J Neurophysiol 88: 2322–2328, 2002; 10.1152/jn.00119.2002. Encoding of Sound Localization Cues by an Identified Auditory Interneuron: Effects of Stimulus Temporal Pattern ANNIE-HE´ LE` NE SAMSON AND GERALD S. POLLACK Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada Received 19 February 2002; accepted in final form 1 August 2002 Samson, Annie-He´le`ne and Gerald S. Pollack. Encoding of sound Two features of the neural response to acoustic stimuli vary localization cues by an identified auditory interneuron: effects of with stimulus intensity: spike count and/or rate increases with stimulus temporal pattern. J Neurophysiol 88: 2322–2328, 2002; increasing intensity and response latency decreases (Imaizumi 10.1152/jn.00119.2002. An important cue for sound localization is binaural comparison of stimulus intensity. Two features of neuronal and Pollack 2001; Kiang et al. 1965). The binaural difference responses, response strength, i.e., spike count and/or rate, and re- in both of these measures varies systematically with sound sponse latency, vary with stimulus intensity, and binaural comparison location, and either or both might code for sound direction of either or both might underlie localization. Previous studies at the (Boyan 1979; Eggermont 1998; Mo¨rchen 1980). receptor-neuron level showed that these response features are affected Crickets localize sound both to find mates and to evade by the stimulus temporal pattern. When sounds are repeated rapidly, predators. Stridulating male crickets produce songs with rela- as occurs in many natural sounds, response strength decreases and tively low carrier frequency (dominant frequency for Te- latency increases, resulting in altered coding of localization cues. In leogryllus oceanicus: 4.5 kHz), consisting of repeated trains of 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 sound pulses at rates varying (for T. oceancius) between 8 and cricket auditory system, with emphasis on the effects of stimulus 32 pulses/s (Balakrishnan and Pollack 1996). Echolocating temporal pattern on binaural response differences. AN2 spike count bats produce ultrasound pulses (20 to Ͼ100 kHz), repeated at decreases with rapidly repeated stimulation and latency increases. rates ranging from a few pulses per second to Ͼ100 pulses/s Both effects depend on stimulus intensity. Because of the difference (Griffin et al. 1960). Behavioral experiments have demon- in intensity at the two ears, binaural differences in spike count and strated that crickets perform positive phonotaxis (movement latency change as stimulation continues. The binaural difference in toward the sound source) in response to cricket-like stimuli and spike count decreases, whereas the difference in latency increases. negative phonotaxis (movement away from the sound source) The proportional changes in response strength and in latency are when presented with bat-like sounds (Moiseff et al. 1978; greater at the interneuron level than at the receptor level, suggesting Nolen and Hoy 1986; Pollack et al. 1984). The importance of that factors in addition to decrement of receptor responses are in- volved. Intracellular recordings reveal that a slowly building, long- the frequency ranges of cricket song and bat sounds is reflected lasting hyperpolarization is established in AN2. At the same time, the by the organization of the cricket’s ear; nearly three-quarters of level of depolarization reached during the excitatory postsynaptic the approximately 70 receptor neurons in each ear are tuned to potential (EPSP) resulting from each sound stimulus decreases. Nei- cricket-like frequencies, and more than one-half of the remain- ther these effects on membrane potential nor the changes in spiking der are most sensitive to ultrasonic frequencies (Imaizumi and response are accounted for by contralateral inhibition. Based on Pollack 1999). comparison of our results with earlier behavioral experiments, it is When sensory neurons are stimulated with a long series of unlikely that crickets use the binaural difference in latency of AN2 rapidly repeated stimuli, their response strength (spike count responses as the main cue for determining sound direction, leaving the and/or rate) decreases for successive stimuli, and response difference in response strength, i.e., spike count and/or rate, as the latency increases (Coro et al. 1998; Givois and Pollack 2000; most likely candidate. Pasztor 1983). In cricket auditory receptors, as in mammals (Eggermont and Spoor 1973), the decline in response strength is greater the higher the sound level (Givois and Pollack 2000). INTRODUCTION This implies that neurons ipsilateral to the sound source, where Sound localization in the horizontal plane is based on com- stimulus intensity is higher, will experience a larger response paring sounds at the two ears. Sound arrives earlier at the ear decrement. This may even lead to a reversal in the sign of the closer to the source, and in general stimulus intensity is higher binaural difference in response strength (contralateral re- at that ear. Some animals can detect the small (tens of micro- sponseϾ ipsilateral; Givois and Pollack 2000). The change in seconds) binaural difference in arrival time (for review, see latency of receptor-neuron responses induced by repeated stim- Carr 1993); others, including insects, rely mainly on binaural ulation does not depend on sound level; thus directional infor- intensity difference as the cue for sound location (for review, mation encoded by binaural latency difference is not affected. see Pollack 1998). Phonotaxis responses are not driven directly by receptor Address for reprint requests: G. S. Pollack, Dept. of Biology, McGill The costs of publication of this article were defrayed in part by the payment University, 1205 Ave. Doctor Penfield, Montreal, Quebec H3A 1B1, Canada of page charges. The article must therefore be hereby marked ‘‘advertisement’’ (E-mail: [email protected]). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2322 0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society www.jn.org TEMPORAL PATTERN AND SOUND LOCALIZATION CUES 2323 neurons, but rather by interneurons that receive input from receptors. Negative phonotaxis in response to ultrasound stim- uli is initiated by the identified, bilaterally paired interneuron AN2 (Nolen and Hoy 1984). The effects of the changes in receptor responses described above might be compensated or exaggerated during signal transmission from receptors to in- terneurons, but as yet this issue has not been studied. In this study, we investigate the effects of rapidly repeated stimulation on encoding of sound localization cues by AN2. METHODS Animals Teleogryllus oceanicus were raised in the laboratory on a diet of Purina Cat Chow and water. Unmated females, ages 12–20 days after the final molt, were used in experiments. Crickets were anesthetized by chilling on ice and were mounted on a wax support, ventral side up, after removing their wings and mid- and hind legs. In most experi- ments, the femur of the fore legs was fixed with a bees’ wax- colophonium mixture, parallel to the body axis, and the tibia and tarsus were held flexed against the femur, simulating their position during flight. When recordings were made from receptor neurons, the legs were rotated around the coxa-trochanter-femur joints so as to project perpendicularly from the body axis, exposing the anterior surface of the femur for electrode implantation (see Electrophysiol- ogy). FIG. 1. Effect of repeated stimulation on spike count (A) and first-spike Electrophysiology latency (B) of AN2 responses. Sound pulses were presented at 16 and 0.3 pps (for clarity, responses to 0.3 pps are shown only for 90 dB). Each point is the For extracellular recordings of AN2, the cervical connectives were response to 1 sound pulse; data are from a single, representative experiment. In exposed and placed on a pair of silver-wire hook electrodes. AN2 A, for time points Ͼ1 s, only every 5th data point is plotted for 16 pps. Solid produces the largest sound-evoked spikes in the cervical connectives, lines (for 16 pps,70 dB in A;70dBinB) are curves fit to an equation of the ϭ ϩ Ϫt/ss ϩ Ϫt/ll and these are easily recognized by visual inspection (Moiseff and Hoy form: r(t) rss ae be , where r(t) is response at time t, rss is the 1983, see inset of Fig. 1A). To record compound action potentials of steady-state response, ss and ll are short and long time constants, and a and b auditory receptor neurons, we inserted a Teflon-coated silver wire are constants. Inset: extracellular recording, from a different experiment, (114 ␮m OD) into the anterio-dorsal surface of the femur, close to the showing AN2 spikes (top; first 8 responses; 90 dB, 16 pps) and stimulus nerve branch carrying the axons of the receptor neurons from their monitor (bottom). origin in the prothoracic tibia to the prothoracic ganglion. The indif- ferent electrode was placed proximally and ventrally in the femur (see the series. Stimuli were produced, using LabWindows programs (Na- Pollack and Faulkes 1998 for further details). For intracellular record- tional Instruments), by a D/A circuit (ATMIO16F5, National Instru- ings of AN2, the prothoracic ganglion was exposed and supported on ments, Austin; D/A update rate 200 kHz) and were attenuated (PA4, a metal platform. The ganglion was submerged in physiological saline Tucker-Davis, Gainesville, FL), amplified (D150A, Amcron, Elkhart, (Strausfeld et al. 1983) and recordings were made using microelec- IN), and broadcast through loudspeakers (RadioShack, 40–1310B) trodes (Ͼ30 M⍀) filled with2MKacetate. Electrophysiological situated on the cricket’s left and right in the horizontal plane, perpen- recordings were digitized (Digidata 1320A, 10 kHz sampling rate, dicular to the longitudinal axis, at a distance of 50 cm. The loud- Axon Instruments) using the program AxoScope 8.0 (Axon Instru- speakers and cricket were housed in a chamber lined with echo- suppressing mineral-wool wedges.