Sound processing in the cricket brain: evidence for a pulse duration filter.

The auditory system of female crickets allows them to specifically recognize and approach the species-specific male calling song, defined by sound pulses and silent intervals. Auditory brain neurons form a delay-line and coincidence detector network tuned to the pulse period of the male song. We analyzed the impact of changes in pulse duration on the behavior and the responses of the auditory neurons and the network. We confirm that the ascending neuron AN1 and the local neuron LN2 copy the temporal structure of the song. During ongoing long sound pulses, the delay-line neuron LN5 shows additional rebound responses and the coincidence detector neuron LN3 can generate additional bursts of activity, indicating that these may be driven by intrinsic oscillations of the network. Moreover, the response of the feature detector neuron LN4 is shaped by a combination of inhibitory and excitatory synaptic inputs, and LN4 responds even to long sound pulses with a short depolarization and burst of spikes, like to a sound pulse of natural duration. This response property of LN4 indicates a selective auditory pulse duration filter mechanism of the pattern recognition network, which is tuned to the duration of natural pulses. Comparing the tuning of the phonotactic behavior with the tuning of the local auditory brain neurons to the same test patterns, we find no evidence that a modulation of the phonotactic behavior is reflected at the level of the feature detector neurons. This rather suggests that steering to nonattractive pulse patterns is organized at the thoracic level.NEW & NOTEWORTHY Pulse period selectivity has been reported for the cricket delay-line and coincidence detector network, whereas pulse duration selectivity is evident from behavioral tests. Pulses of increasing duration elicit responses in the pattern recognition neurons, which do not parallel the behavioral responses and indicate additional processing mechanisms. Long sound pulses elicit rhythmic rebound activity and additional bursts, whereas the feature detector neuron reveals a pulse duration filter, expanding our understanding of the pattern recognition process.

Response properties of spiking and non-spiking brain neurons mirror pulse interval selectivity.

In the bispotted field cricket auditory pulse pattern recognition of the species-specific calling song is based on a delay-line and coincidence detection network, established by the activity and synaptic connections of only 5 auditory neurons in the brain. To obtain a more detailed understanding of the network and the dynamic of the neural activity over time we analyzed the response properties of these neurons to test patterns, in which the pulse duration was kept constant while the duration of specific pulse intervals was systematically altered. We confirm that the ascending interneuron AN1 and the local interneuron LN2 copy the structure of the pulse pattern, however with limited resolution at short pulse intervals, further evident in downstream neural responses. In the non-spiking delay-line interneuron LN5 during long pulse intervals full-blown rebound potentials develop over a time course of 35-70 ms. LN5 also reveals an overall increase in its membrane potential tuned to chirps of the calling song pulse pattern. This may contribute to the pattern recognition process by driving the activity of the coincidence-detector LN3 and may indicate a further function of the delay-line neuron LN5. The activity of LN3 and of the feature detector LN4 match the tuning of the phonotactic behavior and demonstrate an increasingly sparse coding of the calling song pulse patterns as evident in the response of the feature detector LN4. The circuitry reveals a fundamental mechanism of auditory pattern recognition and demonstrates a principle of neuronal coding.

An auditory-responsive interneuron descending from the cricket brain: a new element in the auditory pathway.

Crickets receive auditory information from their environment via ears located on the front legs. Ascending interneurons forward auditory activity to the brain, which houses a pattern recognition network for phonotaxis to conspecific calling songs and which controls negative phonotaxis to high-frequency sound pulses. Descending brain neurons, however, which are clearly involved in controlling these behaviors, have not yet been identified. We describe a descending auditory-responsive brain neuron with an arborization pattern that coincides with the ring-like auditory neuropil in the brain formed by the axonal arborizations of ascending and local interneurons, indicating its close link to auditory processing. Spiking activity of this interneuron occurs with a short latency to calling song patterns and the neuron copies the sound pulse pattern. The neuron preferentially responds to short sound pulses, but its activity appears to be independent of the calling song pattern recognition process. It also receives a weaker synaptic input in response to high-frequency pulses, which may contribute to its short latency spiking responses. This interneuron could be a crucial part in the auditory-to-motor transformation of the nervous system and contribute to the motor control of cricket auditory behavior.

The central organisation of courtship and rivalry behaviour in Gryllus bimaculatus (deGeer) as revealed by lesions of abdominal connectives.

Behaviour is rooted in the organization and activity of an animal's nervous system. As male crickets use their front wings for sound production, the neural circuits underlying singing had been suggested to be housed in the thoracic ganglia. However, systematic lesion experiments of the CNS demonstrated that the abdominal nervous system is essential for their calling song behaviour. As male crickets also generate a courtship and rivalry song, we explored which parts of the abdominal central nervous system control the underlying motor patterns. A combination of systematic lesions to the abdominal nerve cord and video recording of courtship and rivalry behaviour revealed that most components of male courtship and rivalry behaviour were not affected by the lesions, except for the generation of courtship song, rivalry song, and the male's ability to copulate with the female. Any lesion to the abdominal nerve cord abolished copulations. Generation of courtship song initially failed when the connection to abdominal ganglion A6 was severed but in few males recovered after a week. For rivalry song production a central nerve cord extending to abdominal ganglion A4 was sufficient. These findings indicate that in the bispotted cricket the neural organization of courtship song is different from calling and rivalry song, while calling song and rivalry song might share a common network for generating the song patterns.

Tolerant pattern recognition: evidence from phonotactic responses in the cricket Gryllus bimaculatus (de Geer).

When the amplitude modulation of species-specific acoustic signals is distorted in the transmission channel, signals become difficult to recognize by the receiver. Tolerant auditory pattern recognition systems, which after having perceived the correct species-specific signal transiently broaden their acceptance of signals, would be advantageous for animals as an adaptation to the constraints of the environment. Using a well-studied cricket species, Gryllus bimaculatus, we analysed tolerance in auditory steering responses to 'Odd' chirps, mimicking a signal distorted by the transmission channel, and control 'Silent' chirps by employing a fine-scale open-loop trackball system. Odd chirps on their own did not elicit a phonotactic response. However, when inserted into a calling song pattern with attractive Normal chirps, the females' phonotactic response toward these patterns was significantly larger than to patterns with Silent chirps. Moreover, females actively steered toward Odd chirps when these were presented within a sequence of attractive chirps. Our results suggest that crickets employ a tolerant pattern recognition system that, once activated, transiently allows responses to distorted sound patterns, as long as sufficient natural chirps are present. As pattern recognition modulates how crickets process non-attractive acoustic signals, the finding is also relevant for the interpretation of two-choice behavioural experiments.

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.

Wing movements underlying sound production in calling, rivalry, and courtship songs of the cricket Gryllus bimaculatus (DeGeer).

We recorded the wing movements and sound signals during the production of calling, rivalry, and courtship song in the bispotted field cricket Gryllus bimaculatus. Recordings confirm that salient sound pulses during calling and rivalry song are generated during the closing movements of the wings. Wing movements for calling and rivalry song start from an elevated wing position and are performed with a very similar opening-closing movement, indicating that both types of songs may be generated by the same neuronal network. Wing movements for courtship song start from a low wing position; rapid closing movements generate high-frequency ticks and low-amplitude wing oscillations lead to low-amplitude pulses, generated during the opening and closing movements with a carrier frequency corresponding to the calling song. The two types of wing movements underlying courtship song indicate a different motor control as compared to calling song and may represent an early evolutionary phenotype.

Does the choosiness of female crickets change as they age?

For crickets, which approach singing males by phonotaxis, the female choosiness hypothesis postulates that young females should be more selective of male calling song patterns than older individuals. However, there is no information about the behavioural preferences of females over their complete adulthood. We analysed phonotaxis in female Gryllus bimaculatus throughout their entire adult lifetime and measured the impact of sound amplitude, carrier frequency and the temporal pattern of test songs on their auditory response. Females of all ages demonstrated their best responses to male calling songs with a pulse period of 34-42 ms, a carrier frequency of 4.5 kHz and a sound pressure level of 75 dB. The response profile to somewhat less optimal song types did vary with age, but not in a manner consistent with a simple loosening of selectiveness in older females. Age, however, had an effect on the overall strength of phonotaxis, as very old females showed an overall diminishing response to all song types. Our data suggest that although there are minor changes in the relative preferences of crickets to individual song elements as they age, the breadth of song patterns to which they will perform phonotaxis remains similar across age groups.

Lesions of abdominal connectives reveal a conserved organization of the calling song central pattern generator (CPG) network in different cricket species.

Although crickets move their front wings for sound production, the abdominal ganglia house the network of the singing central pattern generator. We compared the effects of specific lesions to the connectives of the abdominal ganglion chain on calling song activity in four different species of crickets, generating very different pulse patterns in their calling songs. In all species, singing activity was abolished after the connectives between the metathoracic ganglion complex and the first abdominal ganglion A3 were severed. The song structure was lost and males generated only single sound pulses when connectives between A3 and A4 were cut. Severing connectives between A4 and A5 had no effect in the trilling species, it led to an extension of chirps in a chirping species and to a loss of the phrase structure in two Teleogryllus species. Cutting the connectives between A5 and A6 caused no or minor changes in singing activity. In spite of the species-specific pulse patterns of calling songs, our data indicate a conserved organisation of the calling song motor pattern generating network. The generation of pulses is controlled by ganglia A3 and A4 while A4 and A5 provide the timing information for the chirp and/or phrase structure of the song.

Modular timer networks: abdominal interneurons controlling the chirp and pulse pattern in a cricket calling song.

Chirping male crickets combine a 30 Hz pulse pattern with a 3 Hz chirp pattern to drive the rhythmic opening-closing movements of the front wings for sound production. Lesion experiments suggest two coupled modular timer-networks located along the chain of abdominal ganglia, a network in A3 and A4 generating the pulse pattern, and a network organized along with ganglia A4-A6 controlling the generation of the chirp rhythm. We analyzed neurons of the timer-networks and their synaptic connections by intracellular recordings and staining. We identified neurons spiking in phase with the chirps and pulses, or that are inhibited during the chirps. Neurons share a similar "gestalt", regarding the position of the cell body, the dendritic arborizations and the contralateral ascending axon. Activating neurons of the pulse-timer network elicits ongoing motor activity driving the generation of pulses; this activity is not structured in the chirp pattern. Activating neurons of the chirp-timer network excites pulse-timer neurons; it drives the generation of chirps and during the chirps the pulse pattern is produced. Our results support the hypothesis that two modular networks along the abdominal ganglion chain control the cricket calling song, a pattern generating network in the mesothoracic ganglion may not be required.

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.

Bilateral auditory processing studied by selective cold-deactivation of cricket hearing organs.

We studied bilateral processing in the auditory ON neurons of crickets using reversible cold-deactivation of the hearing organs by means of Peltier elements. Intracellular recordings of the neurons' activity in response to acoustic stimuli were obtained, while either the ipsilateral or the contralateral hearing organ was cold-deactivated. Afferent activity was abolished at a temperature of approximately 10°C. In ON1, contralateral inhibition had no effect on the latency and amplitude of the phasic onset activity, it enhanced the decline of the onset activity and it decreased the subsequent tonic spiking response to acoustic stimuli. As a consequence, the phasic onset activity became more salient and reciprocal inhibition may support the detection of sound pulses. Contralateral inhibition had a significant impact on the tonic ON1 response, in line with its presumed function to enhance the bilateral auditory contrast. In ON2, experiments confirmed a bilateral excitatory input, with the ipsilateral input dominating the response, and no inhibitory coupling between the ON2 neurons.

Structure, Activity and Function of a Singing CPG Interneuron Controlling Cricket Species-Specific Acoustic Signaling.

The evolution of species-specific song patterns is a driving force in the speciation of acoustic communicating insects. It must be closely linked to adaptations of the neuronal network controlling the underlying singing motor activity. What are the cellular and network properties that allow generating different songs? In five cricket species, we analyzed the structure and activity of the identified abdominal ascending opener interneuron, a homologous key component of the singing central pattern generator. The structure of the interneuron, based on the position of the cell body, ascending axon, dendritic arborization pattern, and dye coupling, is highly similar across species. The neuron's spike activity shows a tight coupling to the singing motor activity. In all species, current injection into the interneuron drives artificial song patterns, highlighting the key functional role of this neuron. However, the pattern of the membrane depolarization during singing, the fine dendritic and axonal ramifications, and the number of dye-coupled neurons indicate species-specific adaptations of the neuronal network that might be closely linked to the evolution of species-specific singing.SIGNIFICANCE STATEMENT A fundamental question in evolutionary neuroscience is how species-specific behaviors arise in closely related species. We demonstrate behavioral, neurophysiological, and morphological evidence for homology of one key identified interneuron of the singing central pattern generator in five cricket species. Across-species differences of this interneuron are also observed, which might be important to the generation of the species-specific song patterns. This work offers a comprehensive and detailed comparative analysis addressing the neuronal basis of species-specific behavior.

Dendritic Ca2+ dynamics and multimodal processing in a cricket antennal interneuron.

The integration of stimuli of different modalities is fundamental to information processing within the nervous system. A descending interneuron in the cricket brain, with prominent dendrites in the deutocerebrum, receives input from three sensory modalities: touch of the antennal flagellum, strain of the antennal base, and visual stimulation. Using calcium imaging, we demonstrate that each modality drives a Ca2+ increase in a different dendritic region. Moreover, touch of the flagellum is represented in a topographic map along the neuron's dendrites. Using intracellular recording, we investigated the effects of Ca2+ on spike shape through the application of the Ca2+ channel antagonist Cd2+ and identified probable Ca2+-dependent K+ currents. NEW & NOTEWORTHY Different dendritic regions of the cricket brain neuron DBNi1-2 showed localized Ca2+ increases when three modalities of stimulation (touch of the flagellum, strain at antennal base, and visual input) were given. Touch stimulation induces localized Ca2+ increases according to a topographic map of the antenna. Ca2+ appears to activate K+ currents in DBNi1-2.

Vestigial singing behaviour persists after the evolutionary loss of song in crickets.

The evolutionary loss of sexual traits is widely predicted. Because sexual signals can arise from the coupling of specialized motor activity with morphological structures, disruption to a single component could lead to overall loss of function. Opportunities to observe this process and characterize any remaining signal components are rare, but could provide insight into the mechanisms, indirect costs and evolutionary consequences of signal loss. We investigated the recent evolutionary loss of a long-range acoustic sexual signal in the Hawaiian field cricket Teleogryllus oceanicus Flatwing males carry mutations that remove sound-producing wing structures, eliminating all acoustic signalling and affording protection against an acoustically-orientating parasitoid fly. We show that flatwing males produce wing movement patterns indistinguishable from those that generate sonorous calling song in normal-wing males. Evolutionary song loss caused by the disappearance of structural components of the sound-producing apparatus has left behind the energetically costly motor behaviour underlying normal singing. These results provide a rare example of a vestigial behaviour and raise the possibility that such traits could be co-opted for novel functions.

Surface electrodes record and label brain neurons in insects.

We used suction electrodes to reliably record the activity of identified ascending auditory interneurons from the anterior surface of the brain in crickets. Electrodes were gently attached to the sheath covering the projection area of the ascending interneurons and the ringlike auditory neuropil in the protocerebrum. The specificity and selectivity of the recordings were determined by the precise electrode location, which could easily be changed without causing damage to the tissue. Different nonauditory fibers were recorded at other spots of the brain surface; stable recordings lasted for several hours. The same electrodes were used to deliver fluorescent tracers into the nervous system by means of electrophoresis. This allowed us to retrograde label the recorded auditory neurons and to reveal their cell body and dendritic structure in the first thoracic ganglion. By adjusting the amount of dye injected, we specifically stained the ringlike auditory neuropil in the brain, demonstrating the clusters of cell bodies contributing to it. Our data provide a proof that surface electrodes are a versatile tool to analyze neural processing in small brains of invertebrates.NEW & NOTEWORTHY We show that surface suction electrodes can be used to monitor the activity of auditory neurons in the cricket brain. They also allow delivering electrophoretically a fluorescent tracer to label the structure of the recorded neurons and the local neuropil to which the electrode was attached. This new extracellular recording and labeling technique is a versatile and useful method to explore neural processing in invertebrate sensory and motor systems.

Song pattern recognition in crickets based on a delay-line and coincidence-detector mechanism.

Acoustic communication requires filter mechanisms to process and recognize key features of the perceived signals. We analysed such a filter mechanism in field crickets (Gryllus bimaculatus), which communicate with species-specific repetitive patterns of sound pulses and chirps. A delay-line and coincidence-detection mechanism, in which each sound pulse has an impact on the processing of the following pulse, is implicated to underlie the recognition of the species-specific pulse pattern. Based on this concept, we hypothesized that altering the duration of a single pulse or inter-pulse interval in three-pulse chirps will lead to different behavioural responses. Phonotaxis was tested in female crickets walking on a trackball exposed to different sound paradigms. Changing the duration of either the first, second or third pulse of the chirps led to three different characteristic tuning curves. Long first pulses decreased the phonotactic response whereas phonotaxis remained strong when the third pulse was long. Chirps with three pulses of increasing duration of 5, 20 and 50 ms elicited phonotaxis, but the chirps were not attractive when played in reverse order. This demonstrates specific, pulse duration-dependent effects while sequences of pulses are processed. The data are in agreement with a mechanism in which processing of a sound pulse has an effect on the processing of the subsequent pulse, as outlined in the flow of activity in a delay-line and coincidence-detector circuit. Additionally our data reveal a substantial increase in the gain of phonotaxis, when the number of pulses of a chirp is increased from two to three.

Electrophoresis of polar fluorescent tracers through the nerve sheath labels neuronal populations for anatomical and functional imaging.

The delivery of tracers into populations of neurons is essential to visualize their anatomy and analyze their function. In some model systems genetically-targeted expression of fluorescent proteins is the method of choice; however, these genetic tools are not available for most organisms and alternative labeling methods are very limited. Here we describe a new method for neuronal labelling by electrophoretic dye delivery from a suction electrode directly through the neuronal sheath of nerves and ganglia in insects. Polar tracer molecules were delivered into the locust auditory nerve without destroying its function, simultaneously staining peripheral sensory structures and central axonal projections. Local neuron populations could be labelled directly through the surface of the brain, and in-vivo optical imaging of sound-evoked activity was achieved through the electrophoretic delivery of calcium indicators. The method provides a new tool for studying how stimuli are processed in peripheral and central sensory pathways and is a significant advance for the study of nervous systems in non-model organisms.

Behavioural integration of auditory and antennal stimulation during phonotaxis in the field cricket Gryllus bimaculatus.

Animals need to flexibly respond to stimuli from their environment without compromising behavioural consistency. For example, female crickets orienting toward a conspecific male's calling song in search of a mating partner need to stay responsive to other signals that provide information about obstacles and predators. Here, we investigate how spontaneously walking crickets and crickets engaging in acoustically guided goal-directed navigation, i.e. phonotaxis, respond to mechanosensory stimuli detected by their long antennae. We monitored walking behaviour of female crickets on a trackball during lateral antennal stimulation, which was achieved by moving a wire mesh transiently into reach of one antenna. During antennal stimulation alone, females reduced their walking speed, oriented toward the object and actively explored it with antennal movements. Additionally, some crickets initially turned away from the approaching object. Females responded in a similar way when the antennal stimulus was presented during ongoing phonotaxis: forward velocity was reduced and phonotactic steering was suppressed while the females turned toward and explored the object. Further, rapid steering bouts to individual chirps, typical for female phonotaxis, no longer occurred. Our data reveal that in this experimental situation, antennal stimulation overrides phonotaxis for extended time periods. Phonotaxis in natural environments, which require the integration of multiple sensory cues, may therefore be more variable than phonotaxis measured under ideal laboratory conditions. Combining this new behavioural paradigm with neurophysiological methods will show where the sensory-motor integration of antennal and acoustic stimulation occurs and how this is achieved on a mechanistic level.