Extracellular recordings of the discharges of a prominent interneuron, the AN1-neuron, were used to examine both the tuning of the hearing system and the peripheral directionality. Action potential activity was recorded with extracellular tungsten hook-electrodes placed at the connectives between prothoracic and suboesophageal ganglia. Insects were shortly anesthesized with chlorethylene and fixed ventral side up on a thin (1 mm) platform with dental wax. The forelegs were fixed in the natural walking position onto thin wires (diameter 0.6 mm). The preparation was sealed with petroleum jelly to prevent desiccation of the connectives. AN1-discharges were amplified by a custom-made amplifier, visualized on an oscilloscope (Agilent 54616B) and monitored through headphones. The threshold for each stimulus was defined as the SPL which elicited at least one AP in each syllable in at least three out of five stimulations.
Neurophysiological experiments were performed in an acoustically isolated Faraday-cage at room temperature between 21–23°C. A speaker (Raveland MHX 138) was placed at a distance of 40 cm, at an angular separation of 30° left or 30° right of the longitudinal body axis. Model songs identical to those used in behaviour were generated, amplified and attenuated in steps of 1 dB using a Tucker-Davis system (Alachua, Florida). The tuning of AN1 was determined with ipsilateral stimulation at frequencies ranging from 3.5 to 6 kHz, in increments of 100 or 500 Hz. In order to measure only the directionality provided by the anatomical arrangement of the acoustic tracheae in the periphery, inhibitory central nervous interactions were eliminated by cutting the contralateral leg nerve with the afferent auditory fibres of the opposite ear. IIDs were calculated by measuring the thresholds of the AN1-neuron for frequencies between 3.5 to 6 kHz with ipsilateral and contralateral stimulation at an angular frontal deviation of 30°. The threshold differences between ipsi- and contralateral stimulation represent the IID for a given frequency at a stimulation angle of ±30°, similar to biophysical measurements using laser-vibrometry [27] ). Pronotum width was measured as an index of structural body size to the nearest 0.1 mm, using a digital calliper.
Neurophysiological experiments were performed in an acoustically isolated Faraday-cage at room temperature between 21–23°C. A speaker (Raveland MHX 138) was placed at a distance of 40 cm, at an angular separation of 30° left or 30° right of the longitudinal body axis. Model songs identical to those used in behaviour were generated, amplified and attenuated in steps of 1 dB using a Tucker-Davis system (Alachua, Florida). The tuning of AN1 was determined with ipsilateral stimulation at frequencies ranging from 3.5 to 6 kHz, in increments of 100 or 500 Hz. In order to measure only the directionality provided by the anatomical arrangement of the acoustic tracheae in the periphery, inhibitory central nervous interactions were eliminated by cutting the contralateral leg nerve with the afferent auditory fibres of the opposite ear. IIDs were calculated by measuring the thresholds of the AN1-neuron for frequencies between 3.5 to 6 kHz with ipsilateral and contralateral stimulation at an angular frontal deviation of 30°. The threshold differences between ipsi- and contralateral stimulation represent the IID for a given frequency at a stimulation angle of ±30°, similar to biophysical measurements using laser-vibrometry [27] ). Pronotum width was measured as an index of structural body size to the nearest 0.1 mm, using a digital calliper.
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