Auditory DUM neurons in a bush-cricket: inhibited inhibitors.

Thoracic ganglia of many hearing insects house the first level of auditory processing. In bush-crickets, the largest population of local auditory neurons in the prothoracic processing centre are dorsal unpaired median (DUM) neurons. It has been suggested that DUM neurons are inhibitory using γ-aminobutyric acid (GABA) as transmitter. Immunohistochemistry reveals a population of about 35-50 GABA-positive somata in the posterior medial cluster of the prothoracic ganglion. Only very few small somata in this cluster remain unstained. At least 10 neurites from 10 neurons can be identified. Intracellularly stained auditory DUM neurons have their soma in the cluster of median GABA positive cells and most of them exhibit GABA-immunoreactivity. Responses of certain DUM neurons show obvious signs of inhibition. Application of picrotoxin (PTX), a chloride-channel blocker in insects, changes the responses of many DUM neurons. They become broader in frequency tuning and broader or narrower in temporal pattern tuning. Furthermore, inhibitory postsynaptic potentials (IPSPs) may be replaced by excitatory postsynaptic potentials. Loss of an IPSP in the rising graded potential after PTX-application leads to a significant reduction of first-spike latency. Therefore, auditory DUM neurons receive effective inhibition and are the best candidates for inhibition in DUM neurons and other auditory interneurons.

Temporal processing properties of auditory DUM neurons in a bush-cricket.

Insects with ears process sounds and respond to conspecific signals or predator cues. Axons of auditory sensory cells terminate in mechanosensory neuropils from which auditory interneurons project into (brain-) areas to prepare response behaviors. In the prothoracic ganglion of a bush-cricket, a cluster of local DUM (dorsal unpaired median) neurons has recently been described and constitutes a filter bank for carrier frequency. Here, we demonstrate that these neurons also constitute a filter bank for temporal patterns. The majority of DUM neurons showed pronounced phasic-tonic responses. The transitions from phasic to tonic activation had different time constants in different DUM neurons. Time constants of the membrane potential were shorter in most DUM neurons than in auditory sensory neurons. Patterned stimuli with known behavioral relevance evoked a broad range of responses in DUM neurons: low-pass, band-pass, and high-pass characteristics were encountered. Temporal and carrier frequency processing were not correlated. Those DUM neurons producing action potentials showed divergent processing of temporal patterns when the graded potential or the spiking was analyzed separately. The extent of membrane potential fluctuations mimicking the patterned stimuli was different between otherwise similarly responding neurons. Different kinds of inhibition were apparent and their relevance for temporal processing is discussed.

The Auditory System of the Dipteran Parasitoid Emblemasoma auditrix (Sarcophagidae).

Several taxa of insects evolved a tympanate ear at different body positions, whereby the ear is composed of common parts: a scolopidial sense organ, a tracheal air space, and a tympanal membrane. Here, we analyzed the anatomy and physiology of the ear at the ventral prothorax of the sarcophagid fly, Emblemasoma auditrix (Soper). We used micro-computed tomography to analyze the ear and its tracheal air space in relation to the body morphology. Both tympana are separated by a small cuticular bridge, face in the same frontal direction, and are backed by a single tracheal enlargement. This enlargement is connected to the anterior spiracles at the dorsofrontal thorax and is continuous with the tracheal network in the thorax and in the abdomen. Analyses of responses of auditory afferents and interneurons show that the ear is broadly tuned, with a sensitivity peak at 5 kHz. Single-cell recordings of auditory interneurons indicate a frequency- and intensity-dependent tuning, whereby some neurons react best to 9 kHz, the peak frequency of the host's calling song. The results are compared to the convergently evolved ear in Tachinidae (Diptera).

Selective forces on origin, adaptation and reduction of tympanal ears in insects.

Insect ears evolved many times independently. As a consequence, a striking diversity exists in the location, construction and behavioural implementation of ears. In this review, we first summarise what is known about the evolutionary origin of ears and the presumed precursor organs in the various insect groups. Thereafter, we focus on selective forces for making and keeping an ear: we discuss detecting and localising predators and conspecifics, including establishing new "private" channels for intraspecific communication. More advanced aspects involve judging the distance of conspecifics, or assessing individual quality from songs which makes auditory processing a means for exerting sexual selection on mating partners. We try to identify negative selective forces, mainly in the context of energy expenditure for developing and keeping an ear, but also in conjunction with acoustic communication, which incorporates risks like eavesdropping by predators and parasitoids. We then discuss balancing pressures, which might oppose optimising an ear for a specific task (when it serves different functions, for example). Subsequently, we describe various scenarios that might have led to a reduction or complete loss of ears in evolution. Finally, we describe cases of sex differences in ears and potential reasons for their appearance.

Response differences of intersegmental auditory neurons recorded close to or far away from the presumed spike-generating zone.

Intracellular recordings may give valuable information about processing of a neuron and possibly its input from the network. Impalement with an electrode causes injury to the cell and depolarization from intrusion of extracellular fluid. Thus, penetration artefacts may contaminate recordings and conceal or even alter relevant information. These penetration artefacts may have the strongest impact close to the spike-generating zone near the dendrites. Recordings in axonal portions might therefore be less vulnerable while providing insufficient information about the synaptic input. In this study, we present data of five previously identified intersegmental auditory neurons of a bushcricket independently recorded in their dendrites (prothorax) and axon (brain). Generally, responses to acoustic pulses of the same parameter combination were similar within a neuronal class at the two recording sites. However, all neuronal classes showed significantly higher response variability and a tendency for higher spike activity when recorded in the dendrites. Unexpectedly, the combined activity of two neurons (Ascending Neurons 1 and 2) recorded in the brain provides a better fit to song recognition than when recorded in the thorax. Axonal recordings of T-shaped Neuron 1 revealed graded potentials originating in the brain and modulating its output in a potentially behaviourally relevant manner.

True katydids (Pseudophyllinae) from Guadeloupe: acoustic signals and functional considerations of song production.

Guadeloupe, the largest of the Leeward Islands, harbors three species of Pseudophyllinae (Orthoptera: Tettigoniidae) belonging to distinct tribes. This study examined the basic aspects of sound production and acousto-vibratory behavior of these species. As the songs of many Pseudophyllinae are complex and peak at high frequencies, they require high quality recordings. Wild specimens were therefore recorded ex situ. Collected specimens were used in structure-function experiments. Karukerana aguilari Bonfils (Pterophyllini) is a large species with a mirror in each tegmen and conspicuous folds over the mirror. It sings 4-6 syllables, each comprising 10-20 pulses, with several peaks in the frequency spectrum between 4 and 20 kHz. The song is among the loudest in Orthoptera (> 125 dB SPL in 10 cm distance). The folds are protective and have no function in song production. Both mirrors may work independently in sound radiation. Nesonotus reticulatus (Fabricius) (Cocconotini) produces verses from two syllables at irregular intervals. The song peaks around 20 kHz. While singing, the males often produce a tremulation signal with the abdomen at about 8-10 Hz. To our knowledge, it is the first record of simultaneous calling song and tremulation in Orthoptera. Other males reply to the tremulation with their own tremulation. Xerophyllopteryx fumosa (Brunner von Wattenwyl) (Pleminiini) is a large, bark-like species, producing a syllable of around 20 pulses. The syllables are produced with irregular rhythms (often two with shorter intervals). The song peaks around 2-3 kHz and 10 kHz. The hind wings are relatively thick and are held between the half opened tegmina during singing. Removal of the hind wings reduces song intensity by about 5 dB, especially of the low frequency component, suggesting that the hind wings have a role in amplifying the song.

Local prothoracic auditory neurons in Ensifera.

A new method for individually staining insect neurons with metal ions was described in the late 60s, closely followed by the introduction of the first bright fluorescent dye, Lucifer Yellow, for the same purpose. These milestones enabled an unprecedented level of detail regarding the neuronal basis of sensory processes such as hearing. Due to their conspicuous auditory behavior, orthopterans rapidly established themselves as a popular model for studies on hearing (first identified auditory neuron: 1974; first local auditory interneuron: 1977). Although crickets (Ensifera, Gryllidae) surpassed grasshoppers (Caelifera) as the main model taxon, surprisingly few neuronal elements have been described in crickets. More auditory neurons are described for bush crickets (Ensifera, Tettigoniidae), but due to their great biodiversity, the described auditory neurons in bush crickets are scattered over distantly related groups, hence being confounded by potential differences in the neuronal pathways themselves. Our review will outline all local auditory elements described in ensiferans so far. We will focus on one bush cricket species, Ancistrura nigrovittata (Phaneropterinae), which has the so-far highest diversity of identified auditory interneurons within Ensifera. We will present one novel and three previously described local prothoracic auditory neuron classes, comparing their morphology and aspects of sensory processing. Finally, we will hypothesize about their functions and evolutionary connections between ensiferan insects.

Are all auditory sensilla of bushcrickets bimodal? Comment on: R. D. Zhantiev and O. S. Korsunovskaya, Functions of chordotonal sensilla in bushcrickets (Orthoptera, Tettigoniidae); Entomological Review, 2021, vol. 101 (6), pp. 755-766.

Detection of sound and substrate vibration is crucial for the survival and reproduction of many animals, particularly insects. Bushcrickets (Orthoptera, Tettigoniidae), developed a large mechanosensory organ complex in their legs to detect such stimuli. As demonstrated by various studies in the past, sensilla in distinct functional groups form specialized vibratory organs (the subgenual organ and the accessory organ), respond sensitively to both vibration and sound (in the intermediate organ [IO]), or mediate hearing (in the crista acustica [CA]; the tympanal hearing organ). In their recent publication, Zhantiev and Korsunovskaya addressed auditory and vibratory sensitivity in the IO and the CA in two species of bushcrickets, using single-cell recording and staining of sensory neurons from their soma in an isolated foreleg. Their main finding was that not only the IO but also the complete CA contains bimodal sensilla responding with high sensitivity to both sound and vibration, which would be a true change in the paradigm of how the auditory/vibratory sense in Orthoptera works. In addition, they revealed vibratory tuning of the IO sensilla, which differs largely from that in previous studies. We propose three major experimental causes of such discrepancies: calibration, experiments with isolated legs, and differences in the sites of recording. To judge the causes of these discrepancies more adequately, a detailed comparison of methods and a number of control experiments are needed. This will deepen our understanding of sensory adaptations and specialization of insect mechanosensory organs to stimuli entering the system by different input pathways.

Timescale-invariant representation of acoustic communication signals by a bursting neuron.

Acoustic communication often involves complex sound motifs in which the relative durations of individual elements, but not their absolute durations, convey meaning. Decoding such signals requires an explicit or implicit calculation of the ratios between time intervals. Using grasshopper communication as a model, we demonstrate how this seemingly difficult computation can be solved in real time by a small set of auditory neurons. One of these cells, an ascending interneuron, generates bursts of action potentials in response to the rhythmic syllable-pause structure of grasshopper calls. Our data show that these bursts are preferentially triggered at syllable onset; the number of spikes within the burst is linearly correlated with the duration of the preceding pause. Integrating the number of spikes over a fixed time window therefore leads to a total spike count that reflects the characteristic syllable-to-pause ratio of the species while being invariant to playing back the call faster or slower. Such a timescale-invariant recognition is essential under natural conditions, because grasshoppers do not thermoregulate; the call of a sender sitting in the shade will be slower than that of a grasshopper in the sun. Our results show that timescale-invariant stimulus recognition can be implemented at the single-cell level without directly calculating the ratio between pulse and interpulse durations.

Timescale-invariant pattern recognition by feedforward inhibition and parallel signal processing.

The timescale-invariant recognition of temporal stimulus sequences is vital for many species and poses a challenge for their sensory systems. Here we present a simple mechanistic model to address this computational task, based on recent observations in insects that use rhythmic acoustic communication signals for mate finding. In the model framework, feedforward inhibition leads to burst-like response patterns in one neuron of the circuit. Integrating these responses over a fixed time window by a readout neuron creates a timescale-invariant stimulus representation. Only two additional processing channels, each with a feature detector and a readout neuron, plus one final coincidence detector for all three parallel signal streams, are needed to account for the behavioral data. In contrast to previous solutions to the general time-warp problem, no time delay lines or sophisticated neural architectures are required. Our results suggest a new computational role for feedforward inhibition and underscore the power of parallel signal processing.

Vibratory interneurons in the non-hearing cave cricket indicate evolutionary origin of sound processing elements in Ensifera.

Tympanal hearing organs in the front tibiae of ensiferan insects supposedly evolved from vibration-sensitive tibial organs (TO), like those in the cave cricket Troglophilus neglectus (Rhaphidophoridae). If this is true, one expects to find interneurons in the cave cricket that are homologous to auditory neurons from hearing Ensifera. Therefore, we examined the central projections of the foreleg TO of the cave cricket, as well as morphology and response properties of interneurons responding to foreleg vibration. Sensory axons of the TO adjoined to the "tympanal nerve" terminate in the equivalent portion of the ring tract neuropile in the prothoracic ganglion as corresponding receptors of crickets and weta. We found nine putatively homologous elements to sound- and/or vibration-sensitive interneurons of Ensifera--one local neuron (unpaired median, DUM), three T-fibres (TN), three descending (DN) and two ascending neurons (AN). Presumable first-order interneurons arborising in the ring tract correspond to a local auditory DUM cell of bush crickets and to TN1, DN1 and AN2 of various Ensifera, respectively. Homologues of some prominent auditory cells, the "omega" neuron(s) and the ascending neuron 1 (AN1), however, were not found. We conclude that (a) T. neglectus interneurons are morphologically primitive with respect to those of hearing taxa, (b) significant changes in the dendritic structure/synaptic connectivity have taken place during the evolution of the most specialised first-order auditory interneurons of Ensifera, (c) the data do not contradict independent evolution of hearing in Grylloidea and Tettigonoidea. Other interneurons appear morpho-physiologically conserved across hearing and non-hearing species, possibly as a part of a multimodal "alert" system.

Evolutionarily conserved coding properties of auditory neurons across grasshopper species.

We investigated encoding properties of identified auditory interneurons in two not closely related grasshopper species (Acrididae). The neurons can be homologized on the basis of their similar morphologies and physiologies. As test stimuli, we used the species-specific stridulation signals of Chorthippus biguttulus, which evidently are not relevant for the other species, Locusta migratoria. We recorded spike trains produced in response to these signals from several neuron types at the first levels of the auditory pathway in both species. Using a spike train metric to quantify differences between neuronal responses, we found a high similarity in the responses of homologous neurons: interspecific differences between the responses of homologous neurons in the two species were not significantly larger than intraspecific differences (between several specimens of a neuron in one species). These results suggest that the elements of the thoracic auditory pathway have been strongly conserved during the evolutionary divergence of these species. According to the 'efficient coding' hypothesis, an adaptation of the thoracic auditory pathway to the specific needs of acoustic communication could be expected. We conclude that there must have been stabilizing selective forces at work that conserved coding characteristics and prevented such an adaptation.

Auditory DUM neurons in a bush-cricket: A filter bank for carrier frequency.

In bush-crickets the first stage of central auditory processing occurs in the prothoracic ganglion. About 15 to 50 different auditory dorsal unpaired median neurons (DUM neurons) exist but they have not been studied in any detail. These DUM neurons may be classified into seven different morphological types, although, there is only limited correlation between morphology and physiological responses. Ninety seven percent of the stained neurons were local, 3% were intersegmental. About 90% project nearly exclusively into the auditory neuropile, and 45% into restricted areas therein. Lateral extensions overlap with the axons of primary auditory sensory neurons close to their branching point. DUM neurons are typically tuned to frequencies covering the range between 2 and 50 kHz and thereby may establish a filter bank for carrier frequency. Less than 10% of DUM neurons have their branches in adjacent and more posterior regions of the auditory neuropile and are mostly tuned to low frequencies, less sensitive than the other types and respond to vibration. Thirty five percent of DUM show indications of inhibition, either through reduced responses at higher intensities, or by hyperpolarizing responses to sound. Most DUM neurons produce phasic spike responses preferably at higher intensities. Spikes may be elicited by intracellular current injection. Preliminary data suggest that auditory DUM neurons have GABA as transmitter and therefore may inhibit other auditory interneurons. From all known local auditory neurons, only DUM neurons have frequency specific responses which appear suited for local processing relevant for acoustic communication in bush crickets.

Morphology and physiology of the prosternal chordotonal organ of the sarcophagid fly Sarcophaga bullata (Parker).

The anatomy and the physiology of the prosternal chordotonal organ (pCO) within the prothorax of Sarcophaga bullata is analysed. Neuroanatomical studies illustrate that the approximately 35 sensory axons terminate within the median ventral association centre of the different neuromeres of the thoracico-abdominal ganglion. At the single-cell level two classes of receptor cells can be discriminated physiologically and morphologically: receptor cells with dorso-lateral branches in the mesothoracic neuromere are insensitive to frequencies below approximately 1 kHz. Receptor cells without such branches respond most sensitive at lower frequencies. Absolute thresholds vary between 0.2 and 8m/s(2) for different frequencies. The sensory information is transmitted to the brain via ascending interneurons. Functional analyses reveal a mechanical transmission of forced head rotations and of foreleg vibrations to the attachment site of the pCO. In summed action potential recordings a physiological correlate was found to stimuli with parameters of leg vibrations, rather than to those of head rotation. The data represent a first physiological study of a putative predecessor organ of an insect ear.

Brain regions for sound processing and song release in a small grasshopper.

We investigated brain regions - mostly neuropils - that process auditory information relevant for the initiation of response songs of female grasshoppers Chorthippus biguttulus during bidirectional intraspecific acoustic communication. Male-female acoustic duets in the species Ch. biguttulus require the perception of sounds, their recognition as a species- and gender-specific signal and the initiation of commands that activate thoracic pattern generating circuits to drive the sound-producing stridulatory movements of the hind legs. To study sensory-to-motor processing during acoustic communication we used multielectrodes that allowed simultaneous recordings of acoustically stimulated electrical activity from several ascending auditory interneurons or local brain neurons and subsequent electrical stimulation of the recording site. Auditory activity was detected in the lateral protocerebrum (where most of the described ascending auditory interneurons terminate), in the superior medial protocerebrum and in the central complex, that has previously been implicated in the control of sound production. Neural responses to behaviorally attractive sound stimuli showed no or only poor correlation with behavioral responses. Current injections into the lateral protocerebrum, the central complex and the deuto-/tritocerebrum (close to the cerebro-cervical fascicles), but not into the superior medial protocerebrum, elicited species-typical stridulation with high success rate. Latencies and numbers of phrases produced by electrical stimulation were different between these brain regions. Our results indicate three brain regions (likely neuropils) where auditory activity can be detected with two of these regions being potentially involved in song initiation.

Multielectrode recordings from auditory neurons in the brain of a small grasshopper.

BACKGROUND: Grasshoppers have been used as a model system to study the neuronal basis of insect acoustic behavior. Auditory neurons have been described from intracellular recordings. The growing interest to study population activity of neurons has been satisfied so far with artificially combining data from different individuals. NEW METHOD: We for the first time used multielectrode recordings from a small grasshopper brain. We used three 12µm tungsten wires (combined in a multielectrode) to record from local brain neurons and from a population of auditory neurons entering the brain from the thorax. Spikes of the recorded units were separated by sorting algorithms and spike collision analysis. RESULTS: The tungsten wires enabled stable recordings with high signal to noise ratio. Due to the tight temporal coupling of auditory activity to the stimulus spike collisions were frequent and collision analysis retrieved 10-15% of additional spikes. Marking the electrode position was possible using a fluorescent dye or electrocoagulation with high current. Physiological identification of units described from intracellular recordings was hard to achieve. COMPARISON WITH EXISTING METHODS: 12µm tungsten wires gave a better signal to noise ratio than 15µm copper wires previously used in recordings from bees' brains. Recording the population activity of auditory neurons in one individual prevents interindividual and trial-to-trial variability which otherwise reduce the validity of the analysis. Double intracellular recordings have quite low success rate and therefore are rarely achieved and their stability is much lower than that of multielectrode recordings which allows sampling of data for 30min or more.

Useless hearing in male Emblemasoma auditrix (Diptera, Sarcophagidae)--a case of intralocus sexual conflict during evolution of a complex sense organ?

Sensory modalities typically are important for both sexes, although sex-specific functional adaptations may occur frequently. This is true for hearing as well. Consequently, distinct behavioural functions were identified for the different insect hearing systems. Here we describe a first case, where a trait of an evolutionary novelty and a highly specialized hearing organ is adaptive in only one sex. The main function of hearing of the parasitoid fly Emblemasoma auditrix is to locate the host, males of the cicada species Okanagana rimosa, by their calling song. This task is performed by female flies, which deposit larvae into the host. We show that male E. auditrix possess a hearing sense as well. The morphology of the tympanal organ of male E. auditrix is rather similar to the female ear, which is 8% broader than the male ear. In both sexes the physiological hearing threshold is tuned to 5 kHz. Behavioural tests show that males are able to orient towards the host calling song, although phonotaxis often is incomplete. However, despite extensive observations in the field and substantial knowledge of the biology of E. auditrix, no potentially adaptive function of the male auditory sense has been identified. This unique hearing system might represent an intralocus sexual conflict, as the complex sense organ and the behavioural relevant neuronal network is adaptive for only one sex. The correlated evolution of the sense organ in both sexes might impose substantial constraints on the sensory properties of the ear. Similar constraints, although hidden, might also apply to other sensory systems in which behavioural functions differ between sexes.

Frequency processing at consecutive levels in the auditory system of bush crickets (tettigoniidae).

We asked how processing of male signals in the auditory pathway of the bush cricket Ancistrura nigrovittata (Phaneropterinae, Tettigoniidae) changes from the ear to the brain. From 37 sensory neurons in the crista acustica single elements (cells 8 or 9) have frequency tuning corresponding closely to the behavioral tuning of the females. Nevertheless, one-quarter of sensory neurons (approximately cells 9 to 18) excite the ascending neuron 1 (AN1), which is best tuned to the male's song carrier frequency. AN1 receives frequency-dependent inhibition, reducing sensitivity especially in the ultrasound. When recorded in the brain, AN1 shows slightly lower overall activity than when recorded in the prothoracic ganglion close to the spike-generating zone. This difference is significant in the ultrasonic range. The first identified local brain neuron in a bush cricket (LBN1) is described. Its dendrites overlap with some of AN1-terminations in the brain. Its frequency tuning and intensity dependence strongly suggest a direct postsynaptic connection to AN1. Spiking in LBN1 is only elicited after summation of excitatory postsynaptic potentials evoked by individual AN1-action potentials. This serves a filtering mechanism that reduces the sensitivity of LBN1 and also its responsiveness to ultrasound as compared to AN1. Consequently, spike latencies of LBN1 are long (>30 ms) despite its being a second-order interneuron. Additionally, LBN1 receives frequency-specific inhibition, most likely further reducing its responses to ultrasound. This demonstrates that frequency-specific inhibition is redundant in two directly connected interneurons on subsequent levels in the auditory system.

Hearing and frequency dependence of auditory interneurons in the parasitoid fly Homotrixa alleni (Tachinidae: Ormiini).

The parasitoid tachinid fly Homotrixa alleni detects its hosts by their acoustic signals. The tympanal organ of the fly is located at the prothorax and contains scolopidial sensory units of different size and orientation. The tympanal membrane vibrates in the frequency range of approximately 4-35 kHz, which is also reflected in the hearing threshold measured at the neck connective. The auditory organ is not tuned to the peak frequency (5 kHz) of the main host, the bush cricket Sciarasaga quadrata. Auditory afferents project in the three thoracic neuromeres. Most of the ascending interneurons branch in all thoracic neuromeres and terminate in the deutocerebrum of the brain. The interneurons do not differ considerably in frequency tuning, but in their sensitivity with lowest thresholds around 30 dB SPL. Suprathreshold responses of most neurons depend on frequency and intensity, indicating inhibitory influence at higher intensities. Some neurons respond particularly well at low frequency sounds (around 5 kHz) and high intensities (80-90 dB SPL), and thus may be involved in detection of the primary host, S. quadrata. The auditory system of H. alleni contains auditory interneurons reacting in a wide range of temporal patterns from strictly phasic to tonic and with clear differences in frequency responses.

Diversity of intersegmental auditory neurons in a bush cricket.

Various auditory interneurons of the duetting bush cricket Ancistrura nigrovittata with axons ascending to the brain are presented. In this species, more intersegmental sound-activated neurons have been identified than in any other bush cricket so far, among them a new type of ascending neuron with posterior soma in the prothoracic ganglion (AN4). These interneurons show not only morphological differences in the prothoracic ganglion and the brain, but also respond differently to carrier frequencies, intensity and direction. As a set of neurons, they show graded differences for all of these parameters. A response type not described among intersegmental neurons of crickets and other bush crickets so far is found in the AN3 neuron with a tonic response, broad frequency tuning and little directional dependence. All neurons, with the exception of AN3, respond in a relatively similar manner to the temporal patterns of the male song: phasically to high syllable repetitions and rhythmically to low syllable repetitions. The strongest coupling to the temporal pattern is found in TN1. In contrast to behavior the neuronal responses depend little on syllable duration. AN4, AN5 and TN1 respond well to the female song. AN4 (at higher intensities) and TN1 respond well to a complete duet.