Multielectrode array use in insect auditory neuroscience to unravel the spatio-temporal response pattern in the prothoracic ganglion of Mecopoda elongata.

Mechanoreceptors in hearing organs transduce sound-induced mechanical responses into neuronal signals, which are further processed and forwarded to the brain along a chain of neurons in the auditory pathway. Bushcrickets (katydids) have their ears in the front leg tibia, and the first synaptic integration of sound-induced neuronal signals takes place in the primary auditory neuropil of the prothoracic ganglion. By combining intracellular recordings of the receptor activity in the ear, extracellular multichannel array recordings on top of the prothoracic ganglion and hook electrode recordings at the neck connective, we mapped the timing of neuronal responses to tonal sound stimuli along the auditory pathway from the ears towards the brain. The use of the multielectrode array allows the observation of spatio-temporal patterns of neuronal responses within the prothoracic ganglion. By eliminating the sensory input from one ear, we investigated the impact of contralateral projecting interneurons in the prothoracic ganglion and added to previous research on the functional importance of contralateral inhibition for binaural processing. Furthermore, our data analysis demonstrates changes in the signal integration processes at the synaptic level indicated by a long-lasting increase in the local field potential amplitude. We hypothesize that this persistent increase of the local field potential amplitude is important for the processing of complex signals, such as the conspecific song.

A small, computationally flexible network produces the phenotypic diversity of song recognition in crickets.

How neural networks evolved to generate the diversity of species-specific communication signals is unknown. For receivers of the signals, one hypothesis is that novel recognition phenotypes arise from parameter variation in computationally flexible feature detection networks. We test this hypothesis in crickets, where males generate and females recognize the mating songs with a species-specific pulse pattern, by investigating whether the song recognition network in the cricket brain has the computational flexibility to recognize different temporal features. Using electrophysiological recordings from the network that recognizes crucial properties of the pulse pattern on the short timescale in the cricket Gryllus bimaculatus, we built a computational model that reproduces the neuronal and behavioral tuning of that species. An analysis of the model's parameter space reveals that the network can provide all recognition phenotypes for pulse duration and pause known in crickets and even other insects. Phenotypic diversity in the model is consistent with known preference types in crickets and other insects, and arises from computations that likely evolved to increase energy efficiency and robustness of pattern recognition. The model's parameter to phenotype mapping is degenerate - different network parameters can create similar changes in the phenotype - which likely supports evolutionary plasticity. Our study suggests that computationally flexible networks underlie the diverse pattern recognition phenotypes, and we reveal network properties that constrain and support behavioral diversity.

Neuroethology of acoustic communication in field crickets - from signal generation to song recognition in an insect brain.

Field crickets are best known for the loud calling songs produced by males to attract conspecific females. This review aims to summarize the current knowledge of the neurobiological basis underlying the acoustic communication for mate finding in field crickets with emphasis on the recent research progress to understand the neuronal networks for motor pattern generation and auditory pattern recognition of the calling song in Gryllus bimaculatus. Strong scientific interest into the neural mechanisms underlying intraspecific communication has driven persistently advancing research efforts to study the male singing behaviour and female phonotaxis for mate finding in these insects. The growing neurobiological understanding also inspired many studies testing verifiable hypotheses in sensory ecology, bioacoustics and on the genetics and evolution of behaviour. Over last decades, acoustic communication in field crickets served as a very successful neuroethological model system. It has contributed significantly to the scientific process of establishing, reconsidering and refining fundamental concepts in behavioural neurosciences such as command neurons, central motor pattern generation, corollary discharge processing and pattern recognition by sensory feature detection, which are basic building blocks of our modern understanding on how nervous systems control and generate behaviour in all animals.

Correction to: Feedforward discharges couple the singing central pattern generator and ventilation central pattern generator in the cricket abdominal central nervous system.

Authors would like to update one of the references which went incorrect in the original publication and the corrected version is updated here.

Feedforward discharges couple the singing central pattern generator and ventilation central pattern generator in the cricket abdominal central nervous system.

We investigated the central nervous coordination between singing motor activity and abdominal ventilatory pumping in crickets. Fictive singing, with sensory feedback removed, was elicited by eserine-microinjection into the brain, and the motor activity underlying singing and abdominal ventilation was recorded with extracellular electrodes. During singing, expiratory abdominal muscle activity is tightly phase coupled to the chirping pattern. Occasional temporary desynchronization of the two motor patterns indicate discrete central pattern generator (CPG) networks that can operate independently. Intracellular recordings revealed a sub-threshold depolarization in phase with the ventilatory cycle in a singing-CPG interneuron, and in a ventilation-CPG interneuron an excitatory input in phase with each syllable of the chirps. Inhibitory synaptic inputs coupled to the syllables of the singing motor pattern were present in another ventilatory interneuron, which is not part of the ventilation-CPG. Our recordings suggest that the two centrally generated motor patterns are coordinated by reciprocal feedforward discharges from the singing-CPG to the ventilation-CPG and vice versa. Consequently, expiratory contraction of the abdomen usually occurs in phase with the chirps and ventilation accelerates during singing due to entrainment by the faster chirp cycle.

Female perception of copulatory courtship by male titillators in a bushcricket.

Males of the bushcricket Metrioptera roeselii bear paired titillators that are spiny genital structures supposedly functioning as copulatory courtship devices. During copulation, the male inserts its titillators into the female's genital chamber, where they rhythmically tap on the sensilla-covered dorsal surface of the genital fold. Here, we investigated the stimulatory function of male titillators during mating in M. roeselii Tracer backfills of presumptive mechanosensory sensilla at the female genital fold revealed a thick bundle of sensory axons entering the last unfused abdominal ganglion (AG-7). Electrophysiological recordings of abdominal nerves demonstrated that females sense mechanical stimulation at their genital fold. The mechanosensory responses, however, were largely reduced by the insecticide pymetrozine that selectively blocks scolopidia of internal chordotonal organs but not campaniform and hair sensilla on the outer cuticle surface. In mating experiments, the females showed resistance behaviours towards males with asymmetrically shortened titillators, but the resistance was largely reduced when mechanoreceptors at the female's genital fold were either pharmacologically silenced by pymetrozine or mechanically blocked by capping with UV-hardened glue. Our findings support the hypothesis that the male titillators in these bushcrickets may serve as copulatory courtship devices to mechanically stimulate the female genitalia to reduce resistance behaviour.

Functional basis of the sexual dimorphism in the auditory fovea of the duetting bushcricket Ancylecha fenestrata.

From mammals to insects, acoustic communication is in many species crucial for successful reproduction. In the duetting bushcricket Ancylecha fenestrata, the mutual acoustic communication between males and females is asymmetrical. We investigated how those signalling disparities are reflected by sexual dimorphism of their ears. Both sexes have tympanic ears in their forelegs, but male ears possess a significantly longer crista acustica containing 35% more scolopidia. With more sensory cells to cover a similar hearing range, the male hearing organ shows a significantly expanded auditory fovea that is tuned to the dominant frequency of the female reply to facilitate phonotactic mate finding. This sex-specific auditory fovea is demonstrated in the mechanical and neuronal responses along the tonotopically organized crista acustica by laservibrometric and electrophysiological frequency mapping, respectively. Morphometric analysis of the crista acustica revealed an interrupted gradient in organ height solely within this auditory fovea region, whereas all other anatomical parameters decrease continuously from proximal to distal. Combining behavioural, anatomical, biomechanical and neurophysiological information, we demonstrate evidence of a pronounced auditory fovea as a sex-specific adaptation of an insect hearing organ for intraspecific acoustic communication.

Auditory fovea in the ear of a duetting katydid shows male-specific adaptation to the female call.

Convergent evolution has led to surprising functional and mechanistic similarities between the vertebrate cochlea and some katydid ears [1,2]. Here we report on an 'auditory fovea' (Figure 1A) in the duetting katydid Ancylecha fenestrata (Tettigoniidae). The auditory fovea is a specialized inner-ear region with a disproportionate number of receptor cells tuned to a narrow frequency range, and has been described in the cochlea of some vertebrates, such as bats and mole rats [3,4]. In tonotopically organized ears, the location in the hearing organ of the optimal neuronal response to a tone changes gradually with the frequency of the stimulation tone. However, in the ears of A. fenestrata, the sensory cells in the auditory fovea are tuned to the dominant frequency of the female call; this area of the hearing organ is extensively expanded in males to provide an overrepresentation of this behaviorally important auditory input. Vertebrates developed an auditory fovea for improved prey or predator detection. In A. fenestrata, however, the foveal region facilitates acoustic pair finding, and the sexual dimorphism of sound-producing and hearing organs reflects the asymmetry in the mutual communication system between the sexes (Figures 1B, S1).

Gating of Acoustic Transducer Channels Is Shaped by Biomechanical Filter Processes.

Mechanoelectrical transduction of acoustic signals is the fundamental process for hearing in all ears across the animal kingdom. Here, we performed in vivo laser-vibrometric and electrophysiological measurements at the transduction site in an insect ear (Mecopoda elongata) to relate the biomechanical tonotopy along the hearing organ to the frequency tuning of the corresponding sensory cells. Our mechanical and electrophysiological map revealed a biomechanical filter process that considerably sharpens the neuronal response. We demonstrate that the channel gating, which acts on chordotonal stretch receptor neurons, is based on a mechanical directionality of the sound-induced motion. Further, anatomical studies of the transduction site support our finding of a stimulus-relevant tilt. In conclusion, we were able to show, in an insect ear, that directionality of channel gating considerably sharpens the neuronal frequency selectivity at the peripheral level and have identified a mechanism that enhances frequency discrimination in tonotopically organized ears.

Evolution of a Communication System by Sensory Exploitation of Startle Behavior.

New communication signals can evolve by sensory exploitation if signaling taps into preexisting sensory biases in receivers [1, 2]. For mate attraction, signals are typically similar to attractive environmental cues like food [3-6], which amplifies their attractiveness to mates, as opposed to aversive stimuli like predator cues. Female field crickets approach the low-frequency calling song of males, whereas they avoid high-frequency sounds like predatory bat calls [7]. In one group of crickets (Eneopterinae: Lebinthini), however, males produce exceptionally high-frequency calling songs in the range of bat calls [8], a surprising signal in the context of mate attraction. We found that female lebinthines, instead of approaching singing males, produce vibrational responses after male calls, and males track the source of vibrations to find females. We also demonstrate that field cricket species closely related to the Lebinthini show an acoustic startle response to high-frequency sounds that generates substrate vibrations similar to those produced by female lebinthine crickets. Therefore, the startle response is the most likely evolutionary origin of the female lebinthine vibrational signal. In field crickets, the brain receives activity from two auditory interneurons; AN1 tuned to male calling song controls positive phonotaxis, and AN2 tuned to high-frequency bat calls triggers negative phonotaxis [9, 10]. In lebinthine crickets, however, we found that auditory ascending neurons are only tuned to high-frequency sounds, and their tuning matches the thresholds for female vibrational signals. Our results demonstrate how sensory exploitation of anti-predator behavior can evolve into a communication system that benefits both senders and receivers.

An auditory feature detection circuit for sound pattern recognition.

From human language to birdsong and the chirps of insects, acoustic communication is based on amplitude and frequency modulation of sound signals. Whereas frequency processing starts at the level of the hearing organs, temporal features of the sound amplitude such as rhythms or pulse rates require processing by central auditory neurons. Besides several theoretical concepts, brain circuits that detect temporal features of a sound signal are poorly understood. We focused on acoustically communicating field crickets and show how five neurons in the brain of females form an auditory feature detector circuit for the pulse pattern of the male calling song. The processing is based on a coincidence detector mechanism that selectively responds when a direct neural response and an intrinsically delayed response to the sound pulses coincide. This circuit provides the basis for auditory mate recognition in field crickets and reveals a principal mechanism of sensory processing underlying the perception of temporal patterns.

Corollary discharge inhibition of wind-sensitive cercal giant interneurons in the singing field cricket.

Crickets carry wind-sensitive mechanoreceptors on their cerci, which, in response to the airflow produced by approaching predators, triggers escape reactions via ascending giant interneurons (GIs). Males also activate their cercal system by air currents generated due to the wing movements underlying sound production. Singing males still respond to external wind stimulation, but are not startled by the self-generated airflow. To investigate how the nervous system discriminates sensory responses to self-generated and external airflow, we intracellularly recorded wind-sensitive afferents and ventral GIs of the cercal escape pathway in fictively singing crickets, a situation lacking any self-stimulation. GI spiking was reduced whenever cercal wind stimulation coincided with singing motor activity. The axonal terminals of cercal afferents showed no indication of presynaptic inhibition during singing. In two ventral GIs, however, a corollary discharge inhibition occurred strictly in phase with the singing motor pattern. Paired intracellular recordings revealed that this inhibition was not mediated by the activity of the previously identified corollary discharge interneuron (CDI) that rhythmically inhibits the auditory pathway during singing. Cercal wind stimulation, however, reduced the spike activity of this CDI by postsynaptic inhibition. Our study reveals how precisely timed corollary discharge inhibition of ventral GIs can prevent self-generated airflow from triggering inadvertent escape responses in singing crickets. The results indicate that the responsiveness of the auditory and wind-sensitive pathway is modulated by distinct CDIs in singing crickets and that the corollary discharge inhibition in the auditory pathway can be attenuated by cercal wind stimulation.

Cellular basis for singing motor pattern generation in the field cricket (Gryllus bimaculatus DeGeer).

The singing behavior of male crickets allows analyzing a central pattern generator (CPG) that was shaped by sexual selection for reliable production of species-specific communication signals. After localizing the essential ganglia for singing in Gryllus bimaculatus, we now studied the calling song CPG at the cellular level. Fictive singing was initiated by pharmacological brain stimulation. The motor pattern underlying syllables and chirps was recorded as alternating spike bursts of wing-opener and wing-closer motoneurons in a truncated wing nerve; it precisely reflected the natural calling song. During fictive singing, we intracellularly recorded and stained interneurons in thoracic and abdominal ganglia and tested their impact on the song pattern by intracellular current injections. We identified three interneurons of the metathoracic and first unfused abdominal ganglion that rhythmically de- and hyperpolarized in phase with the syllable pattern and spiked strictly before the wing-opener motoneurons. Depolarizing current injection in two of these opener interneurons caused additional rhythmic singing activity, which reliably reset the ongoing chirp rhythm. The closely intermeshing arborizations of the singing interneurons revealed the dorsal midline neuropiles of the metathoracic and three most anterior abdominal neuromeres as the anatomical location of singing pattern generation. In the same neuropiles, we also recorded several closer interneurons that rhythmically hyper- and depolarized in the syllable rhythm and spiked strictly before the wing-closer motoneurons. Some of them received pronounced inhibition at the beginning of each chirp. Hyperpolarizing current injection in the dendrite revealed postinhibitory rebound depolarization as one functional mechanism of central pattern generation in singing crickets.

Neural basis of singing in crickets: central pattern generation in abdominal ganglia.

The neural mechanisms underlying cricket singing behavior have been the focus of several studies, but the central pattern generator (CPG) for singing has not been localized conclusively. To test if the abdominal ganglia contribute to the singing motor pattern and to analyze if parts of the singing CPG are located in these ganglia, we systematically truncated the abdominal nerve cord of fictively singing crickets while recording the singing motor pattern from a front-wing nerve. Severing the connectives anywhere between terminal ganglion and abdominal ganglion A3 did not preclude singing, although the motor pattern became more variable and failure-prone as more ganglia were disconnected. Singing terminated immediately and permanently after transecting the connectives between the metathoracic ganglion complex and the first unfused abdominal ganglion A3. The contribution of abdominal ganglia for singing pattern generation was confirmed by intracellular interneuron recordings and current injections. During fictive singing, an ascending interneuron with its soma and dendrite in A3 depolarized rhythmically. It spiked 10 ms before the wing-opener activity and hyperpolarized in phase with the wing-closer activity. Depolarizing current injection elicited rhythmic membrane potential oscillations and spike bursts that elicited additional syllables and reliably reset the ongoing chirp rhythm. Our results disclose that the abdominal ganglion A3 is directly involved in generating the singing motor pattern, whereas the more posterior ganglia seem to provide only stabilizing feedback to the CPG circuit. Localizing the singing CPG in the anterior abdominal neuromeres now allows analyzing its circuitry at the level of identified interneurons in subsequent studies.

Neuronal organization of a fast-mediating cephalothoracic pathway for antennal-tactile information in the cricket (Gryllus bimaculatus DeGeer).

Crickets use their long antennae as tactile sensors. Confronted with obstacles, conspecifics, or predators, antennal contacts trigger short-latency motor responses. To reveal the neuronal pathway underlying these antennal-guided locomotory reactions we identified descending interneurons that rapidly transmit antennal-tactile information from the head to the thorax in the cricket Gryllus bimaculatus. Antennae were stimulated with forces approximating those of naturally occurring antennal contacts. Responding interneurons were individually identified by intracellular axon recordings in the pro-mesothoracic connective and subsequent tracer injection. Simultaneous with the intracellular recordings, the overall spike response in the neck connectives was recorded extracellularly to reveal the precise response-timing of each individual neuron within the collective multiunit response. Here we describe four descending brain neurons and two with the soma in the subesophageal ganglion. All antennal-touch elicited action potentials apparent in the neck connective recordings within 10 ms after antennal-contact are generated by these six interneurons. Their dendrites ramify in primary antennal-mechanosensory neuropils of the head ganglia. Each of them consistently generated action potentials in response to antennal touching and three of them responded also to different visual stimulation (light-off, movement). Their descending axons conduct action potentials with 3-5 m/s to the thoracic ganglia where they send off side branches in dorsal neuropils. Their physiological and anatomical properties qualify them as descending giant fibers in the cricket and suggest an involvement in evoking fast locomotory reactions. They form a fast-mediating cephalo-thoracic pathway for antennal-tactile information, whereas all other antennal-tactile interneurons had response latencies exceeding 40 ms.

Hyperacute directional hearing and phonotactic steering in the cricket (Gryllus bimaculatus deGeer).

BACKGROUND: Auditory mate or prey localisation is central to the lifestyle of many animals and requires precise directional hearing. However, when the incident angle of sound approaches 0° azimuth, interaural time and intensity differences gradually vanish. This poses a demanding challenge to animals especially when interaural distances are small. To cope with these limitations imposed by the laws of acoustics, crickets employ a frequency tuned peripheral hearing system. Although this enhances auditory directionality the actual precision of directional hearing and phonotactic steering has never been studied in the behaviourally important frontal range. PRINCIPAL FINDINGS: Here we analysed the directionality of phonotaxis in female crickets (Gryllus bimaculatus) walking on an open-loop trackball system by measuring their steering accuracy towards male calling song presented at frontal angles of incidence. Within the range of ±30°, females reliably discriminated the side of acoustic stimulation, even when the sound source deviated by only 1° from the animal's length axis. Moreover, for angles of sound incidence between 1° and 6° the females precisely walked towards the sound source. Measuring the tympanic membrane oscillations of the front leg ears with a laser vibrometer revealed between 0° and 30° a linear increasing function of interaural amplitude differences with a slope of 0.4 dB/°. Auditory nerve recordings closely reflected these bilateral differences in afferent response latency and intensity that provide the physiological basis for precise auditory steering. CONCLUSIONS: Our experiments demonstrate that an insect hearing system based on a frequency-tuned pressure difference receiver achieves directional hyperacuity which easily rivals best directional hearing in mammals and birds. Moreover, this directional accuracy of the cricket's hearing system is reflected in the animal's phonotactic motor response.