing with different tempos.

We compare the temporal and directional processing properties of an identified auditory interneuron, ON1, between species with calling songs containing relatively low and high pulse rates (Teleogryllus oceanicus and Gryllus texensis, respectively). Using information theory, we find that the ON1 of G. texensis encodes higher amplitude-modulation frequencies than that of T. oceanicus. Bilateral differences in ON1 responses are also more pronounced in G. texensis, particularly for rapid, G. texensis-like stimuli. We show that brief silent intervals in a pulse train, such as those that occur in the natural calling song of G. texensis, enhance the representation of the stimulus pulse pattern as well as bilateral differences in activity. Our results suggest that the characteristics of an identified neuron vary, across cricket species, in accordance with the temporal structures of their communication signals.

Antibodies against the PER protein of Drosophila label neurons in the optic lobe, central brain, and thoracic ganglia of the crickets Teleogryllus commodus and Teleogryllus oceanicus.

We describe labeling of neurons in the central nervous system of two cricket species, Teleogryllus commodus and T. oceanicus, with both mono- and polyclonal antibodies against the PER protein. Western blots reveal that the monoclonal antibodies recognize a single protein with a molecular weight of approximately 94 kDa, i.e., similar to that of the PER protein of the moth, Anterea pernii. Neurons and their processes are labeled both in the optic lobes and in the central brain. Processes occur in the accessory medulla, the medulla, and proximal lamina, in the central complex, in the non-glomerular neuropil, and in the retrocerebral complex, suggesting that PER-containing neurons form a widely distributed network. Neurons and processes were also labeled in the meso- and metathoracic ganglia. Four to six PER-immunoreactive (ir) neurons with processes in the accessory medulla were double labeled by an antibody against pigment-dispersion factor (PDF), a peptide that is implicated in circadian rhythmicity in Drosophila. In the central brain, projections of fibers labeled by the anti-PER and anti-PDF antibodies were mainly distinct, with overlap only in a few restricted regions. In most neurons, including those projecting into the accessory medulla, PER labeling was restricted to the cytoplasm and there was no indication of circadian variation in the intensity of staining.

Encoding of sound localization cues by an identified auditory interneuron: effects of stimulus temporal pattern.

An important cue for sound localization is binaural comparison of stimulus intensity. Two features of neuronal responses, response strength, i.e., spike count and/or rate, and response latency, vary with stimulus intensity, and binaural comparison of either or both might underlie localization. Previous studies at the receptor-neuron level showed that these response features are affected by the stimulus temporal pattern. When sounds are repeated rapidly, as occurs in many natural sounds, response strength decreases and latency increases, resulting in altered coding of localization cues. In this study we analyze binaural cues for sound localization at the level of an identified pair of interneurons (the left and right AN2) in the cricket auditory system, with emphasis on the effects of stimulus temporal pattern on binaural response differences. AN2 spike count decreases with rapidly repeated stimulation and latency increases. Both effects depend on stimulus intensity. Because of the difference in intensity at the two ears, binaural differences in spike count and latency change as stimulation continues. The binaural difference in spike count decreases, whereas the difference in latency increases. The proportional changes in response strength and in latency are greater at the interneuron level than at the receptor level, suggesting that factors in addition to decrement of receptor responses are involved. Intracellular recordings reveal that a slowly building, long-lasting hyperpolarization is established in AN2. At the same time, the level of depolarization reached during the excitatory postsynaptic potential (EPSP) resulting from each sound stimulus decreases. Neither these effects on membrane potential nor the changes in spiking response are accounted for by contralateral inhibition. Based on comparison of our results with earlier behavioral experiments, it is unlikely that crickets use the binaural difference in latency of AN2 responses as the main cue for determining sound direction, leaving the difference in response strength, i.e., spike count and/or rate, as the most likely candidate.